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Bioavailability of organic phosphorus in a shallow hypereutrophic lake

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Title:
Bioavailability of organic phosphorus in a shallow hypereutrophic lake
Creator:
Newman, Susan, 1963-
Publication Date:
Language:
English
Physical Description:
xiii, 180 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Bodies of water ( jstor )
Chlorophylls ( jstor )
Enzymes ( jstor )
Lakes ( jstor )
Nutrients ( jstor )
Phosphatases ( jstor )
Phosphorus ( jstor )
Phytoplankton ( jstor )
Plankton ( jstor )
Sediments ( jstor )
Dissertations, Academic -- Soil Science -- UF
Freshwater plankton -- Florida -- Apopka, Lake ( lcsh )
Lakes -- Florida -- Apopka, Lake ( lcsh )
Soil Science thesis Ph. D
Water -- Phosphorus content -- Florida -- Apopka, Lake ( lcsh )
Apopka, Lake (Fla.) ( lcsh )
Lake Apopka ( local )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1991.
Bibliography:
Includes bibliographical references (leaves 168-179).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Susan Newman.

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University of Florida
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University of Florida
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The University of Florida George A. Smathers Libraries respect the intellectual property rights of others and do not claim any copyright interest in this item. This item may be protected by copyright but is made available here under a claim of fair use (17 U.S.C. §107) for non-profit research and educational purposes. Users of this work have responsibility for determining copyright status prior to reusing, publishing or reproducing this item for purposes other than what is allowed by fair use or other copyright exemptions. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder. The Smathers Libraries would like to learn more about this item and invite individuals or organizations to contact the RDS coordinator (ufdissertations@uflib.ufl.edu) with any additional information they can provide.
Resource Identifier:
027762968 ( ALEPH )
26483324 ( OCLC )

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BIOAVAILABILITY OF ORGANIC PHOSPHORUS
IN A SHALLOW HYPEREUTROPHIC LAKE
















By

SUSAN NEWMAN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1991














ACKNOWLEDGEMENTS


I would like to thank Dr. Reddy, my major advisor, whose help and guidance made this undertaking an enjoyable learning experience. I would also like to thank the members of my committee, who were always willing to share their expertize. Appreciation is also expressed to Mr. Rick Aldridge whose assistance and lively discussion enabled me to conduct the nutrient enrichment experiments.
I would also like to thank my friends and colleagues in the Soil

Science Department, particularly those associated with the Wetland Soils Laboratory, whose encouragement, assistance and cooperation made this project flow more smoothly.
I would like to thank my parents, Joyce and Chris Newman, whose love and support enabled me to complete my Ph.D. Without their encouragement I would not have continued on to higher education.

Last but not least, I would like to thank Tom, whose love,

companionship and support boosted my morale numerous times throughout this study.











ii















TABLE OF CONTENTS


ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . .

LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . .


LIST OF FIGURES ........ .........................

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . .

CHAPTERS

I INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . .

Statement of the Problem .................
Need for Research .. .....................
Organic Phosphorus Mineralization. .............
Alkaline Phosphatase Activity in the Water Column .....
Alkaline Phosphatase Activity in the Sediment .........
Objectives . . . . . . . . . . . . . . . . . . . . . . . .
Dissertation Format ...... ....................

2 SEASONAL VARIABILITY IN ALKALINE PHOSPHATASE ACTIVITY IN A
SHALLOW HYPEREUTROPHIC LAKE .................

Introduction . . . . . . . . . . . . . . . . . . . . . . .
Materials and Methods ..... ....................
Results ........ ..........................
Discussion . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions ........ ........................

3 RESPONSE OF NATURAL PLANKTON POPULATIONS
TO NUTRIENT ENRICHMENT ...............


Introduction ....... Materials and Methods . . . Results .............
Discussion . . . . . . . . Conclusions ..........


iii


. viii


16 18 23
39
48









4 THE EFFECT OF SEDIMENT RESUSPENSION ON ALKALINE PHOSPHATASE
ACTIVITY ....... ....................... ...95

Introduction .... ........ ............ 95
Materials and Methods . . . . . . . . . . . . . . . . . . . . 97
Results ....... ........................... ...105
Discussion .... ..... ......................... 115
Conclusions ....... ......................... ...123
5 THE EFFECT OF SEDIMENT AND WATER COLUMN ANOXIA ON
ORGANIC PHOSPHORUS MINERALIZATION ............. ...124

Introduction .... ... ........................ . 124
Materials and Methods ....... .................... 127
Results ....... ........................... ...134
Discussion .... ..... ......................... 145
Conclusions ....... ......................... ...151

6 ORGANIC PHOSPHORUS CYCLING IN LAKE APOPKA ............ ..152

APPENDICES

A LORAN COORDINATES ........ ...................... 156

B CONCENTRATIONS OF SELECTED WATER CHEMISTRY
PARAMETERS DETERMINED BIMONTHLY FROM
APRIL 1989 THROUGH FEBRUARY 1990,
AT 8 SITES IN LAKE APOPKA ... ............... ..157
REFERENCE LIST ........ .......................... ..168

BIOGRAPHICAL SKETCH ....... ........................ ..180















LIST OF TABLES


Table gg

2-1. Means of selected weather data measured at the central
station (mean � 1 SE) ........ .................... 24

2-2. Correlation coefficients for chlorophyll and alkaline
phosphatase activity measured bimonthly at 7 sites in
Lake Apopka (significant at a=O.05, n=7) .... ........... 32

2-3. Correlation coefficients between alkaline phosphatase
activity and selected parameters measured bimonthly at
8 sites in Lake Apopka (significant at a=O.05, n=6) ........ 33

2-4. Correlation coefficients of selected water chemistry
parameters determined at 7 sites in October 1989
(significant at a=O.05, n=7) .... .. ................. 42

3-1. Nutrient additions made to diluted lake water collected
in November 1989 ....... ....................... 54

3-2. Nutrient additions made to diluted lake water collected
in April and August 1990 ....... ................... 56

3-3. Initial concentrations of selected parameters measured in
diluted lake water prior to nutrient addition
(triplicate samples) in November 1989 (mean � i SE) ........ 61

3-4. Chlorophyll a and specific alkaline phosphatase activity
measured in natural plankton populations collected in
November 1989, 72 h after receiving nitrogen and
phosphorus additions ...... ..................... .62

3-5. Initial concentrations of selected parameters measured
in diluted lake water prior to nutrient addition
(triplicate samples) (mean � 1 SE) ... .............. .66

3-6. Phosphorus uptake rates for natural plankton populations
collected in August 1990, 30 min after receiving nitrogen
and phosphorus additions (mean � I SE) .... ............ 74

3-7. Hot water extractable phosphorus concentrations of composite
lake water samples collected in April 1990, 216 h after
nutrient additions .... .. .. ...................... 78









pae


3-8. Specific hot water extractable phosphorus measured over time in natural plankton populations collected in August 1990
after receiving nitrogen and phosphorus additions
(mean � I SE) ....... .. ......................... 81
3-9. Specific hot water extractable phosphorus measured over time in natural plankton populations collected in August 1990
after receiving nitrogen and phosphorus additions
(mean � 1 SE) ....... ... ....................... 83

3-10. Specific alkaline phosphatase activity measured over time in
natural plankton populations collected in August 1990
after receiving nitrogen and phosphorus additions
(mean � I SE) ........ ......................... 81

4-1. Distribution of selected parameters measured in May 1989
within the water column at the center of Lake Apopka
(n=3) ....... ... ............................ 106
4-2. Concentrations of parameters measured within the water
column of cores collected in September 1989 from the
center of Lake Apopka (mean � I SE) ... ............. ...109

4-3. The distribution of alkaline phosphatase activity in
disturbed and undisturbed sediment cores collected from the
center of Lake Apopka in January 1990 (mean � 1 SE) ....... 118

5-1. Concentrations of selected parameters measured in Lake
Apopka water in July 1990 (mean � 1 SE) .... ........... 135

5-2. Concentrations of selected parameters measured on sediments
incubated under six different redox levels for one
month (mean � I SE) ....... ..................... 140

5-3. Concentrations of selected parameters measured on sediments
incubated under six different redox levels for one
month (mean � 1 SE) ....... ..................... 143
5-4. Selected correlation coefficients between phosphorus
compounds and alkaline phosphatase activity measured in
sediments incubated for one month under six different
redox levels ...... .... ........................ 144


APPENDIX TABLES

A. Loran coordinates of sampling sites on Lake Apopka
Group repetition interval:7980, Southeast USA ... ........ 156









Dage

B-1. Temperature ......... ......................... 157

B-2. Secchi depth transparency ..... .................. ..158

B-3. Dissolved oxygen ........ ...................... 159

B-4. pH ........ ............................. ...160

B-5. Total solids ....... ........................ . 161

B-6. Total suspended solids ....... ................... 162

B-i. Chlorophyll a ...... ........................ ...163

B-8. Total organic carbon ...... .................... ..164

B-9. Total Kjeldahl nitrogen ..... ................... ...165

B-10. Total and soluble alkaline phosphatase activity ......... ..166

B-11. Phosphorus ......... ......................... 167


vii














LIST OF FIGURES

Figure Page

1-1. Diagram of the phosphorus cycle in Lake Apopka ... ........ 4

2-1. Location of Lake Apopka and sampling sites .. .......... .19

2-2. Seasonal variability of selected parameters determined
bimonthly at 7 sites in Lake Apopka ..... .............. 26

2-3. Seasonal variability of selected parameters determined
bimonthly at 7 sites in Lake Apopka ..... .............. 27

2-4. Size fractionation of alkaline phosphatase activity
determined bimonthly at 3 sites in Lake Apopka ... ........ 29

2-5. Size fractionation of phosphorus concentrations determined
bimonthly at 3 sites in Lake Apopka ..... .............. 30

2-6. Diel variation of selected parameters measured February
6-7, 1990 at the center of Lake Apopka (site 8) .......... .35

2-7. Diel variation of selected parameters measured February
6-7, 1990 at the center of Lake Apopka (site 8) .......... .36

2-8. Diel variation of selected parameters measured February
6-7, 1990 at the center of Lake Apopka (site 8) .......... 37

2-9. Distribution of phosphorus compounds determined in whole
lake water at 8 sites in October 1989 ............... ..38

2-10. Distribution of phosphorus compounds determined in
filtered lake water at 8 sites in October 1989 ... ........ 40

2-11. Distribution of suspended phosphorus compounds determined
by difference between whole and soluble phosphorus measured
at 8 sites in October 1989 ..... .................. .41

3-1. Map showing the location of Lake Apopka and sampling sites.
Water was collected from site 1 in November 1989 and from
site 2 in April and August 1990 .... ................ .53


viii









oae
3-2. Time courses of alkaline phosphatase activity following
nutrient enrichment of natural plankton populations
collected in November 1989 ..... ................. ...63

3-3. Time courses of nutrient concentrations following
nutrient enrichment of natural plankton populations
collected in November 1989 ..... .................. .65

3-4. Time courses of chlorophyll a concentrations following
nutrient enrichment of natural plankton populations
collected in April 1990 ...... .................... 68

3-5. Time courses of chlorophyll a concentrations following
nutrient enrichment of natural plankton populations
collected in August 1990 ...... ................... 69
3-6. Time courses of soluble reactive phosphorus concentrations
following nutrient enrichment of natural plankton
populations collected in April 1990 ..... .............. 71
3-7. Time courses of soluble reactive phosphorus concentrations
following nutrient enrichment of natural plankton
populations collected in August 1990 .... ............. 73

3-8. Time courses of nutrient concentrations following nutrient
enrichment of natural plankton populations collected in
April 1990 ...... .. .......................... .75

3-9. Time courses of nutrient concentrations following nutrient
enrichment of natural plankton populations collected in
August 1990 grown at 29�C ...... ................... 76

3-10. Time courses of hot water extractable total soluble
phosphorus following nutrient enrichment of natural plankton
populations collected in August 1990 .... ............. 79

3-11. Time courses of hot water extractable soluble reactive
phosphorus following nutrient enrichment of natural plankton
populations collected in August 1990 ..... ............. 82

3-12. Time courses of alkaline phosphatase activity following
nutrient enrichment of natural plankton populations
collected in April 1990 ...... .................... 84
3-13. Time courses of alkaline phosphatase activity following
nutrient enrichment of natural plankton populations
collected in August 1990 ...... ................... 85

4-1. Map showing the location of Lake Apopka ... ............ 98

4-2. Diagram of the sediment resuspension device ... ......... 101

ix









p2aU


4-3. The depth distribution of selected parameters measured in
triplicate sediment cores collected in Masy 1989 from
the center of Lake Apopka ...... .................. 107
4-4. The depth distribution of selected parameters measured in
triplicate sediment cores collected in May 1989 from
the center of Lake Apopka ...... .................. 108

4-5. The depth distribution of alkaline phosphatase activity in
triplicate resuspended and undisturbed (control) sediment
cores collected in September 1989 from Lake Apopka .. ..... 111

4-6. Concentrations of selected parameters measured in the
overlying water column following sediment resuspension of
0, 2, 5, and 10 cm surficial sediments .... ........... 112

4-7. The total alkaline phosphatase activity measured in the
overlying water column following sediment resuspension of
0, 2, 5, and 10 cm surficial sediments .... ........... 113

4-8. The relationship between alkaline phosphatase activity and
total suspended solids in the overlying water column of
triplicate sediment cores after resuspension of surficial
sediments ........ .......................... 114
4-9. Soluble alkaline phosphatase activity measured in the
overlying water column following sediment resuspension of
0, 2, 5, and 10 cm surficial sediments .... ........... 116

4-10. Soluble reactive phosphorus concentrations measured in the
overlying water column following sediment resuspension of
0, 2, 5, and 10 cm surficial sediments .... ........... 117

5-1. Diagram illustrating the apparatus used to control dissolved
oxygen concentration and redox potential .... .......... 129

5-2. The sediment extraction scheme used to fractionate organic
phosphorus in sediment ...... ................... 131
5-3. Nutrient concentrations in Lake Apopka water incubated
in the dark under aerobic and anaerobic conditions .. ..... 136
5-4. Nutrient concentrations in Lake Apopka water incubated
in the dark under aerobic and anaerobic conditions .. ..... 138
5-5. Alkaline phosphatase activity in Lake Apopka water
incubated in the dark under aerobic and anaerobic
conditions ..... ... ......................... 139











5-6. Concentrations of selected parameters measured in sediments
incubated under six different redox levels for one month . . 141















Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

BIOAVAILABILITY OF ORGANIC PHOSPHORUS IN A SHALLOW HYPEREUTROPHIC LAKE

By

Susan Newman

May 1991
Chairman: K. R. Reddy
Major Department: Soil Science
Field and laboratory studies were conducted to determine the

importance of organic P mineralization in the sediment-water column of Lake Apopka, a shallow hypereutrophic lake located in central Florida. Alkaline phosphatase activity (APA) was used as a tool to indicate the bioavailability of organic P to native plankton populations.

Spatial and temporal variability in total APA occurred in the water column (range=4 to 45 nM min") in response to different water chemistry characteristics. Nutrient enrichment studies demonstrated that APA increased with plankton biomass and specific APA (APA/chlorophyll a) values > I nmol APA Ag chlorophyll a- min- occurred during severe inorganic P limitation. In both the sediment and the water column APA was mainly associated with particulate matter.

The APA of the plankton was inhibited by high inorganic P concentrations. Phosphorus demand of the plankton was high, as evidenced by the rapid uptake of added inorganic P. During the


xii









conditions of P limitation added inorganic P was immediately assimilated and recovered in the surplus P pool within the plankton tissue, as determined by hot water extraction. The plankton apparently utilized this surplus pool of P for growth under low external inorganic P concentrations.
Resuspension of surficial sediments increased the interaction between sediments and the overlying water column, resulting in an immediate increase in APA and total P (TP) in the water column, indicating an increased potential for biological organic P hydrolysis during periods of resuspension. The APA and TP decreased rapidly during settling of suspended solids, following the cessation of turbulence.

Organic P mineralization was greater under aerobic than anaerobic conditions in the sediment and overlying water column. Under aerobic conditions (dissolved oxygen [DO]-6 mg C") APA in the water column increased from 22 to 43 nM min-, while no change was observed under anaerobic conditions (DO=<0.2 mg L-). Sediment APA was a function of Eh, the measured reduction potential of the sediment-water systems. Under aerobic conditions (Eh=480 mV) APA was 10-fold higher than that observed under anaerobic conditions (Ehz-240 mV). Enzymatic hydrolysis of organic P compounds was significantly inhibited under anaerobic conditions.
The results from this study suggest that the P requirement of plankton in a highly productive lake may partially be met through the enzymatic hydrolysis of organic P. Consequently, efforts to reduce nutrient loading and thus reduce eutrophication should also evaluate the bioavailability of organic P compounds in the system.


xiii















CHAPTER 1
INTRODUCTION

Eutrophication may be defined as the nutrient and/or organic matter enrichment that produces high biological productivity (Likens 1972). This process is often accelerated by man, through allochthonous loading to the system from surface runoff, agricultural drainage and wastewater effluent.

Eutrophication of our waterbodies has recently become a major concern due to the ever increasing need for resource conservation. Consequently, efforts are now being made to further understand the process of eutrophication and to identify key management strategies to abate this process.

Statement of the Problem

Lake Apopka is a 12,500 ha lake located in central Florida. It has a mean water depth of 2 m, overlying highly flocculent organic sediments (Reddy and Graetz 1990). Historically, the lake had clear water, submersed macrophytes and supported substantial sport fish populations. However, the physico-chemical properties of the lake have been altered through nutrient enrichment following the construction of the Apopka-Beauclair canal, discharge of sewage to the lake, and back pumping from the surrounding muck farms (USEPA 1979). Following the 1947 hurricane, the submerged vegetation was uprooted, and the first











algal bloom was recorded (USEPA 1979). Since then, sport fish populations have dwindled and have been replaced by rough fish such as shad, gar and catfish. The high algal populations have resulted in the maintenance of a pea-green color in the lake. Lake Apopka has mean chlorophyll a concentrations > 60 Ag L1 and total P concentrations of 200 Ag L- (Canfield 1981; Huber et al. 1982; Reddy and Graetz 1990) and is thus currently classified as hypereutrophic (Forsberg and Ryding 1980). The lake is the first and largest in the Oklawaha chain of lakes, consequently a ripple effect is apparent. The high nutrient concentrations and algal blooms observed in Lake Apopka are evidenced downstream in the other lakes in the chain.


Need for Research

In order to understand the process of eutrophication in Lake Apopka and thus abate this process, it is necessary to determine the cycling of C, N and P in the sediment-water column. In general, N and P are the key elements involved in eutrophication (Chiaudani and Vighi 1982). Carbon fixation has been determined to be the driving force in the productivity of Lake Apopka (Reddy and Graetz 1990). This is apparent by the vast algal populations observed year round in the water column. Settling of senescent algal cells has resulted in a highly organic sediment. Consequently, both the sediment and the water column are dominated by organic matter which results in high levels of organic N and P. However, readily available P, i.e, soluble inorganic P concentrations are low and frequently undetectable (< 1 Ag L-1) (Newman,S., unpublished data, Department of Soil Science, University of











Florida, Gainesville, FL.). Researchers have investigated sorption reactions (Olila, 0., unpublished data, Department of Soil Science, University of Florida, Gainesville, FL.) and have characterized the cycling of inorganic P within the sediment and water column (Pollman, 1983; Reddy and Graetz 1990), but the dynamics of the organic P pool, the dominant form of P, have not been addressed. Studies have demonstrated that significant quantities of organic P may be bloavailable (Bradford and Peters 1987; Kuenzler and Perras 1965), hence the organic P pool may play a significant role in sustaining the vast plankton biomass under apparent inorganic P limitation. Therefore, the potential bioavailability of organic P in Lake Apopka needs to be determined.

Orqanic Phosphorus Mineralization

In aquatic systems, organic P in sediments constitute 15-50% of TP (Bostrom et al. 1982) while in the water column organic P may account for as much as 90% of TP (Rigler 1964). The P cycle is shown in Fig. 1-1. Organic P is generally characterized as total and soluble organic P. Specific identification of organic P constituents may be achieved following chromatographic fractionation and comparison with known compounds (Minear 1972; Weimer and Armstrong 1979), 3'P nuclear magnetic resonance (NMR) (Condron et al. 1985), or via hydrolysis by specific enzymes (Herbes 1974). Only 50% of organic P forms have been identified, including: inositol phosphates, sugar phosphates, phospholipids and nucleic acids (Stevenson 1982). The rate at which these compounds are mineralized is dependent upon their structure. High










































WATER COLUMN





























EXTERNAL




INPUT























Particulate





inorganic P


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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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. . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . * . . . . . . * - . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . * . . . . . . . . * . . . . . . . . . . . . . . . . . . . . . . . . * . . . . . . . . . . . . . . . . . .
.............
. . . . . . . . . . . . . . . . . . . . I . . . . . . . . . . . . . . . .


Figure 1-1. Diagram of the phosphorus cycle in Lake Apopka.









S

molecular weight, complex structures are highly resistant to mineralization, hence they will tend to accumulate, e.g. humic acids. Conversely, simple, low molecular weight organic compounds are more susceptible to hydrolysis and thus more labile, e.g. sugarphosphates. It is these labile compounds which are more likely to undergo rapid enzymatic hydrolysis and thus be bioavailable.
Soluble reactive P (SRP), the most labile form of P, has been the focus of most P cycling research (Hutchinson and Bowen 1950; Rigler 1956). However, SRP concentrations in lakes are generally low while organic P is abundant (Abbott 1957; Rigler 1956). Such observations resulted in investigations to determine whether organic P compounds could act as a source of P available for plankton nutrition. Under inorganic P limiting conditions phytoplankton may utilize organic P compounds for growth (Harvey 1953; Kuenzler 1965). These phytoplankton produce externally acting enzymes, phosphatases which hydrolyze phosphomonoesters (PME) and release SRP (Fitzgerald and Nelson 1966; Kuenzler and Perras 1965). Phytoplankton which do not produccce externally acting enzymes cannot hydrolyze PME compounds and their growth becomes P limited (Kuenzler 1965). Ecologically, the ability of phytoplankton to utilize organic P gives them a competitive advantage over non-phosphatase producers, during inorganic P limitation.
The mode of action of phosphatases (specifically

phosphomonoesterases) is shown below (Coleman and Gettins 1983):

1 2 3 4
ROP + E - ROPE E-P E.P # E + Pi


R = an organic moiety, Pi = inorganic P, E = enzyme.










The steps involved in the reaction are as follows:
1. The phosphomonoester binds non-covalently to the phosphatase

enzyme (ROP.E).
2. The phosphoseryl intermediate forms by covalent binding of

the phosphate group to the phosphatase enzyme (E-P); alcohol is

released during this nucleophilic attack.

3. Water is taken up resulting in the nucleophilic displacement of

serylphosphate to produce a non-covalently bound complex (E.P).
4. Inorganic P is released and the free phosphatase enzyme is

regenerated.


Phosphatases have a high degree of specificity for the P moiety of the P-0-C bond, but little specificity for the C moiety (Reid and Wilson 1971). These enzymes are classified as alkaline or acid depending on the pH range under which they exhibit optimum activity (Reichardt 1971; Torriani 1960). At acid pH, the dephosphorylation of the serylphosphate is the rate limiting step. At alkaline pH, the dissociation of inorganic P from E.P is the rate limiting step (Coleman and Gettins 1983). The alkaline nature of most aquatic systems has resulted in alkaline phosphatase activity (APA) receiving the most emphasis.

More than one type of phosphatase may be present in any plankton population. Five intracellular phosphatases were extracted from a Peridinium bloom (Wynne 1977). The phosphatases produced in response to P limitation do not have the same biochemical characteristics as those observed in normal tissues (Bielski 1974). Although acid phosphatases have the ability to function outside the cell (Kuenzler and Perras 1965;










Price 1962), they are generally intracellular in action (Moller et al. 1975; Wynne 1977). Acid phosphatases function as specific enzymes in metabolic pathways and non-specific reactions (Cembella et al. 1984a). Hence they are constitutive and generally not repressible by inorganic P. Conversely, APA exhibits principly extracellular function. Alkaline phosphatase synthesis may be induced by the presence of organic P (Aaronson and Patni 1976; Kuenzler 1965). Alkaline phosphatase is a repressible enzyme (Jansson et al. 1988), whose synthesis is inhibited by high levels of inorganic P (Elser and Kimmel 1986; Lien and Knutsen 1973; Torriani 1960). Inorganic P is a competitive inhibitor of APA (Coleman and Gettins 1983; Moore 1969; Reid and Wilson 1971). Other factors which affect APA include; temperature (Garen and Levinthal 1960; Torriani 1960), chelators and divalent cations (Cembella et al. 1984a; Healey 1973).

The derepression of APA in response to P limitation has been examined at the cellular level where APA was shown to transport inorganic P. Studies utilizing Escherichia coli have shown that two forms of P transport exist (Rosenberg et al. 1977). One is a low affinity system, phosphate inorganic transport (PIT), which is constitutive and transfers intracellular P pools. The other is a high affinity system, phosphate specific transport (PST), which is activated when internal P concentrations are low. This utilizes a membrane associated protein, APA, to increase P uptake. The high affinity system is inhibited at high concentrations of inorganic P. However, it is the ability of APA to catalyze the hydrolysis of organic P compounds that has received the most study in the aquatic environment.










The intensity of APA is dependent on the severity of P limitation. As much as 6% of the total protein produced under P limiting conditions may be attributed to APA (Garen and Levinthal 1960). The increase of APA in response to inorganic P deficiency has resulted in the use of APA as a tool to assess the P limitation of plankton.

Alkaline Phosphatase Activity in the Water Column

Inverse relationships between APA and SRP have been reported in many species of plankton (Healey 1973; Olsson 1990; Pettersson 1980; Pettersson et al. 1990). Under low SRP concentrations APA is derepressed, and upon replenishment of external inorganic P, APA is inhibited. In some situations no significant relationship is observed between APA and SRP (Berman 1970; Taft et al. 1977). It has been suggested that under these circumstances high concentrations of soluble organic P counteract the inhibition caused by SRP by stimulating induction of APA (Kuenzler 1965; Cembella et al. 1984a). Alternatively, where no correlation exists, APA may reflect P demand rather than P limitation (Taft et al. 1977).

In combination with the depletion of external concentrations of inorganic P, P limitation in plankton is also demonstrated by reduced internal P concentrations (Chr6st and Overbeck 1987; Rhee 1973). Inverse relationships between APA and surplus P have been recorded (Lien and Knutsen 1973; Rhee 1973). Once internal P concentrations have been reduced below critical levels, APA is produced (Chr6st and Overbeck 1987; Fuhs et al. 1972). Alkaline phosphatase activities have been










observed to be 25 times greater under P limitation than under P sufficiency (Fitzgerald and Nelson 1966).

Ecologically, the importance of APA is dependent on the cooccurrence of both substrates and enzymes. Numerous problems have been associated with the determination of PME concentrations. The most common method of determining PME has been to monitor SRP release from filtered lake water following the addition of pure alkaline phosphatase (Strickland and Parsons 1968). The simultaneous occurrence of PME and APA by cyanobacteria blooms has been observed under low SRP concentrations (Heath and Cooke 1975). In some lakes, the rate of inorganic P release from PME equals the rate of P uptake by the plankton. In other lakes a large discrepancy exists between these two rates, with inorganic P release being considerably less than uptake rate (Boavida and Heath 1988; Cotner and Heath 1988; Heath 1986), thus leading these researchers to conclude that APA is not important in P nutrition of plankton. One of the problems associated with this conclusion is the use of filtered lake water in the analyses, therefore the large particulate organic P pool is absent (Wetzel 1983). Phosphatases have also been shown to release P from particulate matter (Jansson 1977). Seventy-four percent of extractable P in phytoplankton is susceptible to enzymatic hydrolysis and 80% of the organisms involved in phytoplankton decomposition produce phosphatases (Halemejko and Chr6st 1984). These results, and the apparent absence of hydrolyzable soluble PME in the water column (Herbes 1974; Herbes et al. 1975; Pettersson 1980) suggest that it is the substrate availability that limits enzymatic P cycling not APA (Jansson et al. 1988).










The interpretation of APA as a measure of P limitation is complicated by the uncertainty of the origin of APA. Bacteria, phytoplankton and zooplankton are considered to be dominant contributors to this pool, and it is suggested that APA of algal origin is the most important in the epilimnion (Jansson et al. 1988). High levels of soluble APA indicate filterable activity and may reflect bacterial associated APA (Stewart and Wetzel 1982), zooplankton excretion (Jansson 1976; Wynne and Gophen 1981) and cell lysis (Berman 1970). In many lakes APA was determined to be mainly associated with phytoplankton, based on co-occurrence of APA and algal blooms (Heath and Cooke 1975), and as shown by correlations with chlorophyll a (Jones 1972a; Matavulj and Flint 1987; Siuda et al. 1982; Smith and Kalff 1981) and size fractionation of phosphatase activity (Chr6st et al. 1989; Jansson 1977). Alkaline phosphatase activity has also been attributed to bacteria through correlations with bacterial numbers (Jones 1972a; Kobori and Taga 1979a). In shallow lakes, a large portion of particulate material may be sedimentary in origin. Concentrations of P compounds and bacterial numbers may be higher in sediments. Interaction between sediment and the overlying water column may significantly affect the mineralization of organic P in the overlying water. Hence, wind events in shallow lakes can significantly affect APA.

Alkaline Phosphatase Activity in the Sediment

In lake sediments as much as 70% of TP can be in organic form

(Weimer and Armstrong 1979). In highly organic sediments, the relative abundance of organic substrates may result in enhanced breakdown of










organic P (Ayyakannu and Chandramohen 1971). Numerous enzymes can be utilized in organic P breakdown but the phosphatases, specifically APA, are the most frequently cited (Halstead and McKercher 1975; Skujins 1976; Speir and Ross 1978).

As observed in the water column, APA in soils is positively correlated with the concentration of organic matter (Harrison 1983; Speir 1976) and age of organic matter (Rojo et al. 1990). Much of the data concerning APA have been developed in upland soils (Geller and Dobrotvorskaya 1961; Juma and Tabatabai 1978; Tabatabai and Bremner 1969); few studies have investigated APA in sediments. However, highly significant APA has been reported in both freshwater (Klotz 1985a; Sayler et al. 1979) and marine sediments (Ayyakannu and Chandramohen 1971; Kobori and Taga 1979b).

Phosphatase activity decreases with soil (Juma and Tabatabai 1978) and sediment depth (Degobbis et al. 1984; Kobori and Taga 1979b). In upland soils, this has been shown to correspond to decreases in microbial biomass, C, N and organic P (Juma and Tabatabai 1978; Speir and Ross 1978; Baligar et al. 1988). In sediments, redox potential decreases significantly with depth, therefore an important distinction between mineralization of organic compounds in sediments versus the overlying water column is the concentration of oxygen. Limited oxygen diffusion and rapid consumption of oxygen results in an oxygenated layer at the sediment-water interface and decreasing oxygen with depth in the sediment (Charlton 1980; Bostr6m et al. 1982). Alkaline phosphatase activity is generally inhibited under anaerobic conditions, resulting in a slower rate of organic P mineralization (Pulford and Tabatabai 1988).








12

However, in shallow lakes, wind induced resuspension of sediments to the oxygenated water column, results in the rapid breakdown of organic P (Pomeroy et al. 1965). A significant positive correlation between SRP released and APA in the water column has been observed during sediment resuspension (Degobbis et al. 1984).

Resuspension can physically transport SRP to the overlying water column (Ryding and Forsberg 1977). It also increases the suspended solids concentration within the overlying water. This particulate material can provide 28-41% of algal available P (Dorich et al. 1985). Consequently, resuspension of sediments can result in enhanced enzyme activity and P availability within the water column. Hence sediments can play a significant role in organic P mineralization in the overlying water column.


Objectives

Bioavailability of organic P occurs through the action of enzymes (Kuenzler and Perras 1965). These enzymes catalyze the release of inorganic P from both soluble and particulate matter. Both APA and organic P concentrations increase with increased eutrophication (Jones 1979b). Hence, Lake Apopka, which is classified hypereutrophic, should support large concentrations of APA and organic P. This study is based on the hypothesis that enzyme mediated P release is used to support the vast algal populations during inorganic P limitation. Without this ability to utilize organic P at times of high P demand and inorganic P limitation, algal and bacterial species are nutrient stressed. Very little is known about the bioavailability of organic P










in sub-tropical lakes, consequently, research investigating the breakdown and utilization of organic P is essential in any assessment of lake eutrophication.
The main components influencing the cycling of organic P within the water column are; water chemistry, plankton uptake and release, and sediment resuspension (Fig.1-1). To understand the role of organic P in Lake Apopka the following questions were addressed.

(1) How is the enzymatic hydrolysis of organic P compounds
affected by other water chemistry parameters?
(2) Is Lake Apopka plankton APA inhibited by inorganic P and is

it produced in response to inorganic P limitation?
(3) What effect does sediment resuspension have upon organic P

mineralization rates?


The overall objective of this study was to evaluate the
significance of organic P compounds in Lake Apopka and determine their potential bioavailability. Specific objectives are listed below.

(1) Determine the seasonal, spatial and diel variability of APA

within the water column.
Alkaline phosphatase activity is produced by organisms, hence any factors such as changes in environmental conditions which affect metabolism may therefore affect APA. The predominant biotic group in Lake Apopka biota are the plankton, hence APA is linked to fluctuations in response to plankton metabolism.
(2) Determine the influence of inorganic P upon the growth of

natural plankton populations.











Soluble inorganic P is the most readily available form of P for plankton nutrition, however; in its absence organic P compounds may be used for growth. The enzymatic hydrolysis of organic P compounds is competitively inhibited by inorganic P. Soluble reactive P concentrations in Lake Apopka are hypothesized to be too low to inhibit APA. There is, however, the issue of internal concentrations of P which may regulate organic P hydrolysis outside the cell.

(3) Evaluate the effect of sediment resuspension upon the

mineralization of organic P in the sediment and overlying

water column.
In shallow lakes, wind induced resuspension of sediments into the overlying water column increases the interaction between these compartments. It is hypothesized that resuspension of sediment increases the concentration of organic substrates and associated microorganisms in the water column and thus increases mineralization.

(4) Determine the effect of anoxia on organic P mineralization

in the sediment and water column.
Mineralization of organic compounds proceeds more rapidly under aerobic than anaerobic conditions. Since a majority of sediments are anaerobic, it is hypothesized that APA will be inhibited under anaerobic conditions, resulting in a reduced mineralization rate.


Dissertation Format

Each chapter within this dissertation is written as an independent manuscript intended for future publication. Chapter 2 focuses upon the concentrations of P compounds and APA within the water column and the









15

various factors which affect them. Chapter 3 examines the P nutrient status of natural plankton populations. Chapter 4 investigates the effect of sediment-water column interactions upon organic P bioavailability. The effect of anaerobic/aerobic conditions on organic P bioavailability is examined in chapter 5. The overall conclusions and the significance of these results are discussed in chapter 6.














CHAPTER 2
SEASONAL VARIABILITY IN ALKALINE PHOSPHATASE ACTIVITY IN A SHALLOW HYPEREUTROPHIC LAKE

Introduction

Phosphorus is the major nutrient limiting plankton production in many temperate lakes (Schindler 1977). Soluble reactive P (SRP) has been the form of P most often studied (Rigler 1956); however, SRP is only a small fraction of the total P (TP) pool. A significant component of TP may be in organic form (Minear 1972; Rigler 1964). In lakes where inorganic P availability is low, plankton may produce phosphatase enzymes which hydrolyze organic P compounds with the release of inorganic P (Fitzgerald and Nelson 1966; Kuenzler and Perras 1965). These enzymes are designated alkaline or acid phosphatase, depending on the pH range of optimum activity (Kuenzler and Perras 1965; Torriani 1960). The alkaline nature of most water bodies has resulted in alkaline phosphatase activity (APA) receiving the most attention. Although some APA has been determined to be constitutive (Kuenzler 1965), plankton produce increased APA under conditions of P limitation. Alkaline phosphatase activity has hence been used as an indicator of P limitation, however, APA may also reflect P demand, as evidenced by a poor relationship between SRP and APA (Taft et al. 1977).

The release of inorganic P mediated by APA is dependent upon the percentage of organic P which is hydrolyzable by the enzyme. Thirty-six











percent of organic P in seawater (Kobori and Taga 1979a) and 32% of organic P in freshwater (Hino 1988) were hydrolyzed by phosphatase enzymes. Organic P of algal origin is particularly sensitive to hydrolysis; 74% of algal extracted P was hydrolyzed by APA, while 80% of organisms involved in the decomposition of plankton produced phosphatases (Halemejko and Chr6st 1984). In eutrophic situations TP and organic P concentrations can be high (Jones 1979b), resulting in higher APA levels than in lower trophic states (Jones 1979b; Pick 1987). Alkaline phosphatase activity may be a significant mechanism of satisfying high P demand in eutrophic situations, as well as a means of overcoming P limitation in nutrient poor environments. The intensity of APA is subject to the physico-chemical conditions in the environment. Enzyme activity is pH dependent and can respond negatively or positively to pH fluctuations (Torriani 1960). Dissolved oxygen (DO) and temperature also influence microbial enzyme activity and metabolism, thus they may directly or indirectly affect APA (Garen and Levinthal 1960). These environmental effects demonstrate the potential for seasonal and diel fluctuations in APA.

This chapter examines the impact of seasonality on APA in one of Florida's largest hypereutrophic lakes, Lake Apopka. Despite low SRP concentrations < I pg L-, chlorophyll a concentrations are regularly > 100 Ag LI (Canfield 1981; Reddy and Graetz 1990). High standing crops may be maintained through the rapid recycling of SRP or by obtaining P from other sources. Total soluble P (TSP) concentrations of 255 jg P L" have been recorded in Lake Apopka (Reddy and Graetz 1990). Total soluble P may represent bioavailable P (Bradford and









18

Peters 1987), hence organic P compounds in this pool may potentially be hydrolyzed by APA and release inorganic P. With the exception of extensive research by Berman and colleagues on Lake Kinneret, Israel (Berman 1970; Wynne and Berman 1980), the majority of studies investigating APA have been conducted in cold temperate zones. Warmer climates with mild winters which result in extended periods of productivity, may result in increased P demand. More studies in warmer climates are necessary.
The primary objective of this study was to examine the seasonal, spatial and diel changes in APA to determine whether it represents P limitation or high P demand. This would also determine what effect the water chemistry has upon APA. Zooplankton, phytoplankton and bacterioplankton may all contribute to the total APA pool (Jansson 1976; Jones 1979a; Kuenzler and Perras 1965; Wynne and Gophen 1981). A second objective was to estimate the relative importance of these contributors based on filter size fractionation. Total soluble P may be used as an indicator of bioavailable P; however, a third objective was to determine the relationship between APA and other components of the TP pool.


Materials and Methods
Site Description
Lake Apopka is a 12,500 ha, located in central Florida, 28" 37' N latitude, 81" 37' W longitude (Fig. 2-1). It has a mean depth of 2 m. Water influxes to the lake include Apopka Springs and backpumping from surrounding agricultural land. Outflow is northward through the Apopka-













ApopkaBeauclair Canal

4 HA Pump house

Beauclair Canal


Oklawaha Chain of Lakes Smiths Island 0/inte

r L Apok Montverde Ar2


k 0lA

,7 Apopka Spring
Lake
Apopka


Florida


Fig. 2-1. Location of Lake Apopka and sampling sites.










Beauclair canal. The St. John's River Water Management District (SJRWMD) weather station is located at the center of the lake (site 8).


Water Sampling
Bimonthly sampling. Lake water was collected bimonthly from April 1989 thru February 1990, from 8 sites in Lake Apopka (Fig. 2-1). Sites 1, 4 and 8 were selected to represent inflow, outflow and the center of the lake. Site 2 was selected to determine littoral zone influences. Site 5 was located close to a pump station and hence represented backpumping from the surrounding agricultural land. Site 7 was established close to an old fishing camp, and site 6 was selected to correspond to extensive sediment studies which were conducted with samples from that site. Site locations were established using Loran coordinates (Appendix A). Three replicate water samples were collected from a depth of 0.3 m using 1 L polyethylene bottles, from each site. Samples were stored on ice until return to the laboratory. Water was filtered through 0.45 im membrane filters (Gelman) and analyzed for soluble APA within 24 h. Other soluble parameters determined were total soluble P and SRP. Soluble particulate P was defined as TSP-SRP (SPP). Whole lake water was analyzed for total APA, total Kjeldahl N (TKN), TP, SRP, total solids (TS), total suspended solids (TSS), total organic carbon (TOC), and chlorophyll a. Seasonal water chemistry data collected at site 8 were compared with the weather data provided by the SJRWMD.
The contributors to the APA pool are frequently determined via filter fractionation (Chr6st and Overbeck 1987; Currie and Kalff 1984; Currie et al. 1986). To distinguish between the contribution of









21
phytoplankton and bacteria, water samples collected from sites 1, 4 and

8 received further filtration. Subsamples of water were filtered through 150, 8, 2.5, 0.45 and 0.2 pm filters. Water from these size fractions was analyzed for chlorophyll a and determined to represent 80, 9, 3, 1 and 0% chlorophyll a distribution. To minimize the filtration time, the filtration was not performed sequentially. Alkaline phosphatase activity and TP were determined on all samples.

Dissolved oxygen and temperature (YSI, model 58), and pH (Orion, model SA 230) were recorded at 0.3 m. Light penetration was estimated by measuring Secchi disk transparency. Samples were collected at approximately the same time at each sampling period to minimize the effects of diel variation.
Diel sampling. Diel studies were conducted on March 21 1989 and February 6 1990. In March 1989 DO and pH of the water were measured from a pontoon boat which was anchored at the central station (site 8) for 24 h. In February 1990, pH and DO measurements were determined by SJRWMD personnel. Water samples at both time periods were collected using an automatic sampler, and kept cool until returned to the laboratory for analysis.
Fractionation of lake water phosphorus. To elucidate the

relationship between P and APA in the water column, the P forms were separated analytically using discrete extraction procedures. In October 1989, water samples from all 8 sites were partitioned into total and soluble; reactive P, acid hydrolyzable and organic P (APHA 1985). Enzyme hydrolyzable P (EHP) was also determined.










Analytical Methods
Alkaline phosphatase activity was determined fluorometrically (Healey and Hendzel 1979). One half mL of substrate, 3-o-methylfluorescein phosphate (Sigma Chemicals), at a concentration determined to be non-limiting, in 0.1 M Tris buffer (Tham, Fisher Scientific) pH 8.6, was added to 4.0 mL of lake water in a cuvette. Both total (whole lake water) and soluble (filtered through 0.45 Pm Gelman membrane filter) APA were determined. The cuvettes were placed in a water bath (25"C). At timed intervals during a 20 min period the cuvettes were placed in the fluorometer and the fluorescence measured. The enzyme activity was measured as an increase in fluorescence as the substrate was enzymatically hydrolyzed to the fluorescent product. Fluorescence units were converted to enzyme activity using a standard calibration curve of 3-o-methylfluorescein (Sigma Chemicals). The fluorescence was measured using a Turner fluorometer No. 110, equipped with Turner lamp no. 110-853, in combination with 47 B primary and 2a12 secondary filters. Autoclaved lake water with substrate added was used as a control.
Chlorophyll a was determined spectrophotometrically following extraction with acetone (APHA (1002-G), 1985). Total organic C was measured using an 0. I. Corporation Model 524C TOC analyzer, following oxidation by potassium persulfate. Total P, TSP, and TKN were determined following Kjeldahl digestion. Soluble reactive P, TSP and TP were analyzed via ascorbic acid using standard methods (APHA 1985). Total solids and TSS were determined by standard methods (APHA 1985).









23
Enzyme hydrolyzable P was determined using the method of Strickland and Parsons (1968).

Statistics
Data were analyzed using SAS for personal computers, version 6 (SAS 1985). Pearson correlation coefficients were determined for all data.


Results

Measured parameters varied seasonally and spatially. Seasonal variability was generally greater than spatial variability (data presented graphically represent means of sites 2-8). Appendix B contains tables presenting data on a site basis and demonstrates spatial variability. Site 1 is the site of a natural spring, 80 ft deep and is not subject to the same wind induced mixing as the rest of the lake. Data from site 1 are discussed separately, to illustrate the effects of spring input to the lake.

Physico-chemical Characteristics
A 14*C range in water temperature was observed over the sampling period (Table 2-1). The highest water temperatures occurred in June and August, and coincided with maximum photosynthetically active radiation (PAR) (Table 2-1). Temperatures were coolest in October and December during the decline of daylength. Data for other physical and chemical characteristics are presented in Appendix B. Increased Secchi transparency was recorded during October and December. Dissolved oxygen concentrations ranged from 7 to 12 mg L1, with a mean of 9.6 mg L-.










Table 2-1. Means of selected weather data measured at the central
station (mean � I SE).


Date Wind speed Water PAR* I m 5 m temp
above surface

ym ---- km h-'---- C Amol m2 sI


8904 2 � 0 NA 23.6 NA

8906 9 � 0 10 � 0 29.8 548 � 29 8908 11 � 0 13 � 0 28.5 508 � 29 8910 11 � 0 20 � 0 19.4 238 � 14 8912 11 � 0 13 � 0 15.5 294 � 18 9002 13 � 0 18 � 0 20.0 282 � 16


PAR indicates photosynthetically
* NA indicates data not available. Source: Stites, D. L., unpublished Management District, Palatka, FL.


active radiation. data, St. John's River Water











Little variation in pH was observed with a range of 0.8 pH units observed between maximum and minimum pH. Total solids were also constant between 300 and 400 mg U1, until February when a particularly high concentration of 500 mg U' was recorded. Total suspended solids accounted for approximately 16% of TS and increased in February at all sites (Fig. 2-2a).
A distinct peak in chlorophyll a concentration was observed in August at 5 of the 7 sites (Fig. 2-2b). This peak was 3 fold higher than the minimum which occurred in February. Two peaks in TOC were apparent. One occurred in August, along with chlorophyll a (Fig. 2-2c) while the other occurred in February and probably corresponded to the increase in TSS which also occurred in February. Total Kjeldahl N tended to be higher in Spring and Summer and decreased in Fall and Winter (Fig. 2-2d).

Alkaline phosphatase activity was mainly associated with
particulate matter (Fig. 2-3a). Soluble APA averaged only 3% of total APA. Total APA peaked in both June and October; in contrast, soluble APA peaked in October and December.

Phosphorus was also determined to be mainly associated with particulate matter. Total P concentrations peaked in October (Fig. 2-3b). Total soluble P concentrations represented from 6 to 37% of TP in August and April respectively. Soluble reactive P concentrations were very low throughout the year (0 to 7 pg L-) and as such were a minor portion of TP.
All parameters measured at site I were generally much lower than those recorded at other stations (Appendix B). In particular DO and pH









































AJ AODF
1989-90


140 120 100

80 60


40 20


AJ AODF
1989-90


Seasonal variability of selected parameters determined bimonthly at 7 sites in Lake Apopka: a) total suspended solids, data were not collected in April and June; b) chlorophyll 4; c) total organic carbon, data were not collected in April; d) total Kjeldahl nitrogen. Vertical bars represent 1 SE.


100

80


-0


z
0 I
z
I

0

! C.)





E


40 20





40 30


20 10


0


Fig. 2-2.












25

20 15 10


5 0 500


400 300

200 100

0


APR JUN AUG OCT
1989-90


Fig. 2-3. Seasonal variability of
bimonthly at 7 sites in activity; b) phosphorus.


M TP
E TSP ; SRP


IL'


DEC FEB


selected parameters determined Lake Apopka: a) alkaline phosphatase Vertical bars represent 1 SE.


I










were considerably lower and Secchi was significantly greater. Apopka spring temperatures tend to be consistent throughout the year, only a 4C fluctuation in water temperature was observed. Algal biomass was significantly lower than observed in the rest of the lake. Annual chlorophyll a concentrations averaged 21.8 Ag L1 at site 1, while values averaged 81 pg L-1 at other sites.

The contribution of the various components in lake water to the total APA pool was determined via filter size fractionation. The distribution of APA followed that of chlorophyll -a, with the majority of the activity associated with the larger size fraction (Fig. 2-4), and the distribution of APA within the different size fractions remained constant throughout the year. The greatest amount of soluble APA occurred in December. In general, a greater proportion of APA was associated with 8 and 2.5 /m filtered samples in spring water at site 1, than was observed in samples from sites 4 and 8. A similar distribution was also determined for P (Fig. 2-5), although a greater proportion was associated with the soluble fraction. Phosphorus concentrations peaked in October at all three sites.
Relationships between Water Chemistry Data and Selected Environmental

Parameters
Seasonal patterns in PAR, wind speed and water temperature
collected at site 8 (lake center) were observed (Table 2-1). Water temperature and PAR peaked in June and August. Wind speed measured 5 m above the surface was generally greater than that measured 1 m above the surface (Table 2-1). Total P was positively correlated with wind speed observed 5 m above the water surface (r=0.88) and SRP was













qe==
I


APR JUN AUG OCT DEC FEB
1989-90


Size fractionation of alkaline phosphatase activity determined bimonthly at 3 sites in Lake Apopka: a) site l=inflow; b)site 4-ouflow; c) site 8-center of lake. Vertical bars represent 1 SE.


SITE 1 * WHOLE MIA 0M
D 150 psm C3 0.45 im M I jam Q O.20 pm


i~t fklam


Fig. 2-4.


50
40 30
20 10
0
50
40 30
20 10
0
50
40 30

20 10
0












0 % v
I


1989-90


Size fractionation of phosphorus concentrations determined bimonthly at 3 sites in Lake Apopka: a) site 1=inflow; b)site 4=ouflow; c) site 8=center of lake. Vertical bars represent 1 SE.


600 500
400 300
200 100
0
600 500
400 300
200
100
0
600
500
400 300
200 100
0


Fig. 2-5.


SITE 4 (b) SITE 8 (0) APR JUN AUG OCT DEC FEB










positively correlated with PAR (r=O.98). Total Kjeldahl N was inversely related to wind speed measured 1 m above the surface (r=-O.94), while TSS was highly positively correlated with the wind speed recorded 1 m above the surface (r=O.97). Total suspended solids were also positively correlated with chlorophyll a, and inversely correlated with TSP and SOP. Neither chlorophyll -a nor APA were correlated with any of the weather data. Relationships between Alkaline Phosphatase Activity, Chlorophyll a and

other Parameters
Alkaline phosphatase activity did not correlate with many water chemistry parameters (Table 2-2). A significant correlation was observed between chlorophyll a and total APA in December (r=0.82), while APA was inversely related to TSP (r=-O.86). Correlations among chlorophyll a, total APA and other measured parameters varied over time. Chlorophyll - correlated with P four out of the six sampling periods and total APA only correlated with P twice (Table 2-2). On a site by site basis, different correlations between APA and water chemistry parameters were observed (Table 2-3). Spatial variability of the factors affecting APA was apparent.

Utilizing annual means, total APA was negatively correlated with TOC (r=-O.97) and chlorophyll a was highly correlated with TS (r=-0.88). Specific APA (ratio of total APA/chlorophyll -a) was not correlated with any of the water chemistry parameters.

Diel Variability

On March 21 1989 and February 6 1990 selected parameters
influencing APA were measured over a 24 h period to determine diel











Table 2-2.


Correlation coefficients for chlorophyll a and alkaline phosphatase activity measured bimonthly at 7 sites in Lake Apopka (significant at a=0.05, n=7).


Month Correlations with

Chlorophyll a Total APA Soluble APA

April TKN 0.88 TP 0.75 NS"
TP 0.85 SPP -0.67
DO -0.74 temp -0.96
June TKN 0.92 pH -0.79
TSP 0.76 soluble APA 0.74 TOC 0.68 DO -0.76
SPP 0.78
August NS NS NS

October TOC -0.70 NS TKN -0.75
TSS 0.73 TP 0.75 SPP -0.66 temp 0.71 TS -0.67
December
total APA 0.82 TSP -0.86 TP 0.70
temp 0.68 SPP -0.86 TSP 0.81
secchi -0.80 SPP 0.81

February TP 0.71 TOC -0.69 NS



NS indicates not significant at a = 0.05.











Table 2-3.


Correlation coefficients between alkaline phosphatase activity and parameters measured bimonthly at 8 sites in Lake Apopka (significant at &=0.05, n=6)


Alkaline phosphatase activity
Site Total Soluble


1 DO 0.87
temp -0.81 temp -0.77
2 TKN -0.78 TKN -0.90
TOC -0.81 TP 0.89 TSP 0.85
SPP 0.86
secchi 0.85
3 soluble APA 0.82 TP 0.75
secchi 0.79 secchi 0.79
4 NS" CHL 0.75 DO 0.92
5 TP -0.75 secchi 0.90

6 TSS -0.93 secchi 0.81

7 NS TP -0.83 pH 0.73
8 TSP 0.83 NS
SPP 0.81
pH 0.80
DO 0.82
TOC -0.92


NS indicates not significant at e = 0.05.











variation. Diel DO changes were observed at both time periods. No significant changes in other measured water chemistry parameters were observed in March 1989, while parameters did exhibit change in February 1990. In February 1990, TKN concentrations remained constant during the first 12 h of sampling and then declined (Fig. 2-6a). Maximum TKN corresponded to high PAR and wind speed (Fig. 2-7a and 2-7b). The decline in TKN corresponded to the decrease of these two parameters. No significant change was observed for SRP, while TP concentrations fluctuated throughout the sampling period (Fig. 2-6b). Similar fluctuations were also observed for total and soluble APA (Fig. 2-8a). As observed for TKN, chlorophyll a concentrations tended to decrease in conjunction with decreased PAR and wind speed, however, the range was only 33 to 37 jg L-' (Fig. 2-8b).

Fractionation of Lake Water Phosphorus
In October, lake water samples were fractionated to determine the different forms of P present. Site variability in chlorophyll a and TSS concentrations was observed (Appendix B). Total organic C remained constant at 30 mg U' for all sites except site 1. Chlorophyll - and TOC were lower at site 1.
As determined above, most of the total APA was in the particulate fraction with the soluble APA contribution varying from site to site (Appendix B). The distribution of the various P forms also exhibited spatial variability (Fig. 2-9). In most sites TOP represented over 80% of TP. Total acid hydrolyzable P contributed 10% and total reactive P (TRP) contributed 3% to the TP pool. A similar distribution of these
















4k


TKN


31-


2



300 280 260


240


220 200


10:30 14:30 18:30 22:30 TIME (h)


02:30 06:30 10:30


Diel variation of selected paramters measured February 6-7, 1990 at the center of Lake Apopka (site 8): a) total Kjeldahl nitrogen; b) total phosphorus. Vertical bars represent 1 SE.


z
0
I

z
w
.)


z
0 l--


I I I


I I I I I


Fig. 2-6.


I I I I I


I


I I I


I I I











1500


1000


500


10:00 14:00 18:00 22:00 02:00 06:00 10:00 TIME (h)


Diel variation of selected parameters measured February 6-7, 1990 at the center of Lake Apopka (site 8): a) photosynthetically active radiation; b) wind speed
1 m above the water surface. Source: Stites, D. L., unpublished data, St. John's River Water Management District, Palatka, FL.


Fig. 2-7.










11.0
10. 0 - CA

9.0

8.0

1.0
@ Total
0.5 jjl



0.0



40

CHLOROPHYLL a (6) 38


36


34


32 30 I 10:30 14:30 18:30 22:30 02:30 06:30 10:30 TIME (h)


Diel variation of selected parameters measured February 6-7, 1990 at the center of Lake Apopka (site 8): a) alkaline phosphatase activity; b) chlorophyll a. Vertical bars represent I SE. Chlorophyll a values were measured on composite samples.


z
0
I

z

z
0
U


Fig. 2-8.







































1 2


3 4 5 6
SITE


7 8


Distribution of phosphorus compounds determined in whole lake water at 8 sites in October 1989. TP=total phosphorus, TOP= total organic phosphorus, TAH=total acid hydrolyzable phosphorus, TRP=total reactive phosphorus. Vertical bars represent 1 SE.


600


TOP

TAH
r TRP


z
0



W e 0,





0
I-


500


400 300


200


100


0


Fig. 2-9.









39

components was also observed in filtered lake water (Fig. 2-10), organic P represented > 90% of TSP. Soluble acid hydrolyzable P was a less significant contributor to the total soluble P pool. Soluble reactive P concentrations were negligible. Total soluble P accounted for 11 to 60% of TP at sites 8 and 7, respectively. The fraction of TP attributed to suspended material was determined by difference between total and soluble P fractions (Fig. 2-11). The distribution of P forms was similar to that observed in whole lake water. Suspended TP represented from 40 to 89% of TP. No enzyme hydrolyzable P was observed.

Correlating all the site means (including site 1), total APA was positively correlated with chlorophyll a (r=0.88), TOC (r=0.84) and TSS (r-0.85). However, plots of the data showed that these correlations were an artifact of low values for water chemistry parameters at site 1. Correlations without site 1 gave different conclusions (Table 2-4). Total APA was observed to be inversely correlated with the acid hydrolyzable fractions. Chlorophyll a was inversely correlated with TOC (r--0.68) and TSP (rz-0.66) and positively with TSS (r=0.73).


Discussion

Lake Apopka is a shallow lake with a surface area of 12,500 ha.

It has a small littoral zone and is subject to considerable wind induced sediment resuspension. Frequent mixing and mild winters may help to explain the lack of seasonality observed for several of the parameters measured. Chlorophyll a, however, did exhibit a seasonal response. Maximum chlorophyll I corresponded to high PAR and higher temperatures. Over all months, chlorophyll a was only significantly and
















200


150 100


50


2 3 4
SITE


Fig. 2-10.


5 6 7 8


Distribution of phosphorus compounds determined in filtered lake water at 8 sites in October 1989. TSP=total soluble phosphorus, SOP=soluble organic phosphorus, SAH-soluble acid hydrolyzable phosphorus, SRP-soluble reactive phosphorus. Vertical bars represent 1 SE.


I.

-J















- / TP
0 TOP
J TAH
TRP


1 2 3


4
SITE


5 6


Fig. 2-11.


Distribution of suspended phosphorus compounds determined by difference between whole and soluble P measured at 8 sites in October 1989. TP-total phosphorus, TOP-total organic phosphorus, TAH-total acid hydrolyzable phosphorus, TRP-total reactive phosphorus. Vertical bars represent 1 SE.


600 500


400 300


-.I


200 100











Table 2-4.


Correlation coefficients of selected water chemistry parameters determined at 7 sites in October 1989 (significant at &=0.05, n=7).


TP TRP TAH TOP TSP SAH SOP TSUSP SUSAHP CHL


TOP 1.00'
SAH 0.92 SOP 1.00 TSUSP 0.95 0.94 SUSRP 0.78
SUSAHP 0.99 0.86 SUSOP 0.97 0.96 0.99 TAPA -0.80 -0.73 -0.84 CHL -0.66* TOC -0.68 TSS -0.78 -0.74 0.73

Blank space indicates not significant at a=O.05.


* Significant at a=0.10.










inversely correlated with one water chemistry parameter, TS. This correlation along with the ratio TS/chlorophyll a show that chlorophyll A was not a dominant component of the solids in Lake Apopka during the sampling period. In a frequently mixed system such as Lake Apopka, the proportion of total solids which may be attributed to phytoplankton biomass will fluctuate considerably. Other contributors to TS include; bacteria, suspended sediment, zooplankton and inorganic and organic compounds. The inverse relationship between chlorophyll a and TS may be interpreted as follows; 1) increased herbivory by high zooplankton populations and 2) light limitation because of high suspended solids.

A peak in TSS was observed in February, which corresponds to the highest wind speed recorded 1 m above the water surface, and thus reflects wind induced sediment resuspension. In a shallow lake, a large proportion of particulate matter within the water column may frequently be attributed to sediment resuspension. The sediments have high P concentrations (Reddy and Graetz 1990) and resuspension will result in increased levels of TP within the water column; TP concentrations were positively correlated with wind speed (5 m above the surface). Conversely, TKN concentrations were inversely related to wind speed (1 m above the surface), and are mainly associated with the algal biomass.

The rate of P exchange between water and sediment increases during suspension of sediment (Pomeroy et al. 1965). Part of this exchange may be biological. Sediment resuspension into the oxygenated water column results in aerobic mineralization of organic P (Lee et al. 1977). Sediments exhibit significant phosphatase activity (Kobori and Taga 1979b; Ayyakannu and Chandramohen 1971) and a positive correlation










between SRP released and APA in the water column has been observed during sediment resuspension to the overlying water (Degobbis et al. 1984). Assuming resuspended sediment were contributing significantly to the predominately particulate APA pool in Lake Apopka, the ratio of APA/TSS is a better measure of enzyme activity than APA alone. Comparing monthly means, total APA/TSS was positively correlated with soluble APA (r=0.99) and inversely correlated with TOC (r=-1.00). The correlation between APA and TSS was only observed when data were compared on a site basis. The overall lack of correlation between TSS and APA in this study may be due to 1) insufficient sampling during periods of sediment resuspension, 2) the relationship is hidden by the variability in other parameters incorporated in the TSS pool, and 3) sediment resuspension does not contribute to APA activity.
Alkaline phosphatase activity has been significantly correlated
with ATP (Pettersson 1980), particulate organic matter (Gage and Gorham 1985) and chlorophyll a (Healey and Hendzel 1979a; Pettersson 1980). In this study, total APA was significantly correlated with chlorophyll a in December and inversely correlated with TOC in February. Comparing annual means, there is a strong inverse relationship between total APA and TOC. This may be explained by examining the components of the TOC pool; one contributor is humic material. Alkaline phosphatase activity can be inhibited by high concentrations of humic materials (Francko 1986). The inverse relationship observed between APA and TOC could be a result of binding of APA to organic material. Attachment of alkaline phosphatase enzymes to particulate matter may decrease activity, but may also increase longevity of the enzyme activity (Burns 1986).











Resuspension of sediment high in organic matter could bind enzymes and/or release sediment bound APA to the overlying water column. Organic inputs, living or dead should be considered when measuring APA (Healey and Hendzel 1980).
Soluble APA is positively related to Secchi (r=0.87) suggesting
that there is a relationship between APA and water quality. But the low proportion of APA observed in the soluble pool suggest that free dissolved enzymes from cell lysis (Berman 1970) and enzymes excreted by zooplankton (Wynne and Gophen 1981) were not as important as particulate associated APA in the system. Organic phosphorus mineralization is mainly be achieved by APA bound to particulate matter. Examining the data from all sites (excluding site 1), APA was infrequently related to chlorophyll a. The overall lack of correlation between APA and chlorophyll a may be hidden due to the frequent mixing of the lake water. The particulate nature of APA as determined by the size fractionation scheme corresponds to the chlorophyll a distribution. The correlation between APA and chlorophyll a leads to the expression of APA as a ratio, i.e., APA/chlorophyll _ (Pettersson 1980). This ratio tends to increase with P limitation and decrease with trophic state (Pick 1987). Combining data from numerous studies, Pettersson (1980) determined that a ratio between 0.2 to 0.7 nmol APA jg chlorophyll fl min- could be used to indicate P limitation. When ambient lake specific APA was consistently < 0.3 nmol APA Ag chlorophyll fl minratios greater than this were determined to indicate P limitation, i.e., elevated specific APA indicates P limitation (IstvAnovics et al. 1990). In this study a ratio of < 0.3 nmol APA jg chlorophyll a-' min" was










consistently observed, suggesting that plankton in Lake Apopka are generally not P limited. This conclusion tends to agree with other research findings from this lake (Aldridge, F.J., personal communication, Department of Fisheries and Aquaculture, University of Florida, Gainesville, FL.; Reddy and Graetz 1990). Specific APA was lowest when chlorophyll a peaked and SRP concentrations of 5 Ag L- were sufficient to support growth.

High particulate APA has been attributed to the location of

alkaline phosphatase in the cell wall of phytoplankton (Kuenzler and Perras 1965). Recent research has suggested that viable phytoplankton do not contribute much to the particulate APA pool (Stewart and Wetzel 1982). Lake Apopka is generally dominated by cyanobacteria, with large numbers of Lyngbya sp. and icrocystis sp. (Shannon and Brezonik 1972; Stites, D. L., personal communication, St. John's River Water Management District, Palatka, FL.) whose mucilaginous layers can support significant bacterial populations. It is likely that particulate APA is attributable to both phytoplankton and the associated bacteria. Use of specific APA (APA/chlorophyll a) to indicate P limitation of phytoplankton should be verified via nutrient enrichment bioassays.

Diel fluctuations in APA were observed, but these do not
correspond to any particular water chemistry parameter. This may be attributed to spatial patchiness and water movement (Berman 1970; Wynne 1981). Unlike TKN concentrations, APA did not settle out of the water column following wind subsidence. Diel variability of APA may be dependent upon the species composition of the phytoplankton biomass. In diel studies neither Tballassiosira pseudonana Hasle and Heimdal (Perry









47
1976), nor Selenastrum capricornutum Prinz (Klotz 1985b) exhibited diel responses. However, Smith and Kalff (1981) have shown that growth demands for P are more important than the species composition in determining APA.
Preliminary studies (data not shown) and April data demonstrated a strong relationship between APA and TP. Consequently TP, which would include cellular P, would indicate the P forms utilized by APA. However, this relationship was not observed the entire year. Total soluble P, has been suggested as a good indicator of bioavailable P in eutrophic lakes (Bradford and Peters 1987). Correlations between total and soluble APA and TSP were apparent in December, and at certain sites (Table 2-3). Both positive and negative relationships were observed. Total and soluble APA are apparently influenced by different parameters at different sites (Table 2-3, Huber et al. 1985).
In October, the P fractionation experiment demonstrated that APA was inversely related to the acid hydrolyzable P fractions. This suggests that APA may be regulated by acid hydrolyzable compounds, or alternatively they could be used as substrates for APA. Acid hydrolyzable P represents the condensed polyphosphates, a storage form of P in phytoplankton and bacteria. Surplus P concentrations within algal cells have been shown to regulate the production of APA (Fitzgerald and Nelson 1966; Lien and Knutsen 1973; Rhee 1973) and this pool is composed of polyphosphates (Rhee 1972, 1973; Elgavish and Elgavish 1980). Hence, the measurement of surplus P combined with APA could provide a better understanding of the system. Another component which may contribute to the understanding of APA is the concentration of










EHP. No EHP was found in Lake Apopka during the October fractionation experiment. However, this does not reflect the absence of these substrates. In some lakes the release rate of P from EHP satisfies the P uptake rate (Chr6st and Overbeck 1987) while in others a large discrepancy exists between these two rates (Heath 1986; Boavida and Heath 1988). Low EHP concentrations have been attributed to the rapid hydrolysis of this fraction (Berman 1970; Taft et al. 1977). Alternatively, this may reflect methodological problems; 1) the method to measure EHP requires the addition of extracted APA from Escherichia coli to filtered lake water. This enzyme was not adapted to this system and consequently may not be as efficient or may require a longer incubation time, 2) filtered lake water does not represent the entire P pool available, as particulate organic P, a large portion of TP, may also be susceptible to enzymatic hydrolysis (Jansson 1977), 3) enzymes added from E. coli were more inhibited by inorganic P additions than natural enzyme populations (Chr6st et al. 1986). The first and third problems were resolved by measuring the increase in SRP in filtered water without the addition of the enzyme (Chr6st et al. 1986). However, in a lake which has high particulate APA, this would not be a true representation of the potential APA.

Conclusions

Seasonal and spatial differences in water chemistry were observed. In general, seasonal variability was greater than spatial variability. The system was highly productive as evidenced by chlorophyll i concentrations > 150 pg U, and an annual mean of 81 jg LC. Annual










means for TP and TKN were 210 pg L1 and 4.8 mg L1, respectively, confirming the highly eutrophic state of the lake. Conversely, SRP concentrations were consistently < 10 Ag L-.

Alkaline phosphatase activity was mainly associated with

particulate matter and was dependent on different water chemistry parameters both seasonally and spatially. In general, APA was not correlated to chlorophyll a. The relationship between these parameters may be hidden as a result of the frequent mixing of the water column in Lake Apopka. Both positive and negative correlations between P and APA were observed. An inverse relationship existed between acid hydrolyzable P and APA, indicating polyphosphates may be controlling APA.


The particulate association of APA would suggest be correlated with TSS, but this was rarely observed. to the variability in the composition of the TSS pool. between APA and particulate may be both beneficial and Binding to particles results in increased longevity of it also may inhibit APA by binding to the active site, the inverse relationship between APA and TOC.
Future research should examine the components of


that APA should This may be due The relationship detrimental. the enzyme, but as indicated by


the TSS pool,


plankton and sediment and their effect on organic P mineralization under controlled conditions.














CHAPTER 3
RESPONSE OF NATURAL PLANKTON POPULATIONS TO NUTRIENT ENRICHMENT


Introduction

Soluble inorganic P is the main form of P utilized directly by plankton. Phytoplankton growth rates close to maximal have been determined in the apparent absence of inorganic P (Fuhs et al. 1972; Smith and Kalff 1981). Methods used to measure soluble inorganic P are for the most part limited in sensitivity (Rigler 1956; Tarapchak et al. 1982). This has resulted in the use of physiological indicators to determine the nutritional status of plankton. Information is obtained through a variety of methods including the determination of P uptake rates (Lean and White 1983; Rigler 1956), the measurement of surplus P concentrations (Fitzgerald and Nelson 1966; Rhee 1973) and the determination of alkaline phosphatase activity (APA) (Berman 1970; Kuenzler and Perras 1965). As external inorganic P concentrations decline, plankton are able to utilize internal pools of surplus P to maintain growth (Fitzgerald and Nelson 1966; Rhee 1972, 1973, 1974; Wynne and Berman 1980). Once this internal source of P has been reduced to a critical level some phytoplankton produce phosphatase enzymes, which hydrolyze organic P compounds to inorganic P, to satisfy nutritional demands (Chr6st and Overbeck 1987; Kuenzler and Perras 1965; Reichardt 1971). An inverse relationship between APA and surplus P has










been reported (Fitzgerald and Nelson 1966; Chr6st and Overbeck 1987). Inorganic P is a competitive inhibitor of APA (Coleman and Gettins 1983; Moore 1969; Reid and Wilson 1971). Upon replenishment of external inorganic P concentrations enzyme activity is inhibited (Lien and Knutsen 1973; Torriani 1960; Perry 1972) and surplus P accumulates (Rhee 1973). Surplus P is measured as hot water extractable P (HEP), and in conjunction with APA has been shown to accurately assess P demand in some lakes (Sproule and Kalff 1978; Pettersson 1980) but not in others (Wynne and Berman 1980). Wynne and Berman (1980) observed that the HEP concentration in Lake Kinneret, Israel, remained stable throughout the year even under conditions of P stress and concluded that HEP was a metabolic intermediate rather than a form of P storage.
Lake Apopka is a hypereutrophic lake with soluble reactive P (SRP) concentrations frequently < 1 jg L1. In contrast, concentrations of total soluble P (TSP) and APA are high (chapter 2). The objectives of this study were to determine whether high APA in Lake Apopka was due to high demand for P or P limitation, and to evaluate the N and P requirements of native plankton.

Materials and Methods

Site Description
Lake Apopka is a 12,500 ha lake located in central Florida (28* 37' N. latitude, 81" 37' W. longitude). It has a mean depth of

2 m. It has been proposed that the nutrient loading from the surrounding agricultural and urban areas has precipitated the current hypereutrophic conditions in the lake (USEPA 1979).










Sampling Procedures

Water samples were collected 30 cm below the water surface from

the west side of the lake on November 16 1989, and from the east side on April 18 and August 21 1990 (Fig. 3-1). Water was stored in polycarbonate and polyethylene carboys in the dark and at ambient laboratory temperature, for no more than 24 h prior to the start of the experiments.

Experimental Design
The effect of inorganic Dhosphorus concentrations upon alkaline
phosphatase activity
Lake water collected in November 1989 was diluted 2:1 (260 mL unfiltered:140 mL filtered) with filtered lake water (0.45 im), to reduce the chlorophyll A concentration. Four hundred mL were placed in each of 15 wide mouth 500 mL erlenmeyer flasks. The experimental design was completely randomized with 5 treatments and 3 replicates. The water was spiked with nutrient additions (Table 3-1). Nitrogen was added at an N:P ratio of 10:1 to avoid N limitation. The flasks were capped with cotton wool and placed on magnetic stir plates, under a black plastic enclosure in the greenhouse. Temperature within the enclosure was maintained using window air conditioning units and fans (mean � 1SE,25*C � 0.21). Light was supplied at 200 p/ol photons m-2 s"1 using cool-white fluorescent lamps. The light:dark schedule was 16:8. The flasks were shaken and aliquots were withdrawn by syringe from treatments 1 and 2 at 0, 24, 72 and 96 h. Aliquots were removed from remaining treatments at 24, 72, and 168 h. Additional sampling times of 268 and 312 h were included for cultures which received 1000 pg 1. Samples requiring










L. Yale


I L Dora

Beauclair Canal
-


Oklawaha Chain of Lakes


Ctrus


Orlando A


Map showing the location of Lake Apopka and sampling sites. Water was collected from site 1 In November 1989, and from site 2 in April and August 1990.


Lake~


Lake-4
Apopka



Florida


Fig. 3-1.











Table 3-1.


Nutrient additions made to diluted lake water collected in November 1989.


Treatment Nutrient addition number
N" P

---- --- -- g L-1 - - - - - -

1 0 0 2 500 0 3 0 10 4 1000 100 5 2500 1000

N and P were added as potassium nitrate and potassium dihydrogen
phosphate, respectively.










filtration were immediately filtered through 25 mm membrane filters (0.45 pm) in polypropylene holders which attached to the syringes (Gelman). The samples were analyzed for chlorophyll a, total and soluble APA, total (TP), total Kjeldahl N (TKN), SRP, NH4-N, and [NO3 + NO2]-N.

Nutrient enrichment of natural Dlankton oooulations
Experiment 1. Whole lake water was diluted 1:1 with filtered lake water (0.45 pim) and 400 mL were placed in each of 15 wide mouth 500 mL erlenmeyer flasks. The basic experimental design was a 22 factorial with an additional 2 fold addition of both N and P included (Table 3-2). The flasks were stoppered with sponge plugs and placed in a clear glass circulating water bath maintained at ambient lake temperature (27"C). The flasks were illuminated from below at an irradiance of 145 /4mol photons m2 s-1. The contents of the flasks were mixed daily and immediately prior to sampling. Two h after the nutrient addition, aliquots were withdrawn by syringe at predetermined intervals and analyzed for total APA, SRP, NH4-N, [NO3 + N02]-N, TSP and chlorophyll a. Samples requiring filtration were filtered immediately as described above. Hot water extractable P (HEP-SRP) was determined at the beginning and conclusion of the experiment. To obtain a sufficient sample size, HEP-SRP was determined on composite samples containing all treatment replicates.
Experiment 2. To both confirm and compare the results with a different plankton population, a second experiment similar to that described above was conducted in August. Water samples were collected











Table 3-2.


Nutrient additions made to diluted lake water collected in April and August 1990.


Treatment Nutrient addition Incubation N" P temperature


-----------------g L---------- C
Experiment I-April

1 0 0 27 2 400 0 27 3 0 40 27 4 400 40 27 5 800 80 27
Experiment 2-August

1 0 0 29 2 400 0 29 3 0 40 29 4 400 40 29 5 800 80 29 6 0 0 19 7 0 40 19

N and P were added as potassium nitrate and potassium dihydrogen
phosphate, respectively.











from the same site, diluted and placed in water baths as described above. The temperature of the bath was set at 29gC to emulate ambient lake water temperature. Another water bath with cultures receiving no nutrient addition and P only was maintained at 19C, to determine the effect of temperature on the measured parameters. Nutrient uptake rates were determined by measuring the disappearance of the nutrient from solution. During the first 2 h following nutrient addition, the cultures were sampled every 1/2 h and analyzed for SRP, NH4-N, and [NO3 + N02]-N (actual sampling times were 0, 30, 67, 98 and 140 min). Immediately following filtration aliquots were analyzed for SRP. Samples to be analyzed for NH4-N and [N03 + N02]-N samples were acidified with concentrated H2S04 and stored at 4"C prior to analysis. Samples taken at 0 and 2 h were also analyzed for HEP-SRP, and hot water extractable TSP (HEP-TSP). Aliquots were subsequently removed at 24, 48 and 96 h and analyzed for SRP, NH4-N, [NO3 + N02]-N, total APA, TSP, HEP-SRP, HEP-TSP and chlorophyll a.

Analytical Methods

Alkaline phosphatase activity was determined fluorometrically (Healey and Hendzel 1979a). One half mL of substrate, 3-o-methylfluorescein phosphate (Sigma Chemicals), at a concentration determined to be non-limiting, in 0.1 M Tris buffer (Tham, Fisher Scientific) pH 8.6, was added to 4.0 mL of lake water in a cuvette. Both total (whole lake water) and soluble (filtered through 0.45 Am Gelman membrane filter) APA were determined. The cuvettes were placed in a water bath (250C). At timed intervals during a 20 min period the










cuvettes were placed in the fluorometer and fluorescence was measured. The enzyme activity was measured as an increase in fluorescence as the substrate was enzymatically hydrolyzed to the fluorescent product. Fluorescence units were converted to enzyme activity using a standard calibration curve of 3-o-methylfluorescein (Sigma Chemicals). The fluorescence was measured using a Sequoia Turner fluorometer Model 110, equipped with Turner lamp no. 110-853, in combination with 47 B excitation and 2a-12 emission filters. Autoclaved lake water with substrate added was used as a control.
Chlorophyll a was determined by measuring in vivo fluorescence, using a Turner Design Model 10 fluorometer equipped with a Turner lamp no. 110-853, in combination with 5-60 excitation and 2-64 emission filters. A Sequoia Turner fluorometer Model 110 equipped with the same light source and filters was used to measure chlorophyll _ fluorescence in the first experiment. Pheophytin _ fluoresces at the same wavelength as chlorophyll a, so chlorophyll a concentrations are uncorrected for pheophytin. The calculation of chlorophyll a was based on equations by Lorenzen (1967);


ABS x (vol. extracted (mL)) x (unit of measure factor) x 1 89 (vol. filtered (mL)) pathlength
(cm)

ABS = absorbance at 664 nm
89 = absorption coefficient for chlorophyll a in 90% acetone Unit of measure factor = 106 for Ag L1.










Initial and final chlorophyll a concentrations were calibrated against chlorophyll a concentrations determined spectrophotometrically following extraction with 90% acetone (APHA 1985).

Total P, TSP, SRP, NH4-N, and [NO3 + N02]-N were determined by standard methods (APHA 1985).

Hot water extractable P was determined in experiment 1 using a modification of the method by Fitzgerald and Nelson (1966). Water samples (50 to 100 mL) were filtered through 47 mm 0.45 pm membrane filters (Millipore). The filters were placed in 60 mL borosilicate boiling tubes, and 20 mL deionized water were added. The tubes were capped and autoclaved at 120�C for 1 h. Upon cooling samples were analyzed for SRP. Blank filters were autoclaved to determine any filter contribution to P analysis.
Hot water extractable P includes both a molybdate reactive form of P and a non-reactive form of P. Both hot water extractable forms were determined in experiment 2, using the method of Krausse and Sheets (1980). Nine mL of sample were filtered though 25 mm 0.45 pm membrane filters (Gelman). The filters were placed in 15 mL polypropylene tubes, and 13 mL of deionized water was added. The tubes were capped and autoclaved at 120�C for 1 h. Filter blanks were also autoclaved. Gelman filters were used instead of Millipore filters because they had a lower background P concentrations. The filtrate was analyzed for SRP, and also digested via persulfate oxidation and analyzed for TSP.











Statistical Methods
Data were analyzed using SAS (Statistical analysis systems) version 6. Balanced data (equal number of observations for each treatment) were analyzed using the repeated measures procedure which accounts for the within replicate correlation over time, due to repeated sampling from the same flasks. Unbalanced data (unequal number of observations per treatment) were analyzed using a split plot design with time as the subplot.

Results

The Effect of Inorqanic Phosphorus Concentrations upon Alkaline
Phosphatase Activity

The majority of N, P and APA were associated with particulate
matter (Table 3-3). Due to chlorophyll a analysis problems arising from fluorometer calibration, only chlorophyll a data from 0 and 72 h are presented and used for statistical analysis (Table 3-4). Increased chlorophyll a concentrations were observed at 72 h in all cultures except those which received no nutrient addition. Increased growth was associated with higher nutrient additions, a 69% increase in chlorophyll a was observed in treatment 5 (N-2500 P=1000) cultures. Chlorophyll a increases of 33% for treatment 4 (N=1000 P=100) and 11 % for treatments 2 (N=500 P=O) and 3 (N=O P=10) cultures were observed. The opposite was observed for both total and soluble APA (Fig. 3-2a and 3-2b). Total and soluble APA decreased significantly over time in cultures receiving the highest P additions (100 and 1000 pg L.). No decrease in total APA was observed for treatments 1 (N=O P=0) and 3 (N=O P=10), but soluble APA











Table 3-3.


Initial concentrations of selected parameters measured in diluted lake water prior to nutrient addition (triplicate samples) in November 1989 (mean � I SE).


Parameter


Concentration


Chlorophyll (pg L-1) TP (pg L). SRP (pg L") TKN (mg 11) NH4-N (mg U) [NO3 + N02]-N (mg L-1) Total APA (nM min-) Soluble APA (nl min-')


31 65

3

3.30 0.36

0.14 13.5

2.2


�6 �4 � 0.3 �0.1 � 0.08 � 0.00 � 0.3 � 0.13










Table 3-4.


Chlorophyll a and specific alkaline phosphatase activity measured in natural plankton populations collected in November 1989, 72 h after receiving nitrogen and phosphorus additions. 1-no nutrient addition, 2=500 Ig N LC, 3=10 jg P L1, 4=1000 pg N L-1 and 100 ug P U1, 5=2500 jg N L" and 1000 pg P L".


Treatment Chlorophyll Specific APA



Ag 1. nmol APA Ag chlorophyll a-' min1 36 a' 0.41 2 40 b 0.46 3 40 b 0.35 4 48 c 0.15 5 61 d 0.03


Numbers in a column followed significantly different at a


by the same letter are not = 0.05.


































0 48


96 144 192 240 288
TIME (h)


Time courses of alkaline phosphatase activity following nutrient enrichment of natural plankton populations collected in November 1989. ON,OP=no nutrient addition; 500N,OP=500 Ag N L-1; ON,1OP=1O Ag P U; IO00N,1OOP=1000 Ag N L-' and 100 Ag P L-1, and 2500N,IOOOP=2500 jg N U1 and 1000 /hg P L1: a) total alkaline phosphatase activity; b) soluble alkaline phosphatase activity. Vertical bars represent 1 SE. No vertical bar indicates SE is smaller than symbol size.


T


0C


0

--


2.5

2.0 1.5 1.0 0.5 0.0


Fig. 3-2.


25

20 15 10

5

0










did decrease. A significant increase in total APA was observed in cultures which received only N additions suggesting the onset of P limitation. This is more apparent when total APA data are presented as specific activity (i.e. total APA/chlorophyll a) (Table 3-4). A 15 fold difference between specific activity of those cultures which received treatment 5 (N-2500 P=1000) and treatment 2 (N=500 P-0) was observed. Initial specific APA was 0.44 nmol APA Ag chlorophyll fl min", hence P addition resulted in a decrease in specific APA. Significant decreases in SRP concentrations were observed within 24 h (Fig. 3-3a). Apart from treatment 5 (N-2500 P-1000), SRP concentrations were the same in all cultures after 2 h. After an initial rapid uptake from 1000 to 673 pg P L" within the first 2 h, SRP concentrations in cultures treated with 1000 pg P L" remained constant until 168 h, and then began to decrease. At 312 h, 377 pg P L", 37.6% of the original concentration remained in treatment 5 (N=2500 P=1000) cultures.

Nitrate concentrations also exhibited significant treatment

differences within 24 h (Fig. 3-3b). The concentrations at 24 h were ranked in descending order of original addition, with 1 and 3 treatments having equivalent [N03 + N02]-N concentrations. In all cultures there was a distinct decrease over time. A similar trend was observed for NH4-N, concentrations appeared to decrease within 24 h (Fig. 3-3c), however, no significant differences were determined. Nutrient Enrichment of Natural Plankton Populations

Growth. Chlorophyll a concentrations and APA were lower in April than in August (Table 3-5). Conversely, TP and TSP were











1200


0 48 96 144 192 240 288 TIME (h)


Time courses of nutrient concentrations following nutrient enrichment of natural plankton populations collected in November 1989. ON,OP=no nutrient addition, 500N,OP=500 Ag N L-1, ON,1OP=]0 jg P L-1, 1000N,100P=1000 jg N 11 and 100 jg P L-1, and 2500N, 1000P=2500 pg N L-1 and 1000 pg P L': a) soluble reactive phosphorus; b) (NO3 + N02]-N; c) NH4-N. Vertical bars represent 1 SE. No vertical bar indicates SE is smaller than symbol size.


-J
0~


Z
I,
N
0
z
+

0
z


800

400 100


0 3


E


0.3

0.2 0.1 0.0


(b)
* ON. OP V 500N. OP W ON. 10P A 1OON, loP
* 2500NJIOOOP


I-


Z

I
Z


Fig. 3-3.










Table 3-5. Initial concentrations of selected parameters measured in
diluted lake water prior to nutrient addition
(triplicate samples) (mean � I SE).



Parameter Samolin period April 1990 August 1990

Chlorophyll a (pg 1) 22.5 1 0 37.4 t 0 TP (pg L') 113 � 12 56 � 9 TSP (pg L') 70 � 10 9 � 0 SRP (g U) 1 � 0 2 � 0 HEP-TSP (pg L") ND* 33 � 3* HEP-SRP (pg U) 7 � 04 15 � 2* NH4-N (mg U) 0.04 � 0 0.05 � 0 [NO3 + N02]-N (mg U') 0 � 0 0.01 � 0 Total APA (nM min-) 9.1 � 0.2 12.7 � 0.1


ND indicates data not determined.
Determined by the method of Krausse and Sheets (1980).
Determined by the method of Fitzgerald and Nelson (1966).










significantly higher in April. Chlorophyll a was used as a means to indicate phytoplankton growth in the cultures. Chlorophyll ! concentrations increased in response to nutrient additions to the water samples collected in both April and August. Although small and hidden due to axis scale, significant treatment differences in chlorophyll 1 occurred within the first 24 h (Fig. 3-4 and 3-5a). After 96 h, maximum chlorophyll a concentrations in April were 80 jg U' and exceeded 180 pg L1 in August. In April, chlorophyll a concentrations were significantly affected by the interaction between concentrations of N and P added. Chlorophyll a concentrations obtained in the presence of both nutrients exceeded those obtained by single nutrient additions (Fig. 3-4). The greatest initial increase in chlorophyll _ occurred in cultures which received treatment 2 (N-400 P-0). Subsequently, growth rates in treatments 4 (N=400 P-40) and 5 (N-800 P-80) exceeded those of treatment 2. This information, combined with the lag in growth observed in cultures receiving only P additions, suggest that phytoplankton were initially N limited and became co-limited by P as they grew. This is confirmed by the significant interaction of added N and P levels upon chlorophyll a concentrations. In contrast, no significant interaction between the levels of N and P was observed in August. In August, those cultures which received only P additions had the same chlorophyll a as those which received both N and P, while cultures receiving only N had the same chlorophyll a as those with no nutrient addition. Increases in chlorophyll a were also recorded in cultures which received no P addition. These observations suggest that some P was still available for plankton growth, but the growth rates were P limited, and thus the

















200

* ON, OP V 400N, OP 160 - ON, 40P
A 40ON, 40P * BOON. 80P 120



80


40


0 I
0 48 96 144 192 TIME (h)


Time courses of chlorophyll a concentrations following nutrient enrichment of natural plankton populations collected in April 1990. ON,OP=no nutrient addition; 400N,OP=400 pg N -1- ON,40P=40 pg P L1; 400N,40P=400 pg N U' and 40 pg P U", and 800N,80P=800 pg N L-1 and 80 pg P L1. Vertical bars indicate 1 SE. No vertical bar indicates SE is smaller than symbol size.


_J

4.

0 ..J



-r
=
0


Fig. 3-4.











200 160

120

80


0401 4-0
::L

0


-r
2.
0
W, 200
0

-r- 160

120

80


24


48


72


96


TIME (h)


Fig. 3-5.


Time courses of chlorophyll a concentrations following
nutrient enrichment of natural plankton populations collected in August 1990. ON,OP=no nutrient addition; 400N,OP=400 pg N L1; ON,40P=40 pg P L-, 400N,40P=400 pg N L- and 40 pg P U', and 800N,80P=800 pg N L" and 80 pg P C': a) plankton grown at 29�C; b) plankton grown at 19"C. Vertical bars indicate 1 SE. No vertical bar indicates SE is smaller than symbol size.


29 0C

0 ON, OP v 4OON, OP E ON. 40P A 400N, 40P
SOON. SOP



(6)


19C DON, 40P






(b)
II!


40W










growth rate increased with increasing P concentration in the growth media. It is suggested that cultures were only slightly limited by P, because cultures with no P enrichment still increased in biomass. During both experiments, the highest nutrient addition (treatment 5, N-800 P=80) resulted in significantly greater chlorophyll a than any other treatment.
Growth of plankton collected in August was inhibited at cooler temperatures (Fig. 3-5b). Over the entire 96 h period chlorophyll a only increased by 8 jg 1. The phytoplankton grown at 19"C with either a P addition or no nutrient addition only achieved 65% of the chlorophyll j of phytoplankton grown at 29�C with no nutrient addition.

Nutrient uptake. In April, the initial sampling for nutrient analyses occurred after 2 h; however, at this time SRP concentrations had been significantly reduced, consequently to give a true representation of the data a time 0 was included along with 2 h to the data set. Time 0 represents the means of initial concentrations measured in cultures with no nutrient addition, plus the respective additions, hence approximate uptake rates can be envisioned (Fig. 3-6). Only the cultures which received treatment 5 (N=800 P=80) had significant SRP concentrations remaining after 2 h (17 jg C'), SRP concentrations in the other cultures were below 3 jg C'. These concentrations remained close to baseline for the remainder of the experiment. The determination of P uptake thus required a more intensive sampling immediately following nutrient addition. This was achieved in experiment 2, with water samples collected in August. Within 30 min SRP had decreased from 81.6 to 37.4 ug L" in treatment 5















90 80

70 60 50

40 30

20 10


48


96
TIME


(h)


144


192


Time courses of soluble reactive phosphorus concentrations following nutrient enrichment of natural plankton populations collected in April 1990. ON,OP-no nutrient addition; 400N,OP=400 Ag N L"; ON,40P-40 / g P L'; 400N,40P-400 jg N U' and 40 Ag P L-, and 800N,80P800 Mg N L" and 80 Mg P L1. Vertical bars indicate 1 SE. No vertical bar indicates SE is smaller than symbol size.


I

Ot

a
cn


* ON. OP V 400N. OP N ON. 40P A 400N.40P
* BOON, OP


-.11


Fig. 3-6.










(N-800 P=80), from 41.6 to 22 jig L- in treatment 4 (N-400 P-40) and from 41.6 to 13.1 jug L1 in treatment 3 (N=O P=40) cultures (Fig. 3-7a). Uptake rates were calculated as the disappearance of SRP within the first 30 min (Table 3-6). This was selected to indicate maximal uptake because the slope changed over time as the P demand decreased (Fig. 3-la). Cultures which received 80 pg L1 had a significantly greater uptake rate than those which received 40 jg L-1. As expected, temperature had a significant effect upon SRP uptake (Fig. 3-7b). The uptake was not as rapid as that for the same treatment at 29"C (a = 0.12), but even with the 10"C difference in temperature all SRP had been depleted to below detection within 2 h. After 1 h, SRP levels in treatments 3 (N=0 P=40) and 4 (N=400 P=40) were no longer significantly different. The SRP concentrations continued to decrease in all the cultures and were all close to baseline in 2 h and were undetectable (<1 jg L") after 24 h. In both experiments TSP decreased within the first

2 h and then remained constant (Fig. 3-8a and 3-9a).
The uptake of N differed between the two sampling periods. In April, [NO3 + N02]-N concentrations in the cultures decreased. The concentration decreased by 25% in all treatments with N additions, within 2 h and continued to decrease over time (Fig. 3-8b). In contrast, NH4-N concentrations did not change in any of the cultures until 216 h, when an increase was observed in control and N cultures (Fig. 3-8c). In August, no significant treatment by time interaction was recorded for [NO3 + N02]-N, and no apparent uptake of [NO3 + N02]-N occurred (Fig. 3-9b). A significant treatment by time interaction was










80\ 60

40

20

0





80 60

40 20

0
0.0


0.5 1.0 1.5 2.0 2.5
TIME (h)


Time courses of soluble reactive phosphorus concentrations following nutrient enrichment of natural plankton populations collected in August 1990. ON,OP=no nutrient addition; 400N,OP=400 Ag N L1; ON,40P-40 #g P L', 400N,40P-400 jg N L-1 and 40 pg P L-1, and 800N,80P= 800 pg N U' and 80 pg P L': a) plankton grown at 29gC; b) plankton grown at 19"C. Vertical bars indicate I SE. No vertical bar indicates SE is smaller than symbol size.


I
-_1


Fig. 3-7.











Table 3-6.


Phosphorus uptake rates for natural plankton populations collected in August 1990, 30 min after receiving nitrogen and phosphorus additions (mean � SE). 3=40 pg P 1.1, 4=400 /g N L" and 40 pg P 1, 5-800 pg N C" and 80 jg P L 7=40 pg P L1 and plankton grown at 19*C.


Uptake Rate


pg P L-C min-'

0.95 � 0.05 a* 0.43 � 0.27 b 1.47 � 0.06 c

0.65 � 0.01 ab*


Numbers in a column followed significantly different at a


by the same letter are not = 0.05.


Uptake rates for treatments 3 and 7 are significantly different at a - 0.12.


Treatment









200


150 100

50

0
0.9 0.6 0.3


0.0 0.3
I.
-'0.2


0.1 0.0


Fig. 3-8.


0 48 96 144 192
TIME (h)


Time courses of nutrient concentrations following nutrient enrichment of natural plankton populations collected in April 1990. ON,OPzno nutrient addition; 400N,OP-400 Ag N L 1- ON,40P-40 Ag P L-I; 400N,40P=400 jg N L-1 and 40 pg P L-1, and 800N,80P=800 pg N L- and 80 pg P L': a) total soluble phosphorus; b) [NO3 + N02]-N; c) NH4-N. Vertical bars indicate 1 SE. No vertical bar indicates SE is smaller than symbol size.


I
.=1


Z

0I
Z
+


r-


0
Z
Lj


Z

Z










200 150 100

50

0


4
0
Z


0
z




I
Z


-J

E


0.9 0.6 0.3 0.0 0.3


I-


E
%v.O.1


0.0


I"


24 48 72 96
TIME (h)


Fig. 3-9. Time courses of nutrient concentrations following nutrient
enrichment of natural plankton populations collected in
August 1990 grown at 29�C. ON,OP=no nutrient addition;
400N,OP=400 /g N L"; ON,40P=40 Ag P U; 400N,40P=400 pg N UI and 40 pg P U, and 800N,80P=800 pg N L- and 80 pg
P L: a) total soluble phosphorus; b) [N03 + N02]-N;
c) NH4-N. Vertical bars indicate I SE. No vertical bar
indicates SE is smaller than symbol size.


(a)
- ON. OP � 400N. OP * ON, 40P , 400N,40P BOON,80P









I LT










observed for NH4-N, however, the data were highly variable, making meaningful interpretation difficult (Fig. 3-9c). Plankton grown at 190C exhibited the same responses for TSP, [NO3 + N02]-N, and NH4-N (data not shown).
Surplus phosphorus. Soluble P extracted from plankton following boiling with deionized water (HEP-SRP) was used as an indicator of surplus P. In April HEP-SRP was determined at both the start and conclusion of the experiment. Hot water extractable P decreased in cultures which did not receive P additions but remained the same in cultures which received 40 pg L1 (Table 3-7). Those cultures which received 80 pg P U' had almost a 3-fold increase in HEP-SRP after 216 h. The role of HEP was examined in more detail in water samples collected in August. Seventy percent of initial TP was accounted for by HEP-TSP and TSP (Table 3-5), with HEP-TSP representing 59% of TP. Within 2 h of P addition, substantial increases in HEP-TSP were observed (Fig. 3-10a). Hot water extractable-TSP tripled from 33.3 to 99.3 pg P L1 upon addition of 80 pg P L1. Addition of 40 pg P L1 resulted in a doubling of HEP-TSP to 65 and 69 pg P L- in cultures which received treatments 3 (N=O P=40) and 4 (N=400 P=40), respectively. Even with a 10"C temperature difference HEP-TSP accumulated to 63.9 pg P L-1 within 2 h (Fig. 3-l0b). Cultures grown at 19�C which did not receive nutrients had greater HEP-TSP concentrations after 2 h than those with no nutrient addition grown at 29"C. At 29"C the HEP-TSP remained constant within 24 h and then decreased by 15 pg P L- for P added treatments. The downward trend continued to 96 h, HEP-TSP











Table 3-7.


Hot water extractable phosphorus concentrations of composite lake water samples collected in April 1990, 216 h after nutrient additions. 1=no nutrient addition, 2=400 jg N L-1, 3=40 Ag P U', 4=400 pg N L1 and 40 pg P L1,5=800 pg N U' and 80 jg P U.


Treatment Hot water extractable SRP



Ag U
1 3.0 2 2.8 3 7.2 4 6.8 5 18.4










120



80


40


19 C 0 ON, Opi
0 ON. 40P


48
TIME (h)


Fig. 3-10.


Time courses of hot water extractable total soluble phosphorus following nutrient enrichment of natural plankton populations collected in Auqust 1990. ON,OP=no nutrient addition;400N,OP=400 jig N U-; ON,40P=40 pg P L-;
400N,40P=400 /g N CI and 40 jug P LI, and 800N,80P=800 jig N LI and 80 /g P L1: a) plankton grown at 29�C; b) plankton grown at 19�C. Vertical bars indicate I SE. No vertical bar indicates SE is smaller than symbol size.


120



80


40


0


24


72


96


I









80

concentrations in treatment 1 (N-O P-O) cultures were lower than initial concentrations while HEP-TSP concentrations in treatments 3 (N=O P-40) and 4 (N=400 P=40) were higher than initial concentrations. Normalizing the data to chlorophyll a, a decrease in HEP-TSP was observed for all treatments following the initial increase at 2 h (Table 3-8). The final ratios were lower than the initial ratios.
The treatment by time response was different for HEP-SRP
(Fig. 3-11a). The relative increases were greater. An increase in HEPSRP concentrations was observed after 48 h in cultures which received P, except for treatment 4 (N=400 P=40). Hot water extractable P in treatments 1 (N=O P=O) and 2 (N=400 P=O) cultures remained constant. A significantly lower increase was observed in treatment 7 (N=O P=40, temperature=19"C) (Fig. 3-11b &=0.06). Normalizing the data to chlorophyll a resulted in a continuous decline in HEP-SRP. No increase after 48 h was observed (Table 3-9). A significant correlation between HEP-SRP and chlorophyll a was observed (r=0.56).
Alkaline phosphatase activity. Considerable differences in the

production of APA were observed between April and August (Fig. 3-12a and 13a). In both experiments, increases in APA were observed, however in August this increase only lasted 48 h for all cultures grown at 290C. In April, APA in cultures receiving both N and P additions was inhibited within 2 h, 28% and 11% inhibition for treatments 5 (N-800 P=80) and 4 (N-400 P=40), respectively. No inhibition was observed in treatments receiving only a P addition. After initial inhibition, which lasted 24 h, APA increased (Fig. 3-12a). A significant interaction between N and P was apparent at 48 h. Cultures receiving only P had a delayed











Table 3-8. Specific hot water extractable phosphorus measured over time
in natural plankton populations collected in August 1990 after receiving nitrogen and phosphorus additions (mean �
1 SE). 1=no nutrient addition, 2=400 jig N L1, 3=40 jg P 11,
4=400 jg N L- and 40 jig P L", 5=800 ug N L' and 80 pg P
L-, and 6=no nutrient addition and 7=40 pg P L- and
plankton grown at 199C.



Time h
Treatment 0 2 24 48 96


--------------------p jg P pig chlorophyll a_
1 0.89 � 0.07 0.73 � 0.04 0.68 � 0.02 0.51 � 0.02 0.36 � 0.02 2 0.92 � 0.03 0.67 � 0.04 0.51 � 0.02 0.33 � 0.02 3 1.75 � 0.03 1.34 � 0.03 0.89 � 0.04 0.45 � 0.01 4 1.85 � 0.06 1.25 � 0.03 0.90 � 0.04 0.44 � 0.02

5 2.66 � 0.05 1.82 � 0.01 1.25 � 0.04 NA* 6 1.00 � 0.07 0.82 � 0.02 0.72 � 0.07 NA 7 1.71 � 0.01 1.59 � 0.03 1.10 � 0.03 NA NA indicates data is not available.











- I -'
29C ON, OP


24 48 72 96


TIME (h)


Fig. 3-11.


Time courses of hot water extractable soluble reactive phosphorus following nutrient enrichment of natural plankton populations collected in Auqust 1990. ON,OP=no nutrient addition;400N,OP-400 Ag N LU; ON,40P-40 Ag P U'; 400N,40P=400 pg N L1 and 40 pg P U', and 800N,80P=800 #g N L1 and 80 pg P L-: a) plankton grown at 29"C; b) plankton grown at 19*C. Vertical bars indicate 1 SE. No vertical bar indicates SE is smaller than symbol size.


ON, OP
400N. OP


U ON, 40P A 400N,40P , 800NSOP


rI


80 60


40 20


-J
0~ 4.


80 60


40 20


0


I


29%C











Table 3-9.


83
Specific hot water extractable phosphorus measured over time in natural plankton populations collected in August 1990 after receiving nitrogen and phosphorus additions (mean �
1 SE). 1=no nutrient addition, 2=400 pg N L-1, 3=40 /tg P L", 4=400 pg N L1 and 40 pg P L-1, 5=800 pg N L- and 80 pg P L-, and 6=no nutrient addition and 7=40 pg P L- and plankton grown at 190C.


Time h
Treatment 0 2 24 48 96

-g P jg chlorophyll a1 0.41 t 0.05 0.48 t 0.07 0.34 � 0.01 0.29 t 0.01 0.22 t 0.00 2 0.53 � 0.01 0.35 � 0.02 0.27 � 0.00 0.19 � 0.01 3 1.14 � 0.06 0.64 � 0.04 0.35 � 0.01 0.28 � 0.01 4 1.07 � 0.06 0.67 � 0.06 0.37 � 0.01 0.21 � 0.02 5 1.79 � 0.07 0.82 t 0.03 0.51 � 0.02 0.24 � 0.00 6 0.54 � 0.02 0.44 � 0.01 0.31 � 0.04 0.35 � 0.05 7 0.87 � 0.05 0.73 � 0.01 0.48 t 0.01 0.47 � 0.03




































48 96 144 192
TIME (h)


Fig. 3-12.


Time courses of alkaline phosphatase activity following nutrient enrichment of natural plankton populations collected in April 1990. ON,OP=no nutrient addition; 400N,OP=400 jg N LI; ON,40P=40 pg P U; 400N,40P=400 pg N l ' and 40 jg P U', and 800N,80P=800 pg N L-1 and 80 jg P U': a) total alkaline phosphatase activity; b) specific alkaline phosphatase activity. Vertical bars indicate 1 SE. No vertical bar indicates SE is smaller than symbol size.


100


80 60

40 20

0


U,


3.0 2.5


S2.0
U
01.5 < 1.0
0.5
E 0.0
0


V
I


I










100

80 60


-~40
.E 20
0
0

0..


< 100
o 0 ON, oP I 19C (b)
I 80 - ON. 40P

60

40

20
0 I I
0 24- 48 72 96 TIME (h)


Fig. 3-13. Time courses of alkaline phosphatase activity following
nutrient enrichment of natural plankton populations
collected in August 1990. ON,OP-no nutrient addition;
400N,OP=400 Ag N L'; ON,40P=40 pg P L; 400N,40P=400 Ag N LI and 40 ug P U', and 800N,80P=800 pg N L-1 and 80 pg
P L): a) plankton grown at 29�C; b) plankton grown at 19'C.
Vertical bars indicate I SE. No vertical bar indicates SE
is smaller than symbol size.










response in APA increase and produced the least APA. A similar trend was observed by phytoplankton cultured at 19C in August (Fig. 3-13b). In August the response of cultures grown at 29gC with only P added mimicked those which received both N and P (Fig. 3-13a). Disregarding temperature effects, APA in cultures with and without P additions exhibited similar trends; increasing up to 48 h and then decreasing (Fig. 3-13a). The initial increase in APA over the first 24 h was on average 64%, greater in those cultures receiving P. The reverse was true for the next 24 h period. Alkaline phosphatase activity measured in treatments I (N=O P=O) and 2 (N=400 P=O) doubled while only a 22% increase was observed in treatments 3 (N=0 P=40) and 4 (N=400 P-40). Cultures which received treatment 5 (N-800 P=80) did not exhibit a change. Both groups subsequently declined by approximately 20 nM minI to 96 h. Transforming the APA data to specific activity results in a different shape curve for April data (Fig. 3-13b) but no change in curve shape in August (Table 3-10). The interpretation from both experiments is the same, .i.e., higher specific APA was apparent in all cultures which did not receive any P addition. While those cultures which received P had significantly lower specific APA. The highest specific APA was recorded in April in cultures which received treatments I (N=O P=O) and 2 (N=400 P=O).


Discussion

Nutrient loading from external and internal sources can influence the productivity of phytoplankton and other aquatic biota. The response of phytoplankton growth to nutrient enrichment has been used as an











Table 3-10. Specific alkaline phosphatase activity measured over time
in natural plankton populations collected in August 1990
after receiving nitrogen and phosphorus additions
(mean � 1 SE). 1=no nutrient addition, 2=400 ug N L1, 3=40
jg P L-1, 4=400 jg N L" and 40 pg P L-1, 5=800 pg N L-1 and
80 pg P L, and 6=no nutrient addition and 7=40 jg P L"1 and
plankton grown at 19"C.


Time h
Treatment 0 24 48 96


nmol APA jig chlorophyll a-1 min"1 0.34 1 0.00 0.47 � 0.01 1.07 � 0.03 0.45 � 0.03 2 0.47 � 0.02 1.01 � 0.07 0.51 � 0.06 3 0.63 � 0.01 0.64 � 0.02 0.16 � 0.02 4 0.63 � 0.02 0.70 � 0.03 0.17 � 0.00 5 0.67 � 0.02 0.48 � 0.01 0.07 � 0.00 6 0.22 � 0.01 0.43 � 0.01 1.12 _ 0.03 7 0.32 � 0.02 0.52 � 0.01 1.00 � 0.04




Full Text
TOTAL ALKALINE PHOSPHATASE ACTIVITY
114
(mg L 1)
Fig. 4-8. The relationship between alkaline phosphatase activity and
total suspended solids in the overlying water column of
sediment cores after resuspension of surficial sediments.


SOLUBLE ALKALINE PHOSPHATASE ACTIVITY
116
0.8
0.4
(c)

-
-
J
1
_l
1 1
'
1 0 cm
i i
Fig. 4-9. Soluble alkaline phosphatase activity measured in the
overlying water column of triplicate sediment cores after
resuspension of 0, 2, 5 and 10 cm surficial sediments:
a) 2 cm resuspended; b) 5 cm resuspended; c) 10 cm
resuspended; d) control, no resuspension. Initial data
point indicates the conclusion of resuspension. Absence of
vertical bar indicates symbol size is greater than 1 SE.


151
Conclusions
The results from this study show that short-term DO depletion will
not affect APA in the water column of Lake Apopka. However, extended
periods of anoxia (8 to 24 h) will result in APA inhibition and
decreased enzymatic breakdown of organic P. In the sediment, APA was
high under aerobic conditions and decreased with a decrease in Eh, hence
under anaerobic conditions the rate of organic P mineralization will be
slower. Inverse correlations between APA and porewater TOP and labile
organic P also suggest that these are susceptible to enzymatic
hydrolysis or may be inhibited by high concentrations of these
substrates. Based upon the resistant nature of HA-TP the inverse
relationship between HA and APA was attributed to the binding of the
enzyme in the formation of humic complexes which accumulate under
anaerobic conditions.


14
Soluble inorganic P is the most readily available form of P for
plankton nutrition, however; in its absence organic P compounds may be
used for growth. The enzymatic hydrolysis of organic P compounds is
competitively inhibited by inorganic P. Soluble reactive P
concentrations in Lake Apopka are hypothesized to be too low to inhibit
APA. There is, however, the issue of internal concentrations of P which
may regulate organic P hydrolysis outside the cell.
(3) Evaluate the effect of sediment resuspension upon the
mineralization of organic P in the sediment and overlying
water column.
In shallow lakes, wind induced resuspension of sediments into the
overlying water column increases the interaction between these
compartments. It is hypothesized that resuspension of sediment
increases the concentration of organic substrates and associated
microorganisms in the water column and thus increases mineralization.
(4) Determine the effect of anoxia on organic P mineralization
in the sediment and water column.
Mineralization of organic compounds proceeds more rapidly under
aerobic than anaerobic conditions. Since a majority of sediments are
anaerobic, it is hypothesized that APA will be inhibited under anaerobic
conditions, resulting in a reduced mineralization rate.
Dissertation Format
Each chapter within this dissertation is written as an independent
manuscript intended for future publication. Chapter 2 focuses upon the
concentrations of P compounds and APA within the water column and the


46
consistently observed, suggesting that plankton in Lake Apopka are
generally not P limited. This conclusion tends to agree with other
research findings from this lake (Aldridge, F.J., personal
communication, Department of Fisheries and Aquaculture, University of
Florida, Gainesville, FL.; Reddy and Graetz 1990). Specific APA was
lowest when chlorophyll a peaked and SRP concentrations of 5 pq L'1 were
sufficient to support growth.
High particulate APA has been attributed to the location of
alkaline phosphatase in the cell wall of phytoplankton (Kuenzler and
Perras 1965). Recent research has suggested that viable phytoplankton
do not contribute much to the particulate APA pool (Stewart and Wetzel
1982). Lake Apopka is generally dominated by cyanobacteria, with large
numbers of Lyngbya sp. and Microcystis sp. (Shannon and Brezonik 1972;
Stites, D. L., personal communication, St. John's River Water Management
District, Palatka, FL.) whose mucilaginous layers can support
significant bacterial populations. It is likely that particulate APA is
attributable to both phytoplankton and the associated bacteria. Use of
specific APA (APA/chlorophyll a) to indicate P limitation of
phytoplankton should be verified via nutrient enrichment bioassays.
Diel fluctuations in APA were observed, but these do not
correspond to any particular water chemistry parameter. This may be
attributed to spatial patchiness and water movement (Berman 1970; Wynne
1981). Unlike TKN concentrations, APA did not settle out of the water
column following wind subsidence. Diel variability of APA may be
dependent upon the species composition of the phytoplankton biomass. In
diel studies neither Tballassiosira pseudonana Hasle and Heimdal (Perry


Table 4-2. Concentrations of parameters measured within the water column of cores collected in
September 1989, from the center of Lake Apopka (mean 1 SE) -
APA
Treat
ment
Time

TKN
TP
TSP
SRP
Total
Soluble
TSS
h
L'1
M9
L-1
nM
min'1
mg
l '1
my
L
Control
0
3.55

0.06
100

13
100

28
11

3
8.8
0.3
0.04

0.00
72

2
1
3.65

0.12
90

6
90

3
5

0
8.0
0.6
0.09

0.03
75

4
24
3.89

0.12
150

30
90

10
4

1
6.9
0.3
1.20

0.17
54

3
48
4.31

0.12
180

50
60

3
5

0
12.5
0.7
0.41

0.06
47

3
Resusp-
0
3.79

0.33
80

3
100

8

5
5.6
0.29

0.20
59
+
1
ended
1
96.00

5.65
5733

245
70

10
5

1
109.1
3.0
0.07

0.06
3236

38
24
5.44

0.12
160

23
80

3
4

0
6.1
0.2
0.55

0.08
64

1
48
4.33

0.55
130

3
70

3
5

1
14.5
0.8
0.15

0.04
70

8
* Time 0 represents samples taken prior to resuspension, 1 h represents end of period of suspension.
Only 1 replicate per measurement.


APPENDIX A
LORAN COORDINATES
Table A. Loran coordinates of sampling sites on Lake Apopka
Group repetition interval:7980, Southeast USA.
Site number
Time
Y
delay ns
Z
1
44526.6
62460.6
2
44524.2
62446.4
3
44553.9
62452.3
4
44577.2
62449.9
5
44539.7
62416.8
6
44497.1
62403.0
7
44488.8
62420.2
8
44530.0
62432.2
156


66
Table 3-5. Initial concentrations of selected parameters measured in
diluted lake water prior to nutrient addition
(triplicate samples) (mean 1 SE).
Parameter
SamDlina Deriod
April
1990
August
1990
Chlorophyll a (/ig L'1)
22.5
0
37.4
0
TP (jig L )
113
12
56
9
tsp (/ig L-1)
70
10
9
0
SRP (/xg L'1)
1
0
2
0
HEP-TSP (/ig L'1)
ND*
33
34
HEP-SRP (/ig L'1)
7
0*
15
2*
NH4-N (mg L'1)
0.04
0
0.05
i 0
[N03 + N02]-N (mg L'1)
0
0
0.01
0
Total APA (nM min'1)
9.1
0.2
12.7
0.1
ND indicates data not determined.
Determined by the method of Krausse and Sheets (1980).
Determined by the method of Fitzgerald and Nelson (1966).


105
Results
Physico-chemical Properties
Water. Nutrient concentrations were evenly distributed
throughout the water column (Table 4-1). Chlorophyll a, temperature,
DO, and pH decreased with depth. Total APA in the water column
increased with depth from a surface concentration of 37 to 44 nM min'1
at 1.5 m. Soluble APA was <2% of total APA. A high concentration of
soluble APA was measured at a depth of 1 m.
Sediment. Total APA in the sediments decreased with depth, with
the greatest change occurring within the 20-40 cm depth (Fig. 4-3a).
The water content of the sediments decreased from 98% at the surface to
95% in the 20-40 cm depth (data not shown). The porewater SRP remained
constant within the first 0-20 cm but increased significantly within the
20-40 cm depth from 0.01 mg L1 to 1.4 mg L'1 (Fig. 4-3b). Volatile
solids also increased at this depth (Fig. 4-3c). The opposite effect
was observed for NaOH-extractable P, organic P and TP, which decreased
in the 20-40 cm sediment depth (Fig. 4-4a, b, and c).
Sediment Resuspension Effects on Alkaline Phosphatase Activity
Experiment 1
Water column. Resuspension of the top 10 cm of sediment resulted
in increased TSS from 60 to 3000 mg L'1 Within 30 min after
resuspension, most of the sediment particles settled. Concentrations of
all parameters (excluding TSP) increased in the water column during
resuspension (time 1 h) and decreased to initial concentrations within
24 h (Table 4-2). Soluble parameters, e.g. SRP, exhibited considerable


81
Table 3-8. Specific hot water extractable phosphorus measured over time
in natural plankton populations collected in August 1990
after receiving nitrogen and phosphorus additions (mean
1 SE). l=no nutrient addition, 2=400 ng N L"\ 3=40 ng P L'1,
4=400 ng N L'1 and 40 ng P L'1, 5=800 ng N L'1 and 80 fig P
L'1, and 6=no nutrient addition and 7=40 ng P L'1 and
plankton grown at 19*C.
Time h
Treatment ""0 2 24 48 96
Hg P ng chlorophyll a'1
1
0.89 0.07 0.73

0.04
0.68

0.02
0.51
+
0.02
0.36
0.02
2
0.92

0.03
0.67

0.04
0.51

0.02
0.33
0.02
3
1.75

0.03
1.34

0.03
0.89

0.04
0.45
0.01
4
1.85

0.06
1.25
+
0.03
0.90

0.04
0.44
0.02
5
2.66

0.05
1.82
+
0.01
1.25

0.04
NA'
6
1.00

0.07
0.82

0.02
0.72

0.07
NA
7
1.71
+
0.01
1.59

0.03
1.10

0.03
NA
NA indicates data is not available.


56
Table 3-2. Nutrient additions made to diluted lake water collected in
April and August 1990.
Treatment
Nutrient
N*
addition
P
Incubation
temperature
fi g l
i
*C
Experiment 1-April
1
0
0
27
2
400
0
27
3
0
40
27
4
400
40
27
5
800
80
27
Experiment 2-August
1
0
0
29
2
400
0
29
3
0
40
29
4
400
40
29
5
800
80
29
6
0
0
19
7
0
40
19
N and P were added as potassium nitrate and potassium dihydrogen
phosphate, respectively.


This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
May 1991
cu>k X.
ollege of
culture
Dean, Graduate School


molecular weight, complex structures are highly resistant to
mineralization, hence they will tend to accumulate, e.g. humic acids.
Conversely, simple, low molecular weight organic compounds are more
susceptible to hydrolysis and thus more labile, e.g. sugarphosphates.
It is these labile compounds which are more likely to undergo rapid
enzymatic hydrolysis and thus be bioavailable.
Soluble reactive P (SRP), the most labile form of P, has been the
focus of most P cycling research (Hutchinson and Bowen 1950; Rigler
1956). However, SRP concentrations in lakes are generally low while
organic P is abundant (Abbott 1957; Rigler 1956). Such observations
resulted in investigations to determine whether organic P compounds
could act as a source of P available for plankton nutrition. Under
inorganic P limiting conditions phytoplankton may utilize organic P
compounds for growth (Harvey 1953; Kuenzler 1965). These phytoplankton
produce externally acting enzymes, phosphatases which hydrolyze
phosphomonoesters (PME) and release SRP (Fitzgerald and Nelson 1966;
Kuenzler and Perras 1965). Phytoplankton which do not produccce
externally acting enzymes cannot hydrolyze PME compounds and their
growth becomes P limited (Kuenzler 1965). Ecologically, the ability of
phytoplankton to utilize organic P gives them a competitive advantage
over non-phosphatase producers, during inorganic P limitation.
The mode of action of phosphatases (specifically
phosphomonoesterases) is shown below (Coleman and Gettins 1983):
1 2 3 4
ROP + E ~ ROP-E E-P E-P ~ E + P(
R = an organic moiety, P¡ = inorganic P, E = enzyme.


65
1200
cl7~ 800
^ o 4oo y^
100
0.3

ON.
OP

500N.
OP

ON,
10P

1000N,
100P

2500N.1 OOOP
t^lL
J
(b)
96 144 192
TIME (h)
240 288
Fig. 3-3. Time courses of nutrient concentrations following nutrient
enrichment of natural plankton populations collected in
November 1989. ON,OP=no nutrient addition, 500N,0P=500 ng N
L ON, 10P=10 ng P L1, 1000N, 100P=1000 tig N L1 and 100 fig
P L and 2500N, 1000P=2500 ng N L'1 and 1000 iig P L*1:
a) soluble reactive phosphorus; b) [N03 + N02]-N; c) NH4-N.
Vertical bars represent 1 SE. No vertical bar indicates SE
is smaller than symbol size.


12
However, in shallow lakes, wind induced resuspension of sediments to the
oxygenated water column, results in the rapid breakdown of organic P
(Pomeroy et al. 1965). A significant positive correlation between SRP
released and APA in the water column has been observed during sediment
resuspension (Degobbis et al. 1984).
Resuspension can physically transport SRP to the overlying water
column (Ryding and Forsberg 1977). It also increases the suspended
solids concentration within the overlying water. This particulate
material can provide 28-41% of algal available P (Dorich et al. 1985).
Consequently, resuspension of sediments can result in enhanced enzyme
activity and P availability within the water column. Hence sediments
can play a significant role in organic P mineralization in the overlying
water column.
Objectives
Bioavailability of organic P occurs through the action of enzymes
(Kuenzler and Perras 1965). These enzymes catalyze the release of
inorganic P from both soluble and particulate matter. Both APA and
organic P concentrations increase with increased eutrophication (Jones
1979b). Hence, Lake Apopka, which is classified hypereutrophic,
should support large concentrations of APA and organic P. This study is
based on the hypothesis that enzyme mediated P release is used to
support the vast algal populations during inorganic P limitation.
Without this ability to utilize organic P at times of high P demand and
inorganic P limitation, algal and bacterial species are nutrient
stressed. Very little is known about the bioavailability of organic P


88
indicator of nutrient status. This study examined growth, uptake rate,
surplus P and APA to explain P requirements of phytoplankton and
associated microorganisms in Lake Apopka. Natural plankton populations
were used to include the contributions of bacteria and zooplankton to
the overall nutrient status of the lake. The objective was to determine
the response of phytoplankton biomass, hence chlorophyll a measurements
were used to indicate biomass.
This study demonstrated that APA is immediately and rapidly
inhibited by high inorganic P concentrations (Garen and Levinthal 1960;
Moore 1969; Torriani 1960). The extent of inhibition was dependent upon
the concentration of inorganic P added, internal P concentrations and
the growth of the plankton (Fitzgerald 1969). The most severe
inhibition was caused by 1000 ng P L'1. It has been suggested that 1000
ng P L1 is the minimum requirement for APA inhibition (Jones 1979b).
In combination with external P concentrations, the extent of APA
inhibition may also be dependent upon the initial internal nutrient
content. In April, APA was inhibited by 40 and 80 ng P L'1 within the
first 2 h but these concentrations did not result in such rapid
inhibition in August. Phytoplankton growth as determined by increases
in chlorophyll a concentrations, were initially limited by N and had
sufficient P so additions of P resulted in inhibition of APA. As the
phytoplankton grew and exhausted internal supplies, growth became
limited by both N and P, and APA increased. In August, phytoplankton
were P limited, thus the demand was sufficient that even the addition of
80 ng P L'1 did not inhibit APA. Initial APA was also higher in August
than April, and may reflect the difference between slight P limitation


44
between SRP released and APA in the water column has been observed
during sediment resuspension to the overlying water (Degobbis et al.
1984). Assuming resuspended sediment were contributing significantly to
the predominately particulate APA pool in Lake Apopka, the ratio of
APA/TSS is a better measure of enzyme activity than APA alone.
Comparing monthly means, total APA/TSS was positively correlated with
soluble APA (r=0.99) and inversely correlated with TOC (r=-1.00). The
correlation between APA and TSS was only observed when data were
compared on a site basis. The overall lack of correlation between TSS
and APA in this study may be due to 1) insufficient sampling during
periods of sediment resuspension, 2) the relationship is hidden by the
variability in other parameters incorporated in the TSS pool, and 3)
sediment resuspension does not contribute to APA activity.
Alkaline phosphatase activity has been significantly correlated
with ATP (Pettersson 1980), particulate organic matter (Gage and Gorham
1985) and chlorophyll a (Healey and Hendzel 1979a; Pettersson 1980). In
this study, total APA was significantly correlated with chlorophyll a in
December and inversely correlated with TOC in February. Comparing
annual means, there is a strong inverse relationship between total APA
and TOC. This may be explained by examining the components of the TOC
pool; one contributor is humic material. Alkaline phosphatase activity
can be inhibited by high concentrations of humic materials (Francko
1986). The inverse relationship observed between APA and TOC could be a
result of binding of APA to organic material. Attachment of alkaline
phosphatase enzymes to particulate matter may decrease activity, but may
also increase longevity of the enzyme activity (Burns 1986).


Figure 1-1. Diagram of the phosphorus cycle in Lake Apopka.


70
growth rate increased with increasing P concentration in the growth
media. It is suggested that cultures were only slightly limited by P,
because cultures with no P enrichment still increased in biomass.
During both experiments, the highest nutrient addition (treatment 5,
N=800 P=80) resulted in significantly greater chlorophyll a than any
other treatment.
Growth of plankton collected in August was inhibited at cooler
temperatures (Fig. 3-5b). Over the entire 96 h period chlorophyll a
only increased by 8 ng L'1. The phytoplankton grown at 19C with either
a P addition or no nutrient addition only achieved 65% of the
chlorophyll a of phytoplankton grown at 29C with no nutrient addition.
Nutrient uptake. In April, the initial sampling for nutrient
analyses occurred after 2 h; however, at this time SRP concentrations
had been significantly reduced, consequently to give a true
representation of the data a time 0 was included along with 2 h to the
data set. Time 0 represents the means of initial concentrations
measured in cultures with no nutrient addition, plus the respective
additions, hence approximate uptake rates can be envisioned (Fig. 3-6).
Only the cultures which received treatment 5 (N=800 P=80) had
significant SRP concentrations remaining after 2 h (17 ng L'1), SRP
concentrations in the other cultures were below 3 /xg L'1. These
concentrations remained close to baseline for the remainder of the
experiment. The determination of P uptake thus required a more
intensive sampling immediately following nutrient addition. This was
achieved in experiment 2, with water samples collected in August.
Within 30 min SRP had decreased from 81.6 to 37.4 ng L'1 in treatment 5


54
Table 3-1. Nutrient additions made to diluted lake water collected in
November 1989.
Treatment
number
Nutrient
N*
addition
P
g
L-1 -
1
0
0
2
500
0
3
0
10
4
1000
100
5
2500
1000
N and P were added as potassium nitrate and potassium dihydrogen
phosphate, respectively.


106
Table 4-1. Distribution of selected parameters measured in May 1989,
within the water column at the center of Lake Apopka (n=3).
APA
Depth
Total
Soluble
TKN
TP
SRP
DO
pH
Chi
a Temp Secchi
m
--nM min'1--
mg L'1
--M9
L'1-
mg L'1
M9 L 1
C m
0
37.2
0.9
6.26
210
3
8.5
9.13
99
27.5 0.25
0.5
40.5
0.9
5.55
270
4
8.4
9.14
96
27.0
1.0
43.8
1.5
6.68
270
4
6.7
9.08
86
26.3
1.5
43.8
0.3
7.43
260
4
5.7
9.06
83
26.3


CHLOROPHYLL a (/g
68
200
-* 160
120
80
40
0
0 48 96 144 192
TIME (h)

ON,
OP
T
400N,
OP

ON,
40P
A
400N,
40P

SOON,
SOP
Fig. 3-4. Time courses of chlorophyll a concentrations following
nutrient enrichment of natural plankton populations
collected in April 1990. ON,OP=no nutrient addition,
400N,0P=400 iig N L'\ 0N,40P=40 fig P L'1; 400N,40P=400 ng
N L'1 and 40 ng P L\ and 800N,80P=800 ng N L1 and
80 ng P L'1. Vertical bars indicate 1 SE. No vertical bar
indicates SE is smaller than symbol size.


CHAPTER 2
SEASONAL VARIABILITY IN ALKALINE PHOSPHATASE ACTIVITY IN A
SHALLOW HYPEREUTROPHIC LAKE
Introduction
Phosphorus is the major nutrient limiting plankton production in
many temperate lakes (Schindler 1977). Soluble reactive P (SRP) has
been the form of P most often studied (Rigler 1956); however, SRP is
only a small fraction of the total P (TP) pool. A significant component
of TP may be in organic form (Minear 1972; Rigler 1964). In lakes
where inorganic P availability is low, plankton may produce phosphatase
enzymes which hydrolyze organic P compounds with the release of
inorganic P (Fitzgerald and Nelson 1966; Kuenzler and Perras 1965).
These enzymes are designated alkaline or acid phosphatase, depending on
the pH range of optimum activity (Kuenzler and Perras 1965; Torriani
1960). The alkaline nature of most water bodies has resulted in
alkaline phosphatase activity (APA) receiving the most attention.
Although some APA has been determined to be constitutive (Kuenzler
1965), plankton produce increased APA under conditions of P limitation.
Alkaline phosphatase activity has hence been used as an indicator of P
limitation, however, APA may also reflect P demand, as evidenced by a
poor relationship between SRP and APA (Taft et al. 1977).
The release of inorganic P mediated by APA is dependent upon the
percentage of organic P which is hydrolyzable by the enzyme. Thirty-six
16


SRP (/g
73
Fig. 3-7. Time courses of soluble reactive phosphorus concentrations
following nutrient enrichment of natural plankton
populations collected in August 1990. 0N,0P=no nutrient
addition; 400N,0P=400 ng N L'\ 0N,40P=40 ng P L'1*,
400N,40P=400 /ig N L1 and 40 fig P L\ and 800N,80P=
800 ng N L'1 and 80 \ig P L'1; a) plankton grown at 29*C; b)
plankton grown at 19C. Vertical bars indicate 1 SE. No
vertical bar indicates SE is smaller than symbol size.


CONCENTRATION
37
TIME (h)
Fig. 2-8. Diel variation of selected parameters measured February 6-7,
1990 at the center of Lake Apopka (site 8): a) alkaline
phosphatase activity; b) chlorophyll a. Vertical bars
represent 1 SE. Chlorophyll a values were measured on
composite samples.


BIOGRAPHICAL SKETCH
Susan Newman was born 26 June 1963 in Portsmouth, England. She
graduated with a BSc. Honors degree in management and chemical sciences,
from the University of Manchester Institute of Science and Technology,
in June 1984. Following a 6 month appointment at the Weed Research
Organization (the now defunct WRO), Susan determined it was time to
leave the cloudy skies of England. With emotions split between
excitement and trepidation, Susan began a Master of Science degree in
the Agronomy Department at the University of Florida in January 1985.
With an interest in wetland soils dating back to the making of mud pies
during her childhood, and unable to resist the opportunities of the
Sunshine State, she began a Ph.D. program in the Soil Science Department
at the University of Florida in 1987. The first course she completed
within this department was openwater scuba diving, however, following
that the more traditional courses were taken. Upon completion of her
Ph.D. Susan will continue her aquatic/wetland science interests by
obtaining a research position to investigate the nutrient dynamics in
wetlands.
180


3
Florida, Gainesville, FL.). Researchers have investigated sorption
reactions (Olila, 0., unpublished data, Department of Soil Science,
University of Florida, Gainesville, FL.) and have characterized the
cycling of inorganic P within the sediment and water column (Pollman,
1983; Reddy and Graetz 1990), but the dynamics of the organic P pool,
the dominant form of P, have not been addressed. Studies have
demonstrated that significant quantities of organic P may be
bioavailable (Bradford and Peters 1987; Kuenzler and Perras 1965), hence
the organic P pool may play a significant role in sustaining the vast
plankton biomass under apparent inorganic P limitation. Therefore, the
potential bioavailability of organic P in Lake Apopka needs to be
determined.
Organic Phosphorus Mineralization
In aquatic systems, organic P in sediments constitute 15-50% of TP
(Bostrom et al. 1982) while in the water column organic P may account
for as much as 90% of TP (Rigler 1964). The P cycle is shown in Fig.
1-1. Organic P is generally characterized as total and soluble organic
P. Specific identification of organic P constituents may be achieved
following chromatographic fractionation and comparison with known
compounds (Minear 1972; Weimer and Armstrong 1979), 31P nuclear magnetic
resonance (NMR) (Condron et al. 1985), or via hydrolysis by specific
enzymes (Herbes 1974). Only 50% of organic P forms have been
identified, including: inositol phosphates, sugar phosphates,
phospholipids and nucleic acids (Stevenson 1982). The rate at which
these compounds are mineralized is dependent upon their structure. High


64
did decrease. A significant increase in total APA was observed in
cultures which received only N additions suggesting the onset of P
limitation. This is more apparent when total APA data are presented as
specific activity (i.e. total APA/chlorophyll a) (Table 3-4). A 15 fold
difference between specific activity of those cultures which received
treatment 5 (N=2500 P=1000) and treatment 2 (N=500 P=0) was observed.
Initial specific APA was 0.44 nmol APA nq chlorophyll a'1 min'1, hence P
addition resulted in a decrease in specific APA. Significant decreases
in SRP concentrations were observed within 24 h (Fig. 3-3a). Apart from
treatment 5 (N=2500 P=1000), SRP concentrations were the same in all
cultures after 2 h. After an initial rapid uptake from 1000 to 673 /xg P
L'1 within the first 2 h, SRP concentrations in cultures treated with
1000 nq P L'1 remained constant until 168 h, and then began to
decrease. At 312 h, 377 nq P L'1, 37.6% of the original concentration
remained in treatment 5 (N=2500 P=1000) cultures.
Nitrate concentrations also exhibited significant treatment
differences within 24 h (Fig. 3-3b). The concentrations at 24 h were
ranked in descending order of original addition, with 1 and 3 treatments
having equivalent [N03 + N02]-N concentrations. In all cultures there
was a distinct decrease over time. A similar trend was observed for
NH4-N, concentrations appeared to decrease within 24 h (Fig. 3-3c),
however, no significant differences were determined.
Nutrient Enrichment of Natural Plankton Populations
Growth. Chlorophyll a concentrations and APA were lower in
April than in August (Table 3-5). Conversely, TP and TSP were


34
variation. Diel DO changes were observed at both time periods. No
significant changes in other measured water chemistry parameters were
observed in March 1989, while parameters did exhibit change in February
1990. In February 1990, TKN concentrations remained constant during the
first 12 h of sampling and then declined (Fig. 2-6a). Maximum TKN
corresponded to high PAR and wind speed (Fig. 2-7a and 2-7b). The
decline in TKN corresponded to the decrease of these two parameters. No
significant change was observed for SRP, while TP concentrations
fluctuated throughout the sampling period (Fig. 2-6b). Similar
fluctuations were also observed for total and soluble APA (Fig. 2-8a).
As observed for TKN, chlorophyll a concentrations tended to decrease in
conjunction with decreased PAR and wind speed, however, the range was
only 33 to 37 /xg L'1 (Fig. 2-8b).
Fractionation of Lake Water Phosphorus
In October, lake water samples were fractionated to determine the
different forms of P present. Site variability in chlorophyll a and TSS
concentrations was observed (Appendix B). Total organic C remained
constant at 30 mg L1 for all sites except site 1. Chlorophyll a and
TOC were lower at site 1.
As determined above, most of the total APA was in the particulate
fraction with the soluble APA contribution varying from site to site
(Appendix B). The distribution of the various P forms also exhibited
spatial variability (Fig. 2-9). In most sites TOP represented over 80%
of TP. Total acid hydrolyzable P contributed 10% and total reactive P
(TRP) contributed 3% to the TP pool. A similar distribution of these


Fig. 3-1. Hap showing the location of Lake Apopka and sampling sites. Water was collected from
site 1 in November 1989, and from site 2 in April and August 1990.


conditions of P limitation added inorganic P was immediately assimilated
and recovered in the surplus P pool within the plankton tissue, as
determined by hot water extraction. The plankton apparently utilized
this surplus pool of P for growth under low external inorganic P
concentrations.
Resuspension of surficial sediments increased the interaction
between sediments and the overlying water column, resulting in an
immediate increase in APA and total P (TP) in the water column,
indicating an increased potential for biological organic P hydrolysis
during periods of resuspension. The APA and TP decreased rapidly during
settling of suspended solids, following the cessation of turbulence.
Organic P mineralization was greater under aerobic than anaerobic
conditions in the sediment and overlying water column. Under aerobic
conditions (dissolved oxygen [DO]=6 mg L'1) APA in the water column
increased from 22 to 43 nM min1, while no change was observed under
anaerobic conditions (D0=<0.2 mg L'1). Sediment APA was a function of
Eh, the measured reduction potential of the sediment-water systems.
Under aerobic conditions (Eh=480 mV) APA was 10-fold higher than that
observed under anaerobic conditions (Eh=-240 mV). Enzymatic hydrolysis
of organic P compounds was significantly inhibited under anaerobic
conditions.
The results from this study suggest that the P requirement of
plankton in a highly productive lake may partially be met through the
enzymatic hydrolysis of organic P. Consequently, efforts to reduce
nutrient loading and thus reduce eutrophication should also evaluate the
bioavailability of organic P compounds in the system.
xiii


39
components was also observed in filtered lake water (Fig. 2-10), organic
P represented > 90% of TSP. Soluble acid hydrolyzable P was a less
significant contributor to the total soluble P pool. Soluble reactive P
concentrations were negligible. Total soluble P accounted for 11 to
60% of TP at sites 8 and 7, respectively. The fraction of TP attributed
to suspended material was determined by difference between total and
soluble P fractions (Fig. 2-11). The distribution of P forms was
similar to that observed in whole lake water. Suspended TP represented
from 40 to 89% of TP. No enzyme hydrolyzable P was observed.
Correlating all the site means (including site 1), total APA was
positively correlated with chlorophyll a (r=0.88), TOC (r=0.84) and TSS
(r=0.85). However, plots of the data showed that these correlations
were an artifact of low values for water chemistry parameters at site 1.
Correlations without site 1 gave different conclusions (Table 2-4).
Total APA was observed to be inversely correlated with the acid
hydrolyzable fractions. Chlorophyll a was inversely correlated with TOC
(r=-0.68) and TSP (r=-0.66) and positively with TSS (r=0.73).
Discussion
Lake Apopka is a shallow lake with a surface area of 12,500 ha.
It has a small littoral zone and is subject to considerable wind induced
sediment resuspension. Frequent mixing and mild winters may help to
explain the lack of seasonality observed for several of the parameters
measured. Chlorophyll a, however, did exhibit a seasonal response.
Maximum chlorophyll a corresponded to high PAR and higher temperatures.
Over all months, chlorophyll a was only significantly and


61
Table 3-3. Initial concentrations of selected parameters measured in
diluted lake water prior to nutrient addition
(triplicate samples) in November 1989 (mean 1 SE).
Parameter
Concentration
Chlorophyll (tg L'1)
31
6
TP (Mg I/1).
65
4
SRP (jig L)
3
0.3
TKN (mg L'1)
3.30
+ 0.1
NH4-N (mg L'1)
0.36
0.08
[N03 + N02]-N (mg L'1)
0.14
0.00
Total APA (nM min'1)
13.5
0.3
Soluble APA (nM min'1)
2.2
0.13


130
stoppered, placed on stir plates and attached to redox controllers (Cole
Palmer Model 5997-20) (Fig. 5-lb) modified from Patrick et al. (1973).
All flasks were purged with a mixture of 330 ppm C02 balanced with N2,
for 24 h. The flasks were then allowed to equilibrate for 1 month in
the dark under ambient laboratory conditions at the desired redox
potentials; -250, -100, 0, +100, +250 and +500 mV (actual potentials
measured upon sampling were; -242, -157, -2, +48, +338, +483 mV). After
equilibration, triplicate samples were withdrawn from the flasks and
analyzed for APA and fractionated for organic P using an adaptation of
the method of Sommers et al. (1972) (Fig. 5-2). Bicarbonate extractable
P was determined upon sediment samples after the removal of porewater
for pH determinations (Fig. 5-2). Total P and volatile solids were
measured on dried sediment samples.
Analytical Methods
Water. Alkaline phosphatase activity was determined
fluorometrically (Healey and Hendzel 1979a). One half mL of substrate,
3-o-methylfluorescein phosphate (Sigma Chemicals), at a concentration
determined to be non-limiting, in 0.1 M Tris buffer (Tham, Fisher
Scientific) pH 8.6, was added to 4.0 mL of lake water in a cuvette.
Anoxic water samples were injected into cuvettes which were sealed by a
rubber septum and had been purged with N2 and then evacuated. Both
total (whole lake water) and soluble (filtered through 0.45 im Gelman
membrane filter) APA were determined. The cuvettes were placed in a
water bath (25C). At timed intervals during a 20 min period the


SOLUBLE P CONCENTRATION
40
200
^ 150
w 100
50
0
Fig. 2-10. Distribution of phosphorus compounds determined in filtered
lake water at 8 sites in October 1989. TSP=total soluble
phosphorus, SOP=soluble organic phosphorus, SAH=soluble acid
hydrolyzable phosphorus, SRP=soluble reactive phosphorus.
Vertical bars represent 1 SE.
1 2 3
TSP
SOP
H SAH
M SRP
4 5
SITE
6 7 8


174
release, p.294-306. In H. L. Golterman [ed.], Interactions between
sediments and freshwater. Dr. W. Junk Publishers, The Hague.
Lien, T., and G. Knutsen. 1973. Synchronous cultures of Chiamydomonas
reinhardt: properties and regulation of repressible phosphatases.
Physiol. Plant. 28:291-298.
Likens, G. E. 1972. Eutrophication and aquatic ecosystems, p. 3-13. In
G. E. Likens [ed.], Nutrients and Eutrophication: The limiting
nutrient controversy, vol. 1. Spec. Symp. Limnol. Oceanogr.
Lorenzen, C. J. 1967. Determination of chlorophyll and pheo-pigments:
spectrophotometric equations. Limnol. Oceanogr. 12:343-346.
Matavulj, M., and K. P. Flint. 1987. A model for acid and alkaline
phosphatase activity in a small pond. Microb. Ecol. 13:141-158.
McQueen, D. J., D. R. S. Lean, and M. N. Charlton. 1986. The effects
of hypolimnetic aeration on iron-phosphorus interactions. Water Res.
20:1129-1135.
Minear, R. A. 1972. Characterization of naturally occurring dissolved
organophosphorus compounds. Environ. Sci. Technol. 6:431-437.
Miyake, Y., and K. Saruhashi. 1956. On the vertical distribution of
the dissolved oxygen in the ocean. Deep Sea Res. 3:242-247.
Moller, M., S. Myklestad, and A. Haug. 1975. Alkaline and acid
phosphatases of the marine diatoms Chaetoceros affinis var. Willei
(Gran) Hustedt and Skeletonema costatum (Grev) Cleve. J. Exp. Mar.
Biol. Ecol. 19:217-226.
Moore, H. G. 1969. Studies on the surplus phosphorus uptake phenomenon
in algae. Ph.D Dissertation. Univ. of Texas, Austin. 218 pp.
Moore, P. M. Jr., K. R. Reddy and D. A. Graetz. 1991. Phosphorus
geochemistry in the sediment-water column of a hypereutrophic lake.
J. Environ. Qual. (in press).
Mortimer, C. H. 1941. The exchange of dissolved substances between mud
and water in lakes. I. J. Ecol. 29:280-329.
Olsson, H. 1990. Phosphatase activity in relation to phytoplankton
composition and pH in Swedish lakes. Fresh. Biol. 23:353-362.
Patrick, W. H. Jr., B. G. Williams, and J. T. Moraghan. 1973. A simple
system for controlling redox potential and pH in soil suspensions.
Soil Sci. Soc. Am. Proc. 37:331-332.
Perry, M. J. 1972. Alkaline phosphatase activity in subtropical
central North Pacific waters using a sensitive fluorometric method.
Mar. Biol. 15:113-119.


APPENDIX B
CONCENTRATIONS OF SELECTED WATER CHEMISTRY PARAMETERS
DETERMINED BIMONTHLY FROM APRIL 1989 THROUGH
FEBRUARY 1990, AT 8 SITES IN LAKE APOPKA
Table B-l. Temperature.
Date
Site
Mean
' SE
1
2
3
4
5
6
7
8
c
APR
24.3
27.4
27.4
26.7
26.3
26.1
28.1
23.6
26.5
0.21
JUN
24.2
32.5
30.4
28.5
30.0
29.8
28.1
29.8
29.9
0.20
AUG
24.0
28.7
28.0
27.8
29.5
29.8
29.2
28.5
28.8
0.11
OCT
22.7
21.1
19.9
19.8
19.4
19.9
20.0
19.4
19.9
0.08
DEC
20.5
17.6
16.5
15.8
16.3
15.7
15.0
15.5
16.1
0.12
FEB
22.3
20.1
19.8
20.6
20.8
20.4
21.3
20.0
20.0
0.07
Mean
SE
23.1
0.3
25.5
1.0
24.4
1.0
23.7
0.9
24.3
1.0
24.3
1.0
24.1
1.0
23.4
1.0
Means listed in
this
column
are
means
of sites 2-8.
157


23
Enzyme hydrolyzable P was determined using the method of Strickland and
Parsons (1968).
Statistics
Data were analyzed using SAS for personal computers, version 6
(SAS 1985). Pearson correlation coefficients were determined for all
data.
Results
Measured parameters varied seasonally and spatially. Seasonal
variability was generally greater than spatial variability (data
presented graphically represent means of sites 2-8). Appendix B
contains tables presenting data on a site basis and demonstrates spatial
variability. Site 1 is the site of a natural spring, 80 ft deep and is
not subject to the same wind induced mixing as the rest of the lake.
Data from site 1 are discussed separately, to illustrate the effects of
spring input to the lake.
Physico-chemical Characteristics
A 14*C range in water temperature was observed over the sampling
period (Table 2-1). The highest water temperatures occurred in June and
August, and coincided with maximum photosynthetically active radiation
(PAR) (Table 2-1). Temperatures were coolest in October and December
during the decline of daylength. Data for other physical and chemical
characteristics are presented in Appendix B. Increased Secchi
transparency was recorded during October and December. Dissolved oxygen
concentrations ranged from 7 to 12 mg l1, with a mean of 9.6 mg L'1.


153
indirectly as a result of steric hindrance. Evidence of this was
observed by the inverse relationship between APA and total organic C
(TOC).
Alkaline phosphatase activity was higher in the pelagic zone
(sites 2-8), which was more productive and nutrient rich than the spring
(site 1). The mean annual total APA in the water column was 18 nM
min'1, under non-limiting substrate concentrations; this indicates a
potential inorganic P release rate of 0.6 ng P L'1 min'1. Natural
substrate concentrations are usually lower than those used in the APA
assay, thus in situ release rates will be slower.
(2) Is Lake Apopka plankton APA inhibited by inorganic P and is
it produced in response to inorganic P limitation?
Over 90% of APA within the natural plankton population was
inhibited following the addition of 1000 fig L'1 inorganic P to the
growth medium. Addition of lower inorganic P concentrations
(10 to 100 fig L1) did not produce such complete inhibition, thus
ambient soluble reactive P (SRP) concentrations (< 10 fig L1) in the
lake water will not inhibit APA.
The uptake of added inorganic P was very rapid, indicating high P
demand. The inorganic P was immediately incorporated into the plankton
cells as surplus P, as determined by hot water extraction. In general,
surplus P was inversely related to APA, suggesting that internal
inorganic P levels controlled APA. Under field conditions this was
reflected by an inverse relationship between acid hydrolyzable P and
APA. In batch culture, both hot water extractable inorganic and organic
P were used to provide P for growth.


/mol
36
TIME (h)
Fig. 2-7. Diel variation of selected parameters measured February 6-7,
1990 at the center of Lake Apopka (site 8):
a) photosynthetically active radiation; b) wind speed
1 m above the water surface. Source: Stites, D. L.,
unpublished data, St. John's River Water Management District,
Palatka, FL.


67
significantly higher in April. Chlorophyll a was used as a means to
indicate phytoplankton growth in the cultures. Chlorophyll a
concentrations increased in response to nutrient additions to the water
samples collected in both April and August. Although small and hidden
due to axis scale, significant treatment differences in chlorophyll a
occurred within the first 24 h (Fig. 3-4 and 3-5a). After 96 h, maximum
chlorophyll a concentrations in April were 80 /xg L'1 and exceeded
180 /xg L'1 in August. In April, chlorophyll a concentrations were
significantly affected by the interaction between concentrations of N
and P added. Chlorophyll a concentrations obtained in the presence of
both nutrients exceeded those obtained by single nutrient additions
(Fig. 3-4). The greatest initial increase in chlorophyll a occurred in
cultures which received treatment 2 (N=400 P=0). Subsequently, growth
rates in treatments 4 (N=400 P=40) and 5 (N=800 P=80) exceeded those of
treatment 2. This information, combined with the lag in growth observed
in cultures receiving only P additions, suggest that phytoplankton were
initially N limited and became co-limited by P as they grew. This is
confirmed by the significant interaction of added N and P levels upon
chlorophyll a concentrations. In contrast, no significant interaction
between the levels of N and P was observed in August. In August, those
cultures which received only P additions had the same chlorophyll a as
those which received both N and P, while cultures receiving only N had
the same chlorophyll a as those with no nutrient addition. Increases in
chlorophyll a were also recorded in cultures which received no P
addition. These observations suggest that some P was still available
for plankton growth, but the growth rates were P limited, and thus the


126
result in the inhibition of organic P mineralization by APA in the
sediment (Pulford and Tabatabai 1988).
Regardless of the oxygen status of the surface sediments, redox
potential tends to decrease with sediment depth (Kobori and Taga 1979b;
Degobbis et al. 1984). Bacterial populations were also observed to
decrease with sediment depth, in response to redox potential changes
(Kobori and Taga 1979b). Bacterial numbers have been positively
correlated with APA in sediments (Ayyakannu and Chandramohen 1971),
hence APA also decreases with depth (Kobori and Taga 1979b). An
important effect of reduced Eh conditions may be the accumulation of
soluble organic P compounds due to incomplete mineralization. It is
apparent that oxygen concentrations both in the sediment and in the
water column may play a significant role in determining P
mineralization.
The objective of this study was to determine the effects of anoxia
upon organic P mineralization in the sediment and water column of a
highly productive lake. It is known that APA is a non-specific enzyme
capable of hydrolyzing numerous organic P compounds (Coleman and Gettins
1983; Cembella et al. 1984a). However, specific forms of sedimentary
organic P are mainly unknown; identification is based upon the
susceptibility to different extraction solutions (Bowman and Cole 1978;
Sommers et al. 1972). A second objective of this research was to
determine the forms of extractable P influenced by different redox
conditions, and their effect on the mineralization of organic P by APA.


171
Fuhs, G. W., S. D. Demmerle, E. Canelli, and M. Chen. 1972.
Characterization of phosphorus-limited plankton algae, p. 113-133. In
G. E. Likens [ed.], Nutrients and Eutrophication: The limiting
nutrient controversy, vol. 1. Spec. Symp. Limnol. Oceanogr.
Gchter, R., and A. Mares. 1985. Does settling seston release soluble
reactive phosphorus in the hypolimnion of lakes ? Limnol. Oceanogr.
30:364-371.
Gage, M. A., and E. Gorham. 1985. Alkaline phosphatase activity and
cellular phosphorus as an index of the phosphorus status of
phytoplankton in Minnesota lakes. Freshwater Biol. 15:227-233.
Garen, A., and C. Levinthal. 1960. A fine structure genetic and
chemical study of the enzyme alkaline phosphatase of f. coli. 1.
Purification and characterization of alkaline phosphatase. Biochim.
Biophys. Acta 38:470-483.
Geller, A., and K. N. Dobrotvorskaya. 1961. Phosphatase activity of
soils in beet root-seedling areas. Tr. Inst. Mikrobiol., Akad. Nau.
SSSR 11:215-221. (Chem. Abstrs. 1962 13278d)
Halstead, R. L., and R. B. McKercher. 1975. Biochemistry and cycling
of phosphorus, p.31-63. In E.A. Paul, A.D. McClaren, [eds.], Soil
Biochemistry. Vol. 4. Marcel Dekker Inc., New York.
Halemejko, G. E., and R. J. Chrst. 1984. The role of phosphatases in
phosphorus mineralization during decomposition of lake phytoplankton
blooms. Arch. Hydrobiol. 101:589-502.
Harrison, A. F. 1983. Relationship between intensity of phosphatase
activity and physico-chemical properties in woodland soils. Soil
Biol. Biochem. 15:93-99.
Harvey, H. W. 1953. Note on the absorption of organic phosphorus
compounds by Nitzschia closterium in the dark. J. Mar. Biol. 31:475-
476.
Healey, F. P. 1973. Characteristics of phosphorus deficiency in
Anabaena. J. Phycol. 9:383-394.
Healey, F. P., and L. L. Hendzel. 1979a. Fluorometric measurement of
alkaline phosphatase activity in algae. Freshwater Biol. 9:429-439.
Healey, F. P., and L. L. Hendzel. 1979b. Indicators of phosphorus and
nitrogen deficiency in five algae in culture. J. Fish. Res. Bd. Can.
36:1364-1369.
Healey, F. P., and L. L. Hendzel. 1980. Physiological indicators of
nutrient deficiency in lake phytoplankton. Can. J. Fish. Aquat. Sci.
37:442-453.


90
relationship was not recorded in response to treatment, suggesting that
internal P levels were not sufficiently low to control APA. Comparing
specific HEP-SRP (HEP-SRP/chlorophyll a) values in April, P limited
cultures had ratios < 0.09 fig P'1 fig chlorophyll a'1 while treatments 3
(N=0 P=40) and 5 (N=800 P=80) had ratios > 0.12 fig P L'1
ng chlorophyll a'1. Specific HEP-SRP ratios obtained after 96 h in
August were generally > 0.2 fig P L1 fig chlorophyll a1. In August,
after 96 h, an increase in HEP-SRP occurred at the same time as an
increase in growth rate for treatments 3 and 5. High growth rates in P
limited Scenedesmus sp. were associated with increased surplus P (Rhee
1974). No increase in HEP-SRP at 96 h was observed in the cultures
which received treatment 4. This may be anomalous because measurements
of both HEP-TSP and APA obtained from treatment 4 cultures agree with
those determined in treatment 3 cultures. Increased HEP-SRP
concentrations may not be the only factor regulating growth and P
limitation, because decreased APA was also observed in the other
cultures. Stable low concentrations of HEP-SRP, with rapidly declining
specific APA and uptake rates, combined with increasing chlorophyll a,
have been suggested to indicate that P is immediately utilized for
growth as opposed to P storage (Sproule and Kalff 1978). An inverse
relationship between specific APA and growth rate has been recorded
(Smith and Kalff 1981). Phosphorus required for growth is probably
provided from the hydrolysis of small chain polyphosphates from the
continually decreasing HEP-TSP concentrations. While HEP-TSP
concentrations declined, no increase in any other P parameter measured
was sufficient to account for the disappearance of HEP-TSP. Hence it


11
organic P (Ayyakannu and Chandramohen 1971). Numerous enzymes can be
utilized in organic P breakdown but the phosphatases, specifically APA,
are the most frequently cited (Halstead and McKercher 1975; Skujins
1976; Speir and Ross 1978).
As observed in the water column, APA in soils is positively
correlated with the concentration of organic matter (Harrison 1983;
Speir 1976) and age of organic matter (Rojo et al. 1990). Much of the
data concerning APA have been developed in upland soils (Geller and
Dobrotvorskaya 1961; Juma and Tabatabai 1978; Tabatabai and Bremner
1969); few studies have investigated APA in sediments. However, highly
significant APA has been reported in both freshwater (Klotz 1985a;
Sayler et al. 1979) and marine sediments (Ayyakannu and Chandramohen
1971; Kobori and Taga 1979b).
Phosphatase activity decreases with soil (Juma and Tabatabai 1978)
and sediment depth (Degobbis et al. 1984; Kobori and Taga 1979b). In
upland soils, this has been shown to correspond to decreases in
microbial biomass, C, N and organic P (Juma and Tabatabai 1978; Speir
and Ross 1978; Baligar et al. 1988). In sediments, redox potential
decreases significantly with depth, therefore an important distinction
between mineralization of organic compounds in sediments versus the
overlying water column is the concentration of oxygen. Limited oxygen
diffusion and rapid consumption of oxygen results in an oxygenated layer
at the sediment-water interface and decreasing oxygen with depth in the
sediment (Charlton 1980; Bostrom et aV. 1982). Alkaline phosphatase
activity is generally inhibited under anaerobic conditions, resulting in
a slower rate of organic P mineralization (Pulford and Tabatabai 1988).


(/g L_1) CONCENTRATION
35
TIME (h)
Fig. 2-6. Diel variation of selected paramters measured February 6-7,
1990 at the center of Lake Apopka (site 8): a) total Kjeldahl
nitrogen; b) total phosphorus. Vertical bars represent 1 SE.


159
Table B-3. Dissolved oxygen.
Date
Site
Mean'
SE
1
2
3
4
5
6
7
8
1
my l
APR
4.5
12.7
13.0
11.2
12.3
12.8
13.0
10.7
12.2
0.13
JUN
2.4
7.3
10.5
3.7
5.2
8.0
6.3
9.8
7.3
0.35
AUG
2.5
8.7
9.3
5.8
10.2
11.0
10.8
9.6
9.3
0.25
OCT
4.2
9.7
8.6
8.9
9.1
9.6
10.6
10.2
9.5
0.10
DEC
5.3
9.5
9.0
9.9
9.1
9.9
10.1
9.3
9.5
0.06
FEB
5.5
7.7
10.2
10.0
9.6
10.8
12.1
9.0
9.9
0.20
Mean
3.8
9.6
10.1
7.9
9.2
10.3
10.2
9.9
SE
0.2
0.3
0.3
0.5
0.4
0.3
0.4
0.1
Means listed in this column are means of sites 2-8.


134
experiment were analyzed using repeated measures analysis which accounts
for the inherent within replicate correlation due to repeated sampling
of the same flasks. When data were unbalanced repeated measures
analysis could not be used, a split plot design, with time as the
subplot, was used to produce the analysis of variance and F test. Data
in both experiments were analyzed using Pearson correlation
coefficients.
Results
The Effect of Dissolved Oxygen on Organic Phosphorus Mineralization in
the Water Column
Chlorophyll a concentrations of the water samples collected in
July 1990 were extremely high (Table 5-1) and exceeded previous
measurements (chapter 2). Other parameters measured were within the
same range as previously recorded in Lake Apopka (chapter 2).
The response of the measured parameters over time was highly
varied under both aerobic and anaerobic conditions. During the 24 h
experimental period pH decreased from 8.2 to 7.5 and 8.0 under aerobic
and anaerobic conditions, respectively (data not shown). Total soluble
N exhibited significant differences over time (p=0.02). Under aerobic
conditions TSN increased within 2 h and subsequently declined over time
(Fig. 5-3a). Under anaerobic conditions the only significant difference
observed was the increase at 24 h versus initial concentrations. There
was an apparent decrease in NH4-N concentrations over time (Fig. 5-3b),
however, no statistically significant time or treatment effects were
determined for NH4-N. Both TSP and SRP concentrations also exhibited


59
Initial and final chlorophyll a concentrations were calibrated against
chlorophyll a concentrations determined spectrophotometrically following
extraction with 90% acetone (APHA 1985).
Total P, TSP, SRP, NH4-N, and [N03 + N02]-N were determined by
standard methods (APHA 1985).
Hot water extractable P was determined in experiment 1 using a
modification of the method by Fitzgerald and Nelson (1966). Water
samples (50 to 100 mL) were filtered through 47 mm 0.45 /xm membrane
filters (Millipore). The filters were placed in 60 mL borosilicate
boiling tubes, and 20 mL deionized water were added. The tubes were
capped and autoclaved at 120*C for 1 h. Upon cooling samples were
analyzed for SRP. Blank filters were autoclaved to determine any filter
contribution to P analysis.
Hot water extractable P includes both a molybdate reactive form of
P and a non-reactive form of P. Both hot water extractable forms were
determined in experiment 2, using the method of Krausse and Sheets
(1980). Nine mL of sample were filtered though 25 mm 0.45 nm membrane
filters (Gelman). The filters were placed in 15 mL polypropylene tubes,
and 13 mL of deionized water was added. The tubes were capped and
autoclaved at 120*C for 1 h. Filter blanks were also autoclaved.
Gelman filters were used instead of Millipore filters because they had a
lower background P concentrations. The filtrate was analyzed for SRP,
and also digested via persulfate oxidation and analyzed for TSP.


135
Table 5-1. Concentrations of
Lake Apopka water
selected parameters measured in
in July 1990 (mean 1 SE).
Parameter
Concentration
Chlorophyll a (nq L'1)
220 4
Total APA (nM min'1)
21.9 0.1
Soluble APA (nM min'1)
0.82 0.4
TSP (nq L'1)
15 0
SRP (/xg L'1)
1 0
TSN (mg L'1)
2.48 0.0
NH4-N (mg L'1)
0.05 0



PAGE 1

BIOAVAILABILITY OF ORGANIC PHOSPHORUS IN A SHALLOW HYPEREUTROPHIC LAKE By SUSAN NEWMAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1991

PAGE 2

ACKNOWLEDGEMENTS I would like to thank Dr. Reddy, my major advisor, whose help and guidance made this undertaking an enjoyable learning experience. I would also like to thank the members of my committee, who were always willing to share their expertize. Appreciation is also expressed to Mr. Rick Aldridge whose assistance and lively discussion enabled me to conduct the nutrient enrichment experiments. I would also like to thank my friends and colleagues in the Soil Science Department, particularly those associated with the Wetland Soils Laboratory, whose encouragement, assistance and cooperation made this project flow more smoothly. I would like to thank my parents, Joyce and Chris Newman, whose love and support enabled me to complete my Ph.D. Without their encouragement I would not have continued on to higher education. Last but not least, I would like to thank Tom, whose love, companionship and support boosted my morale numerous times throughout this study. ii

PAGE 3

TABLE OF CONTENTS ACKNOWLEDGEMENTS ii LIST OF TABLES v LIST OF FIGURES viii ABSTRACT xii CHAPTERS 1 INTRODUCTION 1 Statement of the Problem 1 Need for Research 2 Organic Phosphorus Mineralization 3 Alkaline Phosphatase Activity in the Water Column 8 Alkaline Phosphatase Activity in the Sediment 10 Objectives 12 Dissertation Format 14 2 SEASONAL VARIABILITY IN ALKALINE PHOSPHATASE ACTIVITY IN A SHALLOW HYPEREUTROPHIC LAKE 16 Introduction 16 Materials and Methods 18 Results 23 Discussion 39 Conclusions 48 3 RESPONSE OF NATURAL PLANKTON POPULATIONS TO NUTRIENT ENRICHMENT 50 Introduction 50 Materials and Methods 51 Results 60 Discussion 86 Conclusions 94 iii

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4 THE EFFECT OF SEDIMENT RESUSPENSION ON ALKALINE PHOSPHATASE ACTIVITY 95 Introduction 95 Materials and Methods 97 Results 105 Discussion 115 Conclusions 123 5 THE EFFECT OF SEDIMENT AND WATER COLUMN ANOXIA ON ORGANIC PHOSPHORUS MINERALIZATION 124 Introduction 124 Materials and Methods 127 Results 134 Discussion 145 Conclusions 151 6 ORGANIC PHOSPHORUS CYCLING IN LAKE APOPKA 152 APPENDICES A LORAN COORDINATES 156 B CONCENTRATIONS OF SELECTED WATER CHEMISTRY PARAMETERS DETERMINED BIMONTHLY FROM APRIL 1989 THROUGH FEBRUARY 1990, AT 8 SITES IN LAKE APOPKA 157 REFERENCE LIST 1 68 BIOGRAPHICAL SKETCH 180 iv

PAGE 5

LIST OF TABLES Table page 2-1. Means of selected weather data measured at the central station (mean ± 1 SE) 24 2-2. Correlation coefficients for chlorophyll and alkaline phosphatase activity measured bimonthly at 7 sites in Lake Apopka (significant at a=0.05, n=7) 32 2-3. Correlation coefficients between alkaline phosphatase activity and selected parameters measured bimonthly at 8 sites in Lake Apopka (significant at a=0.05, n=6) 33 24. Correlation coefficients of selected water chemistry parameters determined at 7 sites in October 1989 (significant at a=0.05, n=7) 42 31. Nutrient additions made to diluted lake water collected in November 1989 54 3-2. Nutrient additions made to diluted lake water collected in April and August 1990 56 3-3. Initial concentrations of selected parameters measured in diluted lake water prior to nutrient addition (triplicate samples) in November 1989 (mean ± 1 SE) 61 3-4. Chlorophyll a and specific alkaline phosphatase activity measured in natural plankton populations collected in November 1989, 72 h after receiving nitrogen and phosphorus additions 62 3-5. Initial concentrations of selected parameters measured in diluted lake water prior to nutrient addition (triplicate samples) (mean + 1 SE) 66 3-6. Phosphorus uptake rates for natural plankton populations collected in August 1990, 30 min after receiving nitrogen and phosphorus additions (mean ± 1 SE) 74 3-7. Hot water extractable phosphorus concentrations of composite lake water samples collected in April 1990, 216 h after nutrient additions 78 v

PAGE 6

pjge 3-8. Specific hot water extractable phosphorus measured over time in natural plankton populations collected in August 1990 after receiving nitrogen and phosphorus additions (mean ± 1 SE) 81 3-9. Specific hot water extractable phosphorus measured over time in natural plankton populations collected in August 1990 after receiving nitrogen and phosphorus additions (mean + 1 SE) 83 310. Specific alkaline phosphatase activity measured over time in natural plankton populations collected in August 1990 after receiving nitrogen and phosphorus additions (mean ± 1 SE) 87 41. Distribution of selected parameters measured in May 1989 within the water column at the center of Lake Apopka (n=3) 106 4-2. Concentrations of parameters measured within the water column of cores collected in September 1989 from the center of Lake Apopka (mean ± 1 SE) 109 43. The distribution of alkaline phosphatase activity in disturbed and undisturbed sediment cores collected from the center of Lake Apopka in January 1990 (mean ± 1 SE) 118 51. Concentrations of selected parameters measured in Lake Apopka water in July 1990 (mean ± 1 SE) 135 5-2. Concentrations of selected parameters measured on sediments incubated under six different redox levels for one month (mean + 1 SE) 140 5-3. Concentrations of selected parameters measured on sediments incubated under six different redox levels for one month (mean ± 1 SE) 143 5-4. Selected correlation coefficients between phosphorus compounds and alkaline phosphatase activity measured in sediments incubated for one month under six different redox levels 144 APPENDIX TABLES ft. Loran coordinates of sampling sites on Lake Apopka Group repetition interval : 7980, Southeast USA 156 vi

PAGE 7

page B-l. Temperature 157 B-2. Secchi depth transparency 158 B-3. Dissolved oxygen 159 B-4. pH 160 B-5. Total solids 161 B-6. Total suspended solids 162 B-7. Chlorophyll a 163 B-8. Total organic carbon 164 B-9. Total Kjeldahl nitrogen 165 B-10. Total and soluble alkaline phosphatase activity 166 B-l 1 . Phosphorus 167 vii

PAGE 8

LIST OF FIGURES Figure page 11. Diagram of the phosphorus cycle in Lake Apopka 4 21. Location of Lake Apopka and sampling sites 19 2-2. Seasonal variability of selected parameters determined bimonthly at 7 sites in Lake Apopka 26 2-3. Seasonal variability of selected parameters determined bimonthly at 7 sites in Lake Apopka 27 2-4. Size fractionation of alkaline phosphatase activity determined bimonthly at 3 sites in Lake Apopka 29 2-5. Size fractionation of phosphorus concentrations determined bimonthly at 3 sites in Lake Apopka 30 2-6. Diel variation of selected parameters measured February 6-7, 1990 at the center of Lake Apopka (site 8) 35 2-7. Diel variation of selected parameters measured February 6-7, 1990 at the center of Lake Apopka (site 8) 36 2-8. Diel variation of selected parameters measured February 6-7, 1990 at the center of Lake Apopka (site 8) 37 2-9. Distribution of phosphorus compounds determined in whole lake water at 8 sites in October 1989 38 2-10. Distribution of phosphorus compounds determined in filtered lake water at 8 sites in October 1989 40 211. Distribution of suspended phosphorus compounds determined by difference between whole and soluble phosphorus measured at 8 sites in October 1989 41 31. Map showing the location of Lake Apopka and sampling sites. Water was collected from site 1 in November 1989 and from site 2 in April and August 1990 53 viii

PAGE 9

fiage 3-2. Time courses of alkaline phosphatase activity following nutrient enrichment of natural plankton populations collected in November 1989 63 3-3. Time courses of nutrient concentrations following nutrient enrichment of natural plankton populations collected in November 1989 65 3-4. Time courses of chlorophyll a concentrations following nutrient enrichment of natural plankton populations collected in April 1990 68 3-5. Time courses of chlorophyll a concentrations following nutrient enrichment of natural plankton populations collected in August 1990 69 3-6. Time courses of soluble reactive phosphorus concentrations following nutrient enrichment of natural plankton populations collected in April 1990 71 3-7. Time courses of soluble reactive phosphorus concentrations following nutrient enrichment of natural plankton populations collected in August 1990 73 3-8. Time courses of nutrient concentrations following nutrient enrichment of natural plankton populations collected in April 1990 75 3-9. Time courses of nutrient concentrations following nutrient enrichment of natural plankton populations collected in August 1990 grown at 29"C 76 3-10. Time courses of hot water extractable total soluble phosphorus following nutrient enrichment of natural plankton populations collected in August 1990 79 3-11. Time courses of hot water extractable soluble reactive phosphorus following nutrient enrichment of natural plankton populations collected in August 1990 82 3-12. Time courses of alkaline phosphatase activity following nutrient enrichment of natural plankton populations collected in April 1990 84 313. Time courses of alkaline phosphatase activity following nutrient enrichment of natural plankton populations collected in August 1990 85 41. Map showing the location of Lake Apopka 98 4-2. Diagram of the sediment resuspension device 101 ix

PAGE 10

gage 4-3. The depth distribution of selected parameters measured in triplicate sediment cores collected in Masy 1989 from the center of Lake Apopka 107 4-4. The depth distribution of selected parameters measured in triplicate sediment cores collected in May 1989 from the center of Lake Apopka 108 4-5. The depth distribution of alkaline phosphatase activity in triplicate resuspended and undisturbed (control) sediment cores collected in September 1989 from Lake Apopka Ill 4-6. Concentrations of selected parameters measured in the overlying water column following sediment resuspension of 0, 2, 5, and 10 cm surficial sediments 112 4-7. The total alkaline phosphatase activity measured in the overlying water column following sediment resuspension of 0, 2, 5, and 10 cm surficial sediments 113 4-8. The relationship between alkaline phosphatase activity and total suspended solids in the overlying water column of triplicate sediment cores after resuspension of surficial sediments U4 4-9. Soluble alkaline phosphatase activity measured in the overlying water column following sediment resuspension of 0, 2, 5, and 10 cm surficial sediments 116 410. Soluble reactive phosphorus concentrations measured in the overlying water column following sediment resuspension of 0, 2, 5, and 10 cm surficial sediments 117 51. Diagram illustrating the apparatus used to control dissolved oxygen concentration and redox potential 129 5-2. The sediment extraction scheme used to fractionate organic phosphorus in sediment 131 5-3. Nutrient concentrations in Lake Apopka water incubated in the dark under aerobic and anaerobic conditions 136 5-4. Nutrient concentrations in Lake Apopka water incubated in the dark under aerobic and anaerobic conditions 138 5-5. Alkaline phosphatase activity in Lake Apopka water incubated in the dark under aerobic and anaerobic conditions 139 x

PAGE 11

Concentrations of selected parameters measured in sediments incubated under six different redox levels for one month . xi

PAGE 12

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy BIOAVAILABILITY OF ORGANIC PHOSPHORUS IN A SHALLOW HYPEREUTROPHIC LAKE By Susan Newman May 1991 Chairman: K. R. Reddy Major Department: Soil Science Field and laboratory studies were conducted to determine the importance of organic P mineralization in the sediment-water column of Lake Apopka, a shallow hypereutrophic lake located in central Florida. Alkaline phosphatase activity (APA) was used as a tool to indicate the bioavailability of organic P to native plankton populations. Spatial and temporal variability in total APA occurred in the water column (range=4 to 45 nM min" 1 ) in response to different water chemistry characteristics. Nutrient enrichment studies demonstrated that APA increased with plankton biomass and specific APA (APA/chlorophyll a) values > 1 nmol APA /xg chlorophyll a' 1 min' 1 occurred during severe inorganic P limitation. In both the sediment and the water column APA was mainly associated with particulate matter. The APA of the plankton was inhibited by high inorganic P concentrations. Phosphorus demand of the plankton was high, as evidenced by the rapid uptake of added inorganic P. During the xii

PAGE 13

conditions of P limitation added inorganic P was immediately assimilated and recovered in the surplus P pool within the plankton tissue, as determined by hot water extraction. The plankton apparently utilized this surplus pool of P for growth under low external inorganic P concentrations. Resuspension of surficial sediments increased the interaction between sediments and the overlying water column, resulting in an immediate increase in APA and total P (TP) in the water column, indicating an increased potential for biological organic P hydrolysis during periods of resuspension. The APA and TP decreased rapidly during settling of suspended solids, following the cessation of turbulence. Organic P mineralization was greater under aerobic than anaerobic conditions in the sediment and overlying water column. Under aerobic conditions (dissolved oxygen [D0]=6 mg L" 1 ) APA in the water column increased from 22 to 43 nM min" 1 , while no change was observed under anaerobic conditions (D0=<0.2 mg L" 1 ). Sediment APA was a function of Eh, the measured reduction potential of the sediment-water systems. Under aerobic conditions (Eh=480 mV) APA was 10-fold higher than that observed under anaerobic conditions (Eh=-240 mV). Enzymatic hydrolysis of organic P compounds was significantly inhibited under anaerobic conditions. The results from this study suggest that the P requirement of plankton in a highly productive lake may partially be met through the enzymatic hydrolysis of organic P. Consequently, efforts to reduce nutrient loading and thus reduce eutrophication should also evaluate the bioavailability of organic P compounds in the system. xiii

PAGE 14

CHAPTER 1 INTRODUCTION Eutrophication may be defined as the nutrient and/or organic matter enrichment that produces high biological productivity (Likens 1972). This process is often accelerated by man, through allochthonous loading to the system from surface runoff, agricultural drainage and wastewater effluent. Eutrophication of our waterbodies has recently become a major concern due to the ever increasing need for resource conservation. Consequently, efforts are now being made to further understand the process of eutrophication and to identify key management strategies to abate this process. Statement of the Problem Lake Apopka is a 12,500 ha lake located in central Florida. It has a mean water depth of 2 m, overlying highly flocculent organic sediments (Reddy and Graetz 1990). Historically, the lake had clear water, submersed macrophytes and supported substantial sport fish populations. However, the physico-chemical properties of the lake have been altered through nutrient enrichment following the construction of the Apopka-Beauclair canal, discharge of sewage to the lake, and back pumping from the surrounding muck farms (USEPA 1979). Following the 1947 hurricane, the submerged vegetation was uprooted, and the first 1

PAGE 15

algal bloom was recorded (USEPA 1979). Since then, sport fish populations have dwindled and have been replaced by rough fish such as shad, gar and catfish. The high algal populations have resulted in the maintenance of a pea-green color in the lake. Lake Apopka has mean chlorophyll a concentrations > 60 fig L' 1 and total P concentrations of 200 nq L 1 (Canfield 1981; Huber et al . 1982; Reddy and Graetz 1990) and is thus currently classified as hypereutrophic (Forsberg and Ryding 1980). The lake is the first and largest in the Oklawaha chain of lakes, consequently a ripple effect is apparent. The high nutrient concentrations and algal blooms observed in Lake Apopka are evidenced downstream in the other lakes in the chain. Need for Research In order to understand the process of eutrophication in Lake Apopka and thus abate this process, it is necessary to determine the cycling of C, N and P in the sediment-water column. In general, N and P are the key elements involved in eutrophication (Chiaudani and Vighi 1982). Carbon fixation has been determined to be the driving force in the productivity of Lake Apopka (Reddy and Graetz 1990). This is apparent by the vast algal populations observed year round in the water column. Settling of senescent algal cells has resulted in a highly organic sediment. Consequently, both the sediment and the water column are dominated by organic matter which results in high levels of organic N and P. However, readily available P, i.e, soluble inorganic P concentrations are low and frequently undetectable (< 1 /xg L' 1 ) (Newman, S., unpublished data, Department of Soil Science, University of

PAGE 16

3 Florida, Gainesville, FL.). Researchers have investigated sorption reactions (Olila, 0., unpublished data, Department of Soil Science, University of Florida, Gainesville, FL.) and have characterized the cycling of inorganic P within the sediment and water column (Pollman, 1983; Reddy and Graetz 1990), but the dynamics of the organic P pool, the dominant form of P, have not been addressed. Studies have demonstrated that significant quantities of organic P may be bioavailable (Bradford and Peters 1987; Kuenzler and Perras 1965), hence the organic P pool may play a significant role in sustaining the vast plankton biomass under apparent inorganic P limitation. Therefore, the potential bioavailability of organic P in Lake Apopka needs to be determined. Organic Phosphorus Mineralization In aquatic systems, organic P in sediments constitute 15-50% of TP (Bostrom et al . 1982) while in the water column organic P may account for as much as 90% of TP (Rigler 1964). The P cycle is shown in Fig. 1-1. Organic P is generally characterized as total and soluble organic P. Specific identification of organic P constituents may be achieved following chromatographic fractionation and comparison with known compounds (Minear 1972; Weimer and Armstrong 1979), 31 P nuclear magnetic resonance (NMR) (Condron et al . 1985), or via hydrolysis by specific enzymes (Herbes 1974). Only 50% of organic P forms have been identified, including: inositol phosphates, sugar phosphates, phospholipids and nucleic acids (Stevenson 1982). The rate at which these compounds are mineralized is dependent upon their structure. High

PAGE 18

5 molecular weight, complex structures are highly resistant to mineralization, hence they will tend to accumulate, e.g. huraic acids. Conversely, simple, low molecular weight organic compounds are more susceptible to hydrolysis and thus more labile, e.g. sugarphosphates. It is these labile compounds which are more likely to undergo rapid enzymatic hydrolysis and thus be bioavailable. Soluble reactive P (SRP), the most labile form of P, has been the focus of most P cycling research (Hutchinson and Bowen 1950; Rigler 1956). However, SRP concentrations in lakes are generally low while organic P is abundant (Abbott 1957; Rigler 1956). Such observations resulted in investigations to determine whether organic P compounds could act as a source of P available for plankton nutrition. Under inorganic P limiting conditions phytoplankton may utilize organic P compounds for growth (Harvey 1953; Kuenzler 1965). These phytoplankton produce externally acting enzymes, phosphatases which hydrolyze phosphomonoesters (PME) and release SRP (Fitzgerald and Nelson 1966; Kuenzler and Perras 1965). Phytoplankton which do not produccce externally acting enzymes cannot hydrolyze PME compounds and their growth becomes P limited (Kuenzler 1965). Ecologically, the ability of phytoplankton to utilize organic P gives them a competitive advantage over non-phosphatase producers, during inorganic P limitation. The mode of action of phosphatases (specifically phosphomonoesterases) is shown below (Coleman and Gettins 1983): 1 2 3 4 ROP + E ROP-E E-P E-P E + P. R = an organic moiety, P, = inorganic P, E = enzyme.

PAGE 19

6 The steps involved in the reaction are as follows: 1. The phosphomonoester binds non-covalently to the phosphatase enzyme (ROP-E). 2. The phosphoseryl intermediate forms by covalent binding of the phosphate group to the phosphatase enzyme (E-P); alcohol is released during this nucleophilic attack. 3. Water is taken up resulting in the nucleophilic displacement of seryl phosphate to produce a non-covalently bound complex (E«P). 4. Inorganic P is released and the free phosphatase enzyme is regenerated. Phosphatases have a high degree of specificity for the P moiety of the P-O-C bond, but little specificity for the C moiety (Reid and Wilson 1971). These enzymes are classified as alkaline or acid depending on the pH range under which they exhibit optimum activity (Reichardt 1971; Torriani 1960). At acid pH, the dephosphorylation of the seryl phosphate is the rate limiting step. At alkaline pH, the dissociation of inorganic P from E-P is the rate limiting step (Coleman and Gettins 1983). The alkaline nature of most aquatic systems has resulted in alkaline phosphatase activity (APA) receiving the most emphasis. More than one type of phosphatase may be present in any plankton population. Five intracellular phosphatases were extracted from a Peridinium bloom (Wynne 1977). The phosphatases produced in response to P limitation do not have the same biochemical characteristics as those observed in normal tissues (Bielski 1974). Although acid phosphatases have the ability to function outside the cell (Kuenzler and Perras 1965;

PAGE 20

Price 1962), they are generally intracellular in action (Moller et al . 1975; Wynne 1977). Acid phosphatases function as specific enzymes in metabolic pathways and non-specific reactions (Cembella et al . 1984a). Hence they are constitutive and generally not repressible by inorganic P. Conversely, APA exhibits principly extracellular function. Alkaline phosphatase synthesis may be induced by the presence of organic P (Aaronson and Patni 1976; Kuenzler 1965). Alkaline phosphatase is a repressible enzyme (Jansson et al . 1988), whose synthesis is inhibited by high levels of inorganic P (Elser and Kimmel 1986; Lien and Knutsen 1973; Torriani 1960). Inorganic P is a competitive inhibitor of APA (Coleman and Gettins 1983; Moore 1969; Reid and Wilson 1971). Other factors which affect APA include; temperature (Garen and Levinthal 1960; Torriani 1960), chelators and divalent cations (Cembella et al . 1984a; Healey 1973). The derepression of APA in response to P limitation has been examined at the cellular level where APA was shown to transport inorganic P. Studies utilizing Escherichia coli have shown that two forms of P transport exist (Rosenberg et al . 1977). One is a low affinity system, phosphate inorganic transport (PIT), which is constitutive and transfers intracellular P pools. The other is a high affinity system, phosphate specific transport (PST), which is activated when internal P concentrations are low. This utilizes a membrane associated protein, APA, to increase P uptake. The high affinity system is inhibited at high concentrations of inorganic P. However, it is the ability of APA to catalyze the hydrolysis of organic P compounds that has received the most study in the aquatic environment.

PAGE 21

8 The intensity of APA is dependent on the severity of P limitation. As much as 6% of the total protein produced under P limiting conditions may be attributed to APA (Garen and Levinthal 1960). The increase of APA in response to inorganic P deficiency has resulted in the use of APA as a tool to assess the P limitation of plankton. Alkaline Phosphatase Activity in the Water Column Inverse relationships between APA and SRP have been reported in many species of plankton (Healey 1973; Olsson 1990; Pettersson 1980; Pettersson et al . 1990). Under low SRP concentrations APA is derepressed, and upon replenishment of external inorganic P, APA is inhibited. In some situations no significant relationship is observed between APA and SRP (Berman 1970; Taft et al . 1977). It has been suggested that under these circumstances high concentrations of soluble organic P counteract the inhibition caused by SRP by stimulating induction of APA (Kuenzler 1965; Cembella et al . 1984a). Alternatively, where no correlation exists, APA may reflect P demand rather than P limitation (Taft et al . 1977). In combination with the depletion of external concentrations of inorganic P, P limitation in plankton is also demonstrated by reduced internal P concentrations (Chrost and Overbeck 1987; Rhee 1973). Inverse relationships between APA and surplus P have been recorded (Lien and Knutsen 1973; Rhee 1973). Once internal P concentrations have been reduced below critical levels, APA is produced (Chrost and Overbeck 1987; Fuhs et al . 1972). Alkaline phosphatase activities have been

PAGE 22

9 observed to be 25 times greater under P limitation than under P sufficiency (Fitzgerald and Nelson 1966). Ecologically, the importance of APA is dependent on the cooccurrence of both substrates and enzymes. Numerous problems have been associated with the determination of PME concentrations. The most common method of determining PME has been to monitor SRP release from filtered lake water following the addition of pure alkaline phosphatase (Strickland and Parsons 1968). The simultaneous occurrence of PME and APA by cyanobacteria blooms has been observed under low SRP concentrations (Heath and Cooke 1975). In some lakes, the rate of inorganic P release from PME equals the rate of P uptake by the plankton. In other lakes a large discrepancy exists between these two rates, with inorganic P release being considerably less than uptake rate (Boavida and Heath 1988; Cotner and Heath 1988; Heath 1986), thus leading these researchers to conclude that APA is not important in P nutrition of plankton. One of the problems associated with this conclusion is the use of filtered lake water in the analyses, therefore the large particulate organic P pool is absent (Wetzel 1983). Phosphatases have also been shown to release P from particulate matter (Jansson 1977). Seventy-four percent of extractable P in phytoplankton is susceptible to enzymatic hydrolysis and 80% of the organisms involved in phytoplankton decomposition produce phosphatases (Halemejko and Chrdst 1984). These results, and the apparent absence of hydrolyzable soluble PME in the water column (Herbes 1974; Herbes et al . 1975; Pettersson 1980) suggest that it is the substrate availability that limits enzymatic P cycling not APA (Jansson et al . 1988).

PAGE 23

10 The interpretation of APA as a measure of P limitation is complicated by the uncertainty of the origin of APA. Bacteria, phytoplankton and zooplankton are considered to be dominant contributors to this pool, and it is suggested that APA of algal origin is the most important in the epilimnion (Jansson et al . 1988). High levels of soluble APA indicate filterable activity and may reflect bacterial associated APA (Stewart and Wetzel 1982), zooplankton excretion (Jansson 1976; Wynne and Gophen 1981) and cell lysis (Berman 1970). In many lakes APA was determined to be mainly associated with phytoplankton, based on co-occurrence of APA and algal blooms (Heath and Cooke 1975), and as shown by correlations with chlorophyll a (Jones 1972a; Matavulj and Flint 1987; Siuda et al . 1982; Smith and Kalff 1981) and size fractionation of phosphatase activity (Chrost et al . 1989; Jansson 1977). Alkaline phosphatase activity has also been attributed to bacteria through correlations with bacterial numbers (Jones 1972a; Kobori and Taga 1979a). In shallow lakes, a large portion of particulate material may be sedimentary in origin. Concentrations of P compounds and bacterial numbers may be higher in sediments. Interaction between sediment and the overlying water column may significantly affect the mineralization of organic P in the overlying water. Hence, wind events in shallow lakes can significantly affect APA. Alkaline Phosphatase Activity in the Sediment In lake sediments as much as 70% of TP can be in organic form (Weimer and Armstrong 1979). In highly organic sediments, the relative abundance of organic substrates may result in enhanced breakdown of

PAGE 24

organic P (Ayyakannu and Chandramohen 1971). Numerous enzymes can be utilized in organic P breakdown but the phosphatases, specifically APA, are the most frequently cited (Halstead and McKercher 1975; Skujins 1976; Speir and Ross 1978). As observed in the water column, APA in soils is positively correlated with the concentration of organic matter (Harrison 1983; Speir 1976) and age of organic matter (Rojo et al . 1990). Much of the data concerning APA have been developed in upland soils (Geller and Dobrotvorskaya 1961; Juma and Tabatabai 1978; Tabatabai and Bremner 1969); few studies have investigated APA in sediments. However, highly significant APA has been reported in both freshwater (Klotz 1985a; Sayler et al . 1979) and marine sediments (Ayyakannu and Chandramohen 1971; Kobori and Taga 1979b). Phosphatase activity decreases with soil (Juma and Tabatabai 1978) and sediment depth (Degobbis et al . 1984; Kobori and Taga 1979b). In upland soils, this has been shown to correspond to decreases in microbial biomass, C, N and organic P (Juma and Tabatabai 1978; Speir and Ross 1978; Baligar et al . 1988). In sediments, redox potential decreases significantly with depth, therefore an important distinction between mineralization of organic compounds in sediments versus the overlying water column is the concentration of oxygen. Limited oxygen diffusion and rapid consumption of oxygen results in an oxygenated layer at the sediment-water interface and decreasing oxygen with depth in the sediment (Charlton 1980; Bostrom et ah 1982). Alkaline phosphatase activity is generally inhibited under anaerobic conditions, resulting in a slower rate of organic P mineralization (Pulford and Tabatabai 1988).

PAGE 25

12 However, in shallow lakes, wind induced resuspension of sediments to the oxygenated water column, results in the rapid breakdown of organic P (Pomeroy et al . 1965). A significant positive correlation between SRP released and APA in the water column has been observed during sediment resuspension (Degobbis et al . 1984). Resuspension can physically transport SRP to the overlying water column (Ryding and Forsberg 1977). It also increases the suspended solids concentration within the overlying water. This particulate material can provide 28-41% of algal available P (Dorich et al . 1985). Consequently, resuspension of sediments can result in enhanced enzyme activity and P availability within the water column. Hence sediments can play a significant role in organic P mineralization in the overlying water column. Objectives Bioavailability of organic P occurs through the action of enzymes (Kuenzler and Perras 1965). These enzymes catalyze the release of inorganic P from both soluble and particulate matter. Both APA and organic P concentrations increase with increased eutrophication (Jones 1979b). Hence, Lake Apopka, which is classified hypereutrophic, should support large concentrations of APA and organic P. This study is based on the hypothesis that enzyme mediated P release is used to support the vast algal populations during inorganic P limitation. Without this ability to utilize organic P at times of high P demand and inorganic P limitation, algal and bacterial species are nutrient stressed. Very little is known about the bioavailability of organic P

PAGE 26

13 in sub-tropical lakes, consequently, research investigating the breakdown and utilization of organic P is essential in any assessment of lake eutrophication. The main components influencing the cycling of organic P within the water column are; water chemistry, plankton uptake and release, and sediment resuspension (Fig. 1-1). To understand the role of organic P in Lake Apopka the following questions were addressed. (1) How is the enzymatic hydrolysis of organic P compounds affected by other water chemistry parameters? (2) Is Lake Apopka plankton APA inhibited by inorganic P and is it produced in response to inorganic P limitation? (3) What effect does sediment resuspension have upon organic P mineralization rates? The overall objective of this study was to evaluate the significance of organic P compounds in Lake Apopka and determine their potential bioavailability. Specific objectives are listed below. (1) Determine the seasonal, spatial and diel variability of APA within the water column. Alkaline phosphatase activity is produced by organisms, hence any factors such as changes in environmental conditions which affect metabolism may therefore affect APA. The predominant biotic group in Lake Apopka biota are the plankton, hence APA is linked to fluctuations in response to plankton metabolism. (2) Determine the influence of inorganic P upon the growth of natural plankton populations.

PAGE 27

14 Soluble inorganic P is the most readily available form of P for plankton nutrition, however; in its absence organic P compounds may be used for growth. The enzymatic hydrolysis of organic P compounds is competitively inhibited by inorganic P. Soluble reactive P concentrations in Lake Apopka are hypothesized to be too low to inhibit APA. There is, however, the issue of internal concentrations of P which may regulate organic P hydrolysis outside the cell. (3) Evaluate the effect of sediment resuspension upon the mineralization of organic P in the sediment and overlying water column. In shallow lakes, wind induced resuspension of sediments into the overlying water column increases the interaction between these compartments. It is hypothesized that resuspension of sediment increases the concentration of organic substrates and associated microorganisms in the water column and thus increases mineralization. (4) Determine the effect of anoxia on organic P mineralization in the sediment and water column. Mineralization of organic compounds proceeds more rapidly under aerobic than anaerobic conditions. Since a majority of sediments are anaerobic, it is hypothesized that APA will be inhibited under anaerobic conditions, resulting in a reduced mineralization rate. Dissertation Format Each chapter within this dissertation is written as an independent manuscript intended for future publication. Chapter 2 focuses upon the concentrations of P compounds and APA within the water column and the

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15 various factors which affect them. Chapter 3 examines the P nutrient status of natural plankton populations. Chapter 4 investigates the effect of sediment-water column interactions upon organic P bioavailability. The effect of anaerobic/aerobic conditions on organic P bioavailability is examined in chapter 5. The overall conclusions and the significance of these results are discussed in chapter 6.

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CHAPTER 2 SEASONAL VARIABILITY IN ALKALINE PHOSPHATASE ACTIVITY IN A SHALLOW HYPEREUTROPHIC LAKE Introduction Phosphorus is the major nutrient limiting plankton production in many temperate lakes (Schindler 1977). Soluble reactive P (SRP) has been the form of P most often studied (Rigler 1956); however, SRP is only a small fraction of the total P (TP) pool. A significant component of TP may be in organic form (Minear 1972; Rigler 1964). In lakes where inorganic P availability is low, plankton may produce phosphatase enzymes which hydrolyze organic P compounds with the release of inorganic P (Fitzgerald and Nelson 1966; Kuenzler and Perras 1965). These enzymes are designated alkaline or acid phosphatase, depending on the pH range of optimum activity (Kuenzler and Perras 1965; Torriani 1960). The alkaline nature of most water bodies has resulted in alkaline phosphatase activity (APA) receiving the most attention. Although some APA has been determined to be constitutive (Kuenzler 1965), plankton produce increased APA under conditions of P limitation. Alkaline phosphatase activity has hence been used as an indicator of P limitation, however, APA may also reflect P demand, as evidenced by a poor relationship between SRP and APA (Taft et al . 1977). The release of inorganic P mediated by APA is dependent upon the percentage of organic P which is hydrolyzable by the enzyme. Thirty-six 16

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17 percent of organic P in seawater (Kobori and Taga 1979a) and 32% of organic P in freshwater (Hino 1988) were hydrolyzed by phosphatase enzymes. Organic P of algal origin is particularly sensitive to hydrolysis; 74% of algal extracted P was hydrolyzed by APA, while 80% of organisms involved in the decomposition of plankton produced phosphatases (Halemejko and Chrdst 1984). In eutrophic situations TP and organic P concentrations can be high (Jones 1979b), resulting in higher APA levels than in lower trophic states (Jones 1979b; Pick 1987). Alkaline phosphatase activity may be a significant mechanism of satisfying high P demand in eutrophic situations, as well as a means of overcoming P limitation in nutrient poor environments. The intensity of APA is subject to the physico-chemical conditions in the environment. Enzyme activity is pH dependent and can respond negatively or positively to pH fluctuations (Torriani 1960). Dissolved oxygen (DO) and temperature also influence microbial enzyme activity and metabolism, thus they may directly or indirectly affect APA (Garen and Levinthal 1960). These environmental effects demonstrate the potential for seasonal and di el fluctuations in APA. This chapter examines the impact of seasonality on APA in one of Florida's largest hypereutrophic lakes, Lake Apopka. Despite low SRP concentrations < 1 fig L"\ chlorophyll a concentrations are regularly > 100 fig L" 1 (Canfield 1981; Reddy and Graetz 1990). High standing crops may be maintained through the rapid recycling of SRP or by obtaining P from other sources. Total soluble P (TSP) concentrations of 255 fig P L" 1 have been recorded in Lake Apopka (Reddy and Graetz 1990). Total soluble P may represent bioavailable P (Bradford and

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18 Peters 1987), hence organic P compounds in this pool may potentially be hydrolyzed by APA and release inorganic P. With the exception of extensive research by Berman and colleagues on Lake Kinneret, Israel (Berman 1970; Wynne and Berman 1980), the majority of studies investigating APA have been conducted in cold temperate zones. Warmer climates with mild winters which result in extended periods of productivity, may result in increased P demand. More studies in warmer climates are necessary. The primary objective of this study was to examine the seasonal, spatial and diel changes in APA to determine whether it represents P limitation or high P demand. This would also determine what effect the water chemistry has upon APA. Zooplankton, phytoplankton and bacterioplankton may all contribute to the total APA pool (Jansson 1976; Jones 1979a; Kuenzler and Perras 1965; Wynne and Gophen 1981). A second objective was to estimate the relative importance of these contributors based on filter size fractionation. Total soluble P may be used as an indicator of bioavailable P; however, a third objective was to determine the relationship between APA and other components of the TP pool. Materials and Methods Site Description Lake Apopka is a 12,500 ha, located in central Florida, 28' 37' N latitude, 81' 37' W longitude (Fig. 2-1). It has a mean depth of 2 m. Water influxes to the lake include Apopka Springs and backpumping from surrounding agricultural land. Outflow is northward through the Apopka-

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19

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20 Beauclair canal. The St. John's River Water Management District (SJRWMD) weather station is located at the center of the lake (site 8). Water Sampl inq Bimonthly sampling. Lake water was collected bimonthly from April 1989 thru February 1990, from 8 sites in Lake Apopka (Fig. 2-1). Sites 1, 4 and 8 were selected to represent inflow, outflow and the center of the lake. Site 2 was selected to determine littoral zone influences. Site 5 was located close to a pump station and hence represented backpumping from the surrounding agricultural land. Site 7 was established close to an old fishing camp, and site 6 was selected to correspond to extensive sediment studies which were conducted with samples from that site. Site locations were established using Loran coordinates (Appendix A). Three replicate water samples were collected from a depth of 0.3 m using 1 L polyethylene bottles, from each site. Samples were stored on ice until return to the laboratory. Water was filtered through 0.45 membrane filters (Gelman) and analyzed for soluble APA within 24 h. Other soluble parameters determined were total soluble P and SRP. Soluble particulate P was defined as TSP-SRP (SPP). Whole lake water was analyzed for total APA, total Kjeldahl N (TKN), TP, SRP, total solids (TS), total suspended solids (TSS), total organic carbon (TOC), and chlorophyll a. Seasonal water chemistry data collected at site 8 were compared with the weather data provided by the SJRWMD. The contributors to the APA pool are frequently determined via filter fractionation (Chrost and Overbeck 1987; Currie and Kalff 1984; Currie et al . 1986). To distinguish between the contribution of

PAGE 34

21 phytopl ankton and bacteria, water samples collected from sites 1, 4 and 8 received further filtration. Subsamples of water were filtered through 150, 8, 2.5, 0.45 and 0.2 /im filters. Water from these size fractions was analyzed for chlorophyll a and determined to represent 80, 9, 3, 1 and 0% chlorophyll a distribution. To minimize the filtration time, the filtration was not performed sequentially. Alkaline phosphatase activity and TP were determined on all samples. Dissolved oxygen and temperature (YSI, model 58), and pH (Orion, model SA 230) were recorded at 0.3 m. Light penetration was estimated by measuring Secchi disk transparency. Samples were collected at approximately the same time at each sampling period to minimize the effects of diel variation. Diel sampling . Diel studies were conducted on March 21 1989 and February 6 1990. In March 1989 DO and pH of the water were measured from a pontoon boat which was anchored at the central station (site 8) for 24 h. In February 1990, pH and DO measurements were determined by SJRWMD personnel. Water samples at both time periods were collected using an automatic sampler, and kept cool until returned to the laboratory for analysis. Fractionation of lake water phosphorus. To elucidate the relationship between P and APA in the water column, the P forms were separated analytically using discrete extraction procedures. In October 1989, water samples from all 8 sites were partitioned into total and soluble; reactive P, acid hydrolyzable and organic P (APHA 1985). Enzyme hydrolyzable P (EHP) was also determined.

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22 Analytical Methods Alkaline phosphatase activity was determined fluorometrically (Healey and Hendzel 1979). One half mL of substrate, 3-o-methyl fluorescein phosphate (Sigma Chemicals), at a concentration determined to be non-limiting, in 0.1 M Tris buffer (Tham, Fisher Scientific) pH 8.6, was added to 4.0 mL of lake water in a cuvette. Both total (whole lake water) and soluble (filtered through 0.45 /xm Gel man membrane filter) APA were determined. The cuvettes were placed in a water bath (25*C). At timed intervals during a 20 min period the cuvettes were placed in the fluorometer and the fluorescence measured. The enzyme activity was measured as an increase in fluorescence as the substrate was enzymatically hydrolyzed to the fluorescent product. Fluorescence units were converted to enzyme activity using a standard calibration curve of 3-o-methyl fluorescein (Sigma Chemicals). The fluorescence was measured using a Turner fluorometer No. 110, equipped with Turner lamp no. 110-853, in combination with 47 B primary and 2a12 secondary filters. Autoclaved lake water with substrate added was used as a control . Chlorophyll a was determined spectrophotometrically following extraction with acetone (APHA (1002-G), 1985). Total organic C was measured using an 0. I. Corporation Model 524C T0C analyzer, following oxidation by potassium persulfate. Total P, TSP, and TKN were determined following Kjeldahl digestion. Soluble reactive P, TSP and TP were analyzed via ascorbic acid using standard methods (APHA 1985). Total solids and TSS were determined by standard methods (APHA 1985).

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23 Enzyme hydrolyzable P was determined using the method of Strickland and Parsons (1968). Statistics Data were analyzed using SAS for personal computers, version 6 (SAS 1985). Pearson correlation coefficients were determined for all data. Results Measured parameters varied seasonally and spatially. Seasonal variability was generally greater than spatial variability (data presented graphically represent means of sites 2-8). Appendix B contains tables presenting data on a site basis and demonstrates spatial variability. Site 1 is the site of a natural spring, 80 ft deep and is not subject to the same wind induced mixing as the rest of the lake. Data from site 1 are discussed separately, to illustrate the effects of spring input to the lake. Physico-chemical Characteristics A 14*C range in water temperature was observed over the sampling period (Table 2-1). The highest water temperatures occurred in June and August, and coincided with maximum photosynthetically active radiation (PAR) (Table 2-1). Temperatures were coolest in October and December during the decline of daylength. Data for other physical and chemical characteristics are presented in Appendix B. Increased Secchi transparency was recorded during October and December. Dissolved oxygen concentrations ranged from 7 to 12 mg L"\ with a mean of 9.6 mg L*\

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24 Table 2-1. Means of selected weather data measured at the central station (mean + 1 SE) . Date Wind speed 1 m 5 m above surface Water temp PAR* ym km h" 1 °C /imol m" 2 s1 8904 2 ± 0 NA* 23.6 NA 8906 9+0 10+0 29.8 548 ± 29 8908 11 ± 0 13 ± 0 28.5 508 ± 29 8910 11 ± 0 20 ± 0 19.4 238 ± 14 8912 11+0 13+0 15.5 294 + 18 9002 13 ± 0 18 ± 0 20.0 282 ± 16 PAR indicates photosynthetically active radiation * NA indicates data not available. Source: Stites, D. L., unpublished data, St. John's River Water Management District, Palatka, FL.

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Little variation in pH was observed with a range of 0.8 pH units observed between maximum and minimum pH. Total solids were also constant between 300 and 400 mg L"\ until February when a particularly high concentration of 500 mg L' 1 was recorded. Total suspended solids accounted for approximately 16% of TS and increased in February at all sites (Fig. 2-2a). A distinct peak in chlorophyll a concentration was observed in August at 5 of the 7 sites (Fig. 2-2b). This peak was 3 fold higher than the minimum which occurred in February. Two peaks in T0C were apparent. One occurred in August, along with chlorophyll a (Fig. 2-2c) while the other occurred in February and probably corresponded to the increase in TSS which also occurred in February. Total Kjeldahl N tended to be higher in Spring and Summer and decreased in Fall and Winter (Fig. 2-2d). Alkaline phosphatase activity was mainly associated with particulate matter (Fig. 2-3a). Soluble APA averaged only 3% of total APA. Total APA peaked in both June and October; in contrast, soluble APA peaked in October and December. Phosphorus was also determined to be mainly associated with particulate matter. Total P concentrations peaked in October (Fig. 2-3b). Total soluble P concentrations represented from 6 to 37% of TP in August and April respectively. Soluble reactive P concentrations were very low throughout the year (0 to 7 ng L 1 ) and as such were a minor portion of TP. All parameters measured at site 1 were generally much lower than those recorded at other stations (Appendix B). In particular DO and pH

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AJAODF AJAODF 1989-90 1989-90 Fig. 2-2. Seasonal variability of selected parameters determined bimonthly at 7 sites in Lake Apopka: a) total suspended solids, data were not collected in April and June; b) chlorophyll a; c) total organic carbon, data were not collected in April; d) total Kjeldahl nitrogen. Vertical bars represent 1 SE.

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27 30 APR JUN AUG OCT DEC FEB 1989-90 ig. 2-3. Seasonal variability of selected parameters determined bimonthly at 7 sites in Lake Apopka: a) alkaline phosphatase activity; b) phosphorus. Vertical bars represent 1 SE.

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28 were considerably lower and Secchi was significantly greater. Apopka spring temperatures tend to be consistent throughout the year, only a 4'C fluctuation in water temperature was observed. Algal biomass was significantly lower than observed in the rest of the lake. Annual chlorophyll a concentrations averaged 21.8 fig L' 1 at site 1, while values averaged 81 fig L" 1 at other sites. The contribution of the various components in lake water to the total APA pool was determined via filter size fractionation. The distribution of APA followed that of chlorophyll a, with the majority of the activity associated with the larger size fraction (Fig. 2-4), and the distribution of APA within the different size fractions remained constant throughout the year. The greatest amount of soluble APA occurred in December. In general, a greater proportion of APA was associated with 8 and 2.5 fm filtered samples in spring water at site 1, than was observed in samples from sites 4 and 8. A similar distribution was also determined for P (Fig. 2-5), although a greater proportion was associated with the soluble fraction. Phosphorus concentrations peaked in October at all three sites. Relations hips between Water Chemistry Data and Selected Environmental Parameters Seasonal patterns in PAR, wind speed and water temperature collected at site 8 (lake center) were observed (Table 2-1). Water temperature and PAR peaked in June and August. Wind speed measured 5 m above the surface was generally greater than that measured 1 m above the surface (Table 2-1). Total P was positively correlated with wind speed observed 5 m above the water surface (r=0.88) and SRP was

PAGE 42

29 c E >> i— < Ld CO < t< Q. LO O Q. Ld < 50 40 30 20 10 0 SITE 1 WHOLE 03 2.5 ISO /tm 0 0.45 Mm IS 8 Mm H 0.20 Mm (o) ttt Hi FthFn_ tl_ APR JUN AUG OCT DEC FEB 1989-90 Fig. 2-4. Size fractionation of alkaline phosphatase activity determined bimonthly at 3 sites in Lake Apopka: a) site l=inflow; b)site 4=ouflow; c) site 8=center of lake. Vertical bars represent 1 SE.

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CJ1 3. < LU O o o in ID 01 o X Q_ 01 o X Q_ 600 500 400 300 200 100 h 0 600 500 400 300 200 r100 0 600 500 400 300 200 100 0 SITE 1 WHOLE Q3 2.9 M m 150 Mm CI 0.4501" IS 8 Mm Q 0.20 ftm (a) iii! APR JUN AUG OCT DEC FEB 1989-90 2-5. Size fractionation of phosphorus concentrations determined bimonthly at 3 sites in Lake Apopka: a) site l=inflow; b)site 4=ouflow; c) site 8=center of lake. Vertical bars represent 1 SE.

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positively correlated with PAR (r=0.98). Total Kjeldahl N was inversely related to wind speed measured 1 m above the surface (r=-0.94), while TSS was highly positively correlated with the wind speed recorded 1 m above the surface (r=0.97). Total suspended solids were also positively correlated with chlorophyll a, and inversely correlated with TSP and SOP. Neither chlorophyll a nor APA were correlated with any of the weather data. Relationships between Alkaline Phosphatase Activity. Chlorophyll a and other Parameters Alkaline phosphatase activity did not correlate with many water chemistry parameters (Table 2-2). A significant correlation was observed between chlorophyll a and total APA in December (r=0.82), while APA was inversely related to TSP (r=-0.86). Correlations among chlorophyll a, total APA and other measured parameters varied over time. Chlorophyll a correlated with P four out of the six sampling periods and total APA only correlated with P twice (Table 2-2). On a site by site basis, different correlations between APA and water chemistry parameters were observed (Table 2-3). Spatial variability of the factors affecting APA was apparent. Utilizing annual means, total APA was negatively correlated with TOC (r=-0.97) and chlorophyll a was highly correlated with TS (r=-0.88). Specific APA (ratio of total APA/chlorophyll a) was not correlated with any of the water chemistry parameters. Diel Variability On March 21 1989 and February 6 1990 selected parameters influencing APA were measured over a 24 h period to determine diel

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Table 2-2. Correlation coefficients for chlorophyll a and alkaline phosphatase activity measured bimonthly at 7 sites in Lake Apopka (significant at a=0.05, n=7). Month Correlations with Chlorophyll a Total A PA Soluble APA April TKN 0.88 TP 0.75 NS* TP 0.85 SPP -0.67 DO -0.74 temp -0.96 June TKN 0.92 pH -0.79 TSP 0.76 soluble APA 0.74 TOC 0.68 DO -0.76 SPP 0.78 August NS NS NS October TOC -0.70 NS TKN -0.75 TSS 0.73 TP 0.75 SPP -0.66 temp 0.71 TS -0.67 December total APA 0.82 TSP -0.86 TP 0.70 temp 0.68 SPP -0.86 TSP 0.81 secchi -0.80 SPP 0.81 February TP 0.71 TOC -0.69 NS NS indicates not significant at a = 0.05.

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33 Table 2-3. Correlation coefficients between alkaline phosphatase activity and parameters measured bimonthly at 8 sites in Lake Apopka (significant at a=0.05, n=6) Alkaline phosphatase activity Site Total Soluble 1 nn 0 87 temp -0.81 temp -0.77 2 TKN -0.78 TKN TOC -0.81 TP 0.89 TSP 0.85 SPP 0.86 secchi 0.85 3 soluble APA 0.82 TP 0.75 secchi 0.79 secchi 0.79 4 NS' CHL 0.75 DO 0.92 5 TP -0.75 secchi 0.90 6 TSS -0.93 secchi 0.81 7 NS TP -0.83 PH 0.73 8 TSP 0.83 NS SPP 0.81 PH 0.80 DO 0.82 TOC -0.92 NS indicates not significant at a = 0.05.

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variation. Diel DO changes were observed at both time periods. No significant changes in other measured water chemistry parameters were observed in March 1989, while parameters did exhibit change in February 1990. In February 1990, TKN concentrations remained constant during the first 12 h of sampling and then declined (Fig. 2-6a). Maximum TKN corresponded to high PAR and wind speed (Fig. 2-7a and 2-7b). The decline in TKN corresponded to the decrease of these two parameters. No significant change was observed for SRP, while TP concentrations fluctuated throughout the sampling period (Fig. 2-6b). Similar fluctuations were also observed for total and soluble APA (Fig. 2-8a). As observed for TKN, chlorophyll a concentrations tended to decrease in conjunction with decreased PAR and wind speed, however, the range was only 33 to 37 nq L" 1 (Fig. 2-8b). Fractionation of Lake Water Phosphorus In October, lake water samples were fractionated to determine the different forms of P present. Site variability in chlorophyll a and TSS concentrations was observed (Appendix B). Total organic C remained constant at 30 mg L" 1 for all sites except site 1. Chlorophyll a and TOC were lower at site 1. As determined above, most of the total APA was in the particulate fraction with the soluble APA contribution varying from site to site (Appendix B). The distribution of the various P forms also exhibited spatial variability (Fig. 2-9). In most sites TOP represented over 80% of TP. Total acid hydrolyzable P contributed 10% and total reactive P (TRP) contributed 3% to the TP pool. A similar distribution of these

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Fig. 2-6. Diel variation of selected paramters measured February 6-7, 1990 at the center of Lake Apopka (site 8): a) total Kjeldahl nitrogen; b) total phosphorus. Vertical bars represent 1 SE.

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36 1500 10:00 14:00 18:00 22:00 02:00 06:00 10:00 TIME (h) Fig. 2-7. Diel variation of selected parameters measured February 6-7, 1990 at the center of Lake Apopka (site 8): a) photosynthetically active radiation; b) wind speed 1 m above the water surface. Source: Stites, D. L., unpublished data, St. John's River Water Management District, Palatka, FL.

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37 < a: 0.5 0.0 • Total V Soluble 7 Ld O 2 O O cn 4. 10:30 14:30 18:30 22:30 02:30 06:30 10:30 TIME (h) Fig. 2-8. Diel variation of selected parameters measured February 6-7, 1990 at the center of Lake Apopka (site 8): a) alkaline phosphatase activity; b) chlorophyll a. Vertical bars represent 1 SE. Chlorophyll a values were measured on composite samples.

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38 600 500 i 400 300 200 100 0 I s i 4 5 SITE 8 -9. Distribution of phosphorus compounds determined in whole lake water at 8 sites in October 1989. TP=total phosphorus, T0P= total organic phosphorus, TAH=total acid hydrolyzable phosphorus, TRP=total reactive phosphorus. Vertical bars represent 1 SE.

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components was also observed in filtered lake water (Fig. 2-10), organic P represented > 90% of TSP. Soluble acid hydrolyzable P was a less significant contributor to the total soluble P pool. Soluble reactive P concentrations were negligible. Total soluble P accounted for 11 to 60% of TP at sites 8 and 7, respectively. The fraction of TP attributed to suspended material was determined by difference between total and soluble P fractions (Fig. 2-11). The distribution of P forms was similar to that observed in whole lake water. Suspended TP represented from 40 to 89% of TP. No enzyme hydrolyzable P was observed. Correlating all the site means (including site 1), total APA was positively correlated with chlorophyll a (r=0.88), TOC (r=0.84) and TSS (r=0.85). However, plots of the data showed that these correlations were an artifact of low values for water chemistry parameters at site f. Correlations without site 1 gave different conclusions (Table 2-4). Total APA was observed to be inversely correlated with the acid hydrolyzable fractions. Chlorophyll a was inversely correlated with TOC (r=-0.68) and TSP (r=-0.66) and positively with TSS (r=0.73). Discussion Lake Apopka is a shallow lake with a surface area of 12,500 ha. It has a small littoral zone and is subject to considerable wind induced sediment resuspension. Frequent mixing and mild winters may help to explain the lack of seasonality observed for several of the parameters measured. Chlorophyll a, however, did exhibit a seasonal response. Maximum chlorophyll i corresponded to high PAR and higher temperatures. Over all months, chlorophyll a was only significantly and

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40 Fig. 2-10. Distribution of phosphorus compounds determined in filtered lake water at 8 sites in October 1989. TSP=total soluble phosphorus, S0P=soluble organic phosphorus, SAH=soluble acid hydrolyzable phosphorus, SRP=soluble reactive phosphorus. Vertical bars represent 1 SE.

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41 ig. 2-11. Distribution of suspended phosphorus compounds determined by difference between whole and soluble P measured at 8 sites in October 1989. TP=total phosphorus, T0P=total organic phosphorus, TAH«total acid hydrolyzable phosphorus, TRP-total reactive phosphorus. Vertical bars represent 1 SE.

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42 Table 2-4. Correlation coefficients of selected water chemistry parameters determined at 7 sites in October 1989 (significant at a=0.05, n=7). TP TRP TAH TOP TSP SAH SOP TSUSP SUSAHP CHL TOP 1.00" SAH 0.92 SOP 1.00 TSUSP 0.95 0.94 SUSRP 0.78 SUSAHP 0.99 0.86 SUSOP 0.97 0.96 0.99 TAPA -0.80 -0.73 -0.84 CHL -0.66* TOC -0.68 TSS -0.78 -0.74 0.73 Blank space indicates not significant at a=0.05. * Significant at a=0.10.

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43 inversely correlated with one water chemistry parameter, TS. This correlation along with the ratio TS/chlorophyll a show that chlorophyll a was not a dominant component of the solids in Lake Apopka during the sampling period. In a frequently mixed system such as Lake Apopka, the proportion of total solids which may be attributed to phytoplankton biomass will fluctuate considerably. Other contributors to TS include; bacteria, suspended sediment, zooplankton and inorganic and organic compounds. The inverse relationship between chlorophyll a and TS may be interpreted as follows; 1) increased herbivory by high zooplankton populations and 2) light limitation because of high suspended solids. A peak in TSS was observed in February, which corresponds to the highest wind speed recorded 1 m above the water surface, and thus reflects wind induced sediment resuspension. In a shallow lake, a large proportion of particulate matter within the water column may frequently be attributed to sediment resuspension. The sediments have high P concentrations (Reddy and Graetz 1990) and resuspension will result in increased levels of TP within the water column; TP concentrations were positively correlated with wind speed (5 m above the surface). Conversely, TKN concentrations were inversely related to wind speed (1 m above the surface), and are mainly associated with the algal biomass. The rate of P exchange between water and sediment increases during suspension of sediment (Pomeroy et al . 1965). Part of this exchange may be biological. Sediment resuspension into the oxygenated water column results in aerobic mineralization of organic P (Lee et al . 1977). Sediments exhibit significant phosphatase activity (Kobori and Taga 1979b; Ayyakannu and Chandramohen 1971) and a positive correlation

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between SRP released and APA in the water column has been observed during sediment resuspension to the overlying water (Degobbis et al . 1984) . Assuming resuspended sediment were contributing significantly to the predominately particulate APA pool in Lake Apopka, the ratio of APA/TSS is a better measure of enzyme activity than APA alone. Comparing monthly means, total APA/TSS was positively correlated with soluble APA (r=0.99) and inversely correlated with TOC (r=-1.00). The correlation between APA and TSS was only observed when data were compared on a site basis. The overall lack of correlation between TSS and APA in this study may be due to 1) insufficient sampling during periods of sediment resuspension, 2) the relationship is hidden by the variability in other parameters incorporated in the TSS pool, and 3) sediment resuspension does not contribute to APA activity. Alkaline phosphatase activity has been significantly correlated with ATP (Pettersson 1980), particulate organic matter (Gage and Gorham 1985) and chlorophyll a (Healey and Hendzel 1979a; Pettersson 1980). In this study, total APA was significantly correlated with chlorophyll a in December and inversely correlated with TOC in February. Comparing annual means, there is a strong inverse relationship between total APA and TOC. This may be explained by examining the components of the TOC pool; one contributor is humic material. Alkaline phosphatase activity can be inhibited by high concentrations of humic materials (Francko 1986) . The inverse relationship observed between APA and TOC could be a result of binding of APA to organic material. Attachment of alkaline phosphatase enzymes to particulate matter may decrease activity, but may also increase longevity of the enzyme activity (Burns 1986).

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45 Resuspension of sediment high in organic matter could bind enzymes and/or release sediment bound APA to the overlying water column. Organic inputs, living or dead should be considered when measuring APA (Healey and Hendzel 1980). Soluble APA is positively related to Secchi (r=0.87) suggesting that there is a relationship between APA and water quality. But the low proportion of APA observed in the soluble pool suggest that free dissolved enzymes from cell lysis (Berman 1970) and enzymes excreted by zooplankton (Wynne and Gophen 1981) were not as important as particulate associated APA in the system. Organic phosphorus mineralization is mainly be achieved by APA bound to particulate matter. Examining the data from all sites (excluding site 1), APA was infrequently related to chlorophyll a. The overall lack of correlation between APA and chlorophyll a may be hidden due to the frequent mixing of the lake water. The particulate nature of APA as determined by the size fractionation scheme corresponds to the chlorophyll a distribution. The correlation between APA and chlorophyll a leads to the expression of APA as a ratio, i.e., APA/chlorophyll a (Pettersson 1980). This ratio tends to increase with P limitation and decrease with trophic state (Pick 1987). Combining data from numerous studies, Pettersson (1980) determined that a ratio between 0.2 to 0.7 nmol APA ng chlorophyll a 1 min" 1 could be used to indicate P limitation. When ambient lake specific APA was consistently < 0.3 nmol APA fig chlorophyll a" 1 min" 1 ratios greater than this were determined to indicate P limitation, i.e., elevated specific APA indicates P limitation (Istvanovics et al . 1990). In this study a ratio of < 0.3 nmol APA /xg chlorophyll a' 1 min" 1 was

PAGE 59

46 consistently observed, suggesting that plankton in Lake Apopka are generally not P limited. This conclusion tends to agree with other research findings from this lake (Aldridge, F.J., personal communication, Department of Fisheries and Aquaculture, University of Florida, Gainesville, FL.; Reddy and Graetz 1990). Specific APA was lowest when chlorophyll a peaked and SRP concentrations of 5 ng L' 1 were sufficient to support growth. High particulate APA has been attributed to the location of alkaline phosphatase in the cell wall of phytoplankton (Kuenzler and Perras 1965). Recent research has suggested that viable phytoplankton do not contribute much to the particulate APA pool (Stewart and Wetzel 1982). Lake Apopka is generally dominated by cyanobacteria, with large numbers of Lyngbya sp. and Microcystis sp. (Shannon and Brezonik 1972; Stites, D. L., personal communication, St. John's River Water Management District, Palatka, FL.) whose mucilaginous layers can support significant bacterial populations. It is likely that particulate APA is attributable to both phytoplankton and the associated bacteria. Use of specific APA (APA/chlorophyll a) to indicate P limitation of phytoplankton should be verified via nutrient enrichment bioassays. Diel fluctuations in APA were observed, but these do not correspond to any particular water chemistry parameter. This may be attributed to spatial patchiness and water movement (Berman 1970; Wynne 1981). Unlike TKN concentrations, APA did not settle out of the water column following wind subsidence. Diel variability of APA may be dependent upon the species composition of the phytoplankton biomass. In diel studies neither Tballassiosira pseudonana Hasle and Heimdal (Perry

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1976), nor Selenastrum capricornutum Prinz (Klotz 1985b) exhibited diel responses. However, Smith and Kalff (1981) have shown that growth demands for P are more important than the species composition in determining APA. Preliminary studies (data not shown) and April data demonstrated a strong relationship between APA and TP. Consequently TP, which would include cellular P, would indicate the P forms utilized by APA. However, this relationship was not observed the entire year. Total soluble P, has been suggested as a good indicator of bioavailable P in eutrophic lakes (Bradford and Peters 1987). Correlations between total and soluble APA and TSP were apparent in December, and at certain sites (Table 2-3). Both positive and negative relationships were observed. Total and soluble APA are apparently influenced by different parameters at different sites (Table 2-3, Huber et al . 1985). In October, the P fractionation experiment demonstrated that APA was inversely related to the acid hydrolyzable P fractions. This suggests that APA may be regulated by acid hydrolyzable compounds, or alternatively they could be used as substrates for APA. Acid hydrolyzable P represents the condensed polyphosphates, a storage form of P in phytoplankton and bacteria. Surplus P concentrations within algal cells have been shown to regulate the production of APA (Fitzgerald and Nelson 1966; Lien and Knutsen 1973; Rhee 1973) and this pool is composed of polyphosphates (Rhee 1972, 1973; Elgavish and Elgavish 1980). Hence, the measurement of surplus P combined with APA could provide a better understanding of the system. Another component which may contribute to the understanding of APA is the concentration of

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48 EHP. No EHP was found in Lake Apopka during the October fractionation experiment. However, this does not reflect the absence of these substrates. In some lakes the release rate of P from EHP satisfies the P uptake rate (Chrost and Overbeck 1987) while in others a large discrepancy exists between these two rates (Heath 1986; Boavida and Heath 1988). Low EHP concentrations have been attributed to the rapid hydrolysis of this fraction (Berman 1970; Taft et al . 1977). Alternatively, this may reflect methodological problems; 1) the method to measure EHP requires the addition of extracted APA from Escherichia coli to filtered lake water. This enzyme was not adapted to this system and consequently may not be as efficient or may require a longer incubation time, 2) filtered lake water does not represent the entire P pool available, as particulate organic P, a large portion of TP, may also be susceptible to enzymatic hydrolysis (Jansson 1977), 3) enzymes added from f. coli were more inhibited by inorganic P additions than natural enzyme populations (Chrost et al . 1986). The first and third problems were resolved by measuring the increase in SRP in filtered water without the addition of the enzyme (Chrost et al . 1986). However, in a lake which has high particulate APA, this would not be a true representation of the potential APA. Conclusions Seasonal and spatial differences in water chemistry were observed. In general, seasonal variability was greater than spatial variability. The system was highly productive as evidenced by chlorophyll a concentrations > 150 /ig L" 1 , and an annual mean of 81 /zg L" 1 . Annual

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49 means for TP and TKN were 210 ng L' 1 and 4.8 mg L"\ respectively, confirming the highly eutrophic state of the lake. Conversely, SRP concentrations were consistently < 10 /xg L*\ Alkaline phosphatase activity was mainly associated with particulate matter and was dependent on different water chemistry parameters both seasonally and spatially. In general, APA was not correlated to chlorophyll a. The relationship between these parameters may be hidden as a result of the frequent mixing of the water column in Lake Apopka. Both positive and negative correlations between P and APA were observed. An inverse relationship existed between acid hydrolyzable P and APA, indicating polyphosphates may be controlling APA. The particulate association of APA would suggest that APA should be correlated with TSS, but this was rarely observed. This may be due to the variability in the composition of the TSS pool. The relationship between APA and particulate may be both beneficial and detrimental. Binding to particles results in increased longevity of the enzyme, but it also may inhibit APA by binding to the active site, as indicated by the inverse relationship between APA and TOC. Future research should examine the components of the TSS pool, plankton and sediment and their effect on organic P mineralization under controlled conditions.

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CHAPTER 3 RESPONSE OF NATURAL PLANKTON POPULATIONS TO NUTRIENT ENRICHMENT Introduction Soluble inorganic P is the main form of P utilized directly by plankton. Phytoplankton growth rates close to maximal have been determined in the apparent absence of inorganic P (Funs et al . 1972; Smith and Kalff 1981). Methods used to measure soluble inorganic P are for the most part limited in sensitivity (Rigler 1956; Tarapchak et al . 1982). This has resulted in the use of physiological indicators to determine the nutritional status of plankton. Information is obtained through a variety of methods including the determination of P uptake rates (Lean and White 1983; Rigler 1956), the measurement of surplus P concentrations (Fitzgerald and Nelson 1966; Rhee 1973) and the determination of alkaline phosphatase activity (APA) (Berman 1970; Kuenzler and Perras 1965). As external inorganic P concentrations decline, plankton are able to utilize internal pools of surplus P to maintain growth (Fitzgerald and Nelson 1966; Rhee 1972, 1973, 1974; Wynne and Berman 1980). Once this internal source of P has been reduced to a critical level some phytoplankton produce phosphatase enzymes, which hydrolyze organic P compounds to inorganic P, to satisfy nutritional demands (Chrost and Overbeck 1987; Kuenzler and Perras 1965; Reichardt 1971). An inverse relationship between APA and surplus P has 50

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been reported (Fitzgerald and Nelson 1966; Chrost and Overbeck 1987). Inorganic Pisa competitive inhibitor of APA (Coleman and Gettins 1983; Moore 1969; Reid and Wilson 1971). Upon replenishment of external inorganic P concentrations enzyme activity is inhibited (Lien and Knutsen 1973; Torriani 1960; Perry 1972) and surplus P accumulates (Rhee 1973). Surplus P is measured as hot water extractable P (HEP), and in conjunction with APA has been shown to accurately assess P demand in some lakes (Sproule and Kalff 1978; Pettersson 1980) but not in others (Wynne and Berman 1980). Wynne and Berman (1980) observed that the HEP concentration in Lake Kinneret, Israel, remained stable throughout the year even under conditions of P stress and concluded that HEP was a metabolic intermediate rather than a form of P storage. Lake Apopka is a hypereutrophic lake with soluble reactive P (SRP) concentrations frequently < 1 nq L"\ In contrast, concentrations of total soluble P (TSP) and APA are high (chapter 2). The objectives of this study were to determine whether high APA in Lake Apopka was due to high demand for P or P limitation, and to evaluate the N and P requirements of native plankton. Materials and Methods Site Description Lake Apopka is a 12,500 ha lake located in central Florida (28* 37' N. latitude, 81* 37' W. longitude). It has a mean depth of 2 m. It has been proposed that the nutrient loading from the surrounding agricultural and urban areas has precipitated the current hypereutrophic conditions in the lake (USEPA 1979).

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52 Sampling Procedures Water samples were collected 30 cm below the water surface from the west side of the lake on November 16 1989, and from the east side on April 18 and August 21 1990 (Fig. 3-1). Water was stored in polycarbonate and polyethylene carboys in the dark and at ambient laboratory temperature, for no more than 24 h prior to the start of the experiments. Experimental Design The effect of inor ganic phosphorus concentrations upon alkaline phosphatase activity Lake water collected in November 1989 was diluted 2:1 (260 mL unfiltered:140 mL filtered) with filtered lake water (0.45 jon), to reduce the chlorophyll a concentration. Four hundred mL were placed in each of 15 wide mouth 500 mL erlenmeyer flasks. The experimental design was completely randomized with 5 treatments and 3 replicates. The water was spiked with nutrient additions (Table 3-1). Nitrogen was added at an N:P ratio of 10:1 to avoid N limitation. The flasks were capped with cotton wool and placed on magnetic stir plates, under a black plastic enclosure in the greenhouse. Temperature within the enclosure was maintained using window air conditioning units and fans (mean ± 1SE,25*C ± 0.21). Light was supplied at 200 /unol photons m" 2 s" 1 using cool -white fluorescent lamps. The light:dark schedule was 16:8. The flasks were shaken and aliquots were withdrawn by syringe from treatments 1 and 2 at 0, 24, 72 and 96 h. Aliquots were removed from remaining treatments at 24, 72, and 168 h. Additional sampling times of 268 and 312 h were included for cultures which received 1000 ng L' 1 . Samples requiring

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54 Table 3-1. Nutrient additions made to diluted lake water collected in November 1989. Treatment Nutrient addition number N" P 0 0 500 0 0 10 1000 100 2500 1000 N and P were added as potassium nitrate and potassium dihydrogen phosphate, respectively.

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55 filtration were immediately filtered through 25 mm membrane filters (0.45 /xm) in polypropylene holders which attached to the syringes (Gelman). The samples were analyzed for chlorophyll i, total and soluble APA, total (TP), total Kjeldahl N (TKN) , SRP, NH 4 -N, and [N0 3 + N0 2 ]-N. Nutrient enrichment of natural plankton populations Experiment 1. Whole lake water was diluted 1:1 with filtered lake water (0.45 urn) and 400 mL were placed in each of 15 wide mouth 500 mL erlenmeyer flasks. The basic experimental design was a 2 2 factorial with an additional 2 fold addition of both N and P included (Table 3-2). The flasks were stoppered with sponge plugs and placed in a clear glass circulating water bath maintained at ambient lake temperature (27'C). The flasks were illuminated from below at an irradiance of 145 /xmol photons m' 2 s'\ The contents of the flasks were mixed daily and immediately prior to sampling. Two h after the nutrient addition, aliquots were withdrawn by syringe at predetermined intervals and analyzed for total APA, SRP, NH 4 -N, [N0 3 + N0 2 ]-N, TSP and chlorophyll a. Samples requiring filtration were filtered immediately as described above. Hot water extractable P (HEP-SRP) was determined at the beginning and conclusion of the experiment. To obtain a sufficient sample size, HEP-SRP was determined on composite samples containing all treatment replicates. Experiment 2. To both confirm and compare the results with a different plankton population, a second experiment similar to that described above was conducted in August. Water samples were collected

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56 Table 3-2. Nutrient additions made to diluted lake water collected in April and August 1990. Treatment Nutrient N addition P Incubation temperature Experiment 1 -April 1 *C 1 0 o 27 2 400 0 27 3 0 40 27 4 400 40 27 5 800 80 27 Experiment 2-August 1 0 0 29 2 400 0 29 3 0 40 29 4 5 400 40 29 800 80 29 6 0 0 19 7 0 40 19 N and P were added as potassium nitrate and potassium di hydrogen phosphate, respectively.

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57 from the same site, diluted and placed in water baths as described above. The temperature of the bath was set at 29*C to emulate ambient lake water temperature. Another water bath with cultures receiving no nutrient addition and P only was maintained at 19*C, to determine the effect of temperature on the measured parameters. Nutrient uptake rates were determined by measuring the disappearance of the nutrient from solution. During the first 2 h following nutrient addition, the cultures were sampled every 1/2 h and analyzed for SRP, NH 4 -N, and [N0 3 + N0 2 ]-N (actual sampling times were 0, 30, 67, 98 and 140 min). Immediately following filtration aliquots were analyzed for SRP. Samples to be analyzed for NH 4 -N and [N0 3 + N0 2 ]-N samples were acidified with concentrated H 2 S0 4 and stored at 4*C prior to analysis. Samples taken at 0 and 2 h were also analyzed for HEP-SRP, and hot water extractable TSP (HEP-TSP). Aliquots were subsequently removed at 24, 48 and 96 h and analyzed for SRP, NH 4 -N, [N0 3 + N0 2 ]-N, total APA, TSP, HEP-SRP, HEP-TSP and chlorophyll a. Analytical Methods Alkaline phosphatase activity was determined fluorometrically (Healey and Hendzel 1979a). One half mL of substrate, 3-o-methyl fluorescein phosphate (Sigma Chemicals), at a concentration determined to be non-limiting, in 0.1 M Tris buffer (Tham, Fisher Scientific) pH 8.6, was added to 4.0 mL of lake water in a cuvette. Both total (whole lake water) and soluble (filtered through 0.45 ym Gelman membrane filter) APA were determined. The cuvettes were placed in a water bath (25*C). At timed intervals during a 20 min period the

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58 cuvettes were placed in the fluorometer and fluorescence was measured. The enzyme activity was measured as an increase in fluorescence as the substrate was enzymatically hydrolyzed to the fluorescent product. Fluorescence units were converted to enzyme activity using a standard calibration curve of 3-o-methylfluorescein (Sigma Chemicals). The fluorescence was measured using a Sequoia Turner fluorometer Model 110, equipped with Turner lamp no. 110-853, in combination with 47 B excitation and 2a-12 emission filters. Autoclaved lake water with substrate added was used as a control. Chlorophyll a was determined by measuring in vivo fluorescence, using a Turner Design Model 10 fluorometer equipped with a Turner lamp no. 110-853, in combination with 5-60 excitation and 2-64 emission filters. A Sequoia Turner fluorometer Model 110 equipped with the same light source and filters was used to measure chlorophyll a fluorescence in the first experiment. Pheophytin a fluoresces at the same wavelength as chlorophyll a, so chlorophyll a concentrations are uncorrected for pheophytin. The calculation of chlorophyll a was based on equations by Lorenzen (1967); ABS x (vol. extracted (mL)) x (unit of measure factor) x 1 89 (vol. filtered (mL)) pathlength (cm) ABS = absorbance at 664 nm 89 = absorption coefficient for chlorophyll a in 90% acetone Unit of measure factor = 10 6 for ng L'\

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59 Initial and final chlorophyll a concentrations were calibrated against chlorophyll a concentrations determined spectrophotometrically following extraction with 90% acetone (APHA 1985). Total P, TSP, SRP, NH 4 -N, and [N0 3 + N0 2 ]-N were determined by standard methods (APHA 1985). Hot water extractable P was determined in experiment 1 using a modification of the method by Fitzgerald and Nelson (1966). Water samples (50 to 100 mL) were filtered through 47 mm 0.45 /un membrane filters (Millipore). The filters were placed in 60 mL borosilicate boiling tubes, and 20 mL deionized water were added. The tubes were capped and autoclaved at 120'C for 1 h. Upon cooling samples were analyzed for SRP. Blank filters were autoclaved to determine any filter contribution to P analysis. Hot water extractable P includes both a molybdate reactive form of P and a non-reactive form of P. Both hot water extractable forms were determined in experiment 2, using the method of Krausse and Sheets (1980). Nine mL of sample were filtered though 25 mm 0.45 /xm membrane filters (Gelman). The filters were placed in 15 mL polypropylene tubes, and 13 mL of deionized water was added. The tubes were capped and autoclaved at 120'C for 1 h. Filter blanks were also autoclaved. Gelman filters were used instead of Millipore filters because they had a lower background P concentrations. The filtrate was analyzed for SRP, and also digested via persulfate oxidation and analyzed for TSP.

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60 Statistical Methods Data were analyzed using SAS (Statistical analysis systems) version 6. Balanced data (equal number of observations for each treatment) were analyzed using the repeated measures procedure which accounts for the within replicate correlation over time, due to repeated sampling from the same flasks. Unbalanced data (unequal number of observations per treatment) were analyzed using a split plot design with time as the subplot. Results The Effect of Inorg anic Phosphorus Concentrations upon Alkaline Phosphatase Activity The majority of N, P and APA were associated with particulate matter (Table 3-3). Due to chlorophyll a analysis problems arising from fluorometer calibration, only chlorophyll a data from 0 and 72 h are presented and used for statistical analysis (Table 3-4). Increased chlorophyll a concentrations were observed at 72 h in all cultures except those which received no nutrient addition. Increased growth was associated with higher nutrient additions, a 69% increase in chlorophyll a was observed in treatment 5 (N=2500 P=1000) cultures. Chlorophyll a increases of 33% for treatment 4 (N=1000 P=100) and 11 % for treatments 2 (N=500 P=0) and 3 (N=0 P=10) cultures were observed. The opposite was observed for both total and soluble APA (Fig. 3-2a and 3-2b). Total and soluble APA decreased significantly over time in cultures receiving the highest P additions (100 and 1000 ng L' 1 ) . No decrease in total APA was observed for treatments 1 (N=0 P=0) and 3 (N=0 P=10), but soluble APA

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Table 3-3. Initial concentrations of selected parameters measured in diluted lake water prior to nutrient addition (triplicate samples) in November 1989 (mean ± 1 SE). Parameter Concentration Chlorophyll (ng L" 1 ) TP (19 L 1 ). SRP (jig L 1 ) TKN (mg L" 1 ) NH 4 -N (mg L' 1 ) [N0 3 + N0 2 ]-N (mg L" 1 ) Total APA (nM min 1 ) Soluble APA (nM min 1 ) 31 ±6 65 ±4 3 ± 0.3 3.30 + 0.1 0.36 i 0.08 0.14 ± 0.00 13.5 ± 0.3 2.2 + 0.13

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62 Table 3-4. Chlorophyll a and specific alkaline phosphatase activity measured in natural plankton populations collected in November 1989, 72 h after receiving nitrogen and phosphorus additions. l=no nutrient addition, 2=500 ng N L'\ 3=10 ng P L'\ 4=1000 ng N L" 1 and 100 ng P L"\ 5=2500 ng N L" 1 and 1000 ng P L \ Treatment Chlorophyll Specific APA nmol APA ng chlorophyll a" 1 min' 1 1 36 a' 0.41 2 40 b 0.46 3 40 b 0.35 4 48 c 0.15 5 61 d 0.03 Numbers in a column followed by the same letter are not significantly different at a = 0.05.

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63 c *mmm E < Q_ < 48 96 144 192 240 288 TIME (h) Fig. 3-2. Time courses of alkaline phosphatase activity following nutrient enrichment of natural plankton populations collected in November 1989. 0N,0P=no nutrient addition? 500N,0P=500 m N L i 0N,10P=10 ng P L" 1 -, 1000N, 100P=1000 ng N L and 100 ng P L" 1 , and 2500N, 1000P=2500 ng N L" 1 and 1000 Mg P L" : a) total alkaline phosphatase activity; b) soluble alkaline phosphatase activity. Vertical bars represent 1 SE. No vertical bar indicates SE is smaller than symbol size.

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64 did decrease. A significant increase in total APA was observed in cultures which received only N additions suggesting the onset of P limitation. This is more apparent when total APA data are presented as specific activity (i.e. total APA/chlorophyll a) (Table 3-4). A 15 fold difference between specific activity of those cultures which received treatment 5 (N=2500 P=1000) and treatment 2 (N=500 P=0) was observed. Initial specific APA was 0.44 nmol APA ng chlorophyll a -1 min"\ hence P addition resulted in a decrease in specific APA. Significant decreases in SRP concentrations were observed within 24 h (Fig. 3-3a). Apart from treatment 5 (N=2500 P=1000), SRP concentrations were the same in all cultures after 2 h. After an initial rapid uptake from 1000 to 673 /ig P L" 1 within the first 2 h, SRP concentrations in cultures treated with 1000 /ig P V\ remained constant until 168 h, and then began to decrease. At 312 h, 377 ng P L*\ 37.6% of the original concentration remained in treatment 5 (N=2500 P=1000) cultures. Nitrate concentrations also exhibited significant treatment differences within 24 h (Fig. 3-3b). The concentrations at 24 h were ranked in descending order of original addition, with 1 and 3 treatments having equivalent [N0 3 + N0 2 ]-N concentrations. In all cultures there was a distinct decrease over time. A similar trend was observed for NH 4 -N, concentrations appeared to decrease within 24 h (Fig. 3-3c), however, no significant differences were determined. Nutrient Enrichment n f Natural Plankton Populations Growth. Chlorophyll a concentrations and APA were lower in April than in August (Table 3-5). Conversely, TP and TSP were

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65 1200 o_ f 800 (/> en 400 V w 100 96 144 192 TIME (h) 240 288 Fig. 3-3. Time courses of nutrient concentrations following nutrient enrichment of natural plankton populations collected in November 1989. ON,OP=no nutrient addition, 500N,0P=500 /xg N 1/ , 0N,10P=10 jig P L"\ 1000N, 100P=1000 fig N V s and 100 fig P L" , and 2500N, 1000P=2500 ng N L" 1 and 1000 fig P L" 1 : a) soluble reactive phosphorus; b) [N0 3 + N0 2 ]-N; c) NH 4 -N. Vertical bars represent 1 SE. No vertical bar indicates SE is smaller than symbol size.

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66 Table 3-5. Initial concentrations of selected parameters measured in diluted lake water prior to nutrient addition (triplicate samples) (mean ± 1 SE). Parameter Samolinq Deriod April 1990 August 1990 Chlorophyll a (/ig L" 1 ) 22.5 ± 0 37.4 ± 0 TP (jig L* 1 ) 113 ± 12 56 ± 9 TSP [liq L" 1 ) 70 + 10 9 ± 0 SRP (jig L" 1 ) 1 ± 0 2 i o HEP-TSP (/xg L" 1 ) ND" 33 ± 3* HEP-SRP (/xg L" 1 ) 7 ± 0* 15 ± 2* NH 4 -N (mg L" 1 ) 0.04 ± 0 0.05 ± 0 [N0 3 + N0 2 ]-N (mg L" 1 ) 0 ± 0 0.01 ± 0 Total APA (nM min" 1 ) 9.1 ± 0.2 12.7 ± 0.1 ND indicates data not determined. Determined by the method of Krausse and Sheets (1980). Determined by the method of Fitzgerald and Nelson (1966).

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significantly higher in April. Chlorophyll a was used as a means to indicate phytoplankton growth in the cultures. Chlorophyll a concentrations increased in response to nutrient additions to the water samples collected in both April and August. Although small and hidden due to axis scale, significant treatment differences in chlorophyll a occurred within the first 24 h (Fig. 3-4 and 3-5a). After 96 h, maximum chlorophyll a concentrations in April were 80 ^xg L" 1 and exceeded 180 fig L" 1 in August. In April, chlorophyll a concentrations were significantly affected by the interaction between concentrations of N and P added. Chlorophyll a concentrations obtained in the presence of both nutrients exceeded those obtained by single nutrient additions (Fig. 3-4). The greatest initial increase in chlorophyll a occurred in cultures which received treatment 2 (N=400 P=0). Subsequently, growth rates in treatments 4 (N=400 P=40) and 5 (N=800 P=80) exceeded those of treatment 2. This information, combined with the lag in growth observed in cultures receiving only P additions, suggest that phytoplankton were initially N limited and became co-limited by P as they grew. This is confirmed by the significant interaction of added N and P levels upon chlorophyll a concentrations. In contrast, no significant interaction between the levels of N and P was observed in August. In August, those cultures which received only P additions had the same chlorophyll a as those which received both N and P, while cultures receiving only N had the same chlorophyll a as those with no nutrient addition. Increases in chlorophyll a were also recorded in cultures which received no P addition. These observations suggest that some P was still available for plankton growth, but the growth rates were P limited, and thus the

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68 200 160 o 120 >x Q_ O OH O _l X o 96 144 TIME (h) 192 Fig. 3-4. Time courses of chlorophyll a concentrations following nutrient enrichment of natural plankton populations collected in April 1990. 0N,0P=no nutrient addition*, 400N,0P=400 ng N L"\ 0N,40P=40 fig P L" 1 -, 400N,40P=400 fig N L" 1 and 40 ng P L'\ and 800N,80P=800 fig N L" 1 and 80 ng P L" 1 . Vertical bars indicate 1 SE. No vertical bar indicates SE is smaller than symbol size.

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69 200 O g 200 x o 160 120 80 40 19°C O ON, OP ON, 40P O (b) 24 48 72 TIME (h) 96 Fig. 3-5. Time courses of chlorophyll a concentrations following nutrient enrichment of natural plankton populations collected in August 1990. ON,OP=no nutrient addition; 400N,0P=400 tig N L j 0N,40P=40 /xg P L"\ 400N,40P=400 ng N I" 1 and 40 ng P L"\ and 800N,80P=800 itg N L" 1 and 80 ng P L" 1 : a) plankton grown at 29'C; b) plankton grown at 19'C. Vertical bars indicate 1 SE. No vertical bar indicates SE is smaller than symbol size.

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growth rate increased with increasing P concentration in the growth media. It is suggested that cultures were only slightly limited by P, because cultures with no P enrichment still increased in biomass. During both experiments, the highest nutrient addition (treatment 5, N=800 P=80) resulted in significantly greater chlorophyll a than any other treatment. Growth of plankton collected in August was inhibited at cooler temperatures (Fig. 3-5b). Over the entire 96 h period chlorophyll a only increased by 8 fig L' 1 . The phytoplankton grown at 19*C with either a P addition or no nutrient addition only achieved 65% of the chlorophyll a of phytoplankton grown at 29'C with no nutrient addition. Nutrient uptake. In April, the initial sampling for nutrient analyses occurred after 2 h; however, at this time SRP concentrations had been significantly reduced, consequently to give a true representation of the data a time 0 was included along with 2 h to the data set. Time 0 represents the means of initial concentrations measured in cultures with no nutrient addition, plus the respective additions, hence approximate uptake rates can be envisioned (Fig. 3-6). Only the cultures which received treatment 5 (N=800 P=80) had significant SRP concentrations remaining after 2 h (17 fig L" 1 ), SRP concentrations in the other cultures were below 3 fig L" 1 . These concentrations remained close to baseline for the remainder of the experiment. The determination of P uptake thus required a more intensive sampling immediately following nutrient addition. This was achieved in experiment 2, with water samples collected in August. Within 30 min SRP had decreased from 81.6 to 37.4 fig L* 1 in treatment 5

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71 Q_ or CO 90 80 70 60 50 40 30 20 10 0 • ON. OP 400N, OP 0N.40P 400N.40P BOON, 80P 48 96 144 192 TIME (h) Fig. 3-6. Time courses of soluble reactive phosphorus concentrations following nutrient enrichment of natural plankton populations collected in April 1990. ON,OP=no nutrient addition; 400N,0P=400 ng N L" 1 -, 0N,40P=40 ng P L' 1 -, 400N,40P=400 ng N L" 1 and 40 ng P L" 1 , and 800N,80P= 800 m N L' 1 and 80 jig P L* 1 . Vertical bars indicate 1 SE. No vertical bar indicates SE is smaller than symbol size.

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72 (N=800 P=80), from 41.6 to 22 ng L" 1 in treatment 4 (N-400 P=40) and from 41.6 to 13.1 /zg L" 1 in treatment 3 (N=0 P=40) cultures (Fig. 3-7a). Uptake rates were calculated as the disappearance of SRP within the first 30 min (Table 3-6). This was selected to indicate maximal uptake because the slope changed over time as the P demand decreased (Fig. 3-7a). Cultures which received 80 ng L" 1 had a significantly greater uptake rate than those which received 40 ng L'\ As expected, temperature had a significant effect upon SRP uptake (Fig. 3-7b). The uptake was not as rapid as that for the same treatment at 29*C (a = 0.12), but even with the 10*C difference in temperature all SRP had been depleted to below detection within 2 h. After 1 h, SRP levels in treatments 3 (N=0 P=40) and 4 (N=400 P=40) were no longer significantly different. The SRP concentrations continued to decrease in all the cultures and were all close to baseline in 2 h and were undetectable (<1 fig L' 1 ) after 24 h. In both experiments TSP decreased within the first 2 h and then remained constant (Fig. 3-8a and 3-9a). The uptake of N differed between the two sampling periods. In April, [N0 3 + N0 2 ]-N concentrations in the cultures decreased. The concentration decreased by 25% in all treatments with N additions, within 2 h and continued to decrease over time (Fig. 3-8b). In contrast, NH 4 -N concentrations did not change in any of the cultures until 216 h, when an increase was observed in control and N cultures (Fig. 3-8c). In August, no significant treatment by time interaction was recorded for [N0 3 + N0 2 ]-N, and no apparent uptake of [N0 3 + N0 2 ]-N occurred (Fig. 3-9b). A significant treatment by time interaction was

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73 29°C (a) • ON, OP T 400N, OP 0N.40P 400N, 40P BOON, 80P CP Q_ or w 80 60 19°C (b) O ON. OP ON, 40P 1.0 1.5 TIME (h) 2.0 2.5 Fig. 3-7. Time courses of soluble reactive phosphorus concentrations following nutrient enrichment of natural plankton populations collected in August 1990. 0N,0P=no nutrient addition; 400N,0P=400 /zg N L j 0N,40P=40 ng P L" 1 -, 400N,40P=400 ng N L' 1 and 40 ng P l'\ and 800N,80P= 800 ng N L" 1 and 80 fig P L" 1 : a) plankton grown at 29'C; b) plankton grown at 19°C. Vertical bars indicate 1 SE. No vertical bar indicates SE is smaller than symbol size.

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74 Table 3-6. Phosphorus uptake rates for natural plankton populations collected in August 1990, 30 min after receiving nitrogen and phosphorus additions (mean ± SE). 3=40 ng P L*\ 4=400 fig N L" 1 and 40 pg P L"\ 5=800 ftg N L' 1 and 80 fig P L 7=40 ng P L" 1 and plankton grown at 19*C. Treatment Uptake Rate ng P L" 1 min' 1 3 0.95 ± 0.05 a" 4 0.43 ± 0.27 b 5 1.47 ± 0.06 c 7 0.65 + 0.01 ab* Numbers in a column followed by the same letter are not significantly different at a = 0.05. Uptake rates for treatments 3 and 7 are significantly different at a = 0.12.

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75 Fig. 3-8. Time courses of nutrient concentrations following nutrient enrichment of natural plankton populations collected in April 1990. 0N,0P=no nutrient addition? 400N,0P=400 fig N L" ; 0N,40P=40 fig P L" 1 ; 400N,40P=400 fig N L' 1 and 40 fig P L , and 800N,80P=800 fig N L"' and 80 fig P L" 1 : a) total soluble phosphorus; b) [N0 3 + N0 2 ]-N; c) NH 4 -N. Vertical bars indicate 1 SE. No vertical bar indicates SE is smaller than symbol size.

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200 TIME (h) Fig. 3-9. Time courses of nutrient concentrations following nutrient enrichment of natural plankton populations collected in August 1990 grown at 29'C. 0N,0P=no nutrient addition; 400N,0P=400 ng N L' 1 ; 0N,40P=40 fig P L" 1 ; 400N,40P=400 ng N L' 1 and 40 /ig P L'\ and 800N,80P=800 ^g N L" 1 and 80 ng P L" 1 : a) total soluble phosphorus; b) [N0 3 + N0 2 ]-N; c) NH 4 -N. Vertical bars indicate 1 SE. No vertical bar indicates SE is smaller than symbol size.

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observed for NH 4 -N, however, the data were highly variable, making meaningful interpretation difficult (Fig. 3-9c). Plankton grown at 19"C exhibited the same responses for TSP, [N0 3 + N0 2 ]-N, and NH 4 -N (data not shown) . Surplus phosphorus . Soluble P extracted from plankton following boiling with deionized water (HEP-SRP) was used as an indicator of surplus P. In April HEP-SRP was determined at both the start and conclusion of the experiment. Hot water extractable P decreased in cultures which did not receive P additions but remained the same in cultures which received 40 fig L" 1 (Table 3-7). Those cultures which received 80 fig P L" 1 had almost a 3-fold increase in HEP-SRP after 216 h. The role of HEP was examined in more detail in water samples collected in August. Seventy percent of initial TP was accounted for by HEP-TSP and TSP (Table 3-5), with HEP-TSP representing 59% of TP. Within 2 h of P addition, substantial increases in HEP-TSP were observed (Fig. 3-10a). Hot water extractable-TSP tripled from 33.3 to 99.3 fig P L" 1 upon addition of 80 fig P L*\ Addition of 40 fig P L" 1 resulted in a doubling of HEP-TSP to 65 and 69 fig P L" 1 in cultures which received treatments 3 (N=0 P=40) and 4 (N=400 P=40), respectively. Even with a 10'C temperature difference HEP-TSP accumulated to 63.9 fig P L' 1 within 2 h (Fig. 3-10b). Cultures grown at 19*C which did not receive nutrients had greater HEP-TSP concentrations after 2 h than those with no nutrient addition grown at 29*C. At 29*C the HEP-TSP remained constant within 24 h and then decreased by 15 fig P L" 1 for P added treatments. The downward trend continued to 96 h, HEP-TSP

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78 Table 3-7. Hot water extractable phosphorus concentrations of composite lake water samples collected in April 1990, 216 h after nutrient additions. l=no nutrient addition, 2=400 ng N L'\ 3=40 ng P L*\ 4=400 ng N L" 1 and 40 /xg P L' 1 , 5=800 ng N L' 1 and 80 ng P L" 1 . Treatment Hot water extractable SRP M9 L' 1 1 3.0 2 2.8 3 7.2 4 6.8 5 18.4

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79 120 0_ CO I& 1 20 80 40 (b) 19°C O ON, OP ON. 40P D **-^ i 24 48 72 96 TIME (h) 3-10. Time courses of hot water extractable total soluble phosphorus following nutrient enrichment of natural plankton populations collected in August 1990. 0N,0P=no nutrient addition;400N,0P=400 /ig N L ; 0N,40P=40 fig P L* 1 ? 400N,40P=400 fig N L" 1 and 40 ng P L"\ and 800N,80P=800 fig N L" 1 and 80 fjg P L" 1 : a) plankton grown at 29°C; b) plankton grown at 19°C . Vertical bars indicate 1 SE. No vertical bar indicates SE is smaller than symbol size.

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80 concentrations in treatment 1 (N=0 P=0) cultures were lower than initial concentrations while HEP-TSP concentrations in treatments 3 (N=0 P=40) and 4 (N=400 P=40) were higher than initial concentrations. Normalizing the data to chlorophyll a, a decrease in HEP-TSP was observed for all treatments following the initial increase at 2 h (Table 3-8). The final ratios were lower than the initial ratios. The treatment by time response was different for HEP-SRP (Fig. 3-lla). The relative increases were greater. An increase in HEPSRP concentrations was observed after 48 h in cultures which received P, except for treatment 4 (N=400 P=40). Hot water extractable P in treatments 1 (N=0 P=0) and 2 (N=400 P=0) cultures remained constant. A significantly lower increase was observed in treatment 7 (N=0 P=40, temperature=19*C) (Fig. 3-llb a=0.06). Normalizing the data to chlorophyll a resulted in a continuous decline in HEP-SRP. No increase after 48 h was observed (Table 3-9). A significant correlation between HEP-SRP and chlorophyll a was observed (r=0.56). Alkaline phosphatase activity. Considerable differences in the production of APA were observed between April and August (Fig. 3-12a and 13a). In both experiments, increases in APA were observed, however in August this increase only lasted 48 h for all cultures grown at 29*C. In April, APA in cultures receiving both N and P additions was inhibited within 2 h, 28% and 11% inhibition for treatments 5 (N=800 P=80) and 4 (N=400 P=40), respectively. No inhibition was observed in treatments receiving only a P addition. After initial inhibition, which lasted 24 h, APA increased (Fig. 3-12a). A significant interaction between N and P was apparent at 48 h. Cultures receiving only P had a delayed

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81 Table 3-8. Specific hot water extractable phosphorus measured over time in natural plankton populations collected in August 1990 after receiving nitrogen and phosphorus additions (mean ± 1 SE). l=no nutrient addition, 2=400 jig N L"\ 3=40 fig P L'\ 4=400 fig N L" 1 and 40 fig P L"\ 5=800 ng N L' 1 and 80 fig P L"\ and 6=no nutrient addition and 7=40 fig P L' 1 and plankton grown at 19'C. Time h Treatment ~6 2 24" 48 96 fig P fig chlorophyll a 1 0.89 ± 0.07 0.73 ± 0.04 0.68 + 0.02 0.51 + 0.02 0.36 + 0.02 2 0.92 + 0.03 0.67 ± 0.04 0.51 ± 0.02 0.33 ± 0.02 3 1.75 ± 0.03 1.34 ± 0.03 0.89 i 0.04 0.45 ± 0.01 4 1.85 ± 0.06 1.25 + 0.03 0.90 ± 0.04 0.44 ± 0.02 5 2.66 ± 0.05 1.82 i 0.01 1.25 ± 0.04 NA" 6 1.00 ± 0.07 0.82 ± 0.02 0.72 + 0.07 NA 7 1.71 ± 0.01 1.59 t 0.03 1.10 t 0.03 NA NA indicates data is not available.

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82 Fig. 3-11. Time courses of hot water extractable soluble reactive phosphorus following nutrient enrichment of natural plankton populations collected in August 1990. 0N,0P=no nutrient addition-,400N,0P=400 ng N L ; 0N,40P=40 ng P L' 1 \ 400N,40P=400 ng N L' 1 and 40 /zg P L*\ and 800N,80P=800 /zg N L" 1 and 80 ng P L" 1 : a) plankton grown at 29*C; b) plankton grown at 19'C. Vertical bars indicate 1 SE. No vertical bar indicates SE is smaller than symbol size.

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83 Table 3-9. Specific hot water extractable phosphorus measured over time in natural plankton populations collected in August 1990 after receiving nitrogen and phosphorus additions (mean ± 1 SE). l=no nutrient addition, 2=400 fig N L*\ 3=40 fig P L"\ 4=400 fig N L" 1 and 40 fig P L'\ 5=800 fig N L" 1 and 80 fig P L" 1 , and 6=no nutrient addition and 7=40 fig P L" 1 and plankton grown at 19*C. Time h Treatment ~~6 2 24 48 96 fig P fig chlorophyll a 1 0.41 ± 0.05 0.48 ± 0.07 0.34 + 0.01 0.29 + 0.01 0.22 ± 0.00 2 0.53 ± 0.01 0.35 ± 0.02 0.27 + 0.00 0.19 + 0.01 3 1.14 + 0.06 0.64 + 0.04 0.35 ± 0.01 0.28 + 0.01 4 1.07 + 0.06 0.67 ± 0.06 0.37 ± 0.01 0.21 ± 0.02 5 1.79 + 0.07 0.82 + 0.03 0.51 ± 0.02 0.24 + 0.00 6 0.54 + 0.02 0.44 + 0.01 0.31 ± 0.04 0.35 + 0.05 7 0.87 + 0.05 0.73 ± 0.01 0.48 ± 0.01 0.47 + 0.03

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100 96 144 TIME (h) 192 g. 3-12. Time courses of alkaline phosphatase activity following nutrient enrichment of natural plankton populations collected in April 1990. 0N,0P=no nutrient addition; 400N,0P=400 M9 N L ; 0N,40P=40 ng P L" 1 ; 400N,40P=400 ng N L and 40 fig P L"\ and 800N,80P=800 ng N L" 1 and 80 fig P L" : a) total alkaline phosphatase activity; b) specific alkaline phosphatase activity. Vertical bars indicate 1 SE. No vertical bar indicates SE is smaller than symbol size.

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85 c • — E < Q_ < O < 100 80 60 40 20 0 0 O on, op ON. 40P 19°C 24 48 72 TIME (h) (b) 96 ig. 3-13. Time courses of alkaline phosphatase activity following nutrient enrichment of natural plankton populations collected in August 1990. ON,OP=no nutrient addition; 400N,0P=400 fig N L ; 0N,40P=40 fig P L' 1 ; 400N,40P=400 fig N L" and 40 fig P L*\ and 800N,80P=800 fig N L' 1 and 80 fig P L' : a) plankton grown at 29°C; b) plankton grown at 19 # C. Vertical bars indicate 1 SE. No vertical bar indicates SE is smaller than symbol size.

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response in APA increase and produced the least APA. A similar trend was observed by phytoplankton cultured at 19'C in August (Fig. 3-13b). In August the response of cultures grown at 29'C with only P added mimicked those which received both N and P (Fig. 3-13a). Disregarding temperature effects, APA in cultures with and without P additions exhibited similar trends; increasing up to 48 h and then decreasing (Fig. 3-13a). The initial increase in APA over the first 24 h was on average 64%, greater in those cultures receiving P. The reverse was true for the next 24 h period. Alkaline phosphatase activity measured in treatments 1 (N=0 P=0) and 2 (N=400 P=0) doubled while only a 22% increase was observed in treatments 3 (N=0 P=40) and 4 (N=400 P=40). Cultures which received treatment 5 (N=800 P=80) did not exhibit a change. Both groups subsequently declined by approximately 20 nM min" 1 to 96 h. Transforming the APA data to specific activity results in a different shape curve for April data (Fig. 3-13b) but no change in curve shape in August (Table 3-10). The interpretation from both experiments is the same, .i.e., higher specific APA was apparent in all cultures which did not receive any P addition. While those cultures which received P had significantly lower specific APA. The highest specific APA was recorded in April in cultures which received treatments 1 (N=0 P=0) and 2 (N=400 P=0). Discussion Nutrient loading from external and internal sources can influence the productivity of phytoplankton and other aquatic biota. The response of phytoplankton growth to nutrient enrichment has been used as an

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87 Table 3-10. Specific alkaline phosphatase activity measured over time in natural plankton populations collected in August 1990 after receiving nitrogen and phosphorus additions (mean ± 1 SE). l=no nutrient addition, 2=400 /xg N L"\ 3=40 HQ P L'\ 4=400 nq N L" 1 and 40 H9 P L"\ 5=800 fig N L" 1 and 80 H9 P L'\ and 6=no nutrient addition and 7=40 fig P L' 1 and plankton grown at 19*C. Time h Treatment 6 24 48" 96 ---nmol APA Hg chlorophyll a' mm 1 0.34 ± 0.00 0.47 + 0.01 1.07 + 0.03 0.45 ± 0.03 2 0.47 ± 0.02 1.01 ± 0.07 0.51 + 0.06 3 0.63 + 0.01 0.64 ± 0.02 0.16 + 0.02 4 0.63 + 0.02 0.70 ± 0.03 0.17 + 0.00 5 0.67 + 0.02 0.48 ± 0.01 0.07 ± 0.00 6 0.22 ± 0.01 0.43 ± 0.01 1.12 ± 0.03 7 0.32 + 0.02 0.52 ± 0.01 1.00 ± 0.04

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88 indicator of nutrient status. This study examined growth, uptake rate, surplus P and APA to explain P requirements of phytoplankton and associated microorganisms in Lake Apopka. Natural plankton populations were used to include the contributions of bacteria and zooplankton to the overall nutrient status of the lake. The objective was to determine the response of phytoplankton biomass, hence chlorophyll a measurements were used to indicate biomass. This study demonstrated that APA is immediately and rapidly inhibited by high inorganic P concentrations (Garen and Levinthal 1960; Moore 1969; Torriani 1960). The extent of inhibition was dependent upon the concentration of inorganic P added, internal P concentrations and the growth of the plankton (Fitzgerald 1969). The most severe inhibition was caused by 1000 P L"\ It has been suggested that 1000 ng PL" 1 is the minimum requirement for APA inhibition (Jones 1979b). In combination with external P concentrations, the extent of APA inhibition may also be dependent upon the initial internal nutrient content. In April, APA was inhibited by 40 and 80 ng P L" 1 within the first 2 h but these concentrations did not result in such rapid inhibition in August. Phytoplankton growth as determined by increases in chlorophyll a concentrations, were initially limited by N and had sufficient P so additions of P resulted in inhibition of APA. As the phytoplankton grew and exhausted internal supplies, growth became limited by both N and P, and APA increased. In August, phytoplankton were P limited, thus the demand was sufficient that even the addition of 80 fig P L' 1 did not inhibit APA. Initial APA was also higher in August than April, and may reflect the difference between slight P limitation

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89 and initial N limitation. Alternatively, the difference in response may be due to different algal populations which have phosphatases with different affinities (Pettersson 1980). In April, the algal population (on a biovolume basis) was dominated by Microcystis sp. and pennate diatoms. In August, the population was dominated by Microcystis sp. and Lyngbya contorta (Aldridge, F. J., personal communication, Department of Fisheries and Aquaculture, University of Florida, Gainesville, FL.). Smith and Kalff (1981) showed that APA is influenced more by the equilibration of plankton growth with nutrient supply, rather than the species composition of the plankton population. The P demand by plankton populations in Lake Apopka was very high as shown by the rapid uptake of SRP in August. These rates are higher than those obtained by similar methods from other lakes, i.e., 0.02 to 0.03 ng P L' 1 min 1 (Rigler 1956) and 0.017 to 0.43 ng P L" 1 min" 1 uptake rates (Lean and White 1983). Within the first 2 h of P enrichment, an increase in HEP-SRP equivalent to 66% of inorganic P added was observed. The HEP-TSP increase accounted for 86% of added inorganic P. A 10°C reduction in temperature decreased percentage uptake to 43 and 78% as HEP-SRP and HEP-TSP, respectively. The concentrations of HEP-TSP decreased as algal biomass increased. At 96 h an increase in HEP-SRP was observed for cultures which received treatments 3 (N=0 P=40) and 5 (N=800 P=80). Internal P concentrations have been shown to regulate APA (Chrdst and Overbeck 1987; Fitzgerald and Nelson 1966; Moore 1969). The increase at 96 h thus could account for the decreased APA. In April, 1989 HEP-SRP concentrations were shown to be inversely related to APA, while in August a strong inverse

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90 relationship was not recorded in response to treatment, suggesting that internal P levels were not sufficiently low to control APA. Comparing specific HEP-SRP (HEP-SRP/chlorophyll a) values in April, P limited cultures had ratios < 0.09 /jg P 1 /xg chlorophyll a' 1 while treatments 3 (N=0 P=40) and 5 (N=800 P=80) had ratios > 0.12 /xg P L" 1 /xg chlorophyll a" 1 . Specific HEP-SRP ratios obtained after 96 h in August were generally > 0.2 ng P L" 1 jig chlorophyll a" 1 . In August, after 96 h, an increase in HEP-SRP occurred at the same time as an increase in growth rate for treatments 3 and 5. High growth rates in P limited Scenedesmus sp. were associated with increased surplus P (Rhee 1974). No increase in HEP-SRP at 96 h was observed in the cultures which received treatment 4. This may be anomalous because measurements of both HEP-TSP and APA obtained from treatment 4 cultures agree with those determined in treatment 3 cultures. Increased HEP-SRP concentrations may not be the only factor regulating growth and P limitation, because decreased APA was also observed in the other cultures. Stable low concentrations of HEP-SRP, with rapidly declining specific APA and uptake rates, combined with increasing chlorophyll a, have been suggested to indicate that P is immediately utilized for growth as opposed to P storage (Sproule and Kalff 1978). An inverse relationship between specific APA and growth rate has been recorded (Smith and Kalff 1981). Phosphorus required for growth is probably provided from the hydrolysis of small chain polyphosphates from the continually decreasing HEP-TSP concentrations. While HEP-TSP concentrations declined, no increase in any other P parameter measured was sufficient to account for the disappearance of HEP-TSP. Hence it

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92 thus indicate that this system is P limited. In April, specific APA of plankton which received P additions remained between 0.3-0.4 nmol APA fig chlorophyll a" 1 min" 1 for the first 96 h. Hence this ratio may represent constitutive APA (Gage and Gorham 1985). Specific APA determined in a eutrophic Swedish lake remained at < 0.3 nmol APA Hg chlorophyll a" 1 min" 1 for most of the year but increased to 0.8 nmol APA ng chlorophyll a' 1 min' 1 during periods of P limitation (Pettersson et al. 1990). Specific APA thus increases with the severity of P limitation (Perry 1976). In this study plankton grown in the absence of P produced specific APA > 1 nmol APA ng chlorophyll a 1 min 1 . Greater fluctuation in specific APA was apparent in August. However, comparing specific APA with increases in chlorophyll a, in general, specific APA measured in cultures which received P was inversely related to growth rate (Rhee 1973; Smith and Kalff 1981). Specific APA determined bimonthly at 7 sites in Lake Apopka from April 1989 to February 1990, was < 0.3 nmol APA fig chlorophyll a" 1 min 1 , this suggests that growth of Lake Apopka plankton was not severely P limited. This agrees with results from other nutrient limitation studies conducted on Lake Apopka (Aldridge, F. J., unpublished data, Department of Florida, University of Florida, Gainesville, FL.). Hence, it would appear that the expression of APA and HEP relative to chlorophyll a is an accurate indicator of the nutrient status of plankton in Lake Apopka. However, it should be re-emphasized that APA and HEP are found in numerous organisms, phytoplankton, bacteria and zooplankton which vary both spatially and temporally, absolute values may vary. Some uncertainty could be removed

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93 by normalizing the data to ATP, and thus express the indicator relative to living biomass (Healey and Hendzel 1980). Although the focus of this research has been on P limitation, Lake Apopka has been found to often be N limited or co-limited by both N and P (Aldridge, F. J., unpublished data, Department of Fisheries and Aquaculture, University of Florida, Gainesville, FL.). The uptake of NO3-N, shown to be the preferred form of N for Lake Apopka phytoplankton (Aldridge, F. J., unpublished data), gives an insight into the N requirements of the system. In April, a situation of co-limitation, [N0 3 + N0 2 ]-N concentrations were shown to decrease over time, and NH 4 -N concentrations tended to increase over time, suggesting that mineralization of organic N occurred. In August, when plankton were established as being slightly P limited, no decrease in [N0 3 + N0 2 ]-N concentrations were observed. The limitation of the analyses used in this study is that APA and HEP concentrations do not differentiate between P limitation and colimitation. Nitrogen limitation parameters, such as ammonium enhancement, should also be determined to assess the N requirements of the plankton and thus enable the assessment of N limitation in conjunction with P limitation (Vincent et al . 1984). The continued measurement of HEP-SRP as a P limitation indicator has been questioned based on its highly dynamic nature (Wynne and Berman 1980; Cembella 1984b). Under ambient conditions such rapid accumulation of HEP-SRP may not occur (Wynne and Berman 1980). Others have shown that HEP-SRP is a good indicator of the nutritional status of plankton (Pettersson 1980; Sproule and Kalff 1978). In this study it

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94 appeared to be a reliable indicator of the nutritional status of the plankton. Conclusions Results from this study show that APA of Lake Apopka plankton is inhibited by high concentrations of inorganic P. Hence, ambient lake water SRP concentrations (<10 ng L" 1 ) will not be sufficient to inhibit APA. Both P limitation and co-limitation of N and P were observed. During conditions of inorganic P limitation, plankton P uptake demand was very high as shown by the rapid uptake of SRP. Uptake rates as high as 1.5 ng L" 1 min" 1 were reported. The first step in the metabolism of the added P was the accumulation of internal P as identified by hot water extraction. After the apparent removal of all SRP from the growth medium, plankton utilized P from the HEP-SRP and HEP-TSP pools for growth. Studies assessing the importance of surplus P frequently only determine HEP-SRP, however, this study highlighted the need to measure both HEP-TSP and HEP-SRP. Alkaline phosphatase activity tended to increase with growth of the plankton, however, the intensity of APA was dependent upon the conditions of P limitation. Severe P limitation was indicated by specific APA values > 1 nmol APA ng chlorophyll a" 1 min" 1 . Specific APA values < 1 nmol APA ng chlorophyll a 1 min 1 may indicate slight P limitation, but APA is associated with numerous organisms in this eutrophic system, which change both spatially and temporally, hence P limitation should be confirmed by nutrient enrichment bioassays.

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CHAPTER 4 THE EFFECT OF SEDIMENT RESUSPENSION ON ALKALINE PHOSPHATASE ACTIVITY Introduction In many lakes, exchange of P between the sediment and the water column is dependent upon diffusion related processes (Stumm and Leckie 1971; Tessenow 1972). However, in shallow lakes, P exchange also occurs due to sediment resuspension during wind events which increase the interaction between the sediment and the overlying water column. It has been estimated that the upper 10 cm of sediment is actively involved in exchange reactions as a result of resuspension with the overlying water column (Tessenow 1972; Schindler et al . 1977). However, the amount of sediment that will mix with the water column is a function of the shear stress and sediment type (Lee, 1970). The immediate result of sediment resuspension is the increase in suspended solids concentration in the water column. The suspended sediment has been shown to provide 28-41% algal available P (Dorich et al. 1985), as well as physically transporting soluble P to the water (Ryding and Forsberg 1977). Sediment resuspension also results in increased exchange of soluble reactive P (SRP) from the sediment to the water column (Holdren and Armstrong 1980; Pollman 1983). The increase in exchange has been associated with biological activity (Pomeroy et al . 1965). Conversely, SRP may be removed from the water column as a result 95

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of sorption to the particulate material (Gachter and Mares 1985; Reddy and Fisher 1990). Resuspension results in increased aeration of the sediments. Under aerobic conditions P release from sediments has been associated with the decomposition of organic matter (Lee et al. 1977). If the sediments are dominated by Fe, aeration results in increased sedimentation of P (McQueen et al . 1986); however, in highly organic sediments, biological processes catalyzed by numerous phosphatase enzymes are likely to dominate (Ayyakkannu and Chandramohen 1971). Organic P mineralization in sediments is regulated by the activity of enzymes such as phosphatases, particularly alkaline phosphatase activity (APA) in sediments at neutral and alkaline pH (Ayyakannu and Chandramohen 1971; Kobori and Taga 1979b). A significant positive correlation between SRP released and phosphatase activity in the water column was observed during resuspension of marine sediments (Degobbis et al. 1984). Thus the level of APA within the sediment will have an effect on the APA subsequently resuspended in the overlying water column. Phosphatase activity decreases with depth of soils (Juma and Tabatabai 1978; Speir and Ross 1978) and sediments (Degobbis et al. 1984; Kobori and Taga 1979b). This corresponds to a decrease in microbial biomass, C, N and organic P with depth (Juma and Tabatabai 1978; Speir and Ross 1978, Baligar et al . 1988). Consequently the depth of material resuspended is significant in influencing APA in the water column.

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97 Following subsidence of the wind event, suspended solids settle thus transporting any associated material from the water column to the sediment, i.e. APA, and results in a decrease in organic P mineralization within the water column. Settling seston is also a sink for SRP (Gachter and Mares 1985). The objectives of this study were to determine; 1) the depth distribution of APA in the sediment and the overlying water column and, 2) the effect of sediment resuspension upon SRP and APA release. It was hypothesized that sediment resuspension may affect APA by 1) increasing SRP levels in the water column and competitively inhibiting activity, 2) releasing alkaline phosphatase from the sediment to the water column, and 3) a combination of 1 and 2. Materials and Methods Site Description Lake Apopka is a 12,500 ha hypertrophic lake, located in central Florida, 28°37' N latitude, 81°37' W longitude (Fig. 4-1). It has a mean depth of 2 m. Chlorophyll a values exceeding 100 ng L" 1 are frequently recorded (chapter 2). Nutrient loading from the surrounding agricultural and urban areas has resulted in the current hypereutrophic conditions in the lake (USEPA 1979). The bottom sediments in the lake have a 30 cm unconsolidated flocculent layer at the surface, underlain by consolidated flocculent material (Reddy and Graetz 1990). The sediments have an alkaline pH, hence the phosphatases of interest are those with maximum activity in the alkaline region, i.e. alkaline phosphatase.

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99 Field sampling procedures Water . Water samples were collected on May 23 1989, from the center of the lake (Fig. 4-1.) using an alpha sampler (Wildco), at depths 0, 0.5, 1 and 1.5 m below the surface. Water was stored in 1 L polyethylene bottles kept on ice until return to the laboratory. Dissolved oxygen (DO) and temperature (YSI, Model 58) and pH (Orion, Model SA 230) were recorded with depth. Light penetration was estimated by measuring the Secchi disk transparency. Within 24 h of return to the laboratory, samples were analyzed for total and soluble APA. Other parameters measured were total Kjeldahl N (TKN), total P (TP), SRP, and chlorophyll a. Sediment . Three intact sediment cores were collected from the deck of a boat in May 1989 from the center of the lake using a 6 cm (I.D.) x 1 m (length) Plexiglass-PVC sediment core sampler (Reddy and Graetz, 1990). The cores were taken to a depth of 40 cm, capped and brought to the laboratory for sectioning. The cores were sectioned at 0-2, 2-5, 5-10, 10-20 and 20-40 cm intervals, placed in 125 mL centrifuge tubes, immediately purged with N 2 and stored at 4'C until analysis. Preliminary studies demonstrated that sediments could be held for at least 3 weeks at 4°C with no change in APA. Alkaline phosphatase activity, porewater SRP, and water content were determined on wet sediment. NaOH-extractable P, an indication of bioavailable inorganic P and labile organic P (Dorich et al . 1985; Young et al . 1985) was also measured. Total P, organic P and volatile solids content of dried sediment were determined.

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100 Experimental Design Experiment 1. Six intact sediment cores were collected by boat from the center of the lake on September 25 1989. The cores were taken to a depth of 30 cm, capped and brought to the laboratory and maintained under ambient light and temperature conditions. Overlying water was siphoned off to leave equal volumes of water in all cores. Previous studies have shown that the concentration of SRP in the porewater of the upper 10 cm sediments remain close to ambient lake water concentrations (Reddy and Graetz 1990). The high water content (98%) associated with the low SRP concentrations of the surface sediments suggest that these sediments are frequently resuspended. Consequently, the surface 10 cm of sediment was resuspended into the overlying water column for 1 h using a sediment resuspension device similar to that described by Wolanski et al . (1989), (Fig. 4-2). This involved the use of a 52 cm long Plexiglas rod to which 19 Plexiglas rings (approximately 1 cm thick) were attached at 2 cm intervals. The rod was oscillated within the core above the sediment surface. The depth of placement of the rod was adjusted so that only the surface 10 cm of sediment was resuspended. The surface sediments in three cores were resuspended and the remaining three cores were left undisturbed as controls. During resuspension a composite 70 mL sample was withdrawn by syringe from the resuspended material (time =1 h). At predetermined intervals, 70 mL composite samples were withdrawn from the water column of all cores. Water samples were analyzed for total and soluble APA, SRP, total soluble P (TSP), TP, TKN and total suspended solids (TSS). At the end of the 24 h sampling period, the surface 10 cm of sediments of the cores

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101

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102 were sectioned into 2.5 cm intervals, placed in 125 mL centrifuge tubes, immediately purged with N 2 and stored at 4'C until analysis. The sediments were analyzed for total APA, TP, organic P and porewater SRP. Experiment 2. A second experiment was conducted in January 1990 to further evaluate the effect of varying depths of bottom sediment resuspension upon APA and associated parameters within the water column. Twelve sediment cores were collected on January 23 1990, to a depth of 20 cm. The overlying water was siphoned off to leave a 45 cm water column. There were three treatments; the top 2, 5 or 10 cm of triplicate cores were resuspended for 15 min using the procedures described in experiment 1. There were 3 replicates for each treatment and 1 undisturbed core to serve as a control. At predetermined intervals, 70 mL water samples were withdrawn. The amount of resettling was measured and the water withdrawn from the midpoint of the exposed water. Alkaline phosphatase activity, TP, TKN, SRP, TSS and total organic carbon (T0C) of the water column were determined. At the end of the sampling period, the surface 2, 5 and 10 cm sediment fractions representing the respective treatments were sectioned in resuspended and control cores, and analyzed as described above. In addition, APA was determined in the porewater using the method described for total sediment APA, to determine whether the activity was predominantly particulate or soluble. Analytical Methods Water. Alkaline phosphatase activity was determined fluorometrically (Healey and Hendzel 1979a). One half mL of substrate,

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103 3-o-methyl fluorescein phosphate (Sigma Chemicals), at a concentration determined to be non-limiting, in 0.1 M Tris buffer (Tham, Fisher Scientific) pH 8.6, was added to 4.0 mL of lake water in a cuvette. Both total (whole lake water) and soluble (filtered through 0.45 /im Gel man membrane filter) APA were determined. The cuvettes were placed in a water bath (25*C). At timed intervals during a 20 min period the cuvettes were placed in the fluorometer and the fluorescence measured. The enzyme activity was measured as an increase in fluorescence as the substrate was enzymatically hydrolyzed to the fluorescent product. Fluorescence units were converted to enzyme activity using a standard calibration curve of 3-o-methylfluorescein (Sigma Chemicals). The fluorescence was measured using a Turner fluorometer No. 110, equipped with Turner lamp no. 110-853, in combination with 47 B primary and 2a-12 secondary filters. Autoclaved lake water with substrate added was used as a control . Chlorophyll a was determined spectrophotometrically following extraction with acetone and correction for pheophytin (APHA (1002-G), 1985). Total P, TSP, TKN, T0C, SRP and TSS were determined by standard methods (APHA 1985). Sediments . Alkaline phosphatase activity was determined by a method adapted from Sayler et al . (1979). Sediment samples (1 g wet sediment) or 1 mL of porewater, were placed in centrifuge tubes. Three mL of 1 M Tris-Tris HC1 buffer (pH 7.6) were added to each tube. The average pH found in Lake Apopka sediments was approximately 7.6. The samples were sonicated (Heatsystems Ultrasonics Model

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W-220F) for 45 s at 30% relative output, to release cell bound phosphatase. The samples were then incubated with 1 mL of 50 mg mL" 1 of p-nitrophenyl phosphate, (Sigma Chemicals) at 25* C for 1 h. After 1 h, 3 mL of 1 M NaOH were added to the tubes to stop the reaction and enhance p-nitrophenol color. Controls to account for substrate color and color release during solubilization of organic matter by the NaOH, were obtained by incubating sediment without the substrate and subsequently adding the substrate along with NaOH at the end of the incubation. All samples were then centrifuged at 7000 rpm (7096 g) for 15 min. The liquid was removed and absorbance at 410 nm (Shimadzu Model UV-160) measured. Concentrations were determined by calibration with a standard curve of p-nitrophenol (Sigma Chemicals). Porewater was extracted by centrifuging the sediment subsample under anaerobic conditions at 5000 rpm (3620 g) for 15 min. The porewater was immediately filtered through 0.45 fjm Gelman membrane filters. Soluble reactive P was measured using standard methods (APHA 1985). Sodium hydroxide extractable P was obtained by shaking 5 g of wet sediment with 20 mL of 0.1 M NaOH for 16 h. Total P and SRP of filtered extracts were then measured using standard methods described for water. Water content was determined by drying a known weight of wet sediment at 70*C to a constant weight. The dried sediment was ground to pass through a 20 mesh screen, using a Spex 8000 grinding mill. Volatile solids were reported as the loss in weight due to ignition of dried sediment at 500*C for 2 h. Total and organic P content of the dried sediment was determined via ignition (Walker and Adams 1958).

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Results 105 Physico-chemical Properties Water . Nutrient concentrations were evenly distributed throughout the water column (Table 4-1). Chlorophyll a, temperature, DO, and pH decreased with depth. Total APA in the water column increased with depth from a surface concentration of 37 to 44 nM min" 1 at 1.5 m. Soluble APA was <2% of total APA. A high concentration of soluble APA was measured at a depth of 1 m. Sediment . Total APA in the sediments decreased with depth, with the greatest change occurring within the 20-40 cm depth (Fig. 4-3a). The water content of the sediments decreased from 98% at the surface to 95% in the 20-40 cm depth (data not shown). The porewater SRP remained constant within the first 0-20 cm but increased significantly within the 20-40 cm depth from 0.01 mg L 1 to 1.4 mg L" 1 (Fig. 4-3b). Volatile solids also increased at this depth (Fig. 4-3c). The opposite effect was observed for NaOH-extractable P, organic P and TP, which decreased in the 20-40 cm sediment depth (Fig. 4-4a, b, and c). Sediment Resuspension Effects on Alkaline Phosphatase Activity Experiment 1 Water column . Resuspension of the top 10 cm of sediment resulted in increased TSS from 60 to 3000 mg L" 1 Within 30 min after resuspension, most of the sediment particles settled. Concentrations of all parameters (excluding TSP) increased in the water column during resuspension (time 1 h) and decreased to initial concentrations within 24 h (Table 4-2). Soluble parameters, e.g. SRP, exhibited considerable

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106 Table 4-1. Distribution of selected parameters measured in May 1989, within the water column at the center of Lake Apopka (n=3). APA Depth Total Soluble TKN TP SRP DO pH Chi a Temp Secchi m --nM min" 1 mg L" 1 --/ig L" 1 mg L" 1 Mg I." 1 °C m 0 37.2 0.9 6.26 210 3 8.5 9.13 99 27.5 0.25 0.5 40.5 0.9 5.55 270 4 8.4 9.14 96 27.0 1.0 43.8 1.5 6.68 270 4 6.7 9.08 86 26.3 1.5 43.8 0.3 7.43 260 4 5.7 9.06 83 26.3

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108 +-> O U i(J t«w C -O 00 to cd cn o ii. z 0 o u +J XI Ul c oo gj • £c oo i r3 -o SO) 01 o -t-> Vl£ A Q. U 0>
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qj o QJ o o oo QJ so o r00 o O i i-H 01 +-> c CO rC jt oj EE 0) ~— M tO J* C CL •«o -C Q. 4-> 00 C iO) 01 (J +-> a> a> E -c m 4-> Sre £ CL O &oo as c oo o cn ITS isa> -t-> -Q C E a> a> u -M c a. o a> o oo csj QJ -Q as 00 00 a. < QJ .a 3 O 00 oo OO QJ ai c sa> T CSJ CO CO 1—1 CO co —J CO CD +1 -H -H -H +1 +1 +H +i CM LO r-». to r-LO tjLO CO LO CSJ CO ] o CO r~lo o LO 00 o O i — i o CSJ o o O , CD o o o o o o o -H H -H +i +i -(-1 -H +1 f— Tt* CTv O cn c» LO LO c •r— O O Csj CSJ o LO E o O — t o o o O o e ro LO CO r-~ o CSJ oo ! i o t — i ( — > f — ^ CO ( — i ^ — * +1 -H -H -H -H 44 4-1 00 O ai LO LO r—t f— t LO 1 00 00 LO CSJ LO Cn LO •dO r— t co o 1 ) o LO Q 4-1 -H -H -H -H +| 4-1 44 I lo LO oo LO «*• LO i i | 00 CO o CO o CO CO csj -H -H -H +1 4-1 -H 4-1 Li LJ i o O O o o O o o o cn CD LO o 00 1 — r— ( 1 ! CO lo O o CO LO CO LO CO CO ! CSJ csj H -H +1 +1 +1 +i +1 H O o o o o CO o o o cn LO 00 00 CO LO CO 1 — ( r— « r~. i — i I — t LO LO CSJ CSJ CSJ CO LO csj LO o CO LO LO _i o o O o o LO o o cn +1 -H -H +1 +1 +i +1 -H E IT) LT) Cn cn o CO in LO 00 CO o CO CO CO CO CO LO LO cn o oo o CO csj CSJ 00 C QJ CL 00 <4O T3 a sQJ QtO T3 £= aj 00 +-> QJ 00 QJ SQQJ So F — l/l c QJ CL I/) oo QJ U O CL QJ QJ QJ E a> s3 oo ro QJ SQJ CL QJ QJ SQJ o o C_J I CL 00 T3 3 a> 00 13 QJ c CC QJ o _ E >• cS

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variation with no apparent change in concentration. The undisturbed control sediment cores maintained constant nutrient concentrations throughout the duration of the experiment. Sediment . The APA in the control sediment cores decreased with sediment depth. No distinct depth profile of APA was observed in the cores where the top 10 cm of sediment was resuspended (Fig. 4-5), although there was a tendency for the APA to be lower in the surface sediment and increase with depth. There was no net change in porewater SRP, TP, organic P and inorganic P concentrations in any of the cores. Experiment 2 Water . The resuspension of different depths of surface sediment into the overlying water column resulted in distinct differences among measured parameters. Resuspension of the surface 10 cm resulted in TSS values of 3000 mg L 1 which decreased to 100 mg L" 1 within 1 h. (Fig. 4-6a). The resuspension of 5 cm of sediment produced TSS values around 1700 mg L" 1 which decreased to 174 mg L' 1 within 1 h. This compares to the settling of the 2 cm depth increment which produced a resuspended TSS of 400 mg L' 1 and decreased more slowly to 140 mg L" 1 within 1 h. As observed in experiment 1, increases in TP, TKN and T0C corresponded to TSS increases (Fig. 4-6). The total APA also increased with TSS, and declined as the suspended matter settled (Fig. 4-7). Combining the data from both resuspension experiments, the relationship between total APA and TSS measured within the water column (Fig. 4-8) was best described by the following power equation: Total APA = 0.831 x (TSS) 0565 ; r 2 = 0.91 n=32

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Ill Fig. 4-5. The depth distribution of alkaline phosphatase activity in triplicate resuspended and undisturbed (control) sediment cores collected in September 1989 from the center of Lake Apopka. Horizontal bars indicate 1 SE. No bar indicates SE is smaller than symbol size.

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112 OH I iii cr> o E z ^> o 10" 10 3 f10" 10' 10" 10" 10' 1 10' 2 TOC • — V 1 1 i i i 1 10 10 10 1 10 10 1 -1 -2 10' 10 10' TP 10 cm (e) T 5 cm • 2 cm control t= t : =* TKN <«0 4 * f i i i i i i 20 25 30 Fig. 4-6. 0 5 10 15 TIME (h) Concentrations of selected parameters measured in the overlying water column following sediment resuspension of 0, 2, 5, and 10 cm surficial sediments: a) total suspended solids; b) total organic carbon; c) total phosphorus; d) total Kjeldahl nitrogen. Initial data point indicates the conclusion of resuspension. Vertical bars indicate 1 SE. No bar indicates SE is smaller than symbol size.

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113 > hO < CO < X Q. CO o X Q_ UJ < _l < —J < I — O I 75 50 25 • \ • • 2 cm 0 i i 75 50 25 I 0 _ 75 50 25 10 15 TIME (h) _ I 1 i i i i (b) _ — 5 cm 1 \ (c) 1 10 cm i i I • Fig. 4-7. The total alkaline phosphatase activity measured in the overlying water column of triplicate sediment cores after resuspension of surficial sediments: a) 2 cm resuspended; b) 5 cm resuspended; c) 10 cm resuspended; d) control, no resuspension. Initial data point indicates the conclusion of resuspension. Vertical bars indicate 1 SE. No bar indicates SE is smaller than symbol size.

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Fig. 4-8. The relationship between alkaline phosphatase activity and total suspended solids in the overlying water column of sediment cores after resuspension of surficial sediments.

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115 The soluble APA measured in the water column after resuspension of 2 cm of sediment exhibited the same trend as observed for total APA, decreasing as the TSS decreased (Fig. 4-9). Soluble APA did not show significant differences over time in the cores in which the surface 5 and 10 cm were resuspended. Soluble reactive P data exhibit such variability that no statistically significant response was observed. However, all cores including undisturbed cores, exhibited a tendency to increase at time 12 h (Fig. 4-10). This would suggest that SRP was slowly desorbed from the underlying sediment. Sediment . Changes in total and porewater APA within the sediment of control and disturbed cores were not significant (Table 4-3). The highly variable porewater APA accounted for less than 1% of the total activity. Discussion The physico-chemical parameters measured within the water column of Lake Apopka showed no distinct stratification, however, DO, pH, temperature and chlorophyll a tended to decrease with depth. The relatively high concentrations of nutrients and chlorophyll a in the water column are characteristic of hypereutrophic systems, although SRP concentrations are very low. The APA is also representative of productive systems (cf. Heath and Cooke 1975). The soluble APA accounted for only 3% of the total APA, therefore, the majority of APA within this system was associated with particulate matter. A possible incomplete settling of particulate matter would explain the increase in

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0.8 0.4 5 0.0 \(<0 . \ • 2 cm — 1. 1 116 10 15 20 TIME (h) Fig. 4-9. Soluble alkaline phosphatase activity measured in the overlying water column of triplicate sediment cores after resuspension of 0, 2, 5 and 10 cm surficial sediments: a) 2 cm resuspended; b) 5 cm resuspended; c) 10 cm resuspended; d) control, no resuspension. Initial data point indicates the conclusion of resuspension. Absence of vertical bar indicates symbol size is greater than 1 SE.

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117 12 8 4 0 in on o X Q_ (f) o X Q_ Ld > IO < Ld DC Ld _J m z> _i o 00 (a) 2 cm 7 0 (c) 8 T 4 A— k — 0 10 cm 10 15 20 TIME (h) 25 30 Fig. 4-10. Soluble reactive phosphorus concentrations measured in the overlying water column of triplicate sediment cores after resuspension of 0, 2, 5 and 10 cm surficial sediments: a) 2 cm resuspended; b) 5 cm resuspended; c) 10 cm resuspended; d) control, no resuspension. Initial data point indicates the conclusion of resuspension. Vertical bar indicates 1 SE. Absence of vertical bar indicates symbol size is greater than SE.

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118 Table 4-3. The distribution of alkaline phosphatase activity in disturbed and undisturbed sediment cores collected from the center of Lake Apopka in January 1990 (means ± 1 SE). Alkaline phosphatase activity Total Porewater Control /xmol g dry wt. 1 h" 1 /wnol L" 1 h •1 2 cm 14.0" 0.91 5 cm 15.3 0.22 10 cm 10.5 1.07 Resuspended 2 cm 17.8 ± 0.2 0.45 ± 0.14 5 cm 14.0 ± 1.5 0.34 ± 0.03 10 cm 9.5 ± 0.5 1.27 ± 0.38 Control sediment cores were not replicated.

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119 APA at the sediment-water interface, while there was no apparent change in soluble APA. In the sediment, APA was shown to decrease with depth. Soluble reactive P is a competitive inhibitor of APA (Coleman and Gettins 1983), hence the increase of porewater SRP with sediment depth may partially explain the decrease in APA, due to inhibition of the production of the enzyme. Organic P and APA have been shown to be positively correlated (Juma and Tabatabai 1978; Speir and Ross 1978). A high enzyme activity may be maintained in the presence of competitive inhibitors by the presence of increased substrate concentrations; however, the decrease in organic and NaOH-extractable P with depth suggests a decrease in substrate concentrations. Alkaline phosphatase activity was also found to have a positive correlation with organic matter (Speir and Ross 1978), therefore the decrease in volatile solids (an indicator of organic matter), may have contributed to the APA decrease. However, the most probable cause for the reduced activity with depth is a decrease in microbial biomass. Decreasing APA with sediment depth has been shown to have a positive correlation with microbial biomass (Sayler et al . 1979; Ayyakkannu and Chandramohen 1971). Microbial biomass was not measured in this study, however, a decrease with depth could be expected in these sediments because of highly reduced (anaerobic) conditions at lower depths in Lake Apopka sediments (Moore et al . 1991). Pulford and Tabatabai (1988) suggested that under anaerobic conditions, the higher solubility of metals such as Fe and Mn results inhibition of APA (Juma and Tabatabai 1978). Lake Apopka sediment is Ca dominated (Moore et al.

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120 1991), thus, inhibition of APA under anaerobic conditions is probably due to reduced microbial numbers and metabolism. The sediment APA observed in this study is in the same range as those reported for other freshwater systems (APA = 6-18 (imol g dry wt." 1 h" 1 ) with fine particulate organic matter (Sayler et al . 1979). The APA of marine sediments was much lower, i.e., 0.2-3.3 ftmol g dry wt." 1 h" 1 (Ayyakkannu and Chandramohen 1971; Degobbis et al . 1984). However, a comparison of the sediment APA values obtained in other studies is difficult due to the lack of standardization in methodology used. Other complications include the pretreatment of samples, e.g. drying of the soil (Tabatabai and Bremner 1969) which may affect the APA values of some soils (Skujins 1976; Speir and Ross 1978). Short-term resuspension of surficial sediments increased TSS, TKN, TP and TOC concentrations. This is expected because all these parameters are interrelated. The concentrations of these species decreased rapidly following the end of resuspension, as a result of particle settling. However, the soluble fraction, i.e. SRP, did not exhibit significant increases as observed previously (Pollman 1983; Reddy and Graetz 1990) in Lake Apopka cores. The porewater SRP concentrations of the cores used in these resuspension experiments was very low (<5 fig L" 1 ), thus the resuspension of these sediments would not result in an increase in the SRP concentration of the water column. Although the TP concentration in the surficial sediments is high (1200 mg kg" 1 ), a major portion is organically bound and is not readily available. The low porewater SRP concentrations suggest that the surface sediments have a high P sorptive capacity. A high C/P ratio of

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121 the surface sediments also suggests that microbial immobilization of inorganic P occurs (Reddy and Graetz 1990). The mechanism of P release from suspended particles will depend on the rate of P desorption from the solid phase to the liquid phase and the physico-chemical properties of the water column. Resuspension has been shown to increase the biological breakdown of organic P (Pomeroy et al. 1965). Resuspension of anaerobic sediments to the overlying oxygenated water column will result in aeration of the sediments. This may make the associated organic matter more susceptible to enzymatic hydrolysis (Pulford and Tabatabai 1988). The pH of the surface sediment was 7, compared to a water column pH of 8-9. Thus any SRP release into the water column upon resuspension could be immediately precipitated as calcium phosphates (Moore et al . 1991). In shallow lakes sediment resuspension may play an important role in P recycling (Ryding and Forsberg 1977). Due to the association of APA with TSS, prolonged resuspension would result in higher APA levels within the water column. As observed by Burns (1986), the attachment of APA at a non-active site would result in increased longevity of the enzyme within the aquatic system. If phosphomonoesters are present SRP would be released. The insignificant, but apparent gradual increase in SRP in all cores at 12 h, may be due to the enzymatic degradation of the more easily hydrolyzed organic P compounds. Hence, APA may enhance the ability of sediments and particulate matter to recycle P. To test the relationship observed between TSS and total APA, seasonal data collected in the field (chapter 2) was fitted to the equation . A good fit was observed when TSS were high (= 100 mg L' 1 ) and chlorophyll a

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122 concentrations were low. However, with normal TSS {70 mg L' 1 ) and high chlorophyll a, the equation underestimated the measured APA. This suggests that the predictive capability of this equation is only valid during periods of resuspension, when sediment particulate matter is the dominant component of the TSS pool. After 48 h an increase in total APA was observed in the water column of both control and resuspended cores, this may be in response to APA production following P limitation of the plankton population. These increases are apparent as the two points above the regression line in Fig. 4-8. Consequently, during quiescent periods other contributors to the TSS pool. e.g. phytoplankton, should be included to predict APA. Apart from the cores in which the surface 2 cm was resuspended, soluble APA was not shown to increase due to resuspension. The sediment porewater APA was greater than the water soluble APA; however, the dilution effect due to resuspension brought the porewater concentration to ambient levels. The APA attributed to porewater exhibited high variability. This variability could be partially due to differences between cores, and also the insensitivity of the method at such low APA. The fluorescent technique used for lake water would be more sensitive, however, different substrate specificities have been observed (Pettersson and Jansson 1978). Even considering the variability in the porewater APA, it was clearly demonstrated that >99% of the APA was associated with the solids portion of the sediment. A recent study demonstrated the association of APA with the larger particulate matter (Rojo et al. 1990) and it has been suggested by Burns (1986) that the

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123 covalent bonding of enzymes to organic matter would result in increased stabilization of the enzyme. Conclusions Most studies investigating APA in aquatic systems have emphasized the contribution of free-living bacteria, phytoplankton and zooplankton. This study demonstrated the contribution of the sediment associated APA to the total APA pool of the water column. Sediment resuspension resulted in increased APA and TP in the overlying water column, and hence there was an increase in potential organic P mineralization. This increased APA remained high only while sediment particles were still suspended in the water column and decreased upon settling of the sediment. This indicates that no APA was released from the sediment into the water column. The APA determined in both sediments and lake water was mainly associated with particulate matter and thus may have increased longevity. The high APA recorded both in the sediment and water columns suggests that organic P mineralization, via APA, may play a significant role in P cycling of Lake Apopka. The importance of this process is dependent upon the concentration of organic substrates. The forms of sediment organic P capable of acting as substrates needs to be evaluated.

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CHAPTER 5 THE EFFECT OF SEDIMENT AND WATER COLUMN ANOXIA ON ORGANIC PHOSPHORUS MINERALIZATION Introduction Organic P constitutes the major component of total P in the sediment and water column of lakes. When soluble inorganic P concentrations within the water column are low plankton may produce phosphatase enzymes, which hydrolyze organic P compounds with the release of inorganic P (Kuenzler and Perras 1965; Reichardt 1971). As a result the measurement of alkaline phosphatase activity (APA) has been used as a tool to indicate P limitation and potential organic P mineralization through enzymatic hydrolysis (Healey and Hendzel 1979b; Gage and Gorham 1985). Under well oxygenated conditions (aerobic), mineralization of organic P is rapid. However, depletion of dissolved oxygen (DO) concentrations can occur in the water column as a result of high respiratory activity. Under reduced DO concentrations the rate of enzymatic hydrolysis of organic P compounds could be reduced as the metabolism of aerobic plankton is reduced. This may be of particular importance at the sediment-water interface where DO concentrations are often depleted. Oxygen concentrations in the water column may vary both diurnally (Howeler 1972; Reddy 1981) and seasonally (Mortimer 1941). In rare circumstances, metal imnetic DO depletion may occur with increasing 124

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125 eutrophication (Shapiro 1960). More often, highly productive aquatic systems exhibit clinograde DO distributions, resulting in the depletion of DO with water depth in response to summer stratification (Miyake and Saruhashi 1956; Wetzel 1983) and consumption during high respiratory activity at the sediment-water interface (Charlton 1980; Bostrom et al . 1982). Diffusion of DO from the water, bioturbation and sediment resuspension can maintain relatively aerobic conditions at the sedimentwater interface. Under these conditions P release from sediments to overlying water can be due to mineralization of organic P (Lee et al . 1977). Mineralization of organic P may be of particular importance in sediments which possess abundant organic substrates, such as peat sediments (Ayyakannu and Chandramohen 1971). During stratification of the water column, the hypolimnion may become anoxic, hence the surface sediment becomes anaerobic. A significant consequence of anaerobic conditions at the sediment-water interface is the increase in water soluble P in the overlying water column (Holdren and Armstrong 1980; Ponnamperuma 1972; Mortimer 1941). The release of P under these conditions is the result of the reduction of ferric phosphates to the more soluble ferrous phosphates (Mortimer 1941). This flux of P from the sediment to the overlying water column may have a significant impact upon APA in the water column. Inorganic P is a competitive inhibitor of APA (Coleman and Gettins 1983), and sedimentary release of P has been shown to inhibit APA within the overlying water column (Pettersson 1980). Anaerobic conditions may also

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126 result in the inhibition of organic P mineralization by APA in the sediment (Pulford and Tabatabai 1988). Regardless of the oxygen status of the surface sediments, redox potential tends to decrease with sediment depth (Kobori and Taga 1979b; Degobbis et al . 1984). Bacterial populations were also observed to decrease with sediment depth, in response to redox potential changes (Kobori and Taga 1979b). Bacterial numbers have been positively correlated with APA in sediments (Ayyakannu and Chandramohen 1971), hence APA also decreases with depth (Kobori and Taga 1979b). An important effect of reduced Eh conditions may be the accumulation of soluble organic P compounds due to incomplete mineralization. It is apparent that oxygen concentrations both in the sediment and in the water column may play a significant role in determining P mineral ization. The objective of this study was to determine the effects of anoxia upon organic P mineralization in the sediment and water column of a highly productive lake. It is known that APA is a non-specific enzyme capable of hydrolyzing numerous organic P compounds (Coleman and Gettins 1983; Cembella et al . 1984a). However, specific forms of sedimentary organic P are mainly unknown; identification is based upon the susceptibility to different extraction solutions (Bowman and Cole 1978; Sommers et al . 1972). A second objective of this research was to determine the forms of extractable P influenced by different redox conditions, and their effect on the mineralization of organic P by APA.

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127 Materials and Methods Site Description Lake Apopka is a 12,500 ha hypereutrophic lake, located in central Florida, 28*37' N latitude, 8r37' W longitude. It has a mean depth of 2 m. Chlorophyll a values exceeding 100 /xg L" 1 are frequently recorded (chapter 2). Nutrient loading from the surrounding agricultural and urban areas has resulted in the current hypereutrophic conditions in the lake (USEPA 1979). The bottom sediments in the lake have an average of 30 cm unconsolidated flocculent layer at the surface, underlain by consolidated flocculent material (Reddy and Graetz 1990). Sampling Procedures Water column. Water was collected on July 2 1990, 30 cm below the water surface from the center of the lake in 15 and 30 L polycarbonate containers. The water used for the first experiment was kept for less than 24 h in the dark under ambient laboratory conditions prior to the start of the experiment. The water used to dilute the sediment was first coarse filtered and stored under ambient laboratory conditions in polycarbonate containers until the start of the experiment. Sediment. Grab samples of the surface 30 cm of sediment were collected on 28 June 1990 from the center of the lake using an Eckman dredge, and placed in polycarbonate containers, which were filled completely to minimize air spaces. The samples were stored under ambient laboratory conditions prior to use in the study.

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128 Experimental Design The effect of dissolved oxygen on organic phosphorus mineralization in the water column . Dissolved 0 2 minima in the water column of lakes occur during the night hours when respiration exceeds photosynthesis. To emulate this condition this experiment was conducted in the dark, under ambient laboratory temperatures. Four hundred mL of lake water were placed in each of six 500 mL erlenmeyer flasks. The flasks were placed on stir plates and continuously stirred. Three flasks were stoppered and maintained under anaerobic conditions (Fig. 5la), while three were maintained under aerobic conditions. One replicate of each treatment had pH and DO probes in constant contact with the water column; it was assumed that all three replicates would respond similarly. Anaerobic conditions were obtained by closing the flasks with rubber stoppers and purging with a mix of 330 ppm C0 2 balanced with N 2 for approximately 1 1/2 h until the DO concentration was <0.2 mg L*\ The pH and DO probes were tightly sealed in the stoppers. A rubber septum was also included in the stopper arrangement to allow sampling. At this point 30 mL water samples were removed by syringe from all flasks. A time series of samples was subsequently removed after 2, 4, 8 and 24 h. Samples were analyzed for total and soluble APA, total soluble P (TSP), total soluble N (TSN), NH 4 -N, and soluble reactive P (SRP). Initial chlorophyll a was also determined. The effect of redox potential on organic phosphorus mineralization in t he sediment . Fresh sediment was diluted with filtered lake water to obtain 10 g dry wt. L" 1 of slurry, with a total volume of 2.5 L and was placed in each of six 2.8 L flasks, which were

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129 pH electrode 00 electrode Purging gas Sampling port Lake water Magnetic Stirring Plate (a) Platinum redox electrodes Calomel half cell Sediment BVpH contfoder pH electrode Air outlet to acid trap Magnetic Stirring Plate (b) Afrpump ' Air inlet Fig. 5-1. Diagram illustrating the apparatus used to control dissolved oxygen concentration and redox potential: a) anoxia control in lake water; b) redox potential control of sediment suspensions.

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130 stoppered, placed on stir plates and attached to redox controllers (Cole Palmer Model 5997-20) (Fig. 5-lb) modified from Patrick et al . (1973). All flasks were purged with a mixture of 330 ppm C0 2 balanced with N 2 , for 24 h. The flasks were then allowed to equilibrate for 1 month in the dark under ambient laboratory conditions at the desired redox potentials; -250, -100, 0, +100, +250 and +500 mV (actual potentials measured upon sampling were; -242, -157, -2, +48, +338, +483 mV). After equilibration, triplicate samples were withdrawn from the flasks and analyzed for APA and fractionated for organic P using an adaptation of the method of Sommers et al . (1972) (Fig. 5-2). Bicarbonate extractable P was determined upon sediment samples after the removal of porewater for pH determinations (Fig. 5-2). Total P and volatile solids were measured on dried sediment samples. Analytical Methods Water. Alkaline phosphatase activity was determined fluorometrically (Healey and Hendzel 1979a). One half mL of substrate, 3-o-methyl fluorescein phosphate (Sigma Chemicals), at a concentration determined to be non-limiting, in 0.1 M Tris buffer (Tham, Fisher Scientific) pH 8.6, was added to 4.0 mL of lake water in a cuvette. Anoxic water samples were injected into cuvettes which were sealed by a rubber septum and had been purged with N 2 and then evacuated. Both total (whole lake water) and soluble (filtered through 0.45 (an Gelman membrane filter) APA were determined. The cuvettes were placed in a water bath (25'C). At timed intervals during a 20 min period the

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131 ORGANIC PHOSPHORUS FRACTIONATION SCHEME I POREWATER SEDIMENT INORGANIC P RESIDUE filtered Add 1M HCI (1:50) liquid (3h) 1 SRP Add 0.5 M NaHCO 3 (1:50) (30 min) filtered liquid RESIDUE TP SRP Wash with D.I. (1 :50) (15 min) T ACID SOLUBLE ORGANIC P TP SRP RESIDUE LABILE ORGANIC P 0.5 M NaOH (1 :50) (16 h) i MODERATELY LABILE ORGANIC P LIQUID ~ I — Acidify (pH 0.2) Centrifuge + decant RESIDUE ALKALI HYDROLYZABLE P RESIDUE i LIQUID Acid soluble P SRP TP SRP Acid Insoluble P FULVIC ACID ORGANIC P HIGHLY RESISTANT HUMIC ACID ORGANIC P ORGANIC P HA-P = TP FA-P MODERATELY RESISTANT ORGANIC F > The extraction scheme used to fractionate organic phosphorus in sediment.

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132 cuvettes were placed in the fluorometer and fluorescence was measured. The enzyme activity was measured as an increase in fluorescence as the substrate was enzymatical ly hydrolyzed to the fluorescent product. Fluorescence units were converted to enzyme activity using a standard calibration curve of 3-o-methyl fluorescein (Sigma Chemicals). The fluorescence was measured using a Turner fluorometer No. 110, equipped with Turner lamp no. 110-853, in combination with 47 B primary and 2a12 secondary filters. Autoclaved lake water with substrate added was used as a control . Chlorophyll a was determined spectrophotometrically following extraction with acetone and correction for pheophytin (APHA (1002-G), 1985). Total soluble P, TSN, NH 4 -N, and SRP were determined by standard methods (APHA 1985). Sediments. Alkaline phosphatase activity was determined by a method adapted from Sayler et al . (1979). Sediment samples (1 g wet sediment) were placed in centrifuge tubes. Three mL of 1 M Tris-Tris HC1 buffer (pH 7.0) were added to each tube. The samples were sonicated (Heatsystems Ultrasonics Model W-220F) for 45 sec at 30% relative output, to release cell bound phosphatase. The samples were then incubated at 25'C, with 1 mL p-nitrophenyl phosphate, at a concentration of 50 mg mL 1 (Sigma Chemicals) for 1 hr. At the end of the incubation 3 mL of 1 M NaOH were added to the tubes to stop the reaction and enhance p-nitrophenol color. To account for color interference controls were obtained by incubating sediment without the substrate and subsequently adding the substrate along with NaOH at the end of the incubation. All samples were then centrifuged at 7000 rpm (7096 g) for

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133 15 min. The liquid was removed and absorbance at 410 nm (Shimadzu Model UV-160) measured. Concentrations were determined by calibration with a standard curve of p-nitrophenol (Sigma Chemicals). Porewater was extracted by centrifuging a subsample of sediment at 6000 rpm (5213 g) for 15 min. The porewater was immediately filtered through 0.45 m Gelman membrane filters. Total P, TSP and SRP were measured using standard methods (APHA 1985). Porewater total organic P (TOP) was defined as the difference between porewater TP and SRP. Bicarbonate extractable P was determined after porewater extraction by shaking the sediment with 0.5 M NaHC0 3 (1:50, on a dry weight basis) for 30 min. The extracted medium was then centrifuged at 6000 rpm (5213 g) for 15 min and the supernatant filtered through Whatman 40 filter paper. Total P and SRP were determined on the filtrate (APHA 1985). Labile inorganic P was calculated as the sum of porewater SRP and HC0 3 extractable SRP. Labile organic P was defined as the difference between total labile P and labile inorganic P. Water content of the sediment was determined by drying a known weight of wet sediment at 70'C to a constant weight. The dried sediment was ground to a powder using a Spex 8000 grinding mill. Total P content of the dried sediment was determined via ignition (Walker and Adams 1958). Volatile solids were reported as the loss in weight due to ignition of dried sediment at 500*C for 2 h. Statistical Analysis Data were analyzed using the Statistical Analysis Systems (SAS 1985), Version 6.03 for personal computers. Data in the first

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experiment were analyzed using repeated measures analysis which accounts for the inherent within replicate correlation due to repeated sampling of the same flasks. When data were unbalanced repeated measures analysis could not be used, a split plot design, with time as the subplot, was used to produce the analysis of variance and F test. Data in both experiments were analyzed using Pearson correlation coefficients. Results The Effect of Disso lved Qxvaen on Organic Phosphorus Mineralization in the Water Column Chlorophyll a concentrations of the water samples collected in July 1990 were extremely high (Table 5-1) and exceeded previous measurements (chapter 2). Other parameters measured were within the same range as previously recorded in Lake Apopka (chapter 2). The response of the measured parameters over time was highly varied under both aerobic and anaerobic conditions. During the 24 h experimental period pH decreased from 8.2 to 7.5 and 8.0 under aerobic and anaerobic conditions, respectively (data not shown). Total soluble N exhibited significant differences over time (p=0.02). Under aerobic conditions TSN increased within 2 h and subsequently declined over time (Fig. 5-3a). Under anaerobic conditions the only significant difference observed was the increase at 24 h versus initial concentrations. There was an apparent decrease in NH 4 -N concentrations over time (Fig. 5-3b), however, no statistically significant time or treatment effects were determined for NH 4 -N. Both TSP and SRP concentrations also exhibited

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135 Table 5-1. Concentrations of selected parameters measured in Lake Apopka water in July 1990 (mean ± 1 SE) . Parameter Concentration Chlorophyll a (ng L' 1 ) 220 ± 4 Total APA (nM min" 1 ) 21.9 ± 0.1 Soluble APA (nM min" 1 ) 0.82 + 0.4 TSP (#cg L' 1 ) 15 ± 0 SRP (pq L" 1 ) 1+0 TSN (mg L' 1 ) 2.48 ± 0.0 NH 4 -N (mg L 1 ) 0.05 ± 0

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136 3.5 TIME (h) Fig. 5-3. Nutrient concentrations in Lake Apopka water incubated in the dark under aerobic and anaerobic conditions: a) total soluble nitrogen; b) NH 4 -N. Vertical bars represent 1 SE. Absence of bar indicates SE is smaller than the symbol size.

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insignificant time and treatment responses (Fig. 5-4a and b), SRP concentrations were below detection (< 1 ng L" 1 ) for both treatments after 24 h. A significant treatment response was observed for total APA. (Fig 5-5a). Under anoxic conditions total APA remained constant over the 24 h sampling period. Under aerobic conditions total APA increased within 2 h and then remained constant until 24 h. At 24 h, the total APA had increased from 27 to 43 nM min\ Soluble APA accounted for 4% of total APA (Table 5-1). No change in soluble APA was observed in either treatment (Fig. 5-5b). Significant correlations among the water chemistry parameters measured were observed. A significant inverse relationship between NH 4 -N and TSP was determined (r=-0.79), and significant positive correlations between SRP and TSN (r=0.67) and NH 4 -N (r=0.70) were observed. The Effect of Redox Potential on O r ganic Phosphorus Mineralization in the Sediment ' The initial pH of the sediment prior to the month long incubation was 7.12. Following equilibration at different Eh levels slight changes in pH values were recorded (Table 5-2). Redox values were expressed as pE + pH to account for the variability in pH among the reaction vessels (pH range 6.35 to 7.15). pE was calculated as; pE = Eh / 59 A significant negative relationship was observed between APA and pE + pH (Fig. 5-6a). Conversely, a significant positive relationship between pE + pH and porewater TOP was recorded (Fig. 5-6b). pE + pH accounted for 77% of the variability in APA and 73% of porewater TOP

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138 Fig. 5-4. Nutrient concentrations in Lake Apopka water incubated in the dark under aerobic and anaerobic conditions: a) total soluble phosphorus; b) soluble reactive phosphorus. Vertical bars represent 1 SE. No vertical bar indicates SE is smaller than the symbol size.

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Fig. 5-5. Alkaline phosphatase activity in Lake Apopka water incubated in the dark under aerobic and anaerobic conditions: a) total alkaline phosphatase activity; b)soluble alkaline phosphatase activity. Vertical bars represent 1 SE. No vertical bar indicates SE is smaller than the symbol size.

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140 Table 5-2. Concentrations of selected parameters measured on sediments incubated under six different redox levels for one month (mean ± 1 SE) Porewater Labile Eh pH pE + pH APA ~SRP TOT" ~J-, P^ pmol mV g dry wt." 1 mg P kg dry wt." 1 h" 1 483 6.35 15 6.89 + 0.21 26 + 0.45 2.1 ± 0.45 48 ± 1 11 ± 3 338 6.49 12 4.01 + 0.65 51 ± 0.78 7.0 ± 2.81 74 t 3 16 + 3 48 6.78 8 3.64 + 0.39 8 ± 1.19 13.8 ± 2.51 23 ± 2 19 + 2 -2 6.55 7 4.23 + 0.31 58 i 0.90 8.3 ± 3.15 75 + 0 10 ± 2 -157 6.78 4 1.07 + 0.59 44 + 1.35 16.1 ± 1.96 66 ± 2 22 ± 2 -242 7.15 3 0.71 + 0.24 111 + 1.80 18.3 ± 0.55 144 ± 2 19 ± 1

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141 I cn ~o E < Q_ < 8 6 4 (a) • • % APA = 0.46(pE + pH) 0.21 1 2 r = I i 0.81 l I T3 cn cn O 20 15 10 5 8 10 12 pE + pH 14 16 Fig. 5-6. Concentration of selected parameters measured in sediments incubated under six different redox levels for one month: a) alkaline phosphatase activity; b) porewater total organic phosphorus.

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142 variability. Non linear redox effects were observed for other P compounds. A significant increase in porewater SRP and labile inorganic P were observed at a pE + pH value of 3 (Table 5-2). In contrast FA-TP decreased at a pE + pH of 3. The HA-TP fraction tended to increase under pE + pH values of 7. However, the majority of P forms measured were not significantly affected by redox potential. Volatile solids were 66% in both aerobic and anaerobic treatments. The sediment TP as determined by ignition was 1047 fig g dry wt." 1 . On average the total recoverable P determined by the summation of HC1-TP, NaOH-TP and porewater TP accounted for 74% of TP as determined by ignition (Table 5-3). Acid hydrolyzable-TP extracted the largest percentage of P (41%), followed by alkali extractable P (28%). Labile inorganic and organic P were a small component of the TP, contributing 6.8 (2-14) and 1.4 (1-2)%, respectively. Readily available, i.e. porewater SRP and TP, also contributed less than 20% of total P. Porewater TP accounted for 5.8 (2-13)% of the total P pool. Total and inorganic P extracted with HC1 were the same, indicating that no organic P was extracted. Porewater TSP and SRP were also equivalent suggesting that no porewater organic P was in the soluble phase; TOP represented particulate organic P. Significant correlations between the parameters measured were observed (Table 5-4). Alkaline phosphatase activity had a high positive correlation with pE + pH (r=0.90) while porewater TOP was strongly inversely related to pE + pH (r=-0.92). Alkaline phosphatase activity was also highly negatively correlated with labile organic P (r=-0.81) and HA-TP (r=-0.74), while porewater TOP was highly positively related

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143 Table 5-3. Concentrations of selected parameters measured on sediments incubated under six different redox levels for one month (mean ± 1 SE). NaOH-TP Eh pH pE + pH HC1-TP "FA^TP RFP mV mg P kg dry wt.' 1 483 6.35 15 490 + 13 171 + 5 132 i 3 338 6.49 12 393 + 26 162 ± 4 134 + 1 48 6.78 8 431 + 4 180 ± 1 134 + 0 -2 6.55 7 414 + 20 146 ± 8 126 + 3 157 6.78 4 421 + 15 150 ± 15 167 ± 2 242 7.15 3 434 + 3 115 ± 2 148 ± 3

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144 i O rC < OI— l < O Qo_ co ro <: 00 crt oo o cn oo oo 00 00 o i 00 00 o o cn cn oo cn cn pH O 00 00 00 oo 00 o 00 LO cn o i cn oo oo cn r-. cn oo cn oo oo 00 cn cn o 00 CNJ cn oo oo 00 cn o to cn oo cn oo oo cn cn oo 00 00 oo 00 o o I 00 D_ Q. o_ ro D_ o Q. 0 1 o Oo_ 00 ri— 1— D_ IT D_ •a: jQ -O i i i o I ro ro 3 3 3 is_ o o ro o a; +-> ro U cn o C •r— o II 0) ts 00 +-> '5 ro c -C C -M r0 O
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to labile organic P (Table 5-4). A strong inverse relationship between APA and porewater TOP was observed (r=-0.95). Other significant inverse correlations include porewater TP and SRP with FA-P and NaOH-TP. Labile inorganic P was also highly inversely correlated to these P forms. No relationship was observed between HC1-TP and any other parameters measured. Discussion The Effect of Diss olved Oxygen on Organic Phosphorus Mineralization in the Water Column Phosphorus concentrations in lake water incubated in the dark for 24 h, did not show significant changes under either anaerobic (<0.2 mg L" 1 ) or aerobic (6 mg L" 1 ) conditions. However, decreases in TSN concentrations suggested that mineralization of organic N occurred, but no increase in NH 4 -N was observed. This may be due to rapid nitrification under aerobic conditions (Reddy and Graetz 1981) or loss through volatilization (Stratton 1968, 1969). Initial TSNrTSP exceeded 200:1 and would suggest that the system may be P limited. Under conditions of inorganic P limitation, phytoplankton may produce APA which hydrolyzes organic P compounds with the release of inorganic P (Kuenzler 1965). Specific APA (APA/chlorophyll a) ratios were determined to be an effective means of determining inorganic P limitation of Lake Apopka plankton (chapter 3). Under SRP limiting conditions, APA and specific APA increase (Kuenzler and Perras 1965; Heath and Cooke 1975; chapter 3). Initial and final specific APA ratios under aerobic conditions were 0.1 and 0.2 nmol APA fig chlorophyll a' 1

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146 min"\ suggesting that the plankton population was not P limited (Pettersson 1980; chapter 3). However, total APA increased from 22 to 43 nM min' 1 . This increase could be a demonstration of either increased bacterial reproduction or alternatively, a response to decreased internal P supply within the plankton (Fitzgerald and Nelson 1966; Pettersson 1980; chapter 3). Internal P reserves, which include small chain polyphosphates (Elgavish and Elgavish 1980) are broken down in response to P demand (Rhee 1972, 1973, 1974, chapter 3). Once internal P concentrations reach a certain critical level, APA is produced (Taft et al. 1977; Sproule and Kalff 1978; Chrdst and Overbeck 1987). Under natural conditions and in continuous culture, P concentrations may be low but they are continuously replenished. In batch culture, a one time P input can result in rapid depletion of SRP, hence internal P sources may be required for metabolism (Rhee 1972). Once this P pool has been reduced to a critical level APA is produced (Chrdst and Overbeck 1987). This would explain the 24 h lag time observed prior to the increase in APA observed under aerobic conditions. In contrast, under anaerobic conditions, metabolism of aerobes is reduced and eventually inhibited completely without the return of DO. The dominant form of phytoplankton in Lake Apopka are the bluegreens, Lyngbya sp. and Microcystis sp. (Shannon and Brezonik 1972; Stites, D. L., unpublished data, St. John's River Water Management District, Palatka, FL.). Under anaerobic conditions, only anaerobes and facultative anaerobes are active; however, research has demonstrated that after acclimatization cyanobacteria grow well under anaerobic conditions with H 2 S acting as an electron donor in the photolysis of

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147 water during photosynthesis (Stewart and Pearson 1970). The production of APA is dependent upon the P stress within system (Fuhs et al . 1972; Kuenzler and Perras 1965). Because of the low P requirements of anaerobic bacteria, APA inhibition may occur at lower SRP levels than under aerobic conditions, where P requirements of aerobic bacteria are high. Enzyme activity did not cease under anaerobic conditions suggesting that the enzyme itself was not inhibited by anaerobic conditions. Although total APA under aerobic conditions differed from total APA under anaerobic conditions within 2 h, no significant increase in APA occurred until 24 h, this suggests that less severe low DO will not affect APA organic P mineralization. Depth profiles of APA with varying DO concentrations were shown to correlate with chlorophyll a and bacterial counts rather than DO concentrations (Jones 1972a), consequently metal imnetic 0 2 depletion is unlikely to affect APA. However, in waters which become stratified the anoxic conditions at the sediment-water interface may result in decreased APA after an extended time period. This may be due to decreased production by anaerobes or as a response to competitive inhibition by SRP released from sediments (Pettersson 1980). Lake Apopka is a frequently mixed system under which extended periods of 0 2 depletion are not likely to occur. Diel changes in 0 2 will be too brief (< 8 h) to affect organic P mineralization. No change in soluble APA was observed under either aerobic or anaerobic conditions. Soluble APA has been shown to be a result of cell lysis (Berman 1970) as well as excretions by zooplankton, bacteria and phytoplankton (Wynne 1981; Pettersson 1980). It is thought that significant cell death and lysis did not occur during the period of

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148 anaerobiosis because increased nutrient concentrations and soluble APA were not observed. The Effect of Redo x Potential on Organic Phosphorus Mineralization in the Sediment A significant effect of increasing anaerobiosis is the change from a predominately aerobic microbial population to a smaller anaerobic population. Under anaerobic conditions organic material accumulates due to reduced mineralization rates. Microbial biomass is highly correlated with APA (Sayler et al . 1979; Ayyakannu and Chandramohen 1971), and APA was significantly inhibited by the decrease in redox potential. Hence organic P mineralization will decrease under reduced conditions. In general, redox potential did not have a significant effect on the various organic P pool sizes. However, Lake Apopka sediments are poorly poised (poorly redox buffered) and the low concentrations of electron acceptors may reduce the rate of mineralization. Only APA and porewater TOP were significantly correlated with pE + pH. More resistant P forms may require longer incubation times to show a response to redox potential . Significantly lower porewater SRP and labile inorganic P concentrations were observed at Eh 48 mV, than either Eh 338 or -2 mV. This may be a problem associated with the control of redox conditions. Redox levels may need to be established by utilizing specific electron acceptors, rather than fluctuating the air input. Redox potential tends to decrease with sediment depth (Kobori and Taga 1979b; DeGobbis et al . 1984), while porewater SRP has often been shown to increase as a function of depth (Mortimer 1941; chapter 4).

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Soluble reactive P concentrations measured at -242 mV were similar to SRP concentrations measured in the 20-40 cm depth of cores collected in January 1990 from the same site in Lake Apopka (chapter 4) indicating that porewater SRP increased in response to reduced redox potential. In Lake Apopka P fluxes were determined to be mainly a function of decomposition at the sediment-water interface (Moore et al . 1991). Aerobic conditions may also result in SRP release. Sediments may be aerated through wind induced resuspension or by diffusion of 0 2 from the hypolimnion. Aerobic release of P from sediments into the overlying water is a function of the mineralization of organic P compounds (Lee et al. 1977). Greater than 90% of organic P extracted from an upland soil was shown to be in the form of phosphomonoesters (Condron et al 1985). Thus a large portion of organic P may potentially be made available through the action of phosphatase enzymes. Alkaline phosphatase activity has been significantly correlated with SRP release in marine sediments (Degobbis et al . 1985). In this study a significant inverse correlation was observed between porewater TOP and sediment APA (r=-0.95), hence high APA may result in the breakdown of porewater TOP, although this may also indicate the inhibition of APA by porewater TOP. Alkaline phosphatase activity was also highly inversely correlated with labile organic P. Porewater TOP, which is already in the soluble fraction of the sediment, may be more susceptible to enzyme hydrolysis than labile organic P and hence will be hydrolyzed first. The porewater TOP pool is subsequently replenished by the labile organic P pool of the sediment. The solubility of the substrate is the factor limiting organic P mineralization not the enzyme activity (Jackman and Black

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150 1952). It was determined that the rate of phytate hydrolysis in solution was over 71 times as important as the phytase activity; a specific acid phosphatase in the limitation of hydrolysis (Jackman and Black 1952). The high volatile solids content of the sediment indicated the high organic matter content. A significant portion of this organic matter was highly resistant to hydrolysis, as apparent by the 26% difference in TP determined by ignition and extraction. In this study, APA was inversely correlated with HA organic P (r=-0.74), a resistant form of P. This inverse relationship may be explained as the possible binding of alkaline phosphatase to the humic substances which may increase enzyme stability, thus resulting in some inhibition of APA (Burns 1986; Kandeler 1990; Wetzel 1991). Organic P compounds may also form complexes with humates (Stewart and Tiessen 1987; Brannon and Sommers 1985). The two main chemical extractants, HC1 and NaOH, were unable to extract organic P compounds incorporated into humic polymers (Brannon and Sommers 1985). This chemically resistant organic P was also resistant to enzymatic hydrolysis. The high negative correlation between labile inorganic P and SRP with FA-TP and NaOH-TP suggest that FA-TP, a component of NaOH-TP, may be a significant contributor to the labile inorganic P pool through enzymatic hydrolysis. It has been suggested that inositol phosphates bound to FA are hydrolyzable by phytase (Herbes et al . 1975).

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151 Conclusions The results from this study show that short-term DO depletion will not affect APA in the water column of Lake Apopka. However, extended periods of anoxia (8 to 24 h) will result in APA inhibition and decreased enzymatic breakdown of organic P. In the sediment, APA was high under aerobic conditions and decreased with a decrease in Eh, hence under anaerobic conditions the rate of organic P mineralization will be slower. Inverse correlations between APA and porewater TOP and labile organic P also suggest that these are susceptible to enzymatic hydrolysis or may be inhibited by high concentrations of these substrates. Based upon the resistant nature of HA-TP the inverse relationship between HA and APA was attributed to the binding of the enzyme in the formation of humic complexes which accumulate under anaerobic conditions.

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CHAPTER 6 ORGANIC PHOSPHORUS CYCLING IN LAKE APOPKA Organic P plays a dominant role in the P cycle of aquatic systems (Fig. 1-1). The bioavailability of organic P to plankton is regulated by the activity of enzymes, types of substrates and associated physicochemical factors in the system. The research presented in this dissertation examined the specific organic P compartments (plankton, water column and sediment) to evaluate the bioavailability of organic P for plankton growth. Results obtained in this study are summarized in the context of addressing key research issues raised in an attempt to understand the P dynamics in Lake Apopka. (1) How is the enzymatic hydrolysis of organic P affected by other water chemistry parameters? Both seasonal and spatial variability of total P (TP), total soluble P (TSP) and alkaline phosphatase activity (APA), were observed in the water column. Alkaline phosphatase activity, an indicator of potential organic P hydrolysis, was dependent upon different water chemistry parameters, both seasonally and spatially. The majority of APA was associated with particulate matter, and soluble APA averaged only 3% of total APA, therefore particulate interactions are a key component of organic P cycling in Lake Apopka. The attachment of enzymes to surfaces may increase enzyme longevity, but may also reduce enzyme activity, either directly through sorption at the active site or 152

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153 indirectly as a result of steric hindrance. Evidence of this was observed by the inverse relationship between APA and total organic C (TOC). Alkaline phosphatase activity was higher in the pelagic zone (sites 2-8), which was more productive and nutrient rich than the spring (site 1). The mean annual total APA in the water column was 18 nM min" 1 , under non-limiting substrate concentrations; this indicates a potential inorganic P release rate of 0.6 /xg P L" 1 min" 1 . Natural substrate concentrations are usually lower than those used in the APA assay, thus in situ release rates will be slower. (2) Is Lake Apopka plankton APA inhibited by inorganic P and is it produced in response to inorganic P limitation? Over 90% of APA within the natural plankton population was inhibited following the addition of 1000 ng L" 1 inorganic P to the growth medium. Addition of lower inorganic P concentrations (10 to 100 /ig L" 1 ) did not produce such complete inhibition, thus ambient soluble reactive P (SRP) concentrations (< 10 /ig L" 1 ) in the lake water will not inhibit APA. The uptake of added inorganic P was very rapid, indicating high P demand. The inorganic P was immediately incorporated into the plankton cells as surplus P, as determined by hot water extraction. In general, surplus P was inversely related to APA, suggesting that internal inorganic P levels controlled APA. Under field conditions this was reflected by an inverse relationship between acid hydrolyzable P and APA. In batch culture, both hot water extractable inorganic and organic P were used to provide P for growth.

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Increased APA was associated with increased plankton biomass; however, the intensity of APA was dependent upon the severity of P limitation. During severe inorganic P limitation Lake Apopka plankton produced specific APA (total APA/chlorophyll a) values > 1 nmol APA ng chlorophyll a" 1 min 1 . Ambient lake water specific APA was < 0.3 nmol APA /xg chlorophyll a" 1 min" 1 , suggesting that Lake Apopka plankton were not severely P limited. (3) What effect does sediment resuspension have upon organic P mineralization rates? Resuspension of surficial sediments resulted in immediate increases in total suspended solids (TSS), total Kjeldahl N (TKN) , TP and APA within the overlying water column. These elevated concentrations decreased rapidly upon particle settling, indicating they remained associated with the sediment particles. During resuspension, increased APA and TP concentrations within the overlying water column may result in increased organic P mineralization. Exposure of anaerobic sediments to the oxygenated water column during resuspension may also increase the rate of organic P mineralization within the sediment itself. Porewater organic P was inversely related to pE + pH, while APA was positively related to pE + pH, indicating reduced mineralization rates of organic P under anaerobic conditions. Alkaline phosphatase enzymes have been shown to bind to humic acid complexes, the inverse relationship between humic acid P (HA-TP) and APA indicates inhibition in response to binding. The overall conclusion is that mineralization rates may be increased as a result of sediment resuspension and aeration, and are reduced under anaerobic conditions.

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155 The results presented in this study demonstrate significant potential for organic P mineralization in Lake Apopka. Substrates hydrolyzable by APA are very labile, hence the rate of organic P mineralization in Lake Apopka is limited by substrate concentrations rather than enzyme activity. Further research should focus more on specific organic P compounds within the sediment-water column and their relative turnover rates. Studies should also include evaluating the change in enzyme kinetics in response to different organic P compounds. To encourage P limitation, emphasis should be placed upon a means to inhibit organic P mineralization.

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APPENDIX A LORAN COORDINATES Table A. Loran coordinates of sampling sites on Lake Apopka Group repetition interval :7980, Southeast USA. Time delay /is Site number Y Z 1 44526.6 62460.6 2 44524.2 62446.4 3 44553.9 62452.3 4 44577.2 62449.9 5 44539.7 62416.8 6 44497.1 62403.0 7 44488.8 62420.2 8 44530.0 62432.2 156

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APPENDIX B CONCENTRATIONS OF SELECTED WATER CHEMISTRY PARAMETERS DETERMINED BIMONTHLY FROM APRIL 1989 THROUGH FEBRUARY 1990, AT 8 SITES IN LAKE APOPKA Table B-l. Temperature. Date Site Mean' SE I 2 3 4 5 6 7 8~ APR 24.3 27.4 27.4 26.7 26.3 26.1 28.1 23.6 26 .5 0 21 JUN 24.2 32.5 30.4 28.5 30.0 29.8 28.1 29.8 29 .9 0. 20 AUG 24.0 28.7 28.0 27.8 29.5 29.8 29.2 28.5 28 8 0. 11 OCT 22.7 21.1 19.9 19.8 19.4 19.9 20.0 19.4 19. 9 0. 08 DEC 20.5 17.6 16.5 15.8 16.3 15.7 15.0 15.5 16. 1 0. 12 FEB 22.3 20.1 19.8 20.6 20.8 20.4 21.3 20.0 20. 0 0. 07 Mean 23.1 25.5 24.4 23.7 24.3 24.3 24.1 23.4 SE 0.3 1.0 1.0 0.9 1.0 1.0 1.0 1.0 Means listed in this column are means of sites 2-8. 157

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Table B-2. Secchi depth transparency. 158 Date Site Mean SE APR 1 .00 0.24 0. 20 0.25 0.23 0.24 0. 23 0.25 0. 23 0 00 JUN 1 .50 0.21 0. 21 0.24 0.24 0.21 0. 24 0.21 0. 22 0 00 AUG 1 .90 0.20 0. 23 0.30 0.25 0.25 0. 23 0.23 0. 24 0 00 OCT 1 .38 0.27 0. 28 0.28 0.27 0.35 0. 24 0.30 0. 28 0 00 DEC 1 .00 0.25 0. 25 0.39 0.31 0.32 0. 25 0.27 0. 29 0 01 FEB 0 .72 0.26 0. 24 0.30 0.25 0.22 0. 25 0.25 0. 25 0 00 Mean SE 1 0 .36 .06 0.23 0.00 0. 0. 23 01 0.29 0.01 0.26 0.01 0.27 0.01 0. 0. 24 00 0.25 0.01 Means listed in this column are means of sites 2-8.

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I 159 Table B-3. Dissolved oxygen. Date Site Mean" SE 1 2 3 4 5 6 7 8 mg L APR 4.5 12.7 13.0 11.2 12.3 12.8 13.0 10.7 12.2 0.13 JUN 2.4 7.3 10.5 3.7 5.2 8.0 6.3 9.8 7.3 0.35 AUG 2.5 8.7 9.3 5.8 10.2 11.0 10.8 9.6 9.3 0.25 OCT 4.2 9.7 8.6 8.9 9.1 9.6 10.6 10.2 9.5 0.10 DEC 5.3 9.5 9.0 9.9 9.1 9.9 10.1 9.3 9.5 0.06 FEB 5.5 7.7 10.2 10.0 9.6 10.8 12.1 9.0 9.9 0.20 Mean 3.8 9.6 10.1 7.9 9.2 10.3 10.2 9.9 SE 0.2 0.3 0.3 0.5 0.4 0.3 0.4 0.1 Means listed in this column are means of sites 2-8.

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160 Table B-4. pH. Date Site Mean* SE 1 2 3 4 5 6 7 8 APR 8.2 9.4 9.3 9.1 9.3 9.4 9.4 9.2 9.3* 0.0 JUN 8.1 8.8 9.0 8.1 8.8 9.0 8.5 9.1 8.6 0.1 AUG 8.1 8.9 9.1 7.8 8.9 9.2 9.3 8.9 8.5 0.1 OCT 8.2 8.7 8.9 8.7 9.0 9.2 9.1 9.0 8.9 0.0 DEC 8.2 9.0 8.8 9.1 8.9 8.9 9.0 9.0 8.9 0.0 FEB 8.1 8.5 8.7 8.7 8.2 8.5 8.8 8.5 8.5 0.0 Mean SE 8.2 0.0 8.8 0.0 8.9 0.0 8.3 0.1 8.7 0.0 8.9 0.0 8.9 0.1 8.9 0.0 Means listed in this column are means of sites 2-8. Means were calculated following the conversion of pH to hydrogen ion concentrations. Standard errors were calculated without pri conversion of pH values.

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161 Table B-5. Total solids. Date Site Mean" SE 1 2 3 4 5 6 7 8~ mg L APR ND* ND ND ND ND ND ND ND JUNE 148 349 373 337 411 411 300 404 369 6 AUG 156 309 317 328 334 335 323 350 328 2 OCT 202 387 396 385 402 395 402 411 397 1 DEC 229 369 369 389 370 373 393 396 380 2 FEB 271 531 499 524 511 459 496 508 504 3 Mean 201 389 391 393 406 395 383 414 SE 10 17 13 16 13 9 15 12 Jleans listed in this column are means of sites 2-8. * ND indicates not determined.

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162 Table B-6. Total suspended solids. Site Mean* SE l 2 3 4 5 6 7 8 APR ND* ND ND ND ND ND ND ND JUN ND ND ND ND ND ND ND ND AUG 6 59 66 42 49 69 72 73 62 2 OCT 5 54 67 52 71 60 64 75 63 1 DEC 15 61 69 48 67 60 58 65 61 1 FEB 17 77 85 109 99 89 92 103 94 2 Mean SE 11 2 63 2 72 2 63 8 72 5 70 3 72 4 79 4 Means listed in this column are means of sites 2-8. * ND indicates not determined.

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Table B-7. Chlorophyll a Site Mean" SE 1 2 3 4 5 6 7 T L APR 33 61 70 68 75 71 52 98 71 2 JUN 24 80 79 88 84 92 55 67 78 2 AUG 24 131 142 87 156 95 158 127 128 4 OCT 16 92 96 75 101 73 85 99 89 2 DEC 26 102 94 60 68 71 79 74 78 2 FEB 7 39 33 49 48 51 40 45 44 1 Mean SE 22 1 84 5 86 6 71 3 89 6 75 3 78 7 85 5 Means listed in this column are means of sites 2-8.

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164 Table B-8. Total organic carbon. Date Site Mean" SE ~1 2 3 4 5 6 7 8 mg L APR ND* ND ND ND ND ND ND ND JUN 0.3 30.6 29.9 29.8 31.6 31.5 24.3 32.2 30 .0 0 .38 AUG 2.4 32.5 34.0 40.5 32.4 31.7 33.1 34.0 34 .0 0 .43 OCT 1.2 28.5 29.8 30.5 29.0 32.9 28.3 29.2 29 .7 0 .22 DEC 9.1 30.1 28.6 29.4 31.1 32.9 27.8 31.9 30 .3 0 .26 FEB 10.6 35.2 36.9 33.7 44.5 32.6 34.0 39.2 36 .6 0 .59 Mean SE 4.7 0.95 31.4 0.51 31.8 0.69 32.8 0.93 33.7 1.23 32.3 0.13 29.5 0.81 33.3 0.75 Means listed in this column are means of sites 2-8. * ND indicates not determined.

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Table B-9. Total Kjeldahl nitrogen. 165 Date Site Mean SE mg L APR 2.57 5 .06 6.12 5.38 5 .71 5.03 5.18 8.11 5 .80 0 .16 JUN 0.18 4 .71 4.72 4.62 4 .78 5.33 3.58 4.48 4 .60 0 .07 AUG 1.30 5 28 6.45 4.86 5 .56 5.53 5.66 5.25 5 .51 0 .07 OCT 0.61 2 08 3.82 3.64 4 68 3.14 4.08 4.28 3 67 0 12 DEC 2.30 4. 19 4.72 3.81 4 13 4.36 4.59 4.45 4. 32 0. 04 FEB 1.87 4. 04 4.12 4.85 5. 30 4.58 5.06 4.00 4. 56 0. 08 Mean SE 1.47 0.16 4. 0. 23 19 4.99 0.18 4.53 0.11 5. 0. 03 10 4.66 0.14 4.69 0.13 5.09 0.26 Means listed in this column are means of sites 2-8.

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166 Table B-10. Total and soluble alkaline phosphatase activity. Date Site Mean SE 8 Total APR 5.9 12.2 12.8 22.0 JUN 3.8 11.8 11.2 44.7 AUG 3.7 10.2 10.6 10.3 OCT 5.7 27.0 28.2 18.2 DEC 7.7 23.8 24.6 19.8 FEB 5.6 9.6 10.3 11.6 Mean 5.4 15.8 16.3 21.1 SE 0.25 1.26 1.33 2.07 nM min" 1 18.7 20.1 20.7 22.8 18 .5 0. 61 29.9 30.6 20.8 19.2 24 .0 1. 70 6.4 17.8 20.8 11.6 12 .5 0. 71 19.6 21.9 25.4 24.9 23 .6 0. 54 18.8 17.9 20.3 17.4 20 .4 0. 40 9.5 10.6 10.9 8.9 10 .2 0. 13 17.2 19.8 19.8 17.5 1.39 1.08 0.80 1.04 Soluble APR 0.16 0 .51 0. 49 0. 34 JUN 0.07 0 .03 0. 05 0. 93 AUG 0.31 0 .10 0. 54 0. 60 OCT 0.74 1 .29 1. 19 0. 64 DEC 0.65 0 .38 0. 62 0. 45 FEB 0.39 0 .56 0. 33 0. 43 Mean 0.39 0 .48 0. 53 0. 57 SE 0.04 0 .08 0. 06 0. 04 0.29 0 .67 0.37 0.30 0. 42 0 .02 0.01 0 .07 0.10 0.20 0. 20 0 .05 0.35 0 .16 0.55 0.64 0. 42 0 .03 0.48 0 .80 0.29 0.27 0. 71 0 .06 1.02 0 .68 0.58 1.34 0. 72 0 .05 0.11 0 .12 0.03 0.09 0. 24 0 .03 0.38 0 .42 0.32 0.47 0.06 0 .06 0.04 0.08 Means listed in this column are means of sites 2-8.

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167 Table B-ll. Phosphorus. Date Mean SE Hg L IE APR 110 140 170 180 200 200 180 260 190 5 JUN 30 150 140 190 150 170 260 140 171 6 A 1 IP HUu ] An 90 240 240 130 120 230 170 9 OCT 300 520 440 350 200 400 270 400 369 15 DLL 20 100 120 110 130 120 150 150 126 3 FEB 70 190 200 250 280 230 240 240 233 4 Mean 93 207 193 220 200 208 203 237 SE 18 26 21 13 9 17 10 16 ISP APR 130 110 70 70 90 80 50 40 73 3 JUN 80 40 70 70 80 60 30 40 56 3 AUG 10 10 10 10 10 10 10 10 10 0 OCT 120 170 70 130 60 140 160 60 113 7 DEC 10 10 10 20 20 10 10 20 14 1 FEB 30 40 40 40 40 20 20 20 31 2 Mean 63 63 45 57 50 53 47 32 SE 9 11 5 7 5 9 10 3 SRP APR 1 3 3 5 3 5 2 3 0 JUN 2 3 4 4 5 3 3 5 0 AUG 4 7 5 4 5 4 4 5 0 OCT 1 1 1 1 1 1 1 1 0 DEC 1 1 1 1 1 1 1 1 0 FEB 0 3 0 1 1 1 2 2 0 Mean 2 3 2 3 3 3 2 3 SE 0 0 0 0 0 0 0 0 Means listed in this column are means of sites 2-8.

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176 Reichardt, W. 1971. Catalytic mobilization of phosphate in lake water and by Cyanophyta. Hydrobiologia 38:377-394. Reid, T. W., and I. B. Wilson. 1971. E. coli alkaline phosphatase, p. 373-415. In P.D. Boyer [ed.], The enzymes. Vol. IV. Academic Press, New York. Rhee, G-Y. 1972. Competition between an alga and an aquatic bacterium for phosphate. Limnol. Oceanogr. 17:505-514. Rhee, G-Y. 1973. A continuous culture study of phosphate uptake, growth rate and polyphosphate in Scenedesmus sp. J. Phycol . 9:495506. Rhee, G-Y. 1974. Phosphate uptake under nitrate limitation by Scenedesmus sp. and its ecological implications. J. Phycol. 10:470-475. Rigler, F. H. 1956. A tracer study of the phosphate cycle in lake water. Ecol . 37:550-562. Rigler, F. H. 1964. The phosphorus fractions and turnover time of inorganic phosphorus in different types of lakes. Limnol. Oceanogr. 6:165-174. Rojo, M. J., S. G. Carcedo, and M. P. Mateos. 1990. Distribution and characterization of phosphatase and organic phosphorus in soil fractions. Soil Biol. Biochem. 22:169-174. Rosenberg, H. , R. G. Gerdes, and K. Chegwidden. 1977. Two systems for the uptake of phosphate in Escherichia coli. J. Bacterid. 131:505511 . Ryding, S-0., and C. Forsberg. 1977. Sediments as a nutrient source in a shallow polluted lake, p. 227-234. In H. L. Golterman [ed.], Interactions between sediments and freshwater. Dr. W. Junk Publishers, The Hague. SAS Institute Inc. 1985. SAS user's guide: statistics, SAS Institute Inc., Cary, N.C. Sayler, G S., M. Puziss and M. Silver. 1979. Alkaline phosphatase assay for freshwater sediments: Application to perturbed sediment systems. Appl . Environ. Microbiol. 38:922-927. Schindler, D. W. 1977. Evolution of phosphorus limitation in lakes. Science. 195:260-262. indler, D. W.,R. Hesslein, and G. Kipphur. 1977. Interactions between sediments and overlying waters in an experimentally eutrophied Precambian Shield Lake, p. 235-243. In H. L. Golterman

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177 [ed.], Interactions between sediments and freshwater. Dr. W. Junk Publishers, The Hague. Shannon, E. E., and P. L. Brezonik. 1972. Limnological characteristics of north and central Florida lakes. Limnol. Oceanogr. 17:97-110. Shapiro, J. 1960. The cause of metal imnetic minimum of dissolved oxygen. Limnol. Oceanogr. 5:216-227. Siuda, W., R. J. Chrost, R. Wcislo, and M. Krupka. 1982. Factors affecting alkaline phosphatase activity in a lake (short term experiments). Acta Hydrobiol. 24:3-20. Skujins, J. 1976. Extracellular enzymes in soil. CRC Crit. Rev. Microbiol. 4:383-421. Smith, R. E. H., and J. Kalff. 1981. The effect of phosphorus limitation on algal growth rates: evidence from alkaline phosphatase. Can. J. Fish. Aquat. Sci. 38:1421-1427. Sommers, L. E., R. F. Harris, J. D. H. Williams, 0. E. Armstrong, and J. K.Syers. 1972. Fractionation of organic phosphorus in lake sediments. Soil Sci. Soc. Amer. Proc. 36:51-54. Speir, T. W. 1976. Studies on a cl imosequence of soils in tussock grasslands. 8. Urease, phosphatase and sulphatase activities of tussock plant materials and of soil. N. Zealand J. Sci. 19:383-387. Speir, T. W., and D. J. Ross. 1978. Soil phosphatases and sulphatases, p. 197-249. In R.G. Burns [ed.], Soil Enzymes. Academic Press, New York. Sproule, J. L. , and J. Kalff. 1978. Seasonal cycles in the phytoplankton phosphorus status of a north temperate zone lake (Lake Memphremagog, Que-Vt), plus a comparison of techniques. Verh. Int. Ver. Limnol. 20:2681-2688. Stevens, R. J., and M. P. Parr. 1977. The significance of alkaline phosphatase activity in Lough Neagh. Fresh. Biol. 7:351-355. Stevenson, F. J. 1982. Humus chemistry genesis, composition, reactions. Wiley-Interscience, New York. Stewart, A. J and R. G. Wetzel. 1982. Phytoplankton contribution to alkaline phosphatase activity. Arch. Hydrobiol. 93:265-271. Stewart, J. W, B., and H. Tiessen. 1987. Dynamics of soil organic phosphorus. Biogeochemistry 4:41-60. Stewart, W. D. P., and H. W. Pearson. 1970. Effects of aerobic and anaerobic conditions on growth and metabolism of blue-green algae. Proc. Roy. Soc. Lond. B. 175:293-311.

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178 Stratton, F. E. 1968. Ammonia nitrogen losses from streams. J. Sanitary Eng. Div. Proc. Am. Soc. Civ. Eng. 94:1085-1092. Stratton, F. E. 1969. Nitrogen losses from alkaline water impoundments. J. Sanitary Eng. Div. Proc. Am. Soc. Civ. Eng. 95:223-231. Strickland, J. D. H., and T. R. Parsons. 1968. A practical handbook of seawater analysis. Bull. 167 Fish. Res. Bd. Can. Stumm, W., and J. 0. Leckie. 1970. Phosphate exchange with sediments; its role in the productivity of surface waters. Fifth Int. Water Polln. Res. Conf. 111:2611-2616. Tabatabai, M. A., and J. M. Bremner. 1969. Use of p-nitrophenyl phosphate for assay of soil phosphatase activity. Soil Biol. Biochem. 1:301-307. Taft, J. L., M. E. Loftus, and W. R. Taylor. 1977. Phosphate uptake from phosphomonoesters by phytoplankton in the Chesapeake Bay. Limnol. Oceanogr. 22:1012-1021. Tarapchak, S. J., S. M. Bigelow, and C. Rubitschum. 1982. Soluble reactive phosphorus measurements in Lake Michigan: filtration artifacts. J. Great Lakes Res. 8:550-557. Tessenow, U. 1972. Losungs-, Diffusionsund Sorptionsprozesse in der Oberschicht von Seesedimenten. 1. Ein Langzeitexperiment unter aseroben und anaeroben Bedingungen in Fleibgleichgewicht. Arch. Hydrobiol. Suppl . 38:353-398. Torriani, A. 1960. Influence of inorganic phosphate in the formation of phosphatases by Escherichia coli. Biochim. Biophys. Acta 38:460U. S. Environmental Protection Agency (USEPA). 1979. Environmental impact statement. Lake Apopka restoration project. Lake and Orange Counties, Florida. (EPA 904/0-8-79-043). U. S. Environmental Protection Agency, EMSL, Cincinnati, OH. Vincent, W. F., W. Wurstbaugh, C. L. Vincent and P. J. Richerson. 1984. Seasonal dynamics of nutrient limitation in a tropical high-altitude lake (Lake Titicaca, Peru-Bolivia): Application of physiological bioassays. Limnol. Oceanogr. 29:540-552. Walker, T. W., and A. F. R. Adams 1958. Organic phosphorus, p. 411413. In A. L. Page, R. H. Miller, and D. R. Keeney [eds.], Methods analvsis > Par t 2: Chemical and microbiological properties. 1982 ASA, SSSA. Madison, WI.

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179 Weimer, W. C, and D. E. Armstrong. 1979. Naturally occurring organic phosphorus compounds in aquatic plants. Environ. Sci. Technol . 13:826-829. Wetzel, R. G. 1983. Limnology. 2nd. Edn. Saunders College, Philadelphia. Wetzel, R. G. 1991. Extracellular enzymatic interactions: storage, redistribution, and interspecific communication, p. 6-28. In R. J. Chrdst [ed.], Microbial enzymes in aquatic environments. Science Tech. Publ . Wolanski, E., T. Asaeda, and J. Imberger. 1989. Mixing across a lutocline. Limnol. Oceanogr. 34:931-938. Wynne, D. 1977. Alterations in activity of phosphatases during the Peridinium bloom in Lake Kinneret. Physiol. Plant. 40:219-224. Wynne, D. 1981. The role of phosphatases in the metabolism of Peridinium cinctum, from Lake Kinneret. Hydrobiol. 83:93-99. Wynne, D., and T. Berman. 1980. Hot water extractable phosphorusan indicator of nutritional status of Peridinium cinctum (Dinophyceae) from Lake Kinneret (Israel)?. J. Phycol . 16:40-46. Wynne, D. , and M. Gophen. 1981. Phosphatase activity in freshwater zooplankton. 0IK0S 37:369-376. Young, T. C, J. V. DePinto, S. C. Martin, and J. S. Bonner. 1985. Algal available particulate phosphorus in the Great Lakes basin. J. Great Lakes Res. 11:434-446.

PAGE 192

BIOGRAPHICAL SKETCH Susan Newman was born 26 June 1963 in Portsmouth, England. She graduated with a BSc. Honors degree in management and chemical sciences, from the University of Manchester Institute of Science and Technology, in June 1984. Following a 6 month appointment at the Weed Research Organization (the now defunct WRO), Susan determined it was time to leave the cloudy skies of England. With emotions split between excitement and trepidation, Susan began a Master of Science degree in the Agronomy Department at the University of Florida in January 1985. With an interest in wetland soils dating back to the making of mud pies during her childhood, and unable to resist the opportunities of the Sunshine State, she began a Ph.D. program in the Soil Science Department at the University of Florida in 1987. The first course she completed within this department was openwater scuba diving, however, following that the more traditional courses were taken. Upon completion of her Ph.D. Susan will continue her aquatic/wetland science interests by obtaining a research position to investigate the nutrient dynamics in wetlands. 180

PAGE 193

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Konda R. Reddy, Chair Professor of Soil Science I certify that I conforms to acceptable adequate, in scope and Doctor of Philosophy. I certify that I conforms to acceptable adequate, in scope and Doctor of Philosophy. have read this study and that in my opinion it standards of scholarly presentation and is fully quality, as a dissertation for the degree of Joseph J. Delfino Professor of Environmental Engineering Sciences have read this study and that in my opinion it standards of scholarly presentation and is fully quality, as a dissertation for the degree of fifirJ&A MAS-, Donald A. Graetz Professor of Soil Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. David H. Hubbell Professor of Soil Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Edward J! Assistant ^Profes^or of Forest Resources and Conservation

PAGE 194

This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. r\ May 1991 J^vy Dean /£ol lege of
PAGE 195

This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. r\ May 1991 J^vy Dean /£ol lege of

47
1976), nor Selenastrum capricornutum Prinz (Klotz 1985b) exhibited diel
responses. However, Smith and Kalff (1981) have shown that growth
demands for P are more important than the species composition in
determining APA.
Preliminary studies (data not shown) and April data demonstrated a
strong relationship between APA and TP. Consequently TP, which would
include cellular P, would indicate the P forms utilized by APA.
However, this relationship was not observed the entire year. Total
soluble P, has been suggested as a good indicator of bioavailable P in
eutrophic lakes (Bradford and Peters 1987). Correlations between total
and soluble APA and TSP were apparent in December, and at certain sites
(Table 2-3). Both positive and negative relationships were observed.
Total and soluble APA are apparently influenced by different parameters
at different sites (Table 2-3, Huber et al. 1985).
In October, the P fractionation experiment demonstrated that APA
was inversely related to the acid hydrolyzable P fractions. This
suggests that APA may be regulated by acid hydrolyzable compounds, or
alternatively they could be used as substrates for APA. Acid
hydrolyzable P represents the condensed polyphosphates, a storage form
of P in phytoplankton and bacteria. Surplus P concentrations within
algal cells have been shown to regulate the production of APA
(Fitzgerald and Nelson 1966; Lien and Knutsen 1973; Rhee 1973) and this
pool is composed of polyphosphates (Rhee 1972, 1973; Elgavish and
Elgavish 1980). Hence, the measurement of surplus P combined with APA
could provide a better understanding of the system. Another component
which may contribute to the understanding of APA is the concentration of


15
various factors which affect them. Chapter 3 examines the P nutrient
status of natural plankton populations. Chapter 4 investigates the
effect of sediment-water column interactions upon organic P
bioavailability. The effect of anaerobic/aerobic conditions on organic
P bioavailability is examined in chapter 5. The overall conclusions and
the significance of these results are discussed in chapter 6.


Mi
3-8. Specific hot water extractable phosphorus measured over time
in natural plankton populations collected in August 1990
after receiving nitrogen and phosphorus additions
(mean 1 SE) 81
3-9. Specific hot water extractable phosphorus measured over time
in natural plankton populations collected in August 1990
after receiving nitrogen and phosphorus additions
(mean 1 SE) 83
3-10. Specific alkaline phosphatase activity measured over time in
natural plankton populations collected in August 1990
after receiving nitrogen and phosphorus additions
(mean 1 SE) 87
4-1. Distribution of selected parameters measured in May 1989
within the water column at the center of Lake Apopka
(n=3) 106
4-2. Concentrations of parameters measured within the water
column of cores collected in September 1989 from the
center of Lake Apopka (mean 1 SE) 109
4-3. The distribution of alkaline phosphatase activity in
disturbed and undisturbed sediment cores collected from the
center of Lake Apopka in January 1990 (mean + 1 SE) 118
5-1. Concentrations of selected parameters measured in Lake
Apopka water in July 1990 (mean 1 SE) 135
5-2. Concentrations of selected parameters measured on sediments
incubated under six different redox levels for one
month (mean 1 SE) 140
5-3. Concentrations of selected parameters measured on sediments
incubated under six different redox levels for one
month (mean 1 SE) 143
5-4. Selected correlation coefficients between phosphorus
compounds and alkaline phosphatase activity measured in
sediments incubated for one month under six different
redox levels 144
APPENDIX TABLES
A. Loran coordinates of sampling sites on Lake Apopka
Group repetition interval:7980, Southeast USA 156
vi


133
15 min. The liquid was removed and absorbance at 410 nm (Shimadzu Model
UV-160) measured. Concentrations were determined by calibration with a
standard curve of p-nitrophenol (Sigma Chemicals).
Porewater was extracted by centrifuging a subsample of sediment at
6000 rpm (5213 g) for 15 min. The porewater was immediately filtered
through 0.45 pm Gelman membrane filters. Total P, TSP and SRP were
measured using standard methods (APHA 1985). Porewater total organic P
(TOP) was defined as the difference between porewater TP and SRP.
Bicarbonate extractable P was determined after porewater extraction by
shaking the sediment with 0.5 M NaHC03 (1:50, on a dry weight basis) for
30 min. The extracted medium was then centrifuged at 6000 rpm (5213 g)
for 15 min and the supernatant filtered through Whatman 40 filter paper.
Total P and SRP were determined on the filtrate (APHA 1985). Labile
inorganic P was calculated as the sum of porewater SRP and HC03
extractable SRP. Labile organic P was defined as the difference between
total labile P and labile inorganic P.
Water content of the sediment was determined by drying a known
weight of wet sediment at 70*C to a constant weight. The dried sediment
was ground to a powder using a Spex 8000 grinding mill. Total P content
of the dried sediment was determined via ignition (Walker and Adams
1958). Volatile solids were reported as the loss in weight due to
ignition of dried sediment at 500*C for 2 h.
Statistical Analysis
Data were analyzed using the Statistical Analysis Systems (SAS
1985), Version 6.03 for personal computers. Data in the first


13
in sub-tropical lakes, consequently, research investigating the
breakdown and utilization of organic P is essential in any assessment of
lake eutrophication.
The main components influencing the cycling of organic P within
the water column are; water chemistry, plankton uptake and release, and
sediment resuspension (Fig.1-1). To understand the role of organic P in
Lake Apopka the following questions were addressed.
(1) How is the enzymatic hydrolysis of organic P compounds
affected by other water chemistry parameters?
(2) Is Lake Apopka plankton APA inhibited by inorganic P and is
it produced in response to inorganic P limitation?
(3) What effect does sediment resuspension have upon organic P
mineralization rates?
The overall objective of this study was to evaluate the
significance of organic P compounds in Lake Apopka and determine their
potential bioavailability. Specific objectives are listed below.
(1) Determine the seasonal, spatial and diel variability of APA
within the water column.
Alkaline phosphatase activity is produced by organisms, hence any
factors such as changes in environmental conditions which affect
metabolism may therefore affect APA. The predominant biotic group in
Lake Apopka biota are the plankton, hence APA is linked to fluctuations
in response to plankton metabolism.
(2) Determine the influence of inorganic P upon the growth of
natural plankton populations.


127
Materials and Methods
Site Description
Lake Apopka is a 12,500 ha hypereutrophic lake, located in central
Florida, 28*37' N latitude, 81*37' W longitude. It has a mean depth of
2 m. Chlorophyll a values exceeding 100 /zg L'1 are frequently recorded
(chapter 2). Nutrient loading from the surrounding agricultural and
urban areas has resulted in the current hypereutrophic conditions in the
lake (USEPA 1979). The bottom sediments in the lake have an average of
30 cm unconsolidated flocculent layer at the surface, underlain by
consolidated flocculent material (Reddy and Graetz 1990).
Sampling Procedures
Water column. Water was collected on July 2 1990, 30 cm below the
water surface from the center of the lake in 15 and 30 L polycarbonate
containers. The water used for the first experiment was kept for less
than 24 h in the dark under ambient laboratory conditions prior to the
start of the experiment. The water used to dilute the sediment was
first coarse filtered and stored under ambient laboratory conditions in
polycarbonate containers until the start of the experiment.
Sediment. Grab samples of the surface 30 cm of sediment were
collected on 28 June 1990 from the center of the lake using an Eckman
dredge, and placed in polycarbonate containers, which were filled
completely to minimize air spaces. The samples were stored under
ambient laboratory conditions prior to use in the study.


Fig. 4-1. Map showing the location of Lake Apopka.
KO
00


149
Soluble reactive P concentrations measured at -242 mV were similar to
SRP concentrations measured in the 20-40 cm depth of cores collected in
January 1990 from the same site in Lake Apopka (chapter 4) indicating
that porewater SRP increased in response to reduced redox potential. In
Lake Apopka P fluxes were determined to be mainly a function of
decomposition at the sediment-water interface (Moore et al. 1991).
Aerobic conditions may also result in SRP release. Sediments may
be aerated through wind induced resuspension or by diffusion of 02 from
the hypolimnion. Aerobic release of P from sediments into the overlying
water is a function of the mineralization of organic P compounds (Lee et
al. 1977). Greater than 90% of organic P extracted from an upland soil
was shown to be in the form of phosphomonoesters (Condron et al 1985).
Thus a large portion of organic P may potentially be made available
through the action of phosphatase enzymes. Alkaline phosphatase
activity has been significantly correlated with SRP release in marine
sediments (Degobbis et al. 1985). In this study a significant inverse
correlation was observed between porewater TOP and sediment APA
(r=-0.95), hence high APA may result in the breakdown of porewater TOP,
although this may also indicate the inhibition of APA by porewater TOP.
Alkaline phosphatase activity was also highly inversely correlated with
labile organic P. Porewater TOP, which is already in the soluble
fraction of the sediment, may be more susceptible to enzyme hydrolysis
than labile organic P and hence will be hydrolyzed first. The porewater
TOP pool is subsequently replenished by the labile organic P pool of the
sediment. The solubility of the substrate is the factor limiting
organic P mineralization not the enzyme activity (Jackman and Black


118
Table 4-3. The distribution of alkaline phosphatase activity in
disturbed and undisturbed sediment cores collected from
the center of Lake Apopka in January 1990 (means 1 SE).
Alkaline phosphatase activity
Total Porewater
/xmol g dry wt. '1 h'1 /xmol L'1 h'1
Control
2 cm
14.0'
0.91
5 cm
15.3
0.22
10 cm
10.5
1.07
ResusDended
2 cm
17.8 0.2
0.45
0.14
5 cm
14.0 1.5
0.34
0.03
10 cm
9.5 0.5
1.27
0.38
Control sediment cores were not replicated.


145
to labile organic P (Table 5-4). A strong inverse relationship between
APA and porewater TOP was observed (r=-0.95).
Other significant inverse correlations include porewater TP and
SRP with FA-P and NaOH-TP. Labile inorganic P was also highly inversely
correlated to these P forms. No relationship was observed between
HC1-TP and any other parameters measured.
Discussion
The Effect of Dissolved Oxygen on Organic Phosphorus Mineralization in
the Water Column
Phosphorus concentrations in lake water incubated in the dark for
24 h, did not show significant changes under either anaerobic
(<0.2 mg L'1) or aerobic (6 mg L'1) conditions. However, decreases in
TSN concentrations suggested that mineralization of organic N occurred,
but no increase in NH4-N was observed. This may be due to rapid
nitrification under aerobic conditions (Reddy and Graetz 1981) or loss
through volatilization (Stratton 1968, 1969). Initial TSN:TSP exceeded
200:1 and would suggest that the system may be P limited.
Under conditions of inorganic P limitation, phytoplankton may
produce APA which hydrolyzes organic P compounds with the release of
inorganic P (Kuenzler 1965). Specific APA (APA/chlorophyll a) ratios
were determined to be an effective means of determining inorganic P
limitation of Lake Apopka plankton (chapter 3). Under SRP limiting
conditions, APA and specific APA increase (Kuenzler and Perras 1965;
Heath and Cooke 1975; chapter 3). Initial and final specific APA ratios
under aerobic conditions were 0.1 and 0.2 nmol APA ng chlorophyll a1


9
observed to be 25 times greater under P limitation than under P
sufficiency (Fitzgerald and Nelson 1966).
Ecologically, the importance of APA is dependent on the co
occurrence of both substrates and enzymes. Numerous problems have been
associated with the determination of PME concentrations. The most
common method of determining PME has been to monitor SRP release from
filtered lake water following the addition of pure alkaline phosphatase
(Strickland and Parsons 1968). The simultaneous occurrence of PME and
APA by cyanobacteria blooms has been observed under low SRP
concentrations (Heath and Cooke 1975). In some lakes, the rate of
inorganic P release from PME equals the rate of P uptake by the
plankton. In other lakes a large discrepancy exists between these two
rates, with inorganic P release being considerably less than uptake rate
(Boavida and Heath 1988; Cotner and Heath 1988; Heath 1986), thus
leading these researchers to conclude that APA is not important in P
nutrition of plankton. One of the problems associated with this
conclusion is the use of filtered lake water in the analyses, therefore
the large particulate organic P pool is absent (Wetzel 1983).
Phosphatases have also been shown to release P from particulate matter
(Jansson 1977). Seventy-four percent of extractable P in phytoplankton
is susceptible to enzymatic hydrolysis and 80% of the organisms involved
in phytoplankton decomposition produce phosphatases (Halemejko and
Chrst 1984). These results, and the apparent absence of hydrolyzable
soluble PME in the water column (Herbes 1974; Herbes et al. 1975;
Pettersson 1980) suggest that it is the substrate availability that
limits enzymatic P cycling not APA (Jansson et al. 1988).


TOTAL APA (nM min
85
Fig. 3-13. Time courses of alkaline phosphatase activity following
nutrient enrichment of natural plankton populations
collected in August 1990. 0N,0P=no nutrient addition;
400N,0P=400 /xg N L'1; 0N,40P=40 nq P L1; 400N,40P=400 /xg
N L'1 and 40 nq P L'\ and 800N,80P=800 /xg N L'1 and 80 /xg
P L'1: a) plankton grown at 29C; b) plankton grown at 19*C.
Vertical bars indicate 1 SE. No vertical bar indicates SE
is smaller than symbol size.


77
observed for NH4-N, however, the data were highly variable, making
meaningful interpretation difficult (Fig. 3-9c). Plankton grown at 19*C
exhibited the same responses for TSP, [N03 + N02]-N, and NH4-N (data not
shown).
Surplus phosphorus. Soluble P extracted from plankton following
boiling with deionized water (HEP-SRP) was used as an indicator of
surplus P. In April HEP-SRP was determined at both the start and
conclusion of the experiment. Hot water extractable P decreased in
cultures which did not receive P additions but remained the same in
cultures which received 40 /xg L'1 (Table 3-7). Those cultures which
received 80 ng P L'1 had almost a 3-fold increase in HEP-SRP after
216 h. The role of HEP was examined in more detail in water samples
collected in August. Seventy percent of initial TP was accounted for by
HEP-TSP and TSP (Table 3-5), with HEP-TSP representing 59% of TP.
Within 2 h of P addition, substantial increases in HEP-TSP were observed
(Fig. 3-10a). Hot water extractable-TSP tripled from 33.3 to
99.3 /xg P L'1 upon addition of 80 /xg P L'1. Addition of 40 /xg P L1
resulted in a doubling of HEP-TSP to 65 and 69 /xg P L1 in cultures
which received treatments 3 (N=0 P=40) and 4 (N=400 P=40), respectively.
Even with a 10*C temperature difference HEP-TSP accumulated to 63.9 /xg P
L'1 within 2 h (Fig. 3-10b). Cultures grown at 19C which did not
receive nutrients had greater HEP-TSP concentrations after 2 h than
those with no nutrient addition grown at 29#C. At 29*C the HEP-TSP
remained constant within 24 h and then decreased by 15 ng P L'1 for P
added treatments. The downward trend continued to 96 h, HEP-TSP


57
from the same site, diluted and placed in water baths as described
above. The temperature of the bath was set at 29*C to emulate ambient
lake water temperature. Another water bath with cultures receiving no
nutrient addition and P only was maintained at 19*C, to determine the
effect of temperature on the measured parameters. Nutrient uptake rates
were determined by measuring the disappearance of the nutrient from
solution. During the first 2 h following nutrient addition, the
cultures were sampled every 1/2 h and analyzed for SRP, NH4-N, and
[N03 + N02]-N (actual sampling times were 0, 30, 67, 98 and 140 min).
Immediately following filtration aliquots were analyzed for SRP.
Samples to be analyzed for NH4-N and [N03 + N02]-N samples were
acidified with concentrated H2S04 and stored at 4*C prior to analysis.
Samples taken at 0 and 2 h were also analyzed for HEP-SRP, and hot water
extractable TSP (HEP-TSP). Aliquots were subsequently removed at 24, 48
and 96 h and analyzed for SRP, NH4-N, [N03 + N02]-N, total APA, TSP,
HEP-SRP, HEP-TSP and chlorophyll a.
Analytical Methods
Alkaline phosphatase activity was determined fluorometrically
(Healey and Hendzel 1979a). One half mL of substrate,
3-o-methylfluorescein phosphate (Sigma Chemicals), at a concentration
determined to be non-limiting, in 0.1 M Tris buffer (Tham, Fisher
Scientific) pH 8.6, was added to 4.0 mL of lake water in a cuvette. Both
total (whole lake water) and soluble (filtered through 0.45 nm Gelman
membrane filter) APA were determined. The cuvettes were placed in a
water bath (25*C). At timed intervals during a 20 min period the


55
filtration were immediately filtered through 25 mm membrane filters
(0.45 nm) in polypropylene holders which attached to the syringes
(Gelman). The samples were analyzed for chlorophyll a, total and
soluble APA, total (TP), total Kjeldahl N (TKN), SRP, NH4-N, and [N03 +
N02]-N.
Nutrient enrichment of natural plankton populations
Experiment 1. Whole lake water was diluted 1:1 with filtered
lake water (0.45 /xm) and 400 mL were placed in each of 15 wide mouth
500 mL erlenmeyer flasks. The basic experimental design was a 22
factorial with an additional 2 fold addition of both N and P included
(Table 3-2). The flasks were stoppered with sponge plugs and placed in
a clear glass circulating water bath maintained at ambient lake
temperature (27'C). The flasks were illuminated from below at an
irradiance of 145 xmol photons m'2 s'1. The contents of the flasks were
mixed daily and immediately prior to sampling. Two h after the nutrient
addition, aliquots were withdrawn by syringe at predetermined intervals
and analyzed for total APA, SRP, NH4-N, [N03 + N02]-N, TSP and
chlorophyll a. Samples requiring filtration were filtered immediately
as described above. Hot water extractable P (HEP-SRP) was determined at
the beginning and conclusion of the experiment. To obtain a sufficient
sample size, HEP-SRP was determined on composite samples containing all
treatment replicates.
Experiment 2. To both confirm and compare the results with a
different plankton population, a second experiment similar to that
described above was conducted in August. Water samples were collected


154
Increased APA was associated with increased plankton biomass;
however, the intensity of APA was dependent upon the severity of P
limitation. During severe inorganic P limitation Lake Apopka plankton
produced specific APA (total APA/chlorophyll a) values > 1 nmol APA
ng chlorophyll a'1 min'1. Ambient lake water specific APA was
< 0.3 nmol APA /xg chlorophyll a'1 min'1, suggesting that Lake Apopka
plankton were not severely P limited.
(3) What effect does sediment resuspension have upon organic P
mineralization rates?
Resuspension of surficial sediments resulted in immediate
increases in total suspended solids (TSS), total Kjeldahl N (TKN), TP
and APA within the overlying water column. These elevated
concentrations decreased rapidly upon particle settling, indicating they
remained associated with the sediment particles. During resuspension,
increased APA and TP concentrations within the overlying water column
may result in increased organic P mineralization. Exposure of anaerobic
sediments to the oxygenated water column during resuspension may also
increase the rate of organic P mineralization within the sediment
itself. Porewater organic P was inversely related to pE + pH, while APA
was positively related to pE + pH, indicating reduced mineralization
rates of organic P under anaerobic conditions. Alkaline phosphatase
enzymes have been shown to bind to humic acid complexes, the inverse
relationship between humic acid P (HA-TP) and APA indicates inhibition
in response to binding. The overall conclusion is that mineralization
rates may be increased as a result of sediment resuspension and
aeration, and are reduced under anaerobic conditions.


2
algal bloom was recorded (USEPA 1979). Since then, sport fish
populations have dwindled and have been replaced by rough fish such as
shad, gar and catfish. The high algal populations have resulted in the
maintenance of a pea-green color in the lake. Lake Apopka has mean
chlorophyll a concentrations > 60 /g L'1 and total P concentrations of
200 ng L1 (Canfield 1981; Huber et al. 1982; Reddy and Graetz 1990) and
is thus currently classified as hypereutrophic (Forsberg and Ryding
1980). The lake is the first and largest in the Oklawaha chain of
lakes, consequently a ripple effect is apparent. The high nutrient
concentrations and algal blooms observed in Lake Apopka are evidenced
downstream in the other lakes in the chain.
Need for Research
In order to understand the process of eutrophication in Lake
Apopka and thus abate this process, it is necessary to determine the
cycling of C, N and P in the sediment-water column. In general, N and P
are the key elements involved in eutrophication (Chiaudani and Vighi
1982). Carbon fixation has been determined to be the driving force in
the productivity of Lake Apopka (Reddy and Graetz 1990). This is
apparent by the vast algal populations observed year round in the water
column. Settling of senescent algal cells has resulted in a highly
organic sediment. Consequently, both the sediment and the water column
are dominated by organic matter which results in high levels of organic
N and P. However, readily available P, i.e, soluble inorganic P
concentrations are low and frequently undetectable (< 1 /g L'1)
(Newman,S., unpublished data, Department of Soil Science, University of


89
and initial N limitation. Alternatively, the difference in response
may be due to different algal populations which have phosphatases with
different affinities (Pettersson 1980). In April, the algal population
(on a biovolume basis) was dominated by Microcystis sp. and pennate
diatoms. In August, the population was dominated by Microcystis sp. and
Lyngbya contorta (Aldridge, F. J., personal communication, Department of
Fisheries and Aquaculture, University of Florida, Gainesville, FL.).
Smith and Kalff (1981) showed that APA is influenced more by the
equilibration of plankton growth with nutrient supply, rather than the
species composition of the plankton population.
The P demand by plankton populations in Lake Apopka was very high
as shown by the rapid uptake of SRP in August. These rates are higher
than those obtained by similar methods from other lakes, i.e.,
0.02 to 0.03 ng P L1 min'1 (Rigler 1956) and 0.017 to 0.43 ng P L'1 min1
uptake rates (Lean and White 1983). Within the first 2 h of P
enrichment, an increase in HEP-SRP equivalent to 66% of inorganic P
added was observed. The HEP-TSP increase accounted for 86% of added
inorganic P. A 100C reduction in temperature decreased percentage
uptake to 43 and 78% as HEP-SRP and HEP-TSP, respectively. The
concentrations of HEP-TSP decreased as algal biomass increased. At 96 h
an increase in HEP-SRP was observed for cultures which received
treatments 3 (N=0 P=40) and 5 (N=800 P=80). Internal P concentrations
have been shown to regulate APA (Chrst and Overbeck 1987; Fitzgerald
and Nelson 1966; Moore 1969). The increase at 96 h thus could account
for the decreased APA. In April, 1989 HEP-SRP concentrations were shown
to be inversely related to APA, while in August a strong inverse


175
Perry, M. J. 1976. Phosphate utilization by an oceanic diatom in
phosphorus-limited chemostat culture and in the oligotrophic waters
of the central north Pacific. Limnol. Oceanogr. 21:88-107.
Pettersson, K. 1980. Alkaline phosphatase activity and algal surplus
phosphorus as phosphorus deficiency indicators in Lake Erken. Arch.
Hydrobiol. 89:54-87.
Pettersson, K., and M. Jansson. 1978. Determination of phosphatase
activity in lake water a study of methods. Verh. Int. Ver. Limnol.
20:1226-1230.
Pettersson, K., V. Istvnovics, and D. Pierson. 1990. Effects of
vertical mixing on phytoplankton phosphorus supply during summer in
Lake Erken. Verh. Int. Ver. Limnol. 24:236-241.
Pick, F. R. 1987. Interpretations of alkaline phosphatase activity in
Lake Ontario. Can. J. Fish. Aquat. Sci. 44:2087-2094.
Pollman, C. D. 1983. Internal loading in shallow lakes. Ph.D.
Dissertation, Univ. of Florida. 191 pp.
Pomeroy, L. R., E. E. Smith, and C. M. Grant. 1965. The exchange of
phosphate between estuarine water and sediments. Limnol. Oceanogr.
10:167-172.
Ponnamperuma, F. N. 1972. The chemistry of submerged soils. Adv.
Agron. 24:29-96.
Price, C. A. 1962. Repression of acid phosphatase synthesis in Euglena
gracilis. Science 135:46.
Pulford, I. D., and M. A. Tabatabai. 1988. Effect of waterlogging on
enzyme activities in soils. Soil Biol. Biochem. 20:215-219.
Reddy, K. R. 1981. Diel variations of certain physico-chemical
parameters of water in selected aquatic systems. Hydrobiol. 85:201-
207.
Reddy, K. R., and M. F. Fisher. 1990. Sediment resuspension effects on
phosphorus fluxes across the sediment-water interface: Laboratory
microcosm studies. Final Report submitted to the South Florida Water
Management District, West Palm Beach, FL.
Reddy, K. R., and D. A. Graetz. 1981. Use of shallow reservoirs and
flooded soil systems for wastewater treatment: nitrogen and
phosphorus transformations. J. Environ. Qual. 10:113-119.
Reddy, K. R., and D. A. Graetz. 1990. Internal nutrient budget for
Lake Apopka. St. Johns River Water Management District. Project no.
15-150-01-SWIM. Palatka, Florida.


LIST OF FIGURES
Figure page
1-1. Diagram of the phosphorus cycle in Lake Apopka 4
2-1. Location of Lake Apopka and sampling sites 19
2-2. Seasonal variability of selected parameters determined
bimonthly at 7 sites in Lake Apopka 26
2-3. Seasonal variability of selected parameters determined
bimonthly at 7 sites in Lake Apopka 27
2-4. Size fractionation of alkaline phosphatase activity
determined bimonthly at 3 sites in Lake Apopka 29
2-5. Size fractionation of phosphorus concentrations determined
bimonthly at 3 sites in Lake Apopka 30
2-6. Diel variation of selected parameters measured February
6-7, 1990 at the center of Lake Apopka (site 8) 35
2-7. Diel variation of selected parameters measured February
6-7, 1990 at the center of Lake Apopka (site 8) 36
2-8. Diel variation of selected parameters measured February
6-7, 1990 at the center of Lake Apopka (site 8) 37
2-9. Distribution of phosphorus compounds determined in whole
lake water at 8 sites in October 1989 38
2-10. Distribution of phosphorus compounds determined in
filtered lake water at 8 sites in October 1989 40
2-11. Distribution of suspended phosphorus compounds determined
by difference between whole and soluble phosphorus measured
at 8 sites in October 1989 41
3-1. Map showing the location of Lake Apopka and sampling sites.
Water was collected from site 1 in November 1989 and from
site 2 in April and August 1990 53
viii


fiage
5-6. Concentrations of selected parameters measured in sediments
incubated under six different redox levels for one month .
141


163
Table B-7. Chlorophyll a
Date
Site
Mean'
SE
1
2
3
4
5
6
7
8
-1
/*y *-
APR
33
61
70
68
75
71
52
98
71
2
JUN
24
80
79
88
84
92
55
67
78
2
AUG
24
131
142
87
156
95
158
127
128
4
OCT
16
92
96
75
101
73
85
99
89
2
DEC
26
102
94
60
68
71
79
74
78
2
FEB
7
39
33
49
48
51
40
45
44
1
Mean
22
84
86
71
89
75
78
85
SE
1
5
6
3
6
3
7
5
Means
listed
in this
column
are
means
of sites 2
-8.


123
covalent bonding of enzymes to organic matter would result in increased
stabilization of the enzyme.
Conclusions
Most studies investigating APA in aquatic systems have emphasized
the contribution of free-living bacteria, phytoplankton and zooplankton.
This study demonstrated the contribution of the sediment associated APA
to the total APA pool of the water column. Sediment resuspension
resulted in increased APA and TP in the overlying water column, and
hence there was an increase in potential organic P mineralization.
This increased APA remained high only while sediment particles were
still suspended in the water column and decreased upon settling of the
sediment. This indicates that no APA was released from the sediment
into the water column. The APA determined in both sediments and lake
water was mainly associated with particulate matter and thus may have
increased longevity. The high APA recorded both in the sediment and
water columns suggests that organic P mineralization, via APA, may play
a significant role in P cycling of Lake Apopka. The importance of this
process is dependent upon the concentration of organic substrates. The
forms of sediment organic P capable of acting as substrates needs to be
evaluated.


147
water during photosynthesis (Stewart and Pearson 1970). The production
of APA is dependent upon the P stress within system (Fuhs et al. 1972;
Kuenzler and Perras 1965). Because of the low P requirements of
anaerobic bacteria, APA inhibition may occur at lower SRP levels than
under aerobic conditions, where P requirements of aerobic bacteria are
high. Enzyme activity did not cease under anaerobic conditions
suggesting that the enzyme itself was not inhibited by anaerobic
conditions. Although total APA under aerobic conditions differed from
total APA under anaerobic conditions within 2 h, no significant increase
in APA occurred until 24 h, this suggests that less severe low DO will
not affect APA organic P mineralization. Depth profiles of APA with
varying DO concentrations were shown to correlate with chlorophyll a and
bacterial counts rather than DO concentrations (Jones 1972a),
consequently metalimnetic 02 depletion is unlikely to affect APA.
However, in waters which become stratified the anoxic conditions at the
sediment-water interface may result in decreased APA after an extended
time period. This may be due to decreased production by anaerobes or as
a response to competitive inhibition by SRP released from sediments
(Pettersson 1980). Lake Apopka is a frequently mixed system under which
extended periods of 02 depletion are not likely to occur. Diel changes
in 02 will be too brief (< 8 h) to affect organic P mineralization.
No change in soluble APA was observed under either aerobic or
anaerobic conditions. Soluble APA has been shown to be a result of cell
lysis (Berman 1970) as well as excretions by zooplankton, bacteria and
phytoplankton (Wynne 1981; Pettersson 1980). It is thought that
significant cell death and lysis did not occur during the period of


166
Table B-10. Total and soluble alkaline phosphatase activity.
Date
Site
Mean" SE
1 2 3 4 5 6 7 8
nM min'1
Total
APR
5.9
12.2
12.8
22.0
18.7
20.1
20.7
22.8 18.5
0.61
JUN
3.8
11.8
11.2
44.7
29.9
30.6
20.8
19.2 24.0
1.70
AUG
3.7
10.2
10.6
10.3
6.4
17.8
20.8
11.6 12.5
0.71
OCT
5.7
27.0
28.2
18.2
19.6
21.9
25.4
24.9 23.6
0.54
DEC
7.7
23.8
24.6
19.8
18.8
17.9
20.3
17.4 20.4
0.40
FEB
5.6
9.6
10.3
11.6
9.5
10.6
10.9
8.9 10.2
0.13
Mean
5.4
15.8
16.3
21.1
17.2
19.8
19.8
17.5
SE
0.25
1.26
1.33
2.07
1.39
1.08
0.80
1.04
Soluble
APR 0.16
0.51
0.49
0.34
0.29
0.67
0.37
0.30 0.42
0.02
JUN
0.07
0.03
0.05
0.93
0.01
0.07
0.10
0.20 0.20
0.05
AUG
0.31
0.10
0.54
0.60
0.35
0.16
0.55
0.64 0.42
0.03
OCT
0.74
1.29
1.19
0.64
0.48
0.80
0.29
0.27 0.71
0.06
DEC
0.65
0.38
0.62
0.45
1.02
0.68
0.58
1.34 0.72
0.05
FEB
0.39
0.56
0.33
0.43
0.11
0.12
0.03
0.09 0.24
0.03
Mean
0.39
0.48
0.53
0.57
0.38
0.42
0.32
0.47
SE
0.04
0.08
0.06
0.04
0.06
0.06
0.04
0.08
Means listed in this column are means of sites 2-8.


43
inversely correlated with one water chemistry parameter, TS. This
correlation along with the ratio TS/chlorophyll a show that chlorophyll
a was not a dominant component of the solids in Lake Apopka during the
sampling period. In a frequently mixed system such as Lake Apopka, the
proportion of total solids which may be attributed to phytoplankton
biomass will fluctuate considerably. Other contributors to TS include;
bacteria, suspended sediment, zooplankton and inorganic and organic
compounds. The inverse relationship between chlorophyll a and TS may be
interpreted as follows; 1) increased herbivory by high zooplankton
populations and 2) light limitation because of high suspended solids.
A peak in TSS was observed in February, which corresponds to the
highest wind speed recorded 1 m above the water surface, and thus
reflects wind induced sediment resuspension. In a shallow lake, a large
proportion of particulate matter within the water column may frequently
be attributed to sediment resuspension. The sediments have high P
concentrations (Reddy and Graetz 1990) and resuspension will result in
increased levels of TP within the water column; TP concentrations were
positively correlated with wind speed (5 m above the surface).
Conversely, TKN concentrations were inversely related to wind speed (1 m
above the surface), and are mainly associated with the algal biomass.
The rate of P exchange between water and sediment increases during
suspension of sediment (Pomeroy et al. 1965). Part of this exchange may
be biological. Sediment resuspension into the oxygenated water column
results in aerobic mineralization of organic P (Lee et al. 1977).
Sediments exhibit significant phosphatase activity (Kobori and Taga
1979b; Ayyakannu and Chandramohen 1971) and a positive correlation


72
(N=800 P=80), from 41.6 to 22 ng L'1 in treatment 4 (N=400 P=40) and
from 41.6 to 13.1 ng L'1 in treatment 3 (N=0 P=40) cultures (Fig. 3-7a).
Uptake rates were calculated as the disappearance of SRP within the
first 30 min (Table 3-6). This was selected to indicate maximal uptake
because the slope changed over time as the P demand decreased
(Fig. 3-7a). Cultures which received 80 ng L'1 had a significantly
greater uptake rate than those which received 40 ng L1. As expected,
temperature had a significant effect upon SRP uptake (Fig. 3-7b). The
uptake was not as rapid as that for the same treatment at 29*C
(a = 0.12), but even with the 10*C difference in temperature all SRP had
been depleted to below detection within 2 h. After 1 h, SRP levels in
treatments 3 (N=0 P=40) and 4 (N=400 P=40) were no longer significantly
different. The SRP concentrations continued to decrease in all the
cultures and were all close to baseline in 2 h and were undetectable (<1
ng L'1) after 24 h. In both experiments TSP decreased within the first
2 h and then remained constant (Fig. 3-8a and 3-9a).
The uptake of N differed between the two sampling periods. In
April, [N03 + N02]-N concentrations in the cultures decreased. The
concentration decreased by 25% in all treatments with N additions,
within 2 h and continued to decrease over time (Fig. 3-8b). In
contrast, NH4-N concentrations did not change in any of the cultures
until 216 h, when an increase was observed in control and N cultures
(Fig. 3-8c). In August, no significant treatment by time interaction
was recorded for [N03 + N02]-N, and no apparent uptake of [N03 + N02]-N
occurred (Fig. 3-9b). A significant treatment by time interaction was


CONCENTRATION
112
en
E
Fig. 4-6. Concentrations of selected parameters measured in the
overlying water column following sediment resuspension of 0,
2, 5, and 10 cm surficial sediments: a) total suspended
solids; b) total organic carbon; c) total phosphorus;
d) total Kjeldahl nitrogen. Initial data point indicates
the conclusion of resuspension. Vertical bars indicate
1 SE. No bar indicates SE is smaller than symbol size.


150
1952). It was determined that the rate of phytate hydrolysis in
solution was over 71 times as important as the phytase activity; a
specific acid phosphatase in the limitation of hydrolysis (Jackman and
Black 1952).
The high volatile solids content of the sediment indicated the
high organic matter content. A significant portion of this organic
matter was highly resistant to hydrolysis, as apparent by the 26%
difference in TP determined by ignition and extraction. In this study,
APA was inversely correlated with HA organic P (r=-0.74), a resistant
form of P. This inverse relationship may be explained as the possible
binding of alkaline phosphatase to the humic substances which may
increase enzyme stability, thus resulting in some inhibition of APA
(Burns 1986; Kandeler 1990; Wetzel 1991). Organic P compounds may also
form complexes with humates (Stewart and Tiessen 1987; Brannon and
Sommers 1985). The two main chemical extractants, HC1 and NaOH, were
unable to extract organic P compounds incorporated into humic polymers
(Brannon and Sommers 1985). This chemically resistant organic P was
also resistant to enzymatic hydrolysis.
The high negative correlation between labile inorganic P and SRP
with FA-TP and NaOH-TP suggest that FA-TP, a component of NaOH-TP, may
be a significant contributor to the labile inorganic P pool through
enzymatic hydrolysis. It has been suggested that inositol phosphates
bound to FA are hydrolyzable by phytase (Herbes et al. 1975).


164
Table B-8. Total organic carbon.
Date
Site
Mean*
SE
1
2
3
4
5
6
7
8
mg L'1
APR
ND*
ND
ND
ND
ND
ND
ND
ND
JUN
0.3
30.6
29.9
29.8
31.6
31.5
24.3
32.2
30.0
0.38
AUG
2.4
32.5
34.0
40.5
32.4
31.7
33.1
34.0
34.0
0.43
OCT
1.2
28.5
29.8
30.5
29.0
32.9
28.3
29.2
29.7
0.22
DEC
9.1
30.1
28.6
29.4
31.1
32.9
27.8
31.9
30.3
0.26
FEB
10.6
35.2
36.9
33.7
44.5
32.6
34.0
39.2
36.6
0.59
Mean
SE
4.7
0.95
31.4
0.51
31.8
0.69
32.8
0.93
33.7
1.23
32.3
0.13
29.5
0.81
33.3
0.75
Means listed in this column are means of sites 2-8.
* ND indicates not determined.


137
insignificant time and treatment responses (Fig. 5-4a and b), SRP
concentrations were below detection (< 1 ng L'1) for both treatments
after 24 h. A significant treatment response was observed for total APA.
(Fig 5-5a). Under anoxic conditions total APA remained constant over
the 24 h sampling period. Under aerobic conditions total APA increased
within 2 h and then remained constant until 24 h. At 24 h, the total
APA had increased from 27 to 43 nM min'1. Soluble APA accounted for 4%
of total APA (Table 5-1). No change in soluble APA was observed in
either treatment (Fig. 5-5b).
Significant correlations among the water chemistry parameters
measured were observed. A significant inverse relationship between
NH4-N and TSP was determined (r=-0.79), and significant positive
correlations between SRP and TSN (r=0.67) and NH4-N (r=0.70) were
observed.
The Effect of Redox Potential on Organic Phosphorus Mineralization in
the Sediment
The initial pH of the sediment prior to the month long incubation
was 7.12. Following equilibration at different Eh levels slight changes
in pH values were recorded (Table 5-2). Redox values were expressed as
pE + pH to account for the variability in pH among the reaction vessels
(pH range 6.35 to 7.15). pE was calculated as;
pE = Eh / 59
A significant negative relationship was observed between APA and
pE + pH (Fig. 5-6a). Conversely, a significant positive relationship
between pE + pH and porewater TOP was recorded (Fig. 5-6b). pE + pH
accounted for 77% of the variability in APA and 73% of porewater TOP


162
Table B-6. Total suspended solids.
Date
Site
Mean"
SE
1
2
3
4
5
6
7
8
1
mg l
APR
ND
4 ND
ND
ND
ND
ND
ND
ND
JUN
ND
ND
ND
ND
ND
ND
ND
ND
AUG
6
59
66
42
49
69
72
73
62
2
OCT
5
54
67
52
71
60
64
75
63
1
DEC
15
61
69
48
67
60
58
65
61
1
FEB
17
77
85
109
99
89
92
103
94
2
Mean
11
63
72
63
72
70
72
79
SE
2
2
2
8
5
3
4
4
Means
listed
in this
column
are
means
of sites 2-8.
4 ND indicates not determined.


24
Table 2-1. Means of selected weather data measured at the central
station (mean 1 SE).
Date
Wind
1 m
above
sDeed
5 m
surface
Water
temp
PAR*
ym
km h'1
C
/xmol m2
s'1
8904
2 0
NA*
23.6
NA
8906
9 0
10 + 0
29.8
548
29
8908
11 0
13 0
28.5
508 +
29
8910
11 0
20 0
19.4
238
14
8912
11 + 0
13 + 0
15.5
294 +
18
9002
13 0
18 + 0
20.0
282
16
PAR indicates photosynthetically active radiation.
* NA indicates data not available.
Source: Stites, D. L., unpublished data, St. John's River Water
Management District, Palatka, FL.


6
The steps involved in the reaction are as follows:
1. The phosphomonoester binds non-covalently to the phosphatase
enzyme (R0PE).
2. The phosphoseryl intermediate forms by covalent binding of
the phosphate group to the phosphatase enzyme (E-P); alcohol is
released during this nucleophilic attack.
3. Water is taken up resulting in the nucleophilic displacement of
serylphosphate to produce a non-covalently bound complex (E*P).
4. Inorganic P is released and the free phosphatase enzyme is
regenerated.
Phosphatases have a high degree of specificity for the P moiety of
the P-O-C bond, but little specificity for the C moiety (Reid and Wilson
1971). These enzymes are classified as alkaline or acid depending on
the pH range under which they exhibit optimum activity (Reichardt 1971;
Torriani 1960). At acid pH, the dephosphorylation of the serylphosphate
is the rate limiting step. At alkaline pH, the dissociation of
inorganic P from E*P is the rate limiting step (Coleman and Gettins
1983). The alkaline nature of most aquatic systems has resulted in
alkaline phosphatase activity (APA) receiving the most emphasis.
More than one type of phosphatase may be present in any plankton
population. Five intracellular phosphatases were extracted from a
Peridinium bloom (Wynne 1977). The phosphatases produced in response to
P limitation do not have the same biochemical characteristics as those
observed in normal tissues (Bielski 1974). Although acid phosphatases
have the ability to function outside the cell (Kuenzler and Perras 1965;


177
[ed.], Interactions between sediments and freshwater. Dr. W. Junk
Publishers, The Hague.
Shannon, E. E., and P. L. Brezonik. 1972. Limnological characteristics
of north and central Florida lakes. Limnol. Oceanogr. 17:97-110.
Shapiro, J. 1960. The cause of metalimnetic minimum of dissolved
oxygen. Limnol. Oceanogr. 5:216-227.
Siuda, W., R. J. Chrst, R. Wcislo, and M. Krupka. 1982. Factors
affecting alkaline phosphatase activity in a lake (short term
experiments). Acta Hydrobiol. 24:3-20.
Skujins, J. 1976. Extracellular enzymes in soil. CRC Crit. Rev.
Microbiol. 4:383-421.
Smith, R. E. H., and J. Kalff. 1981. The effect of phosphorus
limitation on algal growth rates: evidence from alkaline phosphatase.
Can. J. Fish. Aquat. Sci. 38:1421-1427.
Sommers, L. E., R. F. Harris, J. D. H. Williams, D. E. Armstrong, and J.
K.Syers. 1972. Fractionation of organic phosphorus in lake
sediments. Soil Sci. Soc. Amer. Proc. 36:51-54.
Speir, T. W. 1976. Studies on a climosequence of soils in tussock
grasslands. 8. Urease, phosphatase and sulphatase activities of
tussock plant materials and of soil. N. Zealand J. Sci. 19:383-387.
Speir, T. W., and D. J. Ross. 1978. Soil phosphatases and sulphatases,
p. 197-249. In R.G. Burns [ed.], Soil Enzymes. Academic Press, New
York.
Sproule, J. L., and J. Kalff. 1978. Seasonal cycles in the
phytoplankton phosphorus status of a north temperate zone lake (Lake
Memphremagog, Qu-Vt), plus a comparison of techniques. Verh. Int.
Ver. Limnol. 20:2681-2688.
Stevens, R. J., and M. P. Parr. 1977. The significance of alkaline
phosphatase activity in Lough Neagh. Fresh. Biol. 7:351-355.
Stevenson, F. J. 1982. Humus chemistry genesis, composition,
reactions. Wiley-Interscience, New York.
Stewart, A. J., and R. G. Wetzel. 1982. Phytoplankton contribution to
alkaline phosphatase activity. Arch. Hydrobiol. 93:265-271.
Stewart, J. W, B., and H. Tiessen. 1987. Dynamics of soil organic
phosphorus. Biogeochemistry 4:41-60.
Stewart, W. D. P., and H. W. Pearson. 1970. Effects of aerobic and
anaerobic conditions on growth and metabolism of blue-green algae.
Proc. Roy. Soc. Lond. B. 175:293-311.


20
Beauclair canal. The St. John's River Water Management District
(SJRWMD) weather station is located at the center of the lake (site 8).
Water Sampling
Bimonthly sampling. Lake water was collected bimonthly from April
1989 thru February 1990, from 8 sites in Lake Apopka (Fig. 2-1). Sites
1, 4 and 8 were selected to represent inflow, outflow and the center of
the lake. Site 2 was selected to determine littoral zone influences.
Site 5 was located close to a pump station and hence represented
backpumping from the surrounding agricultural land. Site 7 was
established close to an old fishing camp, and site 6 was selected to
correspond to extensive sediment studies which were conducted with
samples from that site. Site locations were established using Loran
coordinates (Appendix A). Three replicate water samples were collected
from a depth of 0.3 m using 1 L polyethylene bottles, from each site.
Samples were stored on ice until return to the laboratory. Water was
filtered through 0.45 nm membrane filters (Gelman) and analyzed for
soluble APA within 24 h. Other soluble parameters determined were total
soluble P and SRP. Soluble particulate P was defined as TSP-SRP (SPP).
Whole lake water was analyzed for total APA, total Kjeldahl N (TKN), TP,
SRP, total solids (TS), total suspended solids (TSS), total organic
carbon (TOC), and chlorophyll a. Seasonal water chemistry data
collected at site 8 were compared with the weather data provided by the
SJRWMD.
The contributors to the APA pool are frequently determined via
filter fractionation (Chrst and Overbeck 1987; Currie and Kalff 1984;
Currie et al. 1986). To distinguish between the contribution of


141
pE + pH
Concentration of selected parameters measured in sediments
incubated under six different redox levels for one month:
a) alkaline phosphatase activity; b) porewater total organic
phosphorus.
Fig. 5-6.


148
anaerobiosis because increased nutrient concentrations and soluble APA
were not observed.
The Effect of Redox Potential on Organic Phosphorus Mineralization in
the Sediment
A significant effect of increasing anaerobiosis is the change from
a predominately aerobic microbial population to a smaller anaerobic
population. Under anaerobic conditions organic material accumulates due
to reduced mineralization rates. Microbial biomass is highly correlated
with APA (Sayler et al. 1979; Ayyakannu and Chandramohen 1971), and APA
was significantly inhibited by the decrease in redox potential. Hence
organic P mineralization will decrease under reduced conditions. In
general, redox potential did not have a significant effect on the
various organic P pool sizes. However, Lake Apopka sediments are poorly
poised (poorly redox buffered) and the low concentrations of electron
acceptors may reduce the rate of mineralization. Only APA and porewater
TOP were significantly correlated with pE + pH. More resistant P forms
may require longer incubation times to show a response to redox
potential.
Significantly lower porewater SRP and labile inorganic P
concentrations were observed at Eh 48 mV, than either Eh 338 or -2 mV.
This may be a problem associated with the control of redox conditions.
Redox levels may need to be established by utilizing specific electron
acceptors, rather than fluctuating the air input.
Redox potential tends to decrease with sediment depth (Kobori and
Taga 1979b; DeGobbis et al. 1984), while porewater SRP has often been
shown to increase as a function of depth (Mortimer 1941; chapter 4).


97
Following subsidence of the wind event, suspended solids settle
thus transporting any associated material from the water column to the
sediment, i.e. APA, and results in a decrease in organic P
mineralization within the water column. Settling seston is also a sink
for SRP (Gchter and Mares 1985).
The objectives of this study were to determine; 1) the depth
distribution of APA in the sediment and the overlying water column and,
2) the effect of sediment resuspension upon SRP and APA release. It was
hypothesized that sediment resuspension may affect APA by 1) increasing
SRP levels in the water column and competitively inhibiting activity, 2)
releasing alkaline phosphatase from the sediment to the water column,
and 3) a combination of 1 and 2.
Materials and Methods
Site Description
Lake Apopka is a 12,500 ha hypereutrophic lake, located in central
Florida, 2837' N latitude, 8137' W longitude (Fig. 4-1). It has a
mean depth of 2 m. Chlorophyll a values exceeding 100 ng L'1 are
frequently recorded (chapter 2). Nutrient loading from the surrounding
agricultural and urban areas has resulted in the current hypereutrophic
conditions in the lake (USEPA 1979). The bottom sediments in the lake
have a 30 cm unconsolidated flocculent layer at the surface, underlain
by consolidated flocculent material (Reddy and Graetz 1990). The
sediments have an alkaline pH, hence the phosphatases of interest are
those with maximum activity in the alkaline region, i.e. alkaline
phosphatase.


165
Table B-9. Total Kjeldahl nitrogen.
Date
Site
Mean
SE
1
2
3
4
5
6
7
8
i
my l
APR
2.57
5.06
6.12
5.38
5.71
5.03
5.18
8.11
5.80
0.16
JUN
0.18
4.71
4.72
4.62
4.78
5.33
3.58
4.48
4.60
0.07
AUG
1.30
5.28
6.45
4.86
5.56
5.53
5.66
5.25
5.51
0.07
OCT
0.61
2.08
3.82
3.64
4.68
3.14
4.08
4.28
3.67
0.12
DEC
2.30
4.19
4.72
3.81
4.13
4.36
4.59
4.45
4.32
0.04
FEB
1.87
4.04
4.12
4.85
5.30
4.58
5.06
4.00
4.56
0.08
Mean
1.47
4.23
4.99
4.53
5.03
4.66
4.69
5.09
SE
0.16
0.19
0.18
0.11
0.10
0.14
0.13
0.26
Means listed in this column are means of sites 2-8.


92
thus indicate that this system is P limited. In April, specific APA of
plankton which received P additions remained between 0.3-0.4 nmol APA
ng chlorophyll a'1 min'1 for the first 96 h. Hence this ratio may
represent constitutive APA (Gage and Gorham 1985). Specific APA
determined in a eutrophic Swedish lake remained at < 0.3 nmol APA
ng chlorophyll a'1 min1 for most of the year but increased to 0.8 nmol
APA ng chlorophyll a1 min1 during periods of P limitation (Pettersson
et al. 1990). Specific APA thus increases with the severity of P
limitation (Perry 1976). In this study plankton grown in the absence of
P produced specific APA > 1 nmol APA ng chlorophyll a1 min'1. Greater
fluctuation in specific APA was apparent in August. However, comparing
specific APA with increases in chlorophyll a, in general, specific APA
measured in cultures which received P was inversely related to growth
rate (Rhee 1973; Smith and Kalff 1981). Specific APA determined
bimonthly at 7 sites in Lake Apopka from April 1989 to February 1990,
was < 0.3 nmol APA ng chlorophyll a'1 min'1, this suggests that growth of
Lake Apopka plankton was not severely P limited. This agrees with
results from other nutrient limitation studies conducted on Lake Apopka
(Aldridge, F. J., unpublished data, Department of Florida, University of
Florida, Gainesville, FL.). Hence, it would appear that the expression
of APA and HEP relative to chlorophyll a is an accurate indicator of
the nutrient status of plankton in Lake Apopka. However, it should be
re-emphasized that APA and HEP are found in numerous organisms, phyto
plankton, bacteria and zooplankton which vary both spatially and
temporally, absolute values may vary. Some uncertainty could be removed


49
means for TP and TKN were 210 ng L'1 and 4.8 mg L'1, respectively,
confirming the highly eutrophic state of the lake. Conversely, SRP
concentrations were consistently < 10 ng L'1.
Alkaline phosphatase activity was mainly associated with
particulate matter and was dependent on different water chemistry
parameters both seasonally and spatially. In general, APA was not
correlated to chlorophyll a. The relationship between these parameters
may be hidden as a result of the frequent mixing of the water column in
Lake Apopka. Both positive and negative correlations between P and APA
were observed. An inverse relationship existed between acid
hydrolyzable P and APA, indicating polyphosphates may be controlling
APA.
The particulate association of APA would suggest that APA should
be correlated with TSS, but this was rarely observed. This may be due
to the variability in the composition of the TSS pool. The relationship
between APA and particulate may be both beneficial and detrimental.
Binding to particles results in increased longevity of the enzyme, but
it also may inhibit APA by binding to the active site, as indicated by
the inverse relationship between APA and TOC.
Future research should examine the components of the TSS pool,
plankton and sediment and their effect on organic P mineralization under
controlled conditions.


128
Experimental Design
The effect of dissolved oxygen on organic phosphorus
mineralization in the water column. Dissolved 02 minima in the water
column of lakes occur during the night hours when respiration exceeds
photosynthesis. To emulate this condition this experiment was conducted
in the dark, under ambient laboratory temperatures. Four hundred mL of
lake water were placed in each of six 500 mL erlenmeyer flasks. The
flasks were placed on stir plates and continuously stirred. Three
flasks were stoppered and maintained under anaerobic conditions (Fig. 5-
la), while three were maintained under aerobic conditions. One
replicate of each treatment had pH and DO probes in constant contact
with the water column; it was assumed that all three replicates would
respond similarly. Anaerobic conditions were obtained by closing the
flasks with rubber stoppers and purging with a mix of 330 ppm C02
balanced with N2 for approximately 1 1/2 h until the DO concentration
was <0.2 mg L'1. The pH and DO probes were tightly sealed in the
stoppers. A rubber septum was also included in the stopper arrangement
to allow sampling. At this point 30 mL water samples were removed by
syringe from all flasks. A time series of samples was subsequently
removed after 2, 4, 8 and 24 h. Samples were analyzed for total and
soluble APA, total soluble P (TSP), total soluble N (TSN), NH4-N, and
soluble reactive P (SRP). Initial chlorophyll a was also determined.
The effect of redox potential on organic phosphorus
mineralization in the sediment. Fresh sediment was diluted with
filtered lake water to obtain 10 g dry wt. L'1 of slurry, with a total
volume of 2.5 L and was placed in each of six 2.8 L flasks, which were


142
variability. Non linear redox effects were observed for other P
compounds. A significant increase in porewater SRP and labile inorganic
P were observed at a pE + pH value of 3 (Table 5-2). In contrast FA-TP
decreased at a pE + pH of 3. The HA-TP fraction tended to increase
under pE + pH values of 7. However, the majority of P forms measured
were not significantly affected by redox potential.
Volatile solids were 66% in both aerobic and anaerobic treatments.
The sediment TP as determined by ignition was 1047 ng g dry wt.'1. On
average the total recoverable P determined by the summation of HC1-TP,
NaOH-TP and porewater TP accounted for 74% of TP as determined by
ignition (Table 5-3). Acid hydrolyzable-TP extracted the largest
percentage of P (41%), followed by alkali extractable P (28%). Labile
inorganic and organic P were a small component of the TP, contributing
6.8 (2-14) and 1.4 (1-2)%, respectively. Readily available, i.e.
porewater SRP and TP, also contributed less than 20% of total P.
Porewater TP accounted for 5.8 (2-13)% of the total P pool.
Total and inorganic P extracted with HC1 were the same, indicating
that no organic P was extracted. Porewater TSP and SRP were also
equivalent suggesting that no porewater organic P was in the soluble
phase; TOP represented particulate organic P.
Significant correlations between the parameters measured were
observed (Table 5-4). Alkaline phosphatase activity had a high positive
correlation with pE + pH (r=0.90) while porewater TOP was strongly
inversely related to pE + pH (r0.92). Alkaline phosphatase activity
was also highly negatively correlated with labile organic P (r=-0.81)
and HA-TP (r=-0.74), while porewater TOP was highly positively related


74
Table 3-6. Phosphorus uptake rates for natural plankton populations
collected in August 1990, 30 min after receiving nitrogen
and phosphorus additions (mean SE). 3=40 /xg P L'1,
4=400 /xg N L1 and 40 jig P L1, 5=800 fig N L'1 and 80 /xg P L'1
7=40 /xg P L1 and plankton grown at 19*C.
Treatment
Uptake Rate
ng P
L'1 min'1
3
0.95
0.05 a*
4
0.43
0.27 b
5
1.47
0.06 c
7
0.65
0.01 ab*
Numbers in a column followed by the same letter are not
significantly different at a = 0.05.
Uptake rates for treatments 3 and 7 are significantly different at
a = 0.12.


86
response in APA increase and produced the least APA. A similar trend
was observed by phytoplankton cultured at 19*C in August (Fig. 3-13b).
In August the response of cultures grown at 290C with only P added
mimicked those which received both N and P (Fig. 3-13a). Disregarding
temperature effects, APA in cultures with and without P additions
exhibited similar trends; increasing up to 48 h and then decreasing
(Fig. 3- 13a). The initial increase in APA over the first 24 h was on
average 64%, greater in those cultures receiving P. The reverse was
true for the next 24 h period. Alkaline phosphatase activity measured
in treatments 1 (N=0 P=0) and 2 (N=400 P=0) doubled while only a 22%
increase was observed in treatments 3 (N=0 P=40) and 4 (N=400 P=40).
Cultures which received treatment 5 (N-800 P=80) did not exhibit a
change. Both groups subsequently declined by approximately
20 nM min'1 to 96 h. Transforming the APA data to specific activity
results in a different shape curve for April data (Fig. 3- 13b) but no
change in curve shape in August (Table 3-10). The interpretation from
both experiments is the same, .i.e., higher specific APA was apparent in
all cultures which did not receive any P addition. While those cultures
which received P had significantly lower specific APA. The highest
specific APA was recorded in April in cultures which received treatments
1 (N=0 P-0) and 2 (N=400 P-0).
Discussion
Nutrient loading from external and internal sources can influence
the productivity of phytoplankton and other aquatic biota. The response
of phytoplankton growth to nutrient enrichment has been used as an


176
Reichardt, W. 1971. Catalytic mobilization of phosphate in lake water
and by Cyanophyta. Hydrobiologia 38:377-394.
Reid, T. W., and I. B. Wilson. 1971. E. coli alkaline phosphatase, p.
373-415. In P.D. Boyer [ed.], The enzymes. Vol. IV. Academic
Press, New York.
Rhee, G-Y. 1972. Competition between an alga and an aquatic bacterium
for phosphate. Limnol. Oceanogr. 17:505-514.
Rhee, G-Y. 1973. A continuous culture study of phosphate uptake,
growth rate and polyphosphate in Scenedesmus sp. J. Phycol. 9:495-
506.
Rhee, G-Y. 1974. Phosphate uptake under nitrate limitation by
Scenedesmus sp. and its ecological implications. J. Phycol.
10:470-475.
Rigler, F. H. 1956. A tracer study of the phosphate cycle in lake
water. Ecol. 37:550-562.
Rigler, F. H. 1964. The phosphorus fractions and turnover time of
inorganic phosphorus in different types of lakes. Limnol. Oceanogr.
6:165-174.
Rojo, M. J., S. G. Carcedo, and M. P. Mateos. 1990. Distribution and
characterization of phosphatase and organic phosphorus in soil
fractions. Soil Biol. Biochem. 22:169-174.
Rosenberg, H., R. G. Gerdes, and K. Chegwidden. 1977. Two systems for
the uptake of phosphate in Escherichia coli. J. Bacteriol. 131:505-
511.
Ryding, S-0., and C. Forsberg. 1977. Sediments as a nutrient source in
a shallow polluted lake, p. 227-234. In H. L. Golterman [ed.],
Interactions between sediments and freshwater. Dr. W. Junk
Publishers, The Hague.
SAS Institute Inc. 1985. SAS user's guide: statistics, SAS Institute
Inc., Cary, N.C.
Sayler, G. S., M. Puziss and M. Silver. 1979. Alkaline phosphatase
assay for freshwater sediments: Application to perturbed sediment
systems. Appl. Environ. Microbiol. 38:922-927.
Schindler, D. W. 1977. Evolution of phosphorus limitation in lakes.
Science. 195:260-262.
Schindler, D. W.,R. Hesslein, and G. Kipphur. 1977. Interactions
between sediments and overlying waters in an experimentally
eutrophied Precambian Shield Lake, p. 235-243. In H. L. Golterman


62
Table 3-4. Chlorophyll a and specific alkaline phosphatase activity
measured in natural plankton populations collected in
November 1989, 72 h after receiving nitrogen and phosphorus
additions. l=no nutrient addition, 2=500 ng N L'1, 3=10 ng P
L \ 4=1000 ng N L'1 and 100 ng P L\ 5=2500 ng N L'1 and
1000 ng P L \
Treatment
Chlorophyll
Specific APA
M L-1
nmol APA ng chlorophyll a'1 min'1
1
36 a*
0.41
2
40 b
0.46
3
40 b
0.35
4
48 c
0.15
5
61 d
0.03
Numbers in a column followed by the same letter are not
significantly different at a = 0.05.


PHOSPHORUS CONCENTRATION (/zg
30
1989-90
Fig. 2-5. Size fractionation of phosphorus concentrations determined
bimonthly at 3 sites in Lake Apopka: a) site l=inflow;
b)site 4=ouflow; c) site 8=center of lake. Vertical bars
represent 1 SE.


7
Price 1962), they are generally intracellular in action (Moller et al.
1975; Wynne 1977). Acid phosphatases function as specific enzymes in
metabolic pathways and non-specific reactions (Cembella et al. 1984a).
Hence they are constitutive and generally not repressible by inorganic
P. Conversely, APA exhibits principly extracellular function. Alkaline
phosphatase synthesis may be induced by the presence of organic P
(Aaronson and Patni 1976; Kuenzler 1965). Alkaline phosphatase is a
repressible enzyme (Jansson et al. 1988), whose synthesis is inhibited
by high levels of inorganic P (Elser and Kimmel 1986; Lien and Knutsen
1973; Torriani 1960). Inorganic P is a competitive inhibitor of APA
(Coleman and Gettins 1983; Moore 1969; Reid and Wilson 1971). Other
factors which affect APA include; temperature (Garen and Levinthal 1960;
Torriani 1960), chelators and divalent cations (Cembella et al. 1984a;
Healey 1973).
The derepression of APA in response to P limitation has been
examined at the cellular level where APA was shown to transport
inorganic P. Studies utilizing Escherichia coli have shown that two
forms of P transport exist (Rosenberg et al. 1977). One is a low
affinity system, phosphate inorganic transport (PIT), which is
constitutive and transfers intracellular P pools. The other is a high
affinity system, phosphate specific transport (PST), which is activated
when internal P concentrations are low. This utilizes a membrane
associated protein, APA, to increase P uptake. The high affinity system
is inhibited at high concentrations of inorganic P. However, it is the
ability of APA to catalyze the hydrolysis of organic P compounds that
has received the most study in the aquatic environment.


ALKALINE PHOSPHATASE ACTIVITY (nM min
29
50
40
~ 30
20
10
0
1989-90
Fig. 2-4. Size fractionation of alkaline phosphatase activity
determined bimonthly at 3 sites in Lake Apopka: a) site
l=inflow; b)site 4=ouflow; c) site 8=center of lake.
Vertical bars represent 1 SE.


25
Little variation in pH was observed with a range of 0.8 pH units
observed between maximum and minimum pH. Total solids were also
constant between 300 and 400 mg L'1, until February when a particularly
high concentration of 500 mg L'1 was recorded. Total suspended solids
accounted for approximately 16% of TS and increased in February at all
sites (Fig. 2-2a).
A distinct peak in chlorophyll a concentration was observed in
August at 5 of the 7 sites (Fig. 2-2b). This peak was 3 fold higher
than the minimum which occurred in February. Two peaks in TOC were
apparent. One occurred in August, along with chlorophyll a (Fig. 2-2c)
while the other occurred in February and probably corresponded to the
increase in TSS which also occurred in February. Total Kjeldahl N
tended to be higher in Spring and Summer and decreased in Fall and
Winter (Fig. 2-2d).
Alkaline phosphatase activity was mainly associated with
particulate matter (Fig. 2-3a). Soluble APA averaged only 3% of total
APA. Total APA peaked in both June and October; in contrast, soluble
APA peaked in October and December.
Phosphorus was also determined to be mainly associated with
particulate matter. Total P concentrations peaked in October
(Fig. 2-3b). Total soluble P concentrations represented from 6 to 37%
of TP in August and April respectively. Soluble reactive P
concentrations were very low throughout the year (0 to 7 ng L'1) and as
such were a minor portion of TP.
All parameters measured at site 1 were generally much lower than
those recorded at other stations (Appendix B). In particular DO and pH


33
Table 2-3. Correlation coefficients between alkaline phosphatase
activity and parameters measured bimonthly at 8
sites in Lake Apopka (significant at a=0.05, n=6)
Alkaline phosphatase activity
Site Total Soluble
1
DO
0.87
temp
-0.81
temp
-0.77
2
TKN
-0.78
TKN
-0.90
TOC
-0.81
TP
0.89
TSP
0.85
SPP
0.86
secchi
0.85
3
soluble APA
0.82
TP
0.75
secchi
0.79
secchi
0.79
4
NS'
CHL
0.75
DO
0.92
5
TP
-0.75
secchi
0.90
6
TSS
-0.93
secchi
0.81
7
NS
TP
-0.83
pH
0.73
8
TSP
0.83
NS
SPP
0.81
pH
0.80
DO
0.82
TOC
-0.92
NS indicates not significant at a = 0.05.


SOLUBLE APA (nM min ) TOTAL APA (nM min
63
TIME (h)
Fig. 3-2. Time courses of alkaline phosphatase activity following
nutrient enrichment of natural plankton populations
collected in November 1989. 0N,0P=no nutrient addition;
500N,0P=500 nq N L'1*, ON, 10P=10 pg P L\ 1000N, 100P=1000 fiq
N L'1 and 100 nq P L'\ and 2500N, 1000P=2500 nq N L1 and 1000
nq P L'1: a) total alkaline phosphatase activity; b) soluble
alkaline phosphatase activity. Vertical bars represent 1
SE. No vertical bar indicates SE is smaller than symbol
size.


31
positively correlated with PAR (r=0.98). Total Kjeldahl N was
inversely related to wind speed measured 1 m above the surface
(r=-0.94), while TSS was highly positively correlated with the wind
speed recorded 1 m above the surface (r=0.97). Total suspended solids
were also positively correlated with chlorophyll a, and inversely
correlated with TSP and SOP. Neither chlorophyll a nor APA were
correlated with any of the weather data.
Relationships between Alkaline Phosphatase Activity. Chlorophyll a and
other Parameters
Alkaline phosphatase activity did not correlate with many water
chemistry parameters (Table 2-2). A significant correlation was
observed between chlorophyll a and total APA in December (r=0.82), while
APA was inversely related to TSP (r=-0.86). Correlations among
chlorophyll a, total APA and other measured parameters varied over time.
Chlorophyll a correlated with P four out of the six sampling periods and
total APA only correlated with P twice (Table 2-2). On a site by site
basis, different correlations between APA and water chemistry parameters
were observed (Table 2-3). Spatial variability of the factors affecting
APA was apparent.
Utilizing annual means, total APA was negatively correlated with
TOC (r=-0.97) and chlorophyll a was highly correlated with TS (r=-0.88).
Specific APA (ratio of total APA/chlorophyll a) was not correlated with
any of the water chemistry parameters.
Piel Variability
On March 21 1989 and February 6 1990 selected parameters
influencing APA were measured over a 24 h period to determine diel


100
Experimental Design
Experiment 1. Six intact sediment cores were collected by boat
from the center of the lake on September 25 1989. The cores were taken
to a depth of 30 cm, capped and brought to the laboratory and maintained
under ambient light and temperature conditions. Overlying water was
siphoned off to leave equal volumes of water in all cores. Previous
studies have shown that the concentration of SRP in the porewater of the
upper 10 cm sediments remain close to ambient lake water concentrations
(Reddy and Graetz 1990). The high water content (98%) associated with
the low SRP concentrations of the surface sediments suggest that these
sediments are frequently resuspended. Consequently, the surface 10 cm
of sediment was resuspended into the overlying water column for 1 h
using a sediment resuspension device similar to that described by
Wolanski et al. (1989), (Fig. 4-2). This involved the use of a 52 cm
long Plexiglas rod to which 19 Plexiglas rings (approximately 1 cm
thick) were attached at 2 cm intervals. The rod was oscillated within
the core above the sediment surface. The depth of placement of the rod
was adjusted so that only the surface 10 cm of sediment was resuspended.
The surface sediments in three cores were resuspended and the remaining
three cores were left undisturbed as controls. During resuspension a
composite 70 mL sample was withdrawn by syringe from the resuspended
material (time =1 h). At predetermined intervals, 70 mL composite
samples were withdrawn from the water column of all cores. Water
samples were analyzed for total and soluble APA, SRP, total soluble P
(TSP), TP, TKN and total suspended solids (TSS). At the end of the 24 h
sampling period, the surface 10 cm of sediments of the cores


BIOAVAILABILITY OF ORGANIC PHOSPHORUS
IN A SHALLOW HYPEREUTROPHIC LAKE
By
SUSAN NEWMAN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1991

ACKNOWLEDGEMENTS
I would like to thank Dr. Reddy, my major advisor, whose help and
guidance made this undertaking an enjoyable learning experience. I
would also like to thank the members of my committee, who were always
willing to share their expertize. Appreciation is also expressed to Mr.
Rick Aldridge whose assistance and lively discussion enabled me to
conduct the nutrient enrichment experiments.
I would also like to thank my friends and colleagues in the Soil
Science Department, particularly those associated with the Wetland Soils
Laboratory, whose encouragement, assistance and cooperation made this
project flow more smoothly.
I would like to thank my parents, Joyce and Chris Newman, whose
love and support enabled me to complete my Ph.D. Without their
encouragement I would not have continued on to higher education.
Last but not least, I would like to thank Tom, whose love,
companionship and support boosted my morale numerous times throughout
this study.
ii
A

TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
LIST OF TABLES v
LIST OF FIGURES viii
ABSTRACT xii
CHAPTERS
1 INTRODUCTION 1
Statement of the Problem 1
Need for Research 2
Organic Phosphorus Mineralization 3
Alkaline Phosphatase Activity in the Water Column 8
Alkaline Phosphatase Activity in the Sediment 10
Objectives 12
Dissertation Format 14
2 SEASONAL VARIABILITY IN ALKALINE PHOSPHATASE ACTIVITY IN A
SHALLOW HYPEREUTROPHIC LAKE 16
Introduction 16
Materials and Methods 18
Results 23
Discussion 39
Conclusions 48
3 RESPONSE OF NATURAL PLANKTON POPULATIONS
TO NUTRIENT ENRICHMENT 50
Introduction 50
Materials and Methods 51
Results 60
Discussion 86
Conclusions 94
iii

4 THE EFFECT OF SEDIMENT RESUSPENSION ON ALKALINE PHOSPHATASE
ACTIVITY 95
Introduction 95
Materials and Methods 97
Results 105
Discussion 115
Conclusions 123
5 THE EFFECT OF SEDIMENT AND WATER COLUMN ANOXIA ON
ORGANIC PHOSPHORUS MINERALIZATION 124
Introduction 124
Materials and Methods 127
Results 134
Discussion 145
Conclusions 151
6 ORGANIC PHOSPHORUS CYCLING IN LAKE APOPKA 152
APPENDICES
A LORAN COORDINATES 156
B CONCENTRATIONS OF SELECTED WATER CHEMISTRY
PARAMETERS DETERMINED BIMONTHLY FROM
APRIL 1989 THROUGH FEBRUARY 1990,
AT 8 SITES IN LAKE APOPKA 157
REFERENCE LIST 168
BIOGRAPHICAL SKETCH 180
iv

LIST OF TABLES
Table page
2-1. Means of selected weather data measured at the central
station (mean 1 SE) 24
2-2. Correlation coefficients for chlorophyll and alkaline
phosphatase activity measured bimonthly at 7 sites in
Lake Apopka (significant at a=0.05, n=7) 32
2-3. Correlation coefficients between alkaline phosphatase
activity and selected parameters measured bimonthly at
8 sites in Lake Apopka (significant at a=0.05, n=6) 33
2-4. Correlation coefficients of selected water chemistry
parameters determined at 7 sites in October 1989
(significant at a=0.05, n=7) 42
3-1. Nutrient additions made to diluted lake water collected
in November 1989 54
3-2. Nutrient additions made to diluted lake water collected
in April and August 1990 56
3-3. Initial concentrations of selected parameters measured in
diluted lake water prior to nutrient addition
(triplicate samples) in November 1989 (mean 1 SE) 61
3-4. Chlorophyll a and specific alkaline phosphatase activity
measured in natural plankton populations collected in
November 1989, 72 h after receiving nitrogen and
phosphorus additions 62
3-5. Initial concentrations of selected parameters measured
in diluted lake water prior to nutrient addition
(triplicate samples) (mean 1 SE) 66
3-6. Phosphorus uptake rates for natural plankton populations
collected in August 1990, 30 min after receiving nitrogen
and phosphorus additions (mean 1 SE) 74
3-7. Hot water extractable phosphorus concentrations of composite
lake water samples collected in April 1990, 216 h after
nutrient additions 78
v

Mi
3-8. Specific hot water extractable phosphorus measured over time
in natural plankton populations collected in August 1990
after receiving nitrogen and phosphorus additions
(mean 1 SE) 81
3-9. Specific hot water extractable phosphorus measured over time
in natural plankton populations collected in August 1990
after receiving nitrogen and phosphorus additions
(mean 1 SE) 83
3-10. Specific alkaline phosphatase activity measured over time in
natural plankton populations collected in August 1990
after receiving nitrogen and phosphorus additions
(mean 1 SE) 87
4-1. Distribution of selected parameters measured in May 1989
within the water column at the center of Lake Apopka
(n=3) 106
4-2. Concentrations of parameters measured within the water
column of cores collected in September 1989 from the
center of Lake Apopka (mean 1 SE) 109
4-3. The distribution of alkaline phosphatase activity in
disturbed and undisturbed sediment cores collected from the
center of Lake Apopka in January 1990 (mean + 1 SE) 118
5-1. Concentrations of selected parameters measured in Lake
Apopka water in July 1990 (mean 1 SE) 135
5-2. Concentrations of selected parameters measured on sediments
incubated under six different redox levels for one
month (mean 1 SE) 140
5-3. Concentrations of selected parameters measured on sediments
incubated under six different redox levels for one
month (mean 1 SE) 143
5-4. Selected correlation coefficients between phosphorus
compounds and alkaline phosphatase activity measured in
sediments incubated for one month under six different
redox levels 144
APPENDIX TABLES
A. Loran coordinates of sampling sites on Lake Apopka
Group repetition interval:7980, Southeast USA 156
vi

page
B-l. Temperature 157
B-2. Secchi depth transparency 158

B-3. Dissolved oxygen 159
B-4. pH 160
B-5. Total solids 161
B-6. Total suspended solids 162
B-7. Chlorophyll a 163
B-8. Total organic carbon 164
B-9. Total Kjeldahl nitrogen 165
B-10. Total and soluble alkaline phosphatase activity 166
B-l 1. Phosphorus 167
vii

LIST OF FIGURES
Figure page
1-1. Diagram of the phosphorus cycle in Lake Apopka 4
2-1. Location of Lake Apopka and sampling sites 19
2-2. Seasonal variability of selected parameters determined
bimonthly at 7 sites in Lake Apopka 26
2-3. Seasonal variability of selected parameters determined
bimonthly at 7 sites in Lake Apopka 27
2-4. Size fractionation of alkaline phosphatase activity
determined bimonthly at 3 sites in Lake Apopka 29
2-5. Size fractionation of phosphorus concentrations determined
bimonthly at 3 sites in Lake Apopka 30
2-6. Diel variation of selected parameters measured February
6-7, 1990 at the center of Lake Apopka (site 8) 35
2-7. Diel variation of selected parameters measured February
6-7, 1990 at the center of Lake Apopka (site 8) 36
2-8. Diel variation of selected parameters measured February
6-7, 1990 at the center of Lake Apopka (site 8) 37
2-9. Distribution of phosphorus compounds determined in whole
lake water at 8 sites in October 1989 38
2-10. Distribution of phosphorus compounds determined in
filtered lake water at 8 sites in October 1989 40
2-11. Distribution of suspended phosphorus compounds determined
by difference between whole and soluble phosphorus measured
at 8 sites in October 1989 41
3-1. Map showing the location of Lake Apopka and sampling sites.
Water was collected from site 1 in November 1989 and from
site 2 in April and August 1990 53
viii

m3
3-2. Time courses of alkaline phosphatase activity following
nutrient enrichment of natural plankton populations
collected in November 1989 63
3-3. Time courses of nutrient concentrations following
nutrient enrichment of natural plankton populations
collected in November 1989 65
3-4. Time courses of chlorophyll a concentrations following
nutrient enrichment of natural plankton populations
collected in April 1990 68
3-5. Time courses of chlorophyll a concentrations following
nutrient enrichment of natural plankton populations
collected in August 1990 69
3-6. Time courses of soluble reactive phosphorus concentrations
following nutrient enrichment of natural plankton
populations collected in April 1990 71
3-7. Time courses of soluble reactive phosphorus concentrations
following nutrient enrichment of natural plankton
populations collected in August 1990 73
3-8. Time courses of nutrient concentrations following nutrient
enrichment of natural plankton populations collected in
April 1990 75
3-9. Time courses of nutrient concentrations following nutrient
enrichment of natural plankton populations collected in
August 1990 grown at 29C 76
3-10. Time courses of hot water extractable total soluble
phosphorus following nutrient enrichment of natural plankton
populations collected in August 1990 79
3-11. Time courses of hot water extractable soluble reactive
phosphorus following nutrient enrichment of natural plankton
populations collected in August 1990 82
3-12. Time courses of alkaline phosphatase activity following
nutrient enrichment of natural plankton populations
collected in April 1990 84
3-13. Time courses of alkaline phosphatase activity following
nutrient enrichment of natural plankton populations
collected in August 1990 85
4-1. Map showing the location of Lake Apopka 98
4-2. Diagram of the sediment resuspension device 101
ix

fiage
4-3. The depth distribution of selected parameters measured in
triplicate sediment cores collected in Masy 1989 from
the center of Lake Apopka 107
4-4. The depth distribution of selected parameters measured in
triplicate sediment cores collected in May 1989 from
the center of Lake Apopka 108
4-5. The depth distribution of alkaline phosphatase activity in
triplicate resuspended and undisturbed (control) sediment
cores collected in September 1989 from Lake Apopka Ill
4-6. Concentrations of selected parameters measured in the
overlying water column following sediment resuspension of
0, 2, 5, and 10 cm surficial sediments 112
4-7. The total alkaline phosphatase activity measured in the
overlying water column following sediment resuspension of
0, 2, 5, and 10 cm surficial sediments 113
4-8. The relationship between alkaline phosphatase activity and
total suspended solids in the overlying water column of
triplicate sediment cores after resuspension of surficial
sediments 114
4-9. Soluble alkaline phosphatase activity measured in the
overlying water column following sediment resuspension of
0, 2, 5, and 10 cm surficial sediments 116
4-10. Soluble reactive phosphorus concentrations measured in the
overlying water column following sediment resuspension of
0, 2, 5, and 10 cm surficial sediments 117
5-1. Diagram illustrating the apparatus used to control dissolved
oxygen concentration and redox potential 129
5-2. The sediment extraction scheme used to fractionate organic
phosphorus in sediment 131
5-3. Nutrient concentrations in Lake Apopka water incubated
in the dark under aerobic and anaerobic conditions 136
5-4. Nutrient concentrations in Lake Apopka water incubated
in the dark under aerobic and anaerobic conditions 138
5-5. Alkaline phosphatase activity in Lake Apopka water
incubated in the dark under aerobic and anaerobic
conditions 139
x

fiage
5-6. Concentrations of selected parameters measured in sediments
incubated under six different redox levels for one month .
141

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
BIOAVAILABILITY OF ORGANIC PHOSPHORUS
IN A SHALLOW HYPEREUTROPHIC LAKE
By
Susan Newman
May 1991
Chairman: K. R. Reddy
Major Department: Soil Science
Field and laboratory studies were conducted to determine the
importance of organic P mineralization in the sediment-water column of
Lake Apopka, a shallow hypereutrophic lake located in central Florida.
Alkaline phosphatase activity (APA) was used as a tool to indicate the
bioavailability of organic P to native plankton populations.
Spatial and temporal variability in total APA occurred in the
water column (range=4 to 45 nM min'1) in response to different water
chemistry characteristics. Nutrient enrichment studies demonstrated
that APA increased with plankton biomass and specific APA
(APA/chlorophyll a) values > 1 nmol APA ng chlorophyll a'1 min'1 occurred
during severe inorganic P limitation. In both the sediment and the
water column APA was mainly associated with particulate matter.
The APA of the plankton was inhibited by high inorganic P
concentrations. Phosphorus demand of the plankton was high, as
evidenced by the rapid uptake of added inorganic P. During the
xii

conditions of P limitation added inorganic P was immediately assimilated
and recovered in the surplus P pool within the plankton tissue, as
determined by hot water extraction. The plankton apparently utilized
this surplus pool of P for growth under low external inorganic P
concentrations.
Resuspension of surficial sediments increased the interaction
between sediments and the overlying water column, resulting in an
immediate increase in APA and total P (TP) in the water column,
indicating an increased potential for biological organic P hydrolysis
during periods of resuspension. The APA and TP decreased rapidly during
settling of suspended solids, following the cessation of turbulence.
Organic P mineralization was greater under aerobic than anaerobic
conditions in the sediment and overlying water column. Under aerobic
conditions (dissolved oxygen [DO]=6 mg L'1) APA in the water column
increased from 22 to 43 nM min1, while no change was observed under
anaerobic conditions (D0=<0.2 mg L'1). Sediment APA was a function of
Eh, the measured reduction potential of the sediment-water systems.
Under aerobic conditions (Eh=480 mV) APA was 10-fold higher than that
observed under anaerobic conditions (Eh=-240 mV). Enzymatic hydrolysis
of organic P compounds was significantly inhibited under anaerobic
conditions.
The results from this study suggest that the P requirement of
plankton in a highly productive lake may partially be met through the
enzymatic hydrolysis of organic P. Consequently, efforts to reduce
nutrient loading and thus reduce eutrophication should also evaluate the
bioavailability of organic P compounds in the system.
xiii

CHAPTER 1
INTRODUCTION
Eutrophication may be defined as the nutrient and/or organic
matter enrichment that produces high biological productivity (Likens
1972). This process is often accelerated by man, through allochthonous
loading to the system from surface runoff, agricultural drainage and
wastewater effluent.
Eutrophication of our waterbodies has recently become a major
concern due to the ever increasing need for resource conservation.
Consequently, efforts are now being made to further understand the
process of eutrophication and to identify key management strategies to
abate this process.
Statement of the Problem
Lake Apopka is a 12,500 ha lake located in central Florida. It
has a mean water depth of 2 m, overlying highly flocculent organic
sediments (Reddy and Graetz 1990). Historically, the lake had clear
water, submersed macrophytes and supported substantial sport fish
populations. However, the physico-chemical properties of the lake have
been altered through nutrient enrichment following the construction of
the Apopka-Beauclair canal, discharge of sewage to the lake, and back
pumping from the surrounding muck farms (USEPA 1979). Following the
1947 hurricane, the submerged vegetation was uprooted, and the first
1

2
algal bloom was recorded (USEPA 1979). Since then, sport fish
populations have dwindled and have been replaced by rough fish such as
shad, gar and catfish. The high algal populations have resulted in the
maintenance of a pea-green color in the lake. Lake Apopka has mean
chlorophyll a concentrations > 60 /g L'1 and total P concentrations of
200 ng L1 (Canfield 1981; Huber et al. 1982; Reddy and Graetz 1990) and
is thus currently classified as hypereutrophic (Forsberg and Ryding
1980). The lake is the first and largest in the Oklawaha chain of
lakes, consequently a ripple effect is apparent. The high nutrient
concentrations and algal blooms observed in Lake Apopka are evidenced
downstream in the other lakes in the chain.
Need for Research
In order to understand the process of eutrophication in Lake
Apopka and thus abate this process, it is necessary to determine the
cycling of C, N and P in the sediment-water column. In general, N and P
are the key elements involved in eutrophication (Chiaudani and Vighi
1982). Carbon fixation has been determined to be the driving force in
the productivity of Lake Apopka (Reddy and Graetz 1990). This is
apparent by the vast algal populations observed year round in the water
column. Settling of senescent algal cells has resulted in a highly
organic sediment. Consequently, both the sediment and the water column
are dominated by organic matter which results in high levels of organic
N and P. However, readily available P, i.e, soluble inorganic P
concentrations are low and frequently undetectable (< 1 /g L'1)
(Newman,S., unpublished data, Department of Soil Science, University of

3
Florida, Gainesville, FL.). Researchers have investigated sorption
reactions (Olila, 0., unpublished data, Department of Soil Science,
University of Florida, Gainesville, FL.) and have characterized the
cycling of inorganic P within the sediment and water column (Pollman,
1983; Reddy and Graetz 1990), but the dynamics of the organic P pool,
the dominant form of P, have not been addressed. Studies have
demonstrated that significant quantities of organic P may be
bioavailable (Bradford and Peters 1987; Kuenzler and Perras 1965), hence
the organic P pool may play a significant role in sustaining the vast
plankton biomass under apparent inorganic P limitation. Therefore, the
potential bioavailability of organic P in Lake Apopka needs to be
determined.
Organic Phosphorus Mineralization
In aquatic systems, organic P in sediments constitute 15-50% of TP
(Bostrom et al. 1982) while in the water column organic P may account
for as much as 90% of TP (Rigler 1964). The P cycle is shown in Fig.
1-1. Organic P is generally characterized as total and soluble organic
P. Specific identification of organic P constituents may be achieved
following chromatographic fractionation and comparison with known
compounds (Minear 1972; Weimer and Armstrong 1979), 31P nuclear magnetic
resonance (NMR) (Condron et al. 1985), or via hydrolysis by specific
enzymes (Herbes 1974). Only 50% of organic P forms have been
identified, including: inositol phosphates, sugar phosphates,
phospholipids and nucleic acids (Stevenson 1982). The rate at which
these compounds are mineralized is dependent upon their structure. High

Figure 1-1. Diagram of the phosphorus cycle in Lake Apopka.

molecular weight, complex structures are highly resistant to
mineralization, hence they will tend to accumulate, e.g. humic acids.
Conversely, simple, low molecular weight organic compounds are more
susceptible to hydrolysis and thus more labile, e.g. sugarphosphates.
It is these labile compounds which are more likely to undergo rapid
enzymatic hydrolysis and thus be bioavailable.
Soluble reactive P (SRP), the most labile form of P, has been the
focus of most P cycling research (Hutchinson and Bowen 1950; Rigler
1956). However, SRP concentrations in lakes are generally low while
organic P is abundant (Abbott 1957; Rigler 1956). Such observations
resulted in investigations to determine whether organic P compounds
could act as a source of P available for plankton nutrition. Under
inorganic P limiting conditions phytoplankton may utilize organic P
compounds for growth (Harvey 1953; Kuenzler 1965). These phytoplankton
produce externally acting enzymes, phosphatases which hydrolyze
phosphomonoesters (PME) and release SRP (Fitzgerald and Nelson 1966;
Kuenzler and Perras 1965). Phytoplankton which do not produccce
externally acting enzymes cannot hydrolyze PME compounds and their
growth becomes P limited (Kuenzler 1965). Ecologically, the ability of
phytoplankton to utilize organic P gives them a competitive advantage
over non-phosphatase producers, during inorganic P limitation.
The mode of action of phosphatases (specifically
phosphomonoesterases) is shown below (Coleman and Gettins 1983):
1 2 3 4
ROP + E ~ ROP-E E-P E-P ~ E + P(
R = an organic moiety, P¡ = inorganic P, E = enzyme.

6
The steps involved in the reaction are as follows:
1. The phosphomonoester binds non-covalently to the phosphatase
enzyme (R0PE).
2. The phosphoseryl intermediate forms by covalent binding of
the phosphate group to the phosphatase enzyme (E-P); alcohol is
released during this nucleophilic attack.
3. Water is taken up resulting in the nucleophilic displacement of
serylphosphate to produce a non-covalently bound complex (E*P).
4. Inorganic P is released and the free phosphatase enzyme is
regenerated.
Phosphatases have a high degree of specificity for the P moiety of
the P-O-C bond, but little specificity for the C moiety (Reid and Wilson
1971). These enzymes are classified as alkaline or acid depending on
the pH range under which they exhibit optimum activity (Reichardt 1971;
Torriani 1960). At acid pH, the dephosphorylation of the serylphosphate
is the rate limiting step. At alkaline pH, the dissociation of
inorganic P from E*P is the rate limiting step (Coleman and Gettins
1983). The alkaline nature of most aquatic systems has resulted in
alkaline phosphatase activity (APA) receiving the most emphasis.
More than one type of phosphatase may be present in any plankton
population. Five intracellular phosphatases were extracted from a
Peridinium bloom (Wynne 1977). The phosphatases produced in response to
P limitation do not have the same biochemical characteristics as those
observed in normal tissues (Bielski 1974). Although acid phosphatases
have the ability to function outside the cell (Kuenzler and Perras 1965;

7
Price 1962), they are generally intracellular in action (Moller et al.
1975; Wynne 1977). Acid phosphatases function as specific enzymes in
metabolic pathways and non-specific reactions (Cembella et al. 1984a).
Hence they are constitutive and generally not repressible by inorganic
P. Conversely, APA exhibits principly extracellular function. Alkaline
phosphatase synthesis may be induced by the presence of organic P
(Aaronson and Patni 1976; Kuenzler 1965). Alkaline phosphatase is a
repressible enzyme (Jansson et al. 1988), whose synthesis is inhibited
by high levels of inorganic P (Elser and Kimmel 1986; Lien and Knutsen
1973; Torriani 1960). Inorganic P is a competitive inhibitor of APA
(Coleman and Gettins 1983; Moore 1969; Reid and Wilson 1971). Other
factors which affect APA include; temperature (Garen and Levinthal 1960;
Torriani 1960), chelators and divalent cations (Cembella et al. 1984a;
Healey 1973).
The derepression of APA in response to P limitation has been
examined at the cellular level where APA was shown to transport
inorganic P. Studies utilizing Escherichia coli have shown that two
forms of P transport exist (Rosenberg et al. 1977). One is a low
affinity system, phosphate inorganic transport (PIT), which is
constitutive and transfers intracellular P pools. The other is a high
affinity system, phosphate specific transport (PST), which is activated
when internal P concentrations are low. This utilizes a membrane
associated protein, APA, to increase P uptake. The high affinity system
is inhibited at high concentrations of inorganic P. However, it is the
ability of APA to catalyze the hydrolysis of organic P compounds that
has received the most study in the aquatic environment.

8
The intensity of APA is dependent on the severity of P limitation.
As much as 6% of the total protein produced under P limiting conditions
may be attributed to APA (Garen and Levinthal 1960). The increase of
APA in response to inorganic P deficiency has resulted in the use of APA
as a tool to assess the P limitation of plankton.
Alkaline Phosphatase Activity in the Water Column
Inverse relationships between APA and SRP have been reported in
many species of plankton (Healey 1973; Olsson 1990; Pettersson 1980;
Pettersson et al. 1990). Under low SRP concentrations APA is
derepressed, and upon replenishment of external inorganic P, APA is
inhibited. In some situations no significant relationship is observed
between APA and SRP (Berman 1970; Taft et al. 1977). It has been
suggested that under these circumstances high concentrations of soluble
organic P counteract the inhibition caused by SRP by stimulating
induction of APA (Kuenzler 1965; Cembella et al. 1984a). Alternatively,
where no correlation exists, APA may reflect P demand rather than P
limitation (Taft et al. 1977).
In combination with the depletion of external concentrations of
inorganic P, P limitation in plankton is also demonstrated by reduced
internal P concentrations (Chrst and Overbeck 1987; Rhee 1973).
Inverse relationships between APA and surplus P have been recorded (Lien
and Knutsen 1973; Rhee 1973). Once internal P concentrations have been
reduced below critical levels, APA is produced (Chrst and Overbeck
1987; Fuhs et al. 1972). Alkaline phosphatase activities have been

9
observed to be 25 times greater under P limitation than under P
sufficiency (Fitzgerald and Nelson 1966).
Ecologically, the importance of APA is dependent on the co
occurrence of both substrates and enzymes. Numerous problems have been
associated with the determination of PME concentrations. The most
common method of determining PME has been to monitor SRP release from
filtered lake water following the addition of pure alkaline phosphatase
(Strickland and Parsons 1968). The simultaneous occurrence of PME and
APA by cyanobacteria blooms has been observed under low SRP
concentrations (Heath and Cooke 1975). In some lakes, the rate of
inorganic P release from PME equals the rate of P uptake by the
plankton. In other lakes a large discrepancy exists between these two
rates, with inorganic P release being considerably less than uptake rate
(Boavida and Heath 1988; Cotner and Heath 1988; Heath 1986), thus
leading these researchers to conclude that APA is not important in P
nutrition of plankton. One of the problems associated with this
conclusion is the use of filtered lake water in the analyses, therefore
the large particulate organic P pool is absent (Wetzel 1983).
Phosphatases have also been shown to release P from particulate matter
(Jansson 1977). Seventy-four percent of extractable P in phytoplankton
is susceptible to enzymatic hydrolysis and 80% of the organisms involved
in phytoplankton decomposition produce phosphatases (Halemejko and
Chrst 1984). These results, and the apparent absence of hydrolyzable
soluble PME in the water column (Herbes 1974; Herbes et al. 1975;
Pettersson 1980) suggest that it is the substrate availability that
limits enzymatic P cycling not APA (Jansson et al. 1988).

10
The interpretation of APA as a measure of P limitation is
complicated by the uncertainty of the origin of APA. Bacteria,
phytoplankton and zooplankton are considered to be dominant contributors
to this pool, and it is suggested that APA of algal origin is the most
important in the epilimnion (Jansson et al. 1988). High levels of
soluble APA indicate filterable activity and may reflect bacterial
associated APA (Stewart and Wetzel 1982), zooplankton excretion (Jansson
1976; Wynne and Gophen 1981) and cell lysis (Berman 1970). In many
lakes APA was determined to be mainly associated with phytoplankton,
based on co-occurrence of APA and algal blooms (Heath and Cooke 1975),
and as shown by correlations with chlorophyll a (Jones 1972a; Matavulj
and Flint 1987; Siuda et al. 1982; Smith and Kalff 1981) and size
fractionation of phosphatase activity (Chrst et al. 1989; Jansson
1977). Alkaline phosphatase activity has also been attributed to
bacteria through correlations with bacterial numbers (Jones 1972a;
Kobori and Taga 1979a). In shallow lakes, a large portion of
particulate material may be sedimentary in origin. Concentrations of P
compounds and bacterial numbers may be higher in sediments. Interaction
between sediment and the overlying water column may significantly affect
the mineralization of organic P in the overlying water. Hence, wind
events in shallow lakes can significantly affect APA.
Alkaline Phosphatase Activity in the Sediment
In lake sediments as much as 70% of TP can be in organic form
(Weimer and Armstrong 1979). In highly organic sediments, the relative
abundance of organic substrates may result in enhanced breakdown of

11
organic P (Ayyakannu and Chandramohen 1971). Numerous enzymes can be
utilized in organic P breakdown but the phosphatases, specifically APA,
are the most frequently cited (Halstead and McKercher 1975; Skujins
1976; Speir and Ross 1978).
As observed in the water column, APA in soils is positively
correlated with the concentration of organic matter (Harrison 1983;
Speir 1976) and age of organic matter (Rojo et al. 1990). Much of the
data concerning APA have been developed in upland soils (Geller and
Dobrotvorskaya 1961; Juma and Tabatabai 1978; Tabatabai and Bremner
1969); few studies have investigated APA in sediments. However, highly
significant APA has been reported in both freshwater (Klotz 1985a;
Sayler et al. 1979) and marine sediments (Ayyakannu and Chandramohen
1971; Kobori and Taga 1979b).
Phosphatase activity decreases with soil (Juma and Tabatabai 1978)
and sediment depth (Degobbis et al. 1984; Kobori and Taga 1979b). In
upland soils, this has been shown to correspond to decreases in
microbial biomass, C, N and organic P (Juma and Tabatabai 1978; Speir
and Ross 1978; Baligar et al. 1988). In sediments, redox potential
decreases significantly with depth, therefore an important distinction
between mineralization of organic compounds in sediments versus the
overlying water column is the concentration of oxygen. Limited oxygen
diffusion and rapid consumption of oxygen results in an oxygenated layer
at the sediment-water interface and decreasing oxygen with depth in the
sediment (Charlton 1980; Bostrom et aV. 1982). Alkaline phosphatase
activity is generally inhibited under anaerobic conditions, resulting in
a slower rate of organic P mineralization (Pulford and Tabatabai 1988).

12
However, in shallow lakes, wind induced resuspension of sediments to the
oxygenated water column, results in the rapid breakdown of organic P
(Pomeroy et al. 1965). A significant positive correlation between SRP
released and APA in the water column has been observed during sediment
resuspension (Degobbis et al. 1984).
Resuspension can physically transport SRP to the overlying water
column (Ryding and Forsberg 1977). It also increases the suspended
solids concentration within the overlying water. This particulate
material can provide 28-41% of algal available P (Dorich et al. 1985).
Consequently, resuspension of sediments can result in enhanced enzyme
activity and P availability within the water column. Hence sediments
can play a significant role in organic P mineralization in the overlying
water column.
Objectives
Bioavailability of organic P occurs through the action of enzymes
(Kuenzler and Perras 1965). These enzymes catalyze the release of
inorganic P from both soluble and particulate matter. Both APA and
organic P concentrations increase with increased eutrophication (Jones
1979b). Hence, Lake Apopka, which is classified hypereutrophic,
should support large concentrations of APA and organic P. This study is
based on the hypothesis that enzyme mediated P release is used to
support the vast algal populations during inorganic P limitation.
Without this ability to utilize organic P at times of high P demand and
inorganic P limitation, algal and bacterial species are nutrient
stressed. Very little is known about the bioavailability of organic P

13
in sub-tropical lakes, consequently, research investigating the
breakdown and utilization of organic P is essential in any assessment of
lake eutrophication.
The main components influencing the cycling of organic P within
the water column are; water chemistry, plankton uptake and release, and
sediment resuspension (Fig.1-1). To understand the role of organic P in
Lake Apopka the following questions were addressed.
(1) How is the enzymatic hydrolysis of organic P compounds
affected by other water chemistry parameters?
(2) Is Lake Apopka plankton APA inhibited by inorganic P and is
it produced in response to inorganic P limitation?
(3) What effect does sediment resuspension have upon organic P
mineralization rates?
The overall objective of this study was to evaluate the
significance of organic P compounds in Lake Apopka and determine their
potential bioavailability. Specific objectives are listed below.
(1) Determine the seasonal, spatial and diel variability of APA
within the water column.
Alkaline phosphatase activity is produced by organisms, hence any
factors such as changes in environmental conditions which affect
metabolism may therefore affect APA. The predominant biotic group in
Lake Apopka biota are the plankton, hence APA is linked to fluctuations
in response to plankton metabolism.
(2) Determine the influence of inorganic P upon the growth of
natural plankton populations.

14
Soluble inorganic P is the most readily available form of P for
plankton nutrition, however; in its absence organic P compounds may be
used for growth. The enzymatic hydrolysis of organic P compounds is
competitively inhibited by inorganic P. Soluble reactive P
concentrations in Lake Apopka are hypothesized to be too low to inhibit
APA. There is, however, the issue of internal concentrations of P which
may regulate organic P hydrolysis outside the cell.
(3) Evaluate the effect of sediment resuspension upon the
mineralization of organic P in the sediment and overlying
water column.
In shallow lakes, wind induced resuspension of sediments into the
overlying water column increases the interaction between these
compartments. It is hypothesized that resuspension of sediment
increases the concentration of organic substrates and associated
microorganisms in the water column and thus increases mineralization.
(4) Determine the effect of anoxia on organic P mineralization
in the sediment and water column.
Mineralization of organic compounds proceeds more rapidly under
aerobic than anaerobic conditions. Since a majority of sediments are
anaerobic, it is hypothesized that APA will be inhibited under anaerobic
conditions, resulting in a reduced mineralization rate.
Dissertation Format
Each chapter within this dissertation is written as an independent
manuscript intended for future publication. Chapter 2 focuses upon the
concentrations of P compounds and APA within the water column and the

15
various factors which affect them. Chapter 3 examines the P nutrient
status of natural plankton populations. Chapter 4 investigates the
effect of sediment-water column interactions upon organic P
bioavailability. The effect of anaerobic/aerobic conditions on organic
P bioavailability is examined in chapter 5. The overall conclusions and
the significance of these results are discussed in chapter 6.

CHAPTER 2
SEASONAL VARIABILITY IN ALKALINE PHOSPHATASE ACTIVITY IN A
SHALLOW HYPEREUTROPHIC LAKE
Introduction
Phosphorus is the major nutrient limiting plankton production in
many temperate lakes (Schindler 1977). Soluble reactive P (SRP) has
been the form of P most often studied (Rigler 1956); however, SRP is
only a small fraction of the total P (TP) pool. A significant component
of TP may be in organic form (Minear 1972; Rigler 1964). In lakes
where inorganic P availability is low, plankton may produce phosphatase
enzymes which hydrolyze organic P compounds with the release of
inorganic P (Fitzgerald and Nelson 1966; Kuenzler and Perras 1965).
These enzymes are designated alkaline or acid phosphatase, depending on
the pH range of optimum activity (Kuenzler and Perras 1965; Torriani
1960). The alkaline nature of most water bodies has resulted in
alkaline phosphatase activity (APA) receiving the most attention.
Although some APA has been determined to be constitutive (Kuenzler
1965), plankton produce increased APA under conditions of P limitation.
Alkaline phosphatase activity has hence been used as an indicator of P
limitation, however, APA may also reflect P demand, as evidenced by a
poor relationship between SRP and APA (Taft et al. 1977).
The release of inorganic P mediated by APA is dependent upon the
percentage of organic P which is hydrolyzable by the enzyme. Thirty-six
16

17
percent of organic P in seawater (Kobori and Taga 1979a) and 32% of
organic P in freshwater (Hino 1988) were hydrolyzed by phosphatase
enzymes. Organic P of algal origin is particularly sensitive to
hydrolysis; 74% of algal extracted P was hydrolyzed by APA, while 80% of
organisms involved in the decomposition of plankton produced
phosphatases (Halemejko and Chrst 1984). In eutrophic situations
TP and organic P concentrations can be high (Jones 1979b), resulting in
higher APA levels than in lower trophic states (Jones 1979b; Pick 1987).
Alkaline phosphatase activity may be a significant mechanism of
satisfying high P demand in eutrophic situations, as well as a means of
overcoming P limitation in nutrient poor environments. The intensity of
APA is subject to the physico-chemical conditions in the environment.
Enzyme activity is pH dependent and can respond negatively or positively
to pH fluctuations (Torriani 1960). Dissolved oxygen (DO) and
temperature also influence microbial enzyme activity and metabolism,
thus they may directly or indirectly affect APA (Garen and Levinthal
1960). These environmental effects demonstrate the potential for
seasonal and diel fluctuations in APA.
This chapter examines the impact of seasonality on APA in one of
Florida's largest hypereutrophic lakes, Lake Apopka. Despite low SRP
concentrations < 1 /ig L'\ chlorophyll a concentrations are
regularly > 100 ng L'1 (Canfield 1981; Reddy and Graetz 1990). High
standing crops may be maintained through the rapid recycling of SRP or
by obtaining P from other sources. Total soluble P (TSP) concentrations
of 255 ng P L'1 have been recorded in Lake Apopka (Reddy and Graetz
1990). Total soluble P may represent bioavailable P (Bradford and

18
Peters 1987), hence organic P compounds in this pool may potentially be
hydrolyzed by APA and release inorganic P. With the exception of
extensive research by Berman and colleagues on Lake Kinneret, Israel
(Berman 1970; Wynne and Berman 1980), the majority of studies
investigating APA have been conducted in cold temperate zones. Warmer
climates with mild winters which result in extended periods of
productivity, may result in increased P demand. More studies in warmer
climates are necessary.
The primary objective of this study was to examine the seasonal,
spatial and diel changes in APA to determine whether it represents P
limitation or high P demand. This would also determine what effect the
water chemistry has upon APA. Zooplankton, phytoplankton and
bacterioplankton may all contribute to the total APA pool (Jansson 1976;
Jones 1979a; Kuenzler and Perras 1965; Wynne and Gophen 1981). A second
objective was to estimate the relative importance of these contributors
based on filter size fractionation. Total soluble P may be used as an
indicator of bioavailable P; however, a third objective was to determine
the relationship between APA and other components of the TP pool.
Materials and Methods
Site Description
Lake Apopka is a 12,500 ha, located in central Florida, 28* 37' N
latitude, 81* 37' W longitude (Fig. 2-1). It has a mean depth of 2 m.
Water influxes to the lake include Apopka Springs and backpumping from
surrounding agricultural land. Outflow is northward through the Apopka-

VO
Fig. 2-1. Location of Lake Apopka and sampling sites.

20
Beauclair canal. The St. John's River Water Management District
(SJRWMD) weather station is located at the center of the lake (site 8).
Water Sampling
Bimonthly sampling. Lake water was collected bimonthly from April
1989 thru February 1990, from 8 sites in Lake Apopka (Fig. 2-1). Sites
1, 4 and 8 were selected to represent inflow, outflow and the center of
the lake. Site 2 was selected to determine littoral zone influences.
Site 5 was located close to a pump station and hence represented
backpumping from the surrounding agricultural land. Site 7 was
established close to an old fishing camp, and site 6 was selected to
correspond to extensive sediment studies which were conducted with
samples from that site. Site locations were established using Loran
coordinates (Appendix A). Three replicate water samples were collected
from a depth of 0.3 m using 1 L polyethylene bottles, from each site.
Samples were stored on ice until return to the laboratory. Water was
filtered through 0.45 nm membrane filters (Gelman) and analyzed for
soluble APA within 24 h. Other soluble parameters determined were total
soluble P and SRP. Soluble particulate P was defined as TSP-SRP (SPP).
Whole lake water was analyzed for total APA, total Kjeldahl N (TKN), TP,
SRP, total solids (TS), total suspended solids (TSS), total organic
carbon (TOC), and chlorophyll a. Seasonal water chemistry data
collected at site 8 were compared with the weather data provided by the
SJRWMD.
The contributors to the APA pool are frequently determined via
filter fractionation (Chrst and Overbeck 1987; Currie and Kalff 1984;
Currie et al. 1986). To distinguish between the contribution of

21
phytoplankton and bacteria, water samples collected from sites 1, 4 and
8 received further filtration. Subsamples of water were filtered
through 150, 8, 2.5, 0.45 and 0.2 /im filters. Water from these size
fractions was analyzed for chlorophyll a and determined to represent 80,
9, 3, 1 and 0% chlorophyll a distribution. To minimize the filtration
time, the filtration was not performed sequentially. Alkaline
phosphatase activity and TP were determined on all samples.
Dissolved oxygen and temperature (YSI, model 58), and pH (Orion,
model SA 230) were recorded at 0.3 m. Light penetration was estimated
by measuring Secchi disk transparency. Samples were collected at
approximately the same time at each sampling period to minimize the
effects of diel variation.
Piel sampling. Diel studies were conducted on March 21 1989 and
February 6 1990. In March 1989 DO and pH of the water were measured
from a pontoon boat which was anchored at the central station (site 8)
for 24 h. In February 1990, pH and DO measurements were determined by
SJRWMD personnel. Water samples at both time periods were collected
using an automatic sampler, and kept cool until returned to the
laboratory for analysis.
Fractionation of lake water phosphorus. To elucidate the
relationship between P and APA in the water column, the P forms were
separated analytically using discrete extraction procedures. In October
1989, water samples from all 8 sites were partitioned into total and
soluble; reactive P, acid hydrolyzable and organic P (APHA 1985).
Enzyme hydrolyzable P (EHP) was also determined.

22
Analytical Methods
Alkaline phosphatase activity was determined fluorometrically
(Healey and Hendzel 1979). One half mL of substrate,
3-o-methylfluorescein phosphate (Sigma Chemicals), at a concentration
determined to be non-limiting, in 0.1 M Tris buffer (Tham, Fisher
Scientific) pH 8.6, was added to 4.0 mL of lake water in a cuvette.
Both total (whole lake water) and soluble (filtered through 0.45 nm
Gelman membrane filter) APA were determined. The cuvettes were placed
in a water bath (25*C). At timed intervals during a 20 min period the
cuvettes were placed in the fluorometer and the fluorescence measured.
The enzyme activity was measured as an increase in fluorescence as the
substrate was enzymatically hydrolyzed to the fluorescent product.
Fluorescence units were converted to enzyme activity using a standard
calibration curve of 3-o-methylfluorescein (Sigma Chemicals). The
fluorescence was measured using a Turner fluorometer No. 110, equipped
with Turner lamp no. 110-853, in combination with 47 B primary and 2a-
12 secondary filters. Autoclaved lake water with substrate added was
used as a control.
Chlorophyll a was determined spectrophotometrically following
extraction with acetone (APHA (1002-G), 1985). Total organic C was
measured using an 0. I. Corporation Model 524C TOC analyzer, following
oxidation by potassium persulfate. Total P, TSP, and TKN were
determined following Kjeldahl digestion. Soluble reactive P, TSP and TP
were analyzed via ascorbic acid using standard methods (APHA 1985).
Total solids and TSS were determined by standard methods (APHA 1985).

23
Enzyme hydrolyzable P was determined using the method of Strickland and
Parsons (1968).
Statistics
Data were analyzed using SAS for personal computers, version 6
(SAS 1985). Pearson correlation coefficients were determined for all
data.
Results
Measured parameters varied seasonally and spatially. Seasonal
variability was generally greater than spatial variability (data
presented graphically represent means of sites 2-8). Appendix B
contains tables presenting data on a site basis and demonstrates spatial
variability. Site 1 is the site of a natural spring, 80 ft deep and is
not subject to the same wind induced mixing as the rest of the lake.
Data from site 1 are discussed separately, to illustrate the effects of
spring input to the lake.
Physico-chemical Characteristics
A 14*C range in water temperature was observed over the sampling
period (Table 2-1). The highest water temperatures occurred in June and
August, and coincided with maximum photosynthetically active radiation
(PAR) (Table 2-1). Temperatures were coolest in October and December
during the decline of daylength. Data for other physical and chemical
characteristics are presented in Appendix B. Increased Secchi
transparency was recorded during October and December. Dissolved oxygen
concentrations ranged from 7 to 12 mg l1, with a mean of 9.6 mg L'1.

24
Table 2-1. Means of selected weather data measured at the central
station (mean 1 SE).
Date
Wind
1 m
above
sDeed
5 m
surface
Water
temp
PAR*
ym
km h'1
C
/xmol m2
s'1
8904
2 0
NA*
23.6
NA
8906
9 0
10 + 0
29.8
548
29
8908
11 0
13 0
28.5
508 +
29
8910
11 0
20 0
19.4
238
14
8912
11 + 0
13 + 0
15.5
294 +
18
9002
13 0
18 + 0
20.0
282
16
PAR indicates photosynthetically active radiation.
* NA indicates data not available.
Source: Stites, D. L., unpublished data, St. John's River Water
Management District, Palatka, FL.

25
Little variation in pH was observed with a range of 0.8 pH units
observed between maximum and minimum pH. Total solids were also
constant between 300 and 400 mg L'1, until February when a particularly
high concentration of 500 mg L'1 was recorded. Total suspended solids
accounted for approximately 16% of TS and increased in February at all
sites (Fig. 2-2a).
A distinct peak in chlorophyll a concentration was observed in
August at 5 of the 7 sites (Fig. 2-2b). This peak was 3 fold higher
than the minimum which occurred in February. Two peaks in TOC were
apparent. One occurred in August, along with chlorophyll a (Fig. 2-2c)
while the other occurred in February and probably corresponded to the
increase in TSS which also occurred in February. Total Kjeldahl N
tended to be higher in Spring and Summer and decreased in Fall and
Winter (Fig. 2-2d).
Alkaline phosphatase activity was mainly associated with
particulate matter (Fig. 2-3a). Soluble APA averaged only 3% of total
APA. Total APA peaked in both June and October; in contrast, soluble
APA peaked in October and December.
Phosphorus was also determined to be mainly associated with
particulate matter. Total P concentrations peaked in October
(Fig. 2-3b). Total soluble P concentrations represented from 6 to 37%
of TP in August and April respectively. Soluble reactive P
concentrations were very low throughout the year (0 to 7 ng L'1) and as
such were a minor portion of TP.
All parameters measured at site 1 were generally much lower than
those recorded at other stations (Appendix B). In particular DO and pH

CONCENTRATION
26
en
E
A J A O D F
1989-90
O*
E
A J A 0 D F
1989-90
Fig. 2-2. Seasonal variability of selected parameters determined
bimonthly at 7 sites in Lake Apopka: a) total suspended
solids, data were not collected in April and June;
b) chlorophyll a; c) total organic carbon, data were not
collected in April; d) total Kjeldahl nitrogen. Vertical bars
represent 1 SE.

PHOSPHORUS (/zg L ) APA (nM min
27
Fig. 2-
30
APR JUN AUG OCT DEC FEB
1989-90
3. Seasonal variability of selected parameters determined
bimonthly at 7 sites in Lake Apopka: a) alkaline phosphatase
activity; b) phosphorus. Vertical bars represent 1 SE.

28
were considerably lower and Secchi was significantly greater. Apopka
spring temperatures tend to be consistent throughout the year, only a
4*C fluctuation in water temperature was observed. Algal biomass was
significantly lower than observed in the rest of the lake. Annual
chlorophyll a concentrations averaged 21.8 /xg L'1 at site 1, while
values averaged 81 ng L1 at other sites.
The contribution of the various components in lake water to the
total APA pool was determined via filter size fractionation. The
distribution of APA followed that of chlorophyll a, with the majority of
the activity associated with the larger size fraction (Fig. 2-4), and
the distribution of APA within the different size fractions remained
constant throughout the year. The greatest amount of soluble APA
occurred in December. In general, a greater proportion of APA was
associated with 8 and 2.5 urn filtered samples in spring water at site 1,
than was observed in samples from sites 4 and 8. A similar distribution
was also determined for P (Fig. 2-5), although a greater proportion was
associated with the soluble fraction. Phosphorus concentrations peaked
in October at all three sites.
Relationships between Water Chemistry Data and Selected Environmental
Parameters
Seasonal patterns in PAR, wind speed and water temperature
collected at site 8 (lake center) were observed (Table 2-1). Water
temperature and PAR peaked in June and August. Wind speed measured 5 m
above the surface was generally greater than that measured 1 m above the
surface (Table 2-1). Total P was positively correlated with wind speed
observed 5 m above the water surface (r=0.88) and SRP was

ALKALINE PHOSPHATASE ACTIVITY (nM min
29
50
40
~ 30
20
10
0
1989-90
Fig. 2-4. Size fractionation of alkaline phosphatase activity
determined bimonthly at 3 sites in Lake Apopka: a) site
l=inflow; b)site 4=ouflow; c) site 8=center of lake.
Vertical bars represent 1 SE.

PHOSPHORUS CONCENTRATION (/zg
30
1989-90
Fig. 2-5. Size fractionation of phosphorus concentrations determined
bimonthly at 3 sites in Lake Apopka: a) site l=inflow;
b)site 4=ouflow; c) site 8=center of lake. Vertical bars
represent 1 SE.

31
positively correlated with PAR (r=0.98). Total Kjeldahl N was
inversely related to wind speed measured 1 m above the surface
(r=-0.94), while TSS was highly positively correlated with the wind
speed recorded 1 m above the surface (r=0.97). Total suspended solids
were also positively correlated with chlorophyll a, and inversely
correlated with TSP and SOP. Neither chlorophyll a nor APA were
correlated with any of the weather data.
Relationships between Alkaline Phosphatase Activity. Chlorophyll a and
other Parameters
Alkaline phosphatase activity did not correlate with many water
chemistry parameters (Table 2-2). A significant correlation was
observed between chlorophyll a and total APA in December (r=0.82), while
APA was inversely related to TSP (r=-0.86). Correlations among
chlorophyll a, total APA and other measured parameters varied over time.
Chlorophyll a correlated with P four out of the six sampling periods and
total APA only correlated with P twice (Table 2-2). On a site by site
basis, different correlations between APA and water chemistry parameters
were observed (Table 2-3). Spatial variability of the factors affecting
APA was apparent.
Utilizing annual means, total APA was negatively correlated with
TOC (r=-0.97) and chlorophyll a was highly correlated with TS (r=-0.88).
Specific APA (ratio of total APA/chlorophyll a) was not correlated with
any of the water chemistry parameters.
Piel Variability
On March 21 1989 and February 6 1990 selected parameters
influencing APA were measured over a 24 h period to determine diel

32
Table 2-2. Correlation coefficients for chlorophyll a and alkaline
phosphatase activity measured bimonthly at 7 sites in Lake
Apopka (significant at a=0.05, n=7).
Month
Correlations with
Chlorophyll a
Total APA
Soluble APA
April
TKN 0.88
TP 0.85
DO -0.74
temp -0.96
TP 0.75
SPP -0.67
NS*
June
TKN 0.92
TSP 0.76
TOC 0.68
SPP 0.78
soluble APA 0.74
DO -0.76
pH -0.79
August
NS
NS
NS
October
TOC -0.70
TSS 0.73
SPP -0.66
NS
TKN -0.75
TP 0.75
temp 0.71
TS -0.67
December
total APA 0.82
temp 0.68
secchi -0.80
TSP -0.86
SPP -0.86
TP 0.70
TSP 0.81
SPP 0.81
February
TP 0.71
TOC -0.69
NS
NS indicates not significant at a = 0.05.

33
Table 2-3. Correlation coefficients between alkaline phosphatase
activity and parameters measured bimonthly at 8
sites in Lake Apopka (significant at a=0.05, n=6)
Alkaline phosphatase activity
Site Total Soluble
1
DO
0.87
temp
-0.81
temp
-0.77
2
TKN
-0.78
TKN
-0.90
TOC
-0.81
TP
0.89
TSP
0.85
SPP
0.86
secchi
0.85
3
soluble APA
0.82
TP
0.75
secchi
0.79
secchi
0.79
4
NS'
CHL
0.75
DO
0.92
5
TP
-0.75
secchi
0.90
6
TSS
-0.93
secchi
0.81
7
NS
TP
-0.83
pH
0.73
8
TSP
0.83
NS
SPP
0.81
pH
0.80
DO
0.82
TOC
-0.92
NS indicates not significant at a = 0.05.

34
variation. Diel DO changes were observed at both time periods. No
significant changes in other measured water chemistry parameters were
observed in March 1989, while parameters did exhibit change in February
1990. In February 1990, TKN concentrations remained constant during the
first 12 h of sampling and then declined (Fig. 2-6a). Maximum TKN
corresponded to high PAR and wind speed (Fig. 2-7a and 2-7b). The
decline in TKN corresponded to the decrease of these two parameters. No
significant change was observed for SRP, while TP concentrations
fluctuated throughout the sampling period (Fig. 2-6b). Similar
fluctuations were also observed for total and soluble APA (Fig. 2-8a).
As observed for TKN, chlorophyll a concentrations tended to decrease in
conjunction with decreased PAR and wind speed, however, the range was
only 33 to 37 /xg L'1 (Fig. 2-8b).
Fractionation of Lake Water Phosphorus
In October, lake water samples were fractionated to determine the
different forms of P present. Site variability in chlorophyll a and TSS
concentrations was observed (Appendix B). Total organic C remained
constant at 30 mg L1 for all sites except site 1. Chlorophyll a and
TOC were lower at site 1.
As determined above, most of the total APA was in the particulate
fraction with the soluble APA contribution varying from site to site
(Appendix B). The distribution of the various P forms also exhibited
spatial variability (Fig. 2-9). In most sites TOP represented over 80%
of TP. Total acid hydrolyzable P contributed 10% and total reactive P
(TRP) contributed 3% to the TP pool. A similar distribution of these

(/g L_1) CONCENTRATION
35
TIME (h)
Fig. 2-6. Diel variation of selected paramters measured February 6-7,
1990 at the center of Lake Apopka (site 8): a) total Kjeldahl
nitrogen; b) total phosphorus. Vertical bars represent 1 SE.

/mol
36
TIME (h)
Fig. 2-7. Diel variation of selected parameters measured February 6-7,
1990 at the center of Lake Apopka (site 8):
a) photosynthetically active radiation; b) wind speed
1 m above the water surface. Source: Stites, D. L.,
unpublished data, St. John's River Water Management District,
Palatka, FL.

CONCENTRATION
37
TIME (h)
Fig. 2-8. Diel variation of selected parameters measured February 6-7,
1990 at the center of Lake Apopka (site 8): a) alkaline
phosphatase activity; b) chlorophyll a. Vertical bars
represent 1 SE. Chlorophyll a values were measured on
composite samples.

TOTAL P CONCENTRATION
38
Fig. 2-9. Distribution of phosphorus compounds determined in whole lake
water at 8 sites in October 1989. TP=total phosphorus, T0P=
total organic phosphorus, TAH=total acid hydrolyzable
phosphorus, TRP=total reactive phosphorus. Vertical bars
represent 1 SE.

39
components was also observed in filtered lake water (Fig. 2-10), organic
P represented > 90% of TSP. Soluble acid hydrolyzable P was a less
significant contributor to the total soluble P pool. Soluble reactive P
concentrations were negligible. Total soluble P accounted for 11 to
60% of TP at sites 8 and 7, respectively. The fraction of TP attributed
to suspended material was determined by difference between total and
soluble P fractions (Fig. 2-11). The distribution of P forms was
similar to that observed in whole lake water. Suspended TP represented
from 40 to 89% of TP. No enzyme hydrolyzable P was observed.
Correlating all the site means (including site 1), total APA was
positively correlated with chlorophyll a (r=0.88), TOC (r=0.84) and TSS
(r=0.85). However, plots of the data showed that these correlations
were an artifact of low values for water chemistry parameters at site 1.
Correlations without site 1 gave different conclusions (Table 2-4).
Total APA was observed to be inversely correlated with the acid
hydrolyzable fractions. Chlorophyll a was inversely correlated with TOC
(r=-0.68) and TSP (r=-0.66) and positively with TSS (r=0.73).
Discussion
Lake Apopka is a shallow lake with a surface area of 12,500 ha.
It has a small littoral zone and is subject to considerable wind induced
sediment resuspension. Frequent mixing and mild winters may help to
explain the lack of seasonality observed for several of the parameters
measured. Chlorophyll a, however, did exhibit a seasonal response.
Maximum chlorophyll a corresponded to high PAR and higher temperatures.
Over all months, chlorophyll a was only significantly and

SOLUBLE P CONCENTRATION
40
200
^ 150
w 100
50
0
Fig. 2-10. Distribution of phosphorus compounds determined in filtered
lake water at 8 sites in October 1989. TSP=total soluble
phosphorus, SOP=soluble organic phosphorus, SAH=soluble acid
hydrolyzable phosphorus, SRP=soluble reactive phosphorus.
Vertical bars represent 1 SE.
1 2 3
TSP
SOP
H SAH
M SRP
4 5
SITE
6 7 8

SUSPENDED P CONCENTRATION
41
SITE
Fig. 2-11. Distribution of suspended phosphorus compounds determined by
difference between whole and soluble P measured at 8 sites in
October 1989. TP=total phosphorus, T0P=total organic
phosphorus, TAH=total acid hydrolyzable phosphorus, TRP=total
reactive phosphorus. Vertical bars represent 1 SE.

42
Table 2-4. Correlation coefficients of selected water chemistry
parameters determined at 7 sites in October 1989
(significant at a=0.05, n=7).
TP
TRP
TAH
TOP
TSP
SAH
SOP TSUSP
SUSAHP
CHL
TOP
O
o
*
SAH
0.92
SOP
TSUSP
SUSRP
SUSAHP
0.95
0.78
0.99
0.94
1.00
0.86
SUSOP
TAPA
CHL
TOC
0.97
-0.80
0.96
-0.66*
-0.73
0.99
-0.84
-0.68
TSS
-0.78
0.74
0.73
Blank space indicates not significant at a=0.05.
* Significant at or=0.10.

43
inversely correlated with one water chemistry parameter, TS. This
correlation along with the ratio TS/chlorophyll a show that chlorophyll
a was not a dominant component of the solids in Lake Apopka during the
sampling period. In a frequently mixed system such as Lake Apopka, the
proportion of total solids which may be attributed to phytoplankton
biomass will fluctuate considerably. Other contributors to TS include;
bacteria, suspended sediment, zooplankton and inorganic and organic
compounds. The inverse relationship between chlorophyll a and TS may be
interpreted as follows; 1) increased herbivory by high zooplankton
populations and 2) light limitation because of high suspended solids.
A peak in TSS was observed in February, which corresponds to the
highest wind speed recorded 1 m above the water surface, and thus
reflects wind induced sediment resuspension. In a shallow lake, a large
proportion of particulate matter within the water column may frequently
be attributed to sediment resuspension. The sediments have high P
concentrations (Reddy and Graetz 1990) and resuspension will result in
increased levels of TP within the water column; TP concentrations were
positively correlated with wind speed (5 m above the surface).
Conversely, TKN concentrations were inversely related to wind speed (1 m
above the surface), and are mainly associated with the algal biomass.
The rate of P exchange between water and sediment increases during
suspension of sediment (Pomeroy et al. 1965). Part of this exchange may
be biological. Sediment resuspension into the oxygenated water column
results in aerobic mineralization of organic P (Lee et al. 1977).
Sediments exhibit significant phosphatase activity (Kobori and Taga
1979b; Ayyakannu and Chandramohen 1971) and a positive correlation

44
between SRP released and APA in the water column has been observed
during sediment resuspension to the overlying water (Degobbis et al.
1984). Assuming resuspended sediment were contributing significantly to
the predominately particulate APA pool in Lake Apopka, the ratio of
APA/TSS is a better measure of enzyme activity than APA alone.
Comparing monthly means, total APA/TSS was positively correlated with
soluble APA (r=0.99) and inversely correlated with TOC (r=-1.00). The
correlation between APA and TSS was only observed when data were
compared on a site basis. The overall lack of correlation between TSS
and APA in this study may be due to 1) insufficient sampling during
periods of sediment resuspension, 2) the relationship is hidden by the
variability in other parameters incorporated in the TSS pool, and 3)
sediment resuspension does not contribute to APA activity.
Alkaline phosphatase activity has been significantly correlated
with ATP (Pettersson 1980), particulate organic matter (Gage and Gorham
1985) and chlorophyll a (Healey and Hendzel 1979a; Pettersson 1980). In
this study, total APA was significantly correlated with chlorophyll a in
December and inversely correlated with TOC in February. Comparing
annual means, there is a strong inverse relationship between total APA
and TOC. This may be explained by examining the components of the TOC
pool; one contributor is humic material. Alkaline phosphatase activity
can be inhibited by high concentrations of humic materials (Francko
1986). The inverse relationship observed between APA and TOC could be a
result of binding of APA to organic material. Attachment of alkaline
phosphatase enzymes to particulate matter may decrease activity, but may
also increase longevity of the enzyme activity (Burns 1986).

45
Resuspension of sediment high in organic matter could bind enzymes
and/or release sediment bound APA to the overlying water column.
Organic inputs, living or dead should be considered when measuring APA
(Healey and Hendzel 1980).
Soluble APA is positively related to Secchi (r=0.87) suggesting
that there is a relationship between APA and water quality. But the low
proportion of APA observed in the soluble pool suggest that free
dissolved enzymes from cell lysis (Berman 1970) and enzymes excreted by
zooplankton (Wynne and Gophen 1981) were not as important as particulate
associated APA in the system. Organic phosphorus mineralization is
mainly be achieved by APA bound to particulate matter. Examining the
data from all sites (excluding site 1), APA was infrequently related to
chlorophyll a. The overall lack of correlation between APA and
chlorophyll a may be hidden due to the frequent mixing of the lake
water. The particulate nature of APA as determined by the size
fractionation scheme corresponds to the chlorophyll a distribution.
The correlation between APA and chlorophyll a leads to the expression of
APA as a ratio, i.e., APA/chlorophyll a (Pettersson 1980). This ratio
tends to increase with P limitation and decrease with trophic state
(Pick 1987). Combining data from numerous studies, Pettersson (1980)
determined that a ratio between 0.2 to 0.7 nmol APA ng chlorophyll a'1
min'1 could be used to indicate P limitation. When ambient lake
specific APA was consistently < 0.3 nmol APA /xg chlorophyll a'1 min'1
ratios greater than this were determined to indicate P limitation, i.e.,
elevated specific APA indicates P limitation (Istvnovics et al. 1990).
In this study a ratio of < 0.3 nmol APA nq chlorophyll a'1 min'1 was

46
consistently observed, suggesting that plankton in Lake Apopka are
generally not P limited. This conclusion tends to agree with other
research findings from this lake (Aldridge, F.J., personal
communication, Department of Fisheries and Aquaculture, University of
Florida, Gainesville, FL.; Reddy and Graetz 1990). Specific APA was
lowest when chlorophyll a peaked and SRP concentrations of 5 pq L'1 were
sufficient to support growth.
High particulate APA has been attributed to the location of
alkaline phosphatase in the cell wall of phytoplankton (Kuenzler and
Perras 1965). Recent research has suggested that viable phytoplankton
do not contribute much to the particulate APA pool (Stewart and Wetzel
1982). Lake Apopka is generally dominated by cyanobacteria, with large
numbers of Lyngbya sp. and Microcystis sp. (Shannon and Brezonik 1972;
Stites, D. L., personal communication, St. John's River Water Management
District, Palatka, FL.) whose mucilaginous layers can support
significant bacterial populations. It is likely that particulate APA is
attributable to both phytoplankton and the associated bacteria. Use of
specific APA (APA/chlorophyll a) to indicate P limitation of
phytoplankton should be verified via nutrient enrichment bioassays.
Diel fluctuations in APA were observed, but these do not
correspond to any particular water chemistry parameter. This may be
attributed to spatial patchiness and water movement (Berman 1970; Wynne
1981). Unlike TKN concentrations, APA did not settle out of the water
column following wind subsidence. Diel variability of APA may be
dependent upon the species composition of the phytoplankton biomass. In
diel studies neither Tballassiosira pseudonana Hasle and Heimdal (Perry

47
1976), nor Selenastrum capricornutum Prinz (Klotz 1985b) exhibited diel
responses. However, Smith and Kalff (1981) have shown that growth
demands for P are more important than the species composition in
determining APA.
Preliminary studies (data not shown) and April data demonstrated a
strong relationship between APA and TP. Consequently TP, which would
include cellular P, would indicate the P forms utilized by APA.
However, this relationship was not observed the entire year. Total
soluble P, has been suggested as a good indicator of bioavailable P in
eutrophic lakes (Bradford and Peters 1987). Correlations between total
and soluble APA and TSP were apparent in December, and at certain sites
(Table 2-3). Both positive and negative relationships were observed.
Total and soluble APA are apparently influenced by different parameters
at different sites (Table 2-3, Huber et al. 1985).
In October, the P fractionation experiment demonstrated that APA
was inversely related to the acid hydrolyzable P fractions. This
suggests that APA may be regulated by acid hydrolyzable compounds, or
alternatively they could be used as substrates for APA. Acid
hydrolyzable P represents the condensed polyphosphates, a storage form
of P in phytoplankton and bacteria. Surplus P concentrations within
algal cells have been shown to regulate the production of APA
(Fitzgerald and Nelson 1966; Lien and Knutsen 1973; Rhee 1973) and this
pool is composed of polyphosphates (Rhee 1972, 1973; Elgavish and
Elgavish 1980). Hence, the measurement of surplus P combined with APA
could provide a better understanding of the system. Another component
which may contribute to the understanding of APA is the concentration of

48
EHP. No EHP was found in Lake Apopka during the October fractionation
experiment. However, this does not reflect the absence of these
substrates. In some lakes the release rate of P from EHP satisfies the
P uptake rate (Chrst and Overbeck 1987) while in others a large
discrepancy exists between these two rates (Heath 1986; Boavida and
Heath 1988). Low EHP concentrations have been attributed to the rapid
hydrolysis of this fraction (Berman 1970; Taft et al. 1977).
Alternatively, this may reflect methodological problems; 1) the method
to measure EHP requires the addition of extracted APA from Escherichia
coli to filtered lake water. This enzyme was not adapted to this system
and consequently may not be as efficient or may require a longer
incubation time, 2) filtered lake water does not represent the entire P
pool available, as particulate organic P, a large portion of TP, may
also be susceptible to enzymatic hydrolysis (Jansson 1977), 3) enzymes
added from E. coli were more inhibited by inorganic P additions than
natural enzyme populations (Chrst et al. 1986). The first and third
problems were resolved by measuring the increase in SRP in filtered
water without the addition of the enzyme (Chrst et al. 1986). However,
in a lake which has high particulate APA, this would not be a true
representation of the potential APA.
Conclusions
Seasonal and spatial differences in water chemistry were observed.
In general, seasonal variability was greater than spatial variability.
The system was highly productive as evidenced by chlorophyll a
concentrations > 150 /g L'1, and an annual mean of 81 /ig L'1. Annual

49
means for TP and TKN were 210 ng L'1 and 4.8 mg L'1, respectively,
confirming the highly eutrophic state of the lake. Conversely, SRP
concentrations were consistently < 10 ng L'1.
Alkaline phosphatase activity was mainly associated with
particulate matter and was dependent on different water chemistry
parameters both seasonally and spatially. In general, APA was not
correlated to chlorophyll a. The relationship between these parameters
may be hidden as a result of the frequent mixing of the water column in
Lake Apopka. Both positive and negative correlations between P and APA
were observed. An inverse relationship existed between acid
hydrolyzable P and APA, indicating polyphosphates may be controlling
APA.
The particulate association of APA would suggest that APA should
be correlated with TSS, but this was rarely observed. This may be due
to the variability in the composition of the TSS pool. The relationship
between APA and particulate may be both beneficial and detrimental.
Binding to particles results in increased longevity of the enzyme, but
it also may inhibit APA by binding to the active site, as indicated by
the inverse relationship between APA and TOC.
Future research should examine the components of the TSS pool,
plankton and sediment and their effect on organic P mineralization under
controlled conditions.

CHAPTER 3
RESPONSE OF NATURAL PLANKTON POPULATIONS
TO NUTRIENT ENRICHMENT
Introduction
Soluble inorganic P is the main form of P utilized directly by
plankton. Phytoplankton growth rates close to maximal have been
determined in the apparent absence of inorganic P (Fuhs et al. 1972;
Smith and Kalff 1981). Methods used to measure soluble inorganic P are
for the most part limited in sensitivity (Rigler 1956; Tarapchak et al.
1982). This has resulted in the use of physiological indicators to
determine the nutritional status of plankton. Information is obtained
through a variety of methods including the determination of P uptake
rates (Lean and White 1983; Rigler 1956), the measurement of surplus P
concentrations (Fitzgerald and Nelson 1966; Rhee 1973) and the
determination of alkaline phosphatase activity (APA) (Berman 1970;
Kuenzler and Perras 1965). As external inorganic P concentrations
decline, plankton are able to utilize internal pools of surplus P to
maintain growth (Fitzgerald and Nelson 1966; Rhee 1972, 1973, 1974;
Wynne and Berman 1980). Once this internal source of P has been
reduced to a critical level some phytoplankton produce phosphatase
enzymes, which hydrolyze organic P compounds to inorganic P, to satisfy
nutritional demands (Chrst and Overbeck 1987; Kuenzler and Perras 1965;
Reichardt 1971). An inverse relationship between APA and surplus P has
50

51
been reported (Fitzgerald and Nelson 1966; Chrst and Overbeck 1987).
Inorganic Pisa competitive inhibitor of APA (Coleman and Gettins 1983;
Moore 1969; Reid and Wilson 1971). Upon replenishment of external
inorganic P concentrations enzyme activity is inhibited (Lien and
Knutsen 1973; Torriani 1960; Perry 1972) and surplus P accumulates (Rhee
1973). Surplus P is measured as hot water extractable P (HEP), and in
conjunction with APA has been shown to accurately assess P demand in
some lakes (Sproule and Kalff 1978; Pettersson 1980) but not in others
(Wynne and Berman 1980). Wynne and Berman (1980) observed that the HEP
concentration in Lake Kinneret, Israel, remained stable throughout the
year even under conditions of P stress and concluded that HEP was a
metabolic intermediate rather than a form of P storage.
Lake Apopka is a hypereutrophic lake with soluble reactive P (SRP)
concentrations frequently < 1 ng L'1. In contrast, concentrations of
total soluble P (TSP) and APA are high (chapter 2). The objectives of
this study were to determine whether high APA in Lake Apopka was due to
high demand for P or P limitation, and to evaluate the N and P
requirements of native plankton.
Materials and Methods
Site Description
Lake Apopka is a 12,500 ha lake located in central Florida (28*
37' N. latitude, 81* 37' W. longitude). It has a mean depth of
2 m. It has been proposed that the nutrient loading from the
surrounding agricultural and urban areas has precipitated the current
hypereutrophic conditions in the lake (USEPA 1979).

52
Sampling Procedures
Water samples were collected 30 cm below the water surface from
the west side of the lake on November 16 1989, and from the east side on
April 18 and August 21 1990 (Fig. 3-1). Water was stored in
polycarbonate and polyethylene carboys in the dark and at ambient
laboratory temperature, for no more than 24 h prior to the start of the
experiments.
Experimental Design
The effect of inorganic phosphorus concentrations upon alkaline
phosphatase activity
Lake water collected in November 1989 was diluted 2:1 (260 mL
unfiltered:140 mL filtered) with filtered lake water (0.45 /im), to
reduce the chlorophyll a concentration. Four hundred mL were placed in
each of 15 wide mouth 500 mL erlenmeyer flasks. The experimental design
was completely randomized with 5 treatments and 3 replicates. The water
was spiked with nutrient additions (Table 3-1). Nitrogen was added at
an N:P ratio of 10:1 to avoid N limitation. The flasks were capped with
cotton wool and placed on magnetic stir plates, under a black plastic
enclosure in the greenhouse. Temperature within the enclosure was
maintained using window air conditioning units and fans (mean 1SE,25*C
+ 0.21). Light was supplied at 200 /xmol photons m'2 s'1 using cool-white
fluorescent lamps. The light:dark schedule was 16:8. The flasks were
shaken and aliquots were withdrawn by syringe from treatments 1 and 2 at
0, 24, 72 and 96 h. Aliquots were removed from remaining treatments at
24, 72, and 168 h. Additional sampling times of 268 and 312 h were
included for cultures which received 1000 nq L'1. Samples requiring

Fig. 3-1. Hap showing the location of Lake Apopka and sampling sites. Water was collected from
site 1 in November 1989, and from site 2 in April and August 1990.

54
Table 3-1. Nutrient additions made to diluted lake water collected in
November 1989.
Treatment
number
Nutrient
N*
addition
P
g
L-1 -
1
0
0
2
500
0
3
0
10
4
1000
100
5
2500
1000
N and P were added as potassium nitrate and potassium dihydrogen
phosphate, respectively.

55
filtration were immediately filtered through 25 mm membrane filters
(0.45 nm) in polypropylene holders which attached to the syringes
(Gelman). The samples were analyzed for chlorophyll a, total and
soluble APA, total (TP), total Kjeldahl N (TKN), SRP, NH4-N, and [N03 +
N02]-N.
Nutrient enrichment of natural plankton populations
Experiment 1. Whole lake water was diluted 1:1 with filtered
lake water (0.45 /xm) and 400 mL were placed in each of 15 wide mouth
500 mL erlenmeyer flasks. The basic experimental design was a 22
factorial with an additional 2 fold addition of both N and P included
(Table 3-2). The flasks were stoppered with sponge plugs and placed in
a clear glass circulating water bath maintained at ambient lake
temperature (27'C). The flasks were illuminated from below at an
irradiance of 145 xmol photons m'2 s'1. The contents of the flasks were
mixed daily and immediately prior to sampling. Two h after the nutrient
addition, aliquots were withdrawn by syringe at predetermined intervals
and analyzed for total APA, SRP, NH4-N, [N03 + N02]-N, TSP and
chlorophyll a. Samples requiring filtration were filtered immediately
as described above. Hot water extractable P (HEP-SRP) was determined at
the beginning and conclusion of the experiment. To obtain a sufficient
sample size, HEP-SRP was determined on composite samples containing all
treatment replicates.
Experiment 2. To both confirm and compare the results with a
different plankton population, a second experiment similar to that
described above was conducted in August. Water samples were collected

56
Table 3-2. Nutrient additions made to diluted lake water collected in
April and August 1990.
Treatment
Nutrient
N*
addition
P
Incubation
temperature
fi g l
i
*C
Experiment 1-April
1
0
0
27
2
400
0
27
3
0
40
27
4
400
40
27
5
800
80
27
Experiment 2-August
1
0
0
29
2
400
0
29
3
0
40
29
4
400
40
29
5
800
80
29
6
0
0
19
7
0
40
19
N and P were added as potassium nitrate and potassium dihydrogen
phosphate, respectively.

57
from the same site, diluted and placed in water baths as described
above. The temperature of the bath was set at 29*C to emulate ambient
lake water temperature. Another water bath with cultures receiving no
nutrient addition and P only was maintained at 19*C, to determine the
effect of temperature on the measured parameters. Nutrient uptake rates
were determined by measuring the disappearance of the nutrient from
solution. During the first 2 h following nutrient addition, the
cultures were sampled every 1/2 h and analyzed for SRP, NH4-N, and
[N03 + N02]-N (actual sampling times were 0, 30, 67, 98 and 140 min).
Immediately following filtration aliquots were analyzed for SRP.
Samples to be analyzed for NH4-N and [N03 + N02]-N samples were
acidified with concentrated H2S04 and stored at 4*C prior to analysis.
Samples taken at 0 and 2 h were also analyzed for HEP-SRP, and hot water
extractable TSP (HEP-TSP). Aliquots were subsequently removed at 24, 48
and 96 h and analyzed for SRP, NH4-N, [N03 + N02]-N, total APA, TSP,
HEP-SRP, HEP-TSP and chlorophyll a.
Analytical Methods
Alkaline phosphatase activity was determined fluorometrically
(Healey and Hendzel 1979a). One half mL of substrate,
3-o-methylfluorescein phosphate (Sigma Chemicals), at a concentration
determined to be non-limiting, in 0.1 M Tris buffer (Tham, Fisher
Scientific) pH 8.6, was added to 4.0 mL of lake water in a cuvette. Both
total (whole lake water) and soluble (filtered through 0.45 nm Gelman
membrane filter) APA were determined. The cuvettes were placed in a
water bath (25*C). At timed intervals during a 20 min period the

58
cuvettes were placed in the fluorometer and fluorescence was measured.
The enzyme activity was measured as an increase in fluorescence as the
substrate was enzymatically hydrolyzed to the fluorescent product.
Fluorescence units were converted to enzyme activity using a standard
calibration curve of 3-o-methylfluorescein (Sigma Chemicals). The
fluorescence was measured using a Sequoia Turner fluorometer Model 110,
equipped with Turner lamp no. 110-853, in combination with 47 B
excitation and 2a-12 emission filters. Autoclaved lake water with
substrate added was used as a control.
Chlorophyll a was determined by measuring in vivo fluorescence,
using a Turner Design Model 10 fluorometer equipped with a Turner lamp
no. 110-853, in combination with 5-60 excitation and 2-64 emission
filters. A Sequoia Turner fluorometer Model 110 equipped with the same
light source and filters was used to measure chlorophyll a fluorescence
in the first experiment. Pheophytin a fluoresces at the same wavelength
as chlorophyll a, so chlorophyll a concentrations are uncorrected for
pheophytin. The calculation of chlorophyll a was based on equations by
Lorenzen (1967);
ABS x (vol. extracted (mL)) x (unit of measure factor) x 1
89 (vol. filtered (mL)) pathlength
(cm)
ABS = absorbance at 664 nm
89 = absorption coefficient for chlorophyll a in 90% acetone
Unit of measure factor = 108 for ng L'1.

59
Initial and final chlorophyll a concentrations were calibrated against
chlorophyll a concentrations determined spectrophotometrically following
extraction with 90% acetone (APHA 1985).
Total P, TSP, SRP, NH4-N, and [N03 + N02]-N were determined by
standard methods (APHA 1985).
Hot water extractable P was determined in experiment 1 using a
modification of the method by Fitzgerald and Nelson (1966). Water
samples (50 to 100 mL) were filtered through 47 mm 0.45 /xm membrane
filters (Millipore). The filters were placed in 60 mL borosilicate
boiling tubes, and 20 mL deionized water were added. The tubes were
capped and autoclaved at 120*C for 1 h. Upon cooling samples were
analyzed for SRP. Blank filters were autoclaved to determine any filter
contribution to P analysis.
Hot water extractable P includes both a molybdate reactive form of
P and a non-reactive form of P. Both hot water extractable forms were
determined in experiment 2, using the method of Krausse and Sheets
(1980). Nine mL of sample were filtered though 25 mm 0.45 nm membrane
filters (Gelman). The filters were placed in 15 mL polypropylene tubes,
and 13 mL of deionized water was added. The tubes were capped and
autoclaved at 120*C for 1 h. Filter blanks were also autoclaved.
Gelman filters were used instead of Millipore filters because they had a
lower background P concentrations. The filtrate was analyzed for SRP,
and also digested via persulfate oxidation and analyzed for TSP.

60
Statistical Methods
Data were analyzed using SAS (Statistical analysis systems)
version 6. Balanced data (equal number of observations for each
treatment) were analyzed using the repeated measures procedure which
accounts for the within replicate correlation over time, due to repeated
sampling from the same flasks. Unbalanced data (unequal number of
observations per treatment) were analyzed using a split plot design with
time as the subplot.
Results
The Effect of Inorganic Phosphorus Concentrations upon Alkaline
Phosphatase Activity
The majority of N, P and APA were associated with particulate
matter (Table 3-3). Due to chlorophyll a analysis problems arising from
fluorometer calibration, only chlorophyll a data from 0 and 72 h are
presented and used for statistical analysis (Table 3-4). Increased
chlorophyll a concentrations were observed at 72 h in all cultures
except those which received no nutrient addition. Increased growth was
associated with higher nutrient additions, a 69% increase in chlorophyll
a was observed in treatment 5 (N=2500 P=1000) cultures. Chlorophyll a
increases of 33% for treatment 4 (N=1000 P=100) and 11 % for treatments
2 (N=500 P=0) and 3 (N=0 P=10) cultures were observed. The opposite was
observed for both total and soluble APA (Fig. 3-2a and 3-2b). Total and
soluble APA decreased significantly over time in cultures receiving the
highest P additions (100 and 1000 ng L'1). No decrease in total APA was
observed for treatments 1 (N=0 P=0) and 3 (N=0 P=10), but soluble APA

61
Table 3-3. Initial concentrations of selected parameters measured in
diluted lake water prior to nutrient addition
(triplicate samples) in November 1989 (mean 1 SE).
Parameter
Concentration
Chlorophyll (tg L'1)
31
6
TP (Mg I/1).
65
4
SRP (jig L)
3
0.3
TKN (mg L'1)
3.30
+ 0.1
NH4-N (mg L'1)
0.36
0.08
[N03 + N02]-N (mg L'1)
0.14
0.00
Total APA (nM min'1)
13.5
0.3
Soluble APA (nM min'1)
2.2
0.13

62
Table 3-4. Chlorophyll a and specific alkaline phosphatase activity
measured in natural plankton populations collected in
November 1989, 72 h after receiving nitrogen and phosphorus
additions. l=no nutrient addition, 2=500 ng N L'1, 3=10 ng P
L \ 4=1000 ng N L'1 and 100 ng P L\ 5=2500 ng N L'1 and
1000 ng P L \
Treatment
Chlorophyll
Specific APA
M L-1
nmol APA ng chlorophyll a'1 min'1
1
36 a*
0.41
2
40 b
0.46
3
40 b
0.35
4
48 c
0.15
5
61 d
0.03
Numbers in a column followed by the same letter are not
significantly different at a = 0.05.

SOLUBLE APA (nM min ) TOTAL APA (nM min
63
TIME (h)
Fig. 3-2. Time courses of alkaline phosphatase activity following
nutrient enrichment of natural plankton populations
collected in November 1989. 0N,0P=no nutrient addition;
500N,0P=500 nq N L'1*, ON, 10P=10 pg P L\ 1000N, 100P=1000 fiq
N L'1 and 100 nq P L'\ and 2500N, 1000P=2500 nq N L1 and 1000
nq P L'1: a) total alkaline phosphatase activity; b) soluble
alkaline phosphatase activity. Vertical bars represent 1
SE. No vertical bar indicates SE is smaller than symbol
size.

64
did decrease. A significant increase in total APA was observed in
cultures which received only N additions suggesting the onset of P
limitation. This is more apparent when total APA data are presented as
specific activity (i.e. total APA/chlorophyll a) (Table 3-4). A 15 fold
difference between specific activity of those cultures which received
treatment 5 (N=2500 P=1000) and treatment 2 (N=500 P=0) was observed.
Initial specific APA was 0.44 nmol APA nq chlorophyll a'1 min'1, hence P
addition resulted in a decrease in specific APA. Significant decreases
in SRP concentrations were observed within 24 h (Fig. 3-3a). Apart from
treatment 5 (N=2500 P=1000), SRP concentrations were the same in all
cultures after 2 h. After an initial rapid uptake from 1000 to 673 /xg P
L'1 within the first 2 h, SRP concentrations in cultures treated with
1000 nq P L'1 remained constant until 168 h, and then began to
decrease. At 312 h, 377 nq P L'1, 37.6% of the original concentration
remained in treatment 5 (N=2500 P=1000) cultures.
Nitrate concentrations also exhibited significant treatment
differences within 24 h (Fig. 3-3b). The concentrations at 24 h were
ranked in descending order of original addition, with 1 and 3 treatments
having equivalent [N03 + N02]-N concentrations. In all cultures there
was a distinct decrease over time. A similar trend was observed for
NH4-N, concentrations appeared to decrease within 24 h (Fig. 3-3c),
however, no significant differences were determined.
Nutrient Enrichment of Natural Plankton Populations
Growth. Chlorophyll a concentrations and APA were lower in
April than in August (Table 3-5). Conversely, TP and TSP were

65
1200
cl7~ 800
^ o 4oo y^
100
0.3

ON.
OP

500N.
OP

ON,
10P

1000N,
100P

2500N.1 OOOP
t^lL
J
(b)
96 144 192
TIME (h)
240 288
Fig. 3-3. Time courses of nutrient concentrations following nutrient
enrichment of natural plankton populations collected in
November 1989. ON,OP=no nutrient addition, 500N,0P=500 ng N
L ON, 10P=10 ng P L1, 1000N, 100P=1000 tig N L1 and 100 fig
P L and 2500N, 1000P=2500 ng N L'1 and 1000 iig P L*1:
a) soluble reactive phosphorus; b) [N03 + N02]-N; c) NH4-N.
Vertical bars represent 1 SE. No vertical bar indicates SE
is smaller than symbol size.

66
Table 3-5. Initial concentrations of selected parameters measured in
diluted lake water prior to nutrient addition
(triplicate samples) (mean 1 SE).
Parameter
SamDlina Deriod
April
1990
August
1990
Chlorophyll a (/ig L'1)
22.5
0
37.4
0
TP (jig L )
113
12
56
9
tsp (/ig L-1)
70
10
9
0
SRP (/xg L'1)
1
0
2
0
HEP-TSP (/ig L'1)
ND*
33
34
HEP-SRP (/ig L'1)
7
0*
15
2*
NH4-N (mg L'1)
0.04
0
0.05
i 0
[N03 + N02]-N (mg L'1)
0
0
0.01
0
Total APA (nM min'1)
9.1
0.2
12.7
0.1
ND indicates data not determined.
Determined by the method of Krausse and Sheets (1980).
Determined by the method of Fitzgerald and Nelson (1966).

67
significantly higher in April. Chlorophyll a was used as a means to
indicate phytoplankton growth in the cultures. Chlorophyll a
concentrations increased in response to nutrient additions to the water
samples collected in both April and August. Although small and hidden
due to axis scale, significant treatment differences in chlorophyll a
occurred within the first 24 h (Fig. 3-4 and 3-5a). After 96 h, maximum
chlorophyll a concentrations in April were 80 /xg L'1 and exceeded
180 /xg L'1 in August. In April, chlorophyll a concentrations were
significantly affected by the interaction between concentrations of N
and P added. Chlorophyll a concentrations obtained in the presence of
both nutrients exceeded those obtained by single nutrient additions
(Fig. 3-4). The greatest initial increase in chlorophyll a occurred in
cultures which received treatment 2 (N=400 P=0). Subsequently, growth
rates in treatments 4 (N=400 P=40) and 5 (N=800 P=80) exceeded those of
treatment 2. This information, combined with the lag in growth observed
in cultures receiving only P additions, suggest that phytoplankton were
initially N limited and became co-limited by P as they grew. This is
confirmed by the significant interaction of added N and P levels upon
chlorophyll a concentrations. In contrast, no significant interaction
between the levels of N and P was observed in August. In August, those
cultures which received only P additions had the same chlorophyll a as
those which received both N and P, while cultures receiving only N had
the same chlorophyll a as those with no nutrient addition. Increases in
chlorophyll a were also recorded in cultures which received no P
addition. These observations suggest that some P was still available
for plankton growth, but the growth rates were P limited, and thus the

CHLOROPHYLL a (/g
68
200
-* 160
120
80
40
0
0 48 96 144 192
TIME (h)

ON,
OP
T
400N,
OP

ON,
40P
A
400N,
40P

SOON,
SOP
Fig. 3-4. Time courses of chlorophyll a concentrations following
nutrient enrichment of natural plankton populations
collected in April 1990. ON,OP=no nutrient addition,
400N,0P=400 iig N L'\ 0N,40P=40 fig P L'1; 400N,40P=400 ng
N L'1 and 40 ng P L\ and 800N,80P=800 ng N L1 and
80 ng P L'1. Vertical bars indicate 1 SE. No vertical bar
indicates SE is smaller than symbol size.

CHLOROPHYLL a (fig
69
Fig. 3-5. Time courses of chlorophyll a concentrations following
nutrient enrichment of natural plankton populations
collected in August 1990. 0N,0P=no nutrient addition;
400N,0P=400 ng N L\ 0N,40P=40 ng P L\ 400N,40P=400 ¡tq
N L'1 and 40 /xg P L'1, and 800N,80P=800 /xg N L1 and
80 ng P L'1: a) plankton grown at 29*C; b) plankton grown at
19#C. Vertical bars indicate 1 SE. No vertical bar
indicates SE is smaller than symbol size.

70
growth rate increased with increasing P concentration in the growth
media. It is suggested that cultures were only slightly limited by P,
because cultures with no P enrichment still increased in biomass.
During both experiments, the highest nutrient addition (treatment 5,
N=800 P=80) resulted in significantly greater chlorophyll a than any
other treatment.
Growth of plankton collected in August was inhibited at cooler
temperatures (Fig. 3-5b). Over the entire 96 h period chlorophyll a
only increased by 8 ng L'1. The phytoplankton grown at 19C with either
a P addition or no nutrient addition only achieved 65% of the
chlorophyll a of phytoplankton grown at 29C with no nutrient addition.
Nutrient uptake. In April, the initial sampling for nutrient
analyses occurred after 2 h; however, at this time SRP concentrations
had been significantly reduced, consequently to give a true
representation of the data a time 0 was included along with 2 h to the
data set. Time 0 represents the means of initial concentrations
measured in cultures with no nutrient addition, plus the respective
additions, hence approximate uptake rates can be envisioned (Fig. 3-6).
Only the cultures which received treatment 5 (N=800 P=80) had
significant SRP concentrations remaining after 2 h (17 ng L'1), SRP
concentrations in the other cultures were below 3 /xg L'1. These
concentrations remained close to baseline for the remainder of the
experiment. The determination of P uptake thus required a more
intensive sampling immediately following nutrient addition. This was
achieved in experiment 2, with water samples collected in August.
Within 30 min SRP had decreased from 81.6 to 37.4 ng L'1 in treatment 5

SRP (/g
71
l
90
80
70
60
50
40
30
20
10
0
0 48 96 144 192
TIME (h)
Fig. 3-6. Time courses of soluble reactive phosphorus concentrations
following nutrient enrichment of natural plankton
populations collected in April 1990. ON,OP=no nutrient
addition; 400N,0P=400 ng N L'1; 0N,40P=40 /xg P L*1;
400N,40P=400 ng N L1 and 40 ng P L*\ and 800N,80P=
800 ng N L'1 and 80 ig P L'1. Vertical bars indicate 1 SE.
No vertical bar indicates SE is smaller than symbol size.

72
(N=800 P=80), from 41.6 to 22 ng L'1 in treatment 4 (N=400 P=40) and
from 41.6 to 13.1 ng L'1 in treatment 3 (N=0 P=40) cultures (Fig. 3-7a).
Uptake rates were calculated as the disappearance of SRP within the
first 30 min (Table 3-6). This was selected to indicate maximal uptake
because the slope changed over time as the P demand decreased
(Fig. 3-7a). Cultures which received 80 ng L'1 had a significantly
greater uptake rate than those which received 40 ng L1. As expected,
temperature had a significant effect upon SRP uptake (Fig. 3-7b). The
uptake was not as rapid as that for the same treatment at 29*C
(a = 0.12), but even with the 10*C difference in temperature all SRP had
been depleted to below detection within 2 h. After 1 h, SRP levels in
treatments 3 (N=0 P=40) and 4 (N=400 P=40) were no longer significantly
different. The SRP concentrations continued to decrease in all the
cultures and were all close to baseline in 2 h and were undetectable (<1
ng L'1) after 24 h. In both experiments TSP decreased within the first
2 h and then remained constant (Fig. 3-8a and 3-9a).
The uptake of N differed between the two sampling periods. In
April, [N03 + N02]-N concentrations in the cultures decreased. The
concentration decreased by 25% in all treatments with N additions,
within 2 h and continued to decrease over time (Fig. 3-8b). In
contrast, NH4-N concentrations did not change in any of the cultures
until 216 h, when an increase was observed in control and N cultures
(Fig. 3-8c). In August, no significant treatment by time interaction
was recorded for [N03 + N02]-N, and no apparent uptake of [N03 + N02]-N
occurred (Fig. 3-9b). A significant treatment by time interaction was

SRP (/g
73
Fig. 3-7. Time courses of soluble reactive phosphorus concentrations
following nutrient enrichment of natural plankton
populations collected in August 1990. 0N,0P=no nutrient
addition; 400N,0P=400 ng N L'\ 0N,40P=40 ng P L'1*,
400N,40P=400 /ig N L1 and 40 fig P L\ and 800N,80P=
800 ng N L'1 and 80 \ig P L'1; a) plankton grown at 29*C; b)
plankton grown at 19C. Vertical bars indicate 1 SE. No
vertical bar indicates SE is smaller than symbol size.

74
Table 3-6. Phosphorus uptake rates for natural plankton populations
collected in August 1990, 30 min after receiving nitrogen
and phosphorus additions (mean SE). 3=40 /xg P L'1,
4=400 /xg N L1 and 40 jig P L1, 5=800 fig N L'1 and 80 /xg P L'1
7=40 /xg P L1 and plankton grown at 19*C.
Treatment
Uptake Rate
ng P
L'1 min'1
3
0.95
0.05 a*
4
0.43
0.27 b
5
1.47
0.06 c
7
0.65
0.01 ab*
Numbers in a column followed by the same letter are not
significantly different at a = 0.05.
Uptake rates for treatments 3 and 7 are significantly different at
a = 0.12.

TSP
75
TIME (h)
Fig. 3-8. Time courses of nutrient concentrations following nutrient
enrichment of natural plankton populations collected in
April 1990. 0N,0P=no nutrient addition; 400N,0P=400 ng
N L1; 0N,40P=40 ng P L'1; 400N,40P=400 ng N L'1 and 40 ng
P L'1, and 800N,80P=800 ng N L'1 and 80 ng P L'1: a) total
soluble phosphorus; b) [N03 + N02]-N; c) NH4-N. Vertical
bars indicate 1 SE. No vertical bar indicates SE is smaller
than symbol size.

TSP
76
l
200
^ 150
_i
2* 100
50
0
0.9
i
cn
E
0.6
0.3
0.0
0.3
0.2
X cn
z E
w0.1
0.0
0 24 48 72 96
TIME (h)
Fig. 3-9. Time courses of nutrient concentrations following nutrient
enrichment of natural plankton populations collected in
August 1990 grown at 29C. ON,OP=no nutrient addition;
400N,0P=400 fig N L*1; 0N,40P=40 fig P L'1; 400N,40P=400 ¡iq
N L1 and 40 fig P L'\ and 800N,80P=800 fig N L'1 and 80 fig
P L'1: a) total soluble phosphorus; b) [N03 + N02]-N;
c) NH4-N. Vertical bars indicate 1 SE. No vertical bar
indicates SE is smaller than symbol size.

77
observed for NH4-N, however, the data were highly variable, making
meaningful interpretation difficult (Fig. 3-9c). Plankton grown at 19*C
exhibited the same responses for TSP, [N03 + N02]-N, and NH4-N (data not
shown).
Surplus phosphorus. Soluble P extracted from plankton following
boiling with deionized water (HEP-SRP) was used as an indicator of
surplus P. In April HEP-SRP was determined at both the start and
conclusion of the experiment. Hot water extractable P decreased in
cultures which did not receive P additions but remained the same in
cultures which received 40 /xg L'1 (Table 3-7). Those cultures which
received 80 ng P L'1 had almost a 3-fold increase in HEP-SRP after
216 h. The role of HEP was examined in more detail in water samples
collected in August. Seventy percent of initial TP was accounted for by
HEP-TSP and TSP (Table 3-5), with HEP-TSP representing 59% of TP.
Within 2 h of P addition, substantial increases in HEP-TSP were observed
(Fig. 3-10a). Hot water extractable-TSP tripled from 33.3 to
99.3 /xg P L'1 upon addition of 80 /xg P L'1. Addition of 40 /xg P L1
resulted in a doubling of HEP-TSP to 65 and 69 /xg P L1 in cultures
which received treatments 3 (N=0 P=40) and 4 (N=400 P=40), respectively.
Even with a 10*C temperature difference HEP-TSP accumulated to 63.9 /xg P
L'1 within 2 h (Fig. 3-10b). Cultures grown at 19C which did not
receive nutrients had greater HEP-TSP concentrations after 2 h than
those with no nutrient addition grown at 29#C. At 29*C the HEP-TSP
remained constant within 24 h and then decreased by 15 ng P L'1 for P
added treatments. The downward trend continued to 96 h, HEP-TSP

78
Table 3-7. Hot water extractable phosphorus concentrations of composite
lake water samples collected in April 1990, 216 h after
nutrient additions. l=no nutrient addition, 2=400 nq N L'1,
3=40 nq P L'1, 4=400 ng N L1 and 40 ng P L'1,5=800 nq N L'1
and 80 nq P L1.
Treatment
Hot water extractable SRP
K L-1
1
3.0
2
2.8
3
7.2
4
6.8
5
18.4

HEP-TSP (fig
79
120
80
40
TIME (h)
Fig. 3-10. Time courses of hot water extractable total soluble
phosphorus following nutrient enrichment of natural plankton
populations collected in August 1990. 0N,0P=no nutrient
addition;400N,0P=400 pg N L ; 0N,40P=40 /xg P L'1;
400N,40P=400 M9 N L'1 and 40 jig P L'1, and 800N,80P=800 ng
N L'1 and 80 /xg P L'1: a) plankton grown at 29C; b)
plankton grown at 19C. Vertical bars indicate 1 SE. No
vertical bar indicates SE is smaller than symbol size.

80
concentrations in treatment 1 (N=0 P=0) cultures were lower than initial
concentrations while HEP-TSP concentrations in treatments 3 (N=0 P=40)
and 4 (N=400 P=40) were higher than initial concentrations. Normalizing
the data to chlorophyll a, a decrease in HEP-TSP was observed for all
treatments following the initial increase at 2 h (Table 3-8). The final
ratios were lower than the initial ratios.
The treatment by time response was different for HEP-SRP
(Fig. 3-11 a). The relative increases were greater. An increase in HEP-
SRP concentrations was observed after 48 h in cultures which received P,
except for treatment 4 (N=400 P=40). Hot water extractable P in
treatments 1 (N=0 P=0) and 2 (N=400 P=0) cultures remained constant. A
significantly lower increase was observed in treatment 7 (N=0 P=40,
temperature=19*C) (Fig. 3-llb a=0.06). Normalizing the data to
chlorophyll a resulted in a continuous decline in HEP-SRP. No increase
after 48 h was observed (Table 3-9). A significant correlation between
HEP-SRP and chlorophyll a was observed (r=0.56).
Alkaline phosphatase activity. Considerable differences in the
production of APA were observed between April and August (Fig. 3-12a and
13a). In both experiments, increases in APA were observed, however in
August this increase only lasted 48 h for all cultures grown at 29*C.
In April, APA in cultures receiving both N and P additions was inhibited
within 2 h, 28% and 11% inhibition for treatments 5 (N=800 P=80) and 4
(N=400 P=40), respectively. No inhibition was observed in treatments
receiving only a P addition. After initial inhibition, which lasted
24 h, APA increased (Fig. 3-12a). A significant interaction between N
and P was apparent at 48 h. Cultures receiving only P had a delayed

81
Table 3-8. Specific hot water extractable phosphorus measured over time
in natural plankton populations collected in August 1990
after receiving nitrogen and phosphorus additions (mean
1 SE). l=no nutrient addition, 2=400 ng N L"\ 3=40 ng P L'1,
4=400 ng N L'1 and 40 ng P L'1, 5=800 ng N L'1 and 80 fig P
L'1, and 6=no nutrient addition and 7=40 ng P L'1 and
plankton grown at 19*C.
Time h
Treatment ""0 2 24 48 96
Hg P ng chlorophyll a'1
1
0.89 0.07 0.73

0.04
0.68

0.02
0.51
+
0.02
0.36
0.02
2
0.92

0.03
0.67

0.04
0.51

0.02
0.33
0.02
3
1.75

0.03
1.34

0.03
0.89

0.04
0.45
0.01
4
1.85

0.06
1.25
+
0.03
0.90

0.04
0.44
0.02
5
2.66

0.05
1.82
+
0.01
1.25

0.04
NA'
6
1.00

0.07
0.82

0.02
0.72

0.07
NA
7
1.71
+
0.01
1.59

0.03
1.10

0.03
NA
NA indicates data is not available.

HEP SRP (/g
82
Fig. 3-11. Time courses of hot water extractable soluble reactive
phosphorus following nutrient enrichment of natural plankton
populations collected in August 1990. 0N,0P=no nutrient
addition-,400N,0P=400 ^g N l/1; 0N,40P=40 /xg P L'1-,
400N,40P=400 ng N L1 and 40 /xg P L*1, and 800N,80P=800 /xg
N L'1 and 80 ng P L'1: a) plankton grown at 29C; b)
plankton grown at 19C. Vertical bars indicate 1 SE. No
vertical bar indicates SE is smaller than symbol size.

83
Table 3-9. Specific hot water extractable phosphorus measured over time
in natural plankton populations collected in August 1990
after receiving nitrogen and phosphorus additions (mean
1 SE). l=no nutrient addition, 2=400 M9 N L1, 3=40 M9 P L'\
4=400 ng N L'1 and 40 M9 P L'1, 5=800 ng N L'1 and 80 ng P
L'1, and 6=no nutrient addition and 7=40 M9 P L'1 and
plankton grown at 19*C.
Time h
Treatment ~~0 2 24 48 96
/ig P M9 chlorophyll a'1
1
0.41 0.05 0.48

0.07
0.34

0.01
0.29

0.01
0.22

0.00
2
0.53

0.01
0.35

0.02
0.27
+
0.00
0.19

0.01
3
1.14
+
0.06
0.64

0.04
0.35

0.01
0.28

0.01
4
1.07
+
0.06
0.67

0.06
0.37
+
0.01
0.21

0.02
5
1.79

0.07
0.82

0.03
0.51

0.02
0.24

0.00
6
0.54
+
0.02
0.44

0.01
0.31

0.04
0.35

0.05
7
0.87
+
0.05
0.73

0.01
0.48

0.01
0.47
+
0.03

84
I
TIME (h)
Fig. 3-12. Time courses of alkaline phosphatase activity following
nutrient enrichment of natural plankton populations
collected in April 1990. 0N,0P=no nutrient addition;
400N,0P=400 nq N L'1; 0N,40P=40 ng P L1; 400N,40P=400 nq
N L'1 and 40 ng P L'1, and 800N,80P=800 nq N L'1 and 80 nq
P L'1: a) total alkaline phosphatase activity; b) specific
alkaline phosphatase activity. Vertical bars indicate 1 SE.
No vertical bar indicates SE is smaller than symbol size.

TOTAL APA (nM min
85
Fig. 3-13. Time courses of alkaline phosphatase activity following
nutrient enrichment of natural plankton populations
collected in August 1990. 0N,0P=no nutrient addition;
400N,0P=400 /xg N L'1; 0N,40P=40 nq P L1; 400N,40P=400 /xg
N L'1 and 40 nq P L'\ and 800N,80P=800 /xg N L'1 and 80 /xg
P L'1: a) plankton grown at 29C; b) plankton grown at 19*C.
Vertical bars indicate 1 SE. No vertical bar indicates SE
is smaller than symbol size.

86
response in APA increase and produced the least APA. A similar trend
was observed by phytoplankton cultured at 19*C in August (Fig. 3-13b).
In August the response of cultures grown at 290C with only P added
mimicked those which received both N and P (Fig. 3-13a). Disregarding
temperature effects, APA in cultures with and without P additions
exhibited similar trends; increasing up to 48 h and then decreasing
(Fig. 3- 13a). The initial increase in APA over the first 24 h was on
average 64%, greater in those cultures receiving P. The reverse was
true for the next 24 h period. Alkaline phosphatase activity measured
in treatments 1 (N=0 P=0) and 2 (N=400 P=0) doubled while only a 22%
increase was observed in treatments 3 (N=0 P=40) and 4 (N=400 P=40).
Cultures which received treatment 5 (N-800 P=80) did not exhibit a
change. Both groups subsequently declined by approximately
20 nM min'1 to 96 h. Transforming the APA data to specific activity
results in a different shape curve for April data (Fig. 3- 13b) but no
change in curve shape in August (Table 3-10). The interpretation from
both experiments is the same, .i.e., higher specific APA was apparent in
all cultures which did not receive any P addition. While those cultures
which received P had significantly lower specific APA. The highest
specific APA was recorded in April in cultures which received treatments
1 (N=0 P-0) and 2 (N=400 P-0).
Discussion
Nutrient loading from external and internal sources can influence
the productivity of phytoplankton and other aquatic biota. The response
of phytoplankton growth to nutrient enrichment has been used as an

87
Table 3-10. Specific alkaline phosphatase activity measured over time
in natural plankton populations collected in August 1990
after receiving nitrogen and phosphorus additions
(mean 1 SE). l=no nutrient addition, 2=400 ng N L'1, 3=40
ng P L'1, 4=400 /xg N L'1 and 40 ng P L'1, 5=800 ng N L'1 and
80 ng P L'\ and 6=no nutrient addition and 7=40 ng P L'1 and
plankton grown at 19C.
Time h
Treatment 0 24 48 96
nmol APA ng chlorophyll a'1 min
1
0.34 0.00
0.47

0.01
1.07
+
0.03
0.45

0.03
2
0.47

0.02
1.01
+
0.07
0.51

0.06
3
0.63

0.01
0.64

0.02
0.16

0.02
4
0.63

0.02
0.70

0.03
0.17

0.00
5
0.67
+
0.02
0.48
+
0.01
0.07

0.00
6
0.22
+
0.01
0.43

0.01
1.12

0.03
7
0.32
+
0.02
0.52

0.01
1.00

0.04

88
indicator of nutrient status. This study examined growth, uptake rate,
surplus P and APA to explain P requirements of phytoplankton and
associated microorganisms in Lake Apopka. Natural plankton populations
were used to include the contributions of bacteria and zooplankton to
the overall nutrient status of the lake. The objective was to determine
the response of phytoplankton biomass, hence chlorophyll a measurements
were used to indicate biomass.
This study demonstrated that APA is immediately and rapidly
inhibited by high inorganic P concentrations (Garen and Levinthal 1960;
Moore 1969; Torriani 1960). The extent of inhibition was dependent upon
the concentration of inorganic P added, internal P concentrations and
the growth of the plankton (Fitzgerald 1969). The most severe
inhibition was caused by 1000 ng P L'1. It has been suggested that 1000
ng P L1 is the minimum requirement for APA inhibition (Jones 1979b).
In combination with external P concentrations, the extent of APA
inhibition may also be dependent upon the initial internal nutrient
content. In April, APA was inhibited by 40 and 80 ng P L'1 within the
first 2 h but these concentrations did not result in such rapid
inhibition in August. Phytoplankton growth as determined by increases
in chlorophyll a concentrations, were initially limited by N and had
sufficient P so additions of P resulted in inhibition of APA. As the
phytoplankton grew and exhausted internal supplies, growth became
limited by both N and P, and APA increased. In August, phytoplankton
were P limited, thus the demand was sufficient that even the addition of
80 ng P L'1 did not inhibit APA. Initial APA was also higher in August
than April, and may reflect the difference between slight P limitation

89
and initial N limitation. Alternatively, the difference in response
may be due to different algal populations which have phosphatases with
different affinities (Pettersson 1980). In April, the algal population
(on a biovolume basis) was dominated by Microcystis sp. and pennate
diatoms. In August, the population was dominated by Microcystis sp. and
Lyngbya contorta (Aldridge, F. J., personal communication, Department of
Fisheries and Aquaculture, University of Florida, Gainesville, FL.).
Smith and Kalff (1981) showed that APA is influenced more by the
equilibration of plankton growth with nutrient supply, rather than the
species composition of the plankton population.
The P demand by plankton populations in Lake Apopka was very high
as shown by the rapid uptake of SRP in August. These rates are higher
than those obtained by similar methods from other lakes, i.e.,
0.02 to 0.03 ng P L1 min'1 (Rigler 1956) and 0.017 to 0.43 ng P L'1 min1
uptake rates (Lean and White 1983). Within the first 2 h of P
enrichment, an increase in HEP-SRP equivalent to 66% of inorganic P
added was observed. The HEP-TSP increase accounted for 86% of added
inorganic P. A 100C reduction in temperature decreased percentage
uptake to 43 and 78% as HEP-SRP and HEP-TSP, respectively. The
concentrations of HEP-TSP decreased as algal biomass increased. At 96 h
an increase in HEP-SRP was observed for cultures which received
treatments 3 (N=0 P=40) and 5 (N=800 P=80). Internal P concentrations
have been shown to regulate APA (Chrst and Overbeck 1987; Fitzgerald
and Nelson 1966; Moore 1969). The increase at 96 h thus could account
for the decreased APA. In April, 1989 HEP-SRP concentrations were shown
to be inversely related to APA, while in August a strong inverse

90
relationship was not recorded in response to treatment, suggesting that
internal P levels were not sufficiently low to control APA. Comparing
specific HEP-SRP (HEP-SRP/chlorophyll a) values in April, P limited
cultures had ratios < 0.09 fig P'1 fig chlorophyll a'1 while treatments 3
(N=0 P=40) and 5 (N=800 P=80) had ratios > 0.12 fig P L'1
ng chlorophyll a'1. Specific HEP-SRP ratios obtained after 96 h in
August were generally > 0.2 fig P L1 fig chlorophyll a1. In August,
after 96 h, an increase in HEP-SRP occurred at the same time as an
increase in growth rate for treatments 3 and 5. High growth rates in P
limited Scenedesmus sp. were associated with increased surplus P (Rhee
1974). No increase in HEP-SRP at 96 h was observed in the cultures
which received treatment 4. This may be anomalous because measurements
of both HEP-TSP and APA obtained from treatment 4 cultures agree with
those determined in treatment 3 cultures. Increased HEP-SRP
concentrations may not be the only factor regulating growth and P
limitation, because decreased APA was also observed in the other
cultures. Stable low concentrations of HEP-SRP, with rapidly declining
specific APA and uptake rates, combined with increasing chlorophyll a,
have been suggested to indicate that P is immediately utilized for
growth as opposed to P storage (Sproule and Kalff 1978). An inverse
relationship between specific APA and growth rate has been recorded
(Smith and Kalff 1981). Phosphorus required for growth is probably
provided from the hydrolysis of small chain polyphosphates from the
continually decreasing HEP-TSP concentrations. While HEP-TSP
concentrations declined, no increase in any other P parameter measured
was sufficient to account for the disappearance of HEP-TSP. Hence it

91
appears that HEP-TSP was utilized metabolically, and supports the
suggestion of Wynne and Berman (1980), that HEP-TSP can be utilized for
nutrition.
It was interesting to note that HEP-TSP concentrations increased
within 2 h in cultures which were not enriched with P. Phytoplankton
have been shown to grow even at SRP concentrations of < 3 /xg L'1
provided this concentration is maintained at the cell surface (Fuhs et
al. 1972). Hence, although SRP concentrations were low and did not
change, phytoplankton may have utilized P at the cell surface. In
conjunction with this, SRP concentrations were at the limit of detection
and not sensitive enough to record further decreases. Thus, it is
apparent that the monitoring of P limitation responses would provide a
more accurate assessment of the nutritional status of the plankton.
Alkaline phosphatase activity has been shown to increase with
eutrophication, in combination with increased plankton biomass (Gorham
and Gage 1985; Jones 1972b). Attempts to associate APA with specific
parameters has resulted in the normalization of APA to protein (Stevens
and Parr 1977), particulate organic matter (Gage and Gorham 1985) and
ATP (Healey and Hendzel 1980; Pettersson 1980). Specific APA (i.e.
normalized to chlorophyll a) has frequently been used as a measure of P
limitation and tends to decrease with trophic status (Pick 1987).
Specific APA values of 0.2-0.7 nmol APA /xg chlorophyll a'1 min'1 under
field conditions and values > 0.08 nmol APA /xg chlorophyll a'1 min'1
obtained in laboratory cultures have been suggested to indicate P
limitation (Pettersson 1980; Healey and Hendzel 1979a). In this study,
initial specific APA (0.3-0.4 nmol APA /xg chlorophyll a'1 min'1) would

92
thus indicate that this system is P limited. In April, specific APA of
plankton which received P additions remained between 0.3-0.4 nmol APA
ng chlorophyll a'1 min'1 for the first 96 h. Hence this ratio may
represent constitutive APA (Gage and Gorham 1985). Specific APA
determined in a eutrophic Swedish lake remained at < 0.3 nmol APA
ng chlorophyll a'1 min1 for most of the year but increased to 0.8 nmol
APA ng chlorophyll a1 min1 during periods of P limitation (Pettersson
et al. 1990). Specific APA thus increases with the severity of P
limitation (Perry 1976). In this study plankton grown in the absence of
P produced specific APA > 1 nmol APA ng chlorophyll a1 min'1. Greater
fluctuation in specific APA was apparent in August. However, comparing
specific APA with increases in chlorophyll a, in general, specific APA
measured in cultures which received P was inversely related to growth
rate (Rhee 1973; Smith and Kalff 1981). Specific APA determined
bimonthly at 7 sites in Lake Apopka from April 1989 to February 1990,
was < 0.3 nmol APA ng chlorophyll a'1 min'1, this suggests that growth of
Lake Apopka plankton was not severely P limited. This agrees with
results from other nutrient limitation studies conducted on Lake Apopka
(Aldridge, F. J., unpublished data, Department of Florida, University of
Florida, Gainesville, FL.). Hence, it would appear that the expression
of APA and HEP relative to chlorophyll a is an accurate indicator of
the nutrient status of plankton in Lake Apopka. However, it should be
re-emphasized that APA and HEP are found in numerous organisms, phyto
plankton, bacteria and zooplankton which vary both spatially and
temporally, absolute values may vary. Some uncertainty could be removed

93
by normalizing the data to ATP, and thus express the indicator relative
to living biomass (Healey and Hendzel 1980).
Although the focus of this research has been on P limitation, Lake
Apopka has been found to often be N limited or co-limited by both N and
P (Aldridge, F. J., unpublished data, Department of Fisheries and
Aquaculture, University of Florida, Gainesville, FL.). The uptake of
N03-N, shown to be the preferred form of N for Lake Apopka phytoplankton
(Aldridge, F. J., unpublished data), gives an insight into the N
requirements of the system. In April, a situation of co-limitation,
[N03 + N02]-N concentrations were shown to decrease over time, and NH4-N
concentrations tended to increase over time, suggesting that
mineralization of organic N occurred. In August, when plankton were
established as being slightly P limited, no decrease in [N03 + N02]-N
concentrations were observed.
The limitation of the analyses used in this study is that APA and
HEP concentrations do not differentiate between P limitation and co
limitation. Nitrogen limitation parameters, such as ammonium
enhancement, should also be determined to assess the N requirements of
the plankton and thus enable the assessment of N limitation in
conjunction with P limitation (Vincent et al. 1984).
The continued measurement of HEP-SRP as a P limitation indicator
has been questioned based on its highly dynamic nature (Wynne and Berman
1980; Cembella 1984b). Under ambient conditions such rapid
accumulation of HEP-SRP may not occur (Wynne and Berman 1980). Others
have shown that HEP-SRP is a good indicator of the nutritional status of
plankton (Pettersson 1980; Sproule and Kalff 1978). In this study it

94
appeared to be a reliable indicator of the nutritional status of the
plankton.
Conclusions
Results from this study show that APA of Lake Apopka plankton is
inhibited by high concentrations of inorganic P. Hence, ambient lake
water SRP concentrations (<10 ng L'1) will not be sufficient to inhibit
APA. Both P limitation and co-limitation of N and P were observed.
During conditions of inorganic P limitation, plankton P uptake
demand was very high as shown by the rapid uptake of SRP. Uptake rates
as high as 1.5 /xg L'1 min'1 were reported. The first step in the
metabolism of the added P was the accumulation of internal P as
identified by hot water extraction. After the apparent removal of all
SRP from the growth medium, plankton utilized P from the HEP-SRP and
HEP-TSP pools for growth. Studies assessing the importance of surplus P
frequently only determine HEP-SRP, however, this study highlighted the
need to measure both HEP-TSP and HEP-SRP.
Alkaline phosphatase activity tended to increase with growth of
the plankton, however, the intensity of APA was dependent upon the
conditions of P limitation. Severe P limitation was indicated by
specific APA values > 1 nmol APA /zg chlorophyll a'1 min'1. Specific APA
values < 1 nmol APA ng chlorophyll a'1 min'1 may indicate slight P
limitation, but APA is associated with numerous organisms in this
eutrophic system, which change both spatially and temporally, hence P
limitation should be confirmed by nutrient enrichment bioassays.

CHAPTER 4
THE EFFECT OF SEDIMENT RESUSPENSION ON ALKALINE PHOSPHATASE ACTIVITY
Introduction
In many lakes, exchange of P between the sediment and the water
column is dependent upon diffusion related processes (Stumm and Leckie
1971; Tessenow 1972). However, in shallow lakes, P exchange also occurs
due to sediment resuspension during wind events which increase the
interaction between the sediment and the overlying water column. It has
been estimated that the upper 10 cm of sediment is actively involved in
exchange reactions as a result of resuspension with the overlying water
column (Tessenow 1972; Schindler et al. 1977). However, the amount of
sediment that will mix with the water column is a function of the shear
stress and sediment type (Lee, 1970).
The immediate result of sediment resuspension is the increase in
suspended solids concentration in the water column. The suspended
sediment has been shown to provide 28-41% algal available P (Dorich et
al. 1985), as well as physically transporting soluble P to the water
(Ryding and Forsberg 1977). Sediment resuspension also results in
increased exchange of soluble reactive P (SRP) from the sediment to the
water column (Holdren and Armstrong 1980; Pollman 1983). The increase
in exchange has been associated with biological activity (Pomeroy et al.
1965). Conversely, SRP may be removed from the water column as a result
95

96
of sorption to the particulate material (Gchter and Mares 1985; Reddy
and Fisher 1990).
Resuspension results in increased aeration of the sediments.
Under aerobic conditions P release from sediments has been associated
with the decomposition of organic matter (Lee et al. 1977). If the
sediments are dominated by Fe, aeration results in increased
sedimentation of P (McQueen et al. 1986); however, in highly organic
sediments, biological processes catalyzed by numerous phosphatase
enzymes are likely to dominate (Ayyakkannu and Chandramohen 1971).
Organic P mineralization in sediments is regulated by the activity of
enzymes such as phosphatases, particularly alkaline phosphatase activity
(APA) in sediments at neutral and alkaline pH (Ayyakannu and
Chandramohen 1971; Kobori and Taga 1979b). A significant positive
correlation between SRP released and phosphatase activity in the water
column was observed during resuspension of marine sediments (Degobbis et
al. 1984). Thus the level of APA within the sediment will have an
effect on the APA subsequently resuspended in the overlying water
column.
Phosphatase activity decreases with depth of soils (Juma and
Tabatabai 1978; Speir and Ross 1978) and sediments (Degobbis et al.
1984; Kobori and Taga 1979b). This corresponds to a decrease in
microbial biomass, C, N and organic P with depth (Juma and Tabatabai
1978; Speir and Ross 1978, Baligar et al. 1988). Consequently the depth
of material resuspended is significant in influencing APA in the water
column.

97
Following subsidence of the wind event, suspended solids settle
thus transporting any associated material from the water column to the
sediment, i.e. APA, and results in a decrease in organic P
mineralization within the water column. Settling seston is also a sink
for SRP (Gchter and Mares 1985).
The objectives of this study were to determine; 1) the depth
distribution of APA in the sediment and the overlying water column and,
2) the effect of sediment resuspension upon SRP and APA release. It was
hypothesized that sediment resuspension may affect APA by 1) increasing
SRP levels in the water column and competitively inhibiting activity, 2)
releasing alkaline phosphatase from the sediment to the water column,
and 3) a combination of 1 and 2.
Materials and Methods
Site Description
Lake Apopka is a 12,500 ha hypereutrophic lake, located in central
Florida, 2837' N latitude, 8137' W longitude (Fig. 4-1). It has a
mean depth of 2 m. Chlorophyll a values exceeding 100 ng L'1 are
frequently recorded (chapter 2). Nutrient loading from the surrounding
agricultural and urban areas has resulted in the current hypereutrophic
conditions in the lake (USEPA 1979). The bottom sediments in the lake
have a 30 cm unconsolidated flocculent layer at the surface, underlain
by consolidated flocculent material (Reddy and Graetz 1990). The
sediments have an alkaline pH, hence the phosphatases of interest are
those with maximum activity in the alkaline region, i.e. alkaline
phosphatase.

Fig. 4-1. Map showing the location of Lake Apopka.
KO
00

99
Field sampling procedures
Water. Water samples were collected on May 23 1989, from the
center of the lake (Fig. 4-1.) using an alpha sampler (Wildco), at
depths 0, 0.5, 1 and 1.5 m below the surface. Water was stored in 1 L
polyethylene bottles kept on ice until return to the laboratory.
Dissolved oxygen (DO) and temperature (YSI, Model 58) and pH (Orion,
Model SA 230) were recorded with depth. Light penetration was estimated
by measuring the Secchi disk transparency. Within 24 h of return to the
laboratory, samples were analyzed for total and soluble APA. Other
parameters measured were total Kjeldahl N (TKN), total P (TP), SRP, and
chlorophyll a.
Sediment. Three intact sediment cores were collected from the
deck of a boat in May 1989 from the center of the lake using a 6 cm
(I.D.) x 1 m (length) Plexiglass-PVC sediment core sampler (Reddy and
Graetz, 1990). The cores were taken to a depth of 40 cm, capped and
brought to the laboratory for sectioning. The cores were sectioned at
0-2, 2-5, 5-10, 10-20 and 20-40 cm intervals, placed in 125 mL
centrifuge tubes, immediately purged with N2 and stored at 4C until
analysis. Preliminary studies demonstrated that sediments could be held
for at least 3 weeks at 4'C with no change in APA. Alkaline phosphatase
activity, porewater SRP, and water content were determined on wet
sediment. NaOH-extractable P, an indication of bioavailable inorganic P
and labile organic P (Dorich et al. 1985; Young et al. 1985) was also
measured. Total P, organic P and volatile solids content of dried
sediment were determined.

100
Experimental Design
Experiment 1. Six intact sediment cores were collected by boat
from the center of the lake on September 25 1989. The cores were taken
to a depth of 30 cm, capped and brought to the laboratory and maintained
under ambient light and temperature conditions. Overlying water was
siphoned off to leave equal volumes of water in all cores. Previous
studies have shown that the concentration of SRP in the porewater of the
upper 10 cm sediments remain close to ambient lake water concentrations
(Reddy and Graetz 1990). The high water content (98%) associated with
the low SRP concentrations of the surface sediments suggest that these
sediments are frequently resuspended. Consequently, the surface 10 cm
of sediment was resuspended into the overlying water column for 1 h
using a sediment resuspension device similar to that described by
Wolanski et al. (1989), (Fig. 4-2). This involved the use of a 52 cm
long Plexiglas rod to which 19 Plexiglas rings (approximately 1 cm
thick) were attached at 2 cm intervals. The rod was oscillated within
the core above the sediment surface. The depth of placement of the rod
was adjusted so that only the surface 10 cm of sediment was resuspended.
The surface sediments in three cores were resuspended and the remaining
three cores were left undisturbed as controls. During resuspension a
composite 70 mL sample was withdrawn by syringe from the resuspended
material (time =1 h). At predetermined intervals, 70 mL composite
samples were withdrawn from the water column of all cores. Water
samples were analyzed for total and soluble APA, SRP, total soluble P
(TSP), TP, TKN and total suspended solids (TSS). At the end of the 24 h
sampling period, the surface 10 cm of sediments of the cores

Sediment Suspension Device
Intact Sediment Core
Turbulence Generator
Fluid-Mud Mixture
(Lutocline)
Consolidated Sediment
(Gyttja)
Fig. 4-2. Diagram of the sediment resuspension device (Source: Reddy and Fisher 1990).

102
were sectioned into 2.5 cm intervals, placed in 125 mL centrifuge tubes,
immediately purged with N2 and stored at 4*C until analysis. The
sediments were analyzed for total APA, TP, organic P and porewater SRP.
Experiment 2. A second experiment was conducted in January 1990
to further evaluate the effect of varying depths of bottom sediment
resuspension upon APA and associated parameters within the water column.
Twelve sediment cores were collected on January 23 1990, to a depth of
20 cm. The overlying water was siphoned off to leave a 45 cm water
column. There were three treatments; the top 2, 5 or 10 cm of
triplicate cores were resuspended for 15 min using the procedures
described in experiment 1. There were 3 replicates for each treatment
and 1 undisturbed core to serve as a control. At predetermined
intervals, 70 mL water samples were withdrawn. The amount of resettling
was measured and the water withdrawn from the midpoint of the exposed
water. Alkaline phosphatase activity, TP, TKN, SRP, TSS and total
organic carbon (TOC) of the water column were determined. At the end of
the sampling period, the surface 2, 5 and 10 cm sediment fractions
representing the respective treatments were sectioned in resuspended and
control cores, and analyzed as described above. In addition, APA was
determined in the porewater using the method described for total
sediment APA, to determine whether the activity was predominantly
particulate or soluble.
Analytical Methods
Water. Alkaline phosphatase activity was determined
fluorometrically (Healey and Hendzel 1979a). One half mL of substrate,

103
3-o-methylfluorescein phosphate (Sigma Chemicals), at a concentration
determined to be non-limiting, in 0.1 M Tris buffer (Tham, Fisher
Scientific) pH 8.6, was added to 4.0 mL of lake water in a cuvette.
Both total (whole lake water) and soluble (filtered through 0.45 nm
Gel man membrane filter) APA were determined. The cuvettes were placed
in a water bath (25*C). At timed intervals during a 20 min period the
cuvettes were placed in the fluorometer and the fluorescence measured.
The enzyme activity was measured as an increase in fluorescence as the
substrate was enzymatically hydrolyzed to the fluorescent product.
Fluorescence units were converted to enzyme activity using a standard
calibration curve of 3-o-methylfluorescein (Sigma Chemicals). The
fluorescence was measured using a Turner fluorometer No. 110, equipped
with Turner lamp no. 110-853, in combination with 47 B primary and 2a-12
secondary filters. Autoclaved lake water with substrate added was used
as a control.
Chlorophyll a was determined spectrophotometrically following
extraction with acetone and correction for pheophytin (APHA (1002-G),
1985). Total P, TSP, TKN, TOC, SRP and TSS were determined by standard
methods (APHA 1985).
Sediments. Alkaline phosphatase activity was determined by a
method adapted from Sayler et al. (1979). Sediment samples (1 g wet
sediment) or 1 mL of porewater, were placed in centrifuge tubes.
Three mL of 1 M Tris-Tris HC1 buffer (pH 7.6) were added to each tube.
The average pH found in Lake Apopka sediments was approximately 7.6.
The samples were sonicated (Heatsystems Ultrasonics Model

104
W-220F) for 45 s at 30% relative output, to release cell bound
phosphatase. The samples were then incubated with 1 mL of 50 mg mL 1 of
p-nitrophenyl phosphate, (Sigma Chemicals) at 25* C for 1 h. After
1 h, 3 mL of 1 M NaOH were added to the tubes to stop the reaction and
enhance p-nitrophenol color. Controls to account for substrate color
and color release during solubilization of organic matter by the NaOH,
were obtained by incubating sediment without the substrate and
subsequently adding the substrate along with NaOH at the end of the
incubation. All samples were then centrifuged at 7000 rpm (7096 g) for
15 min. The liquid was removed and absorbance at 410 nm (Shimadzu Model
UV-160) measured. Concentrations were determined by calibration with a
standard curve of p-nitrophenol (Sigma Chemicals).
Porewater was extracted by centrifuging the sediment subsample
under anaerobic conditions at 5000 rpm (3620 g) for 15 min. The
porewater was immediately filtered through 0.45 pm Gelman membrane
filters. Soluble reactive P was measured using standard methods (APHA
1985). Sodium hydroxide extractable P was obtained by shaking 5 g of
wet sediment with 20 mL of 0.1 M NaOH for 16 h. Total P and SRP of
filtered extracts were then measured using standard methods described
for water.
Water content was determined by drying a known weight of wet
sediment at 70*C to a constant weight. The dried sediment was ground to
pass through a 20 mesh screen, using a Spex 8000 grinding mill.
Volatile solids were reported as the loss in weight due to ignition of
dried sediment at 500*C for 2 h. Total and organic P content of the
dried sediment was determined via ignition (Walker and Adams 1958).

105
Results
Physico-chemical Properties
Water. Nutrient concentrations were evenly distributed
throughout the water column (Table 4-1). Chlorophyll a, temperature,
DO, and pH decreased with depth. Total APA in the water column
increased with depth from a surface concentration of 37 to 44 nM min'1
at 1.5 m. Soluble APA was <2% of total APA. A high concentration of
soluble APA was measured at a depth of 1 m.
Sediment. Total APA in the sediments decreased with depth, with
the greatest change occurring within the 20-40 cm depth (Fig. 4-3a).
The water content of the sediments decreased from 98% at the surface to
95% in the 20-40 cm depth (data not shown). The porewater SRP remained
constant within the first 0-20 cm but increased significantly within the
20-40 cm depth from 0.01 mg L1 to 1.4 mg L'1 (Fig. 4-3b). Volatile
solids also increased at this depth (Fig. 4-3c). The opposite effect
was observed for NaOH-extractable P, organic P and TP, which decreased
in the 20-40 cm sediment depth (Fig. 4-4a, b, and c).
Sediment Resuspension Effects on Alkaline Phosphatase Activity
Experiment 1
Water column. Resuspension of the top 10 cm of sediment resulted
in increased TSS from 60 to 3000 mg L'1 Within 30 min after
resuspension, most of the sediment particles settled. Concentrations of
all parameters (excluding TSP) increased in the water column during
resuspension (time 1 h) and decreased to initial concentrations within
24 h (Table 4-2). Soluble parameters, e.g. SRP, exhibited considerable

106
Table 4-1. Distribution of selected parameters measured in May 1989,
within the water column at the center of Lake Apopka (n=3).
APA
Depth
Total
Soluble
TKN
TP
SRP
DO
pH
Chi
a Temp Secchi
m
--nM min'1--
mg L'1
--M9
L'1-
mg L'1
M9 L 1
C m
0
37.2
0.9
6.26
210
3
8.5
9.13
99
27.5 0.25
0.5
40.5
0.9
5.55
270
4
8.4
9.14
96
27.0
1.0
43.8
1.5
6.68
270
4
6.7
9.08
86
26.3
1.5
43.8
0.3
7.43
260
4
5.7
9.06
83
26.3

DEPTH (cm)
APA
. J 1 u~1
/imol g d.w. h
0 2 4 6 8 10
SRP VOLATILE SOLIDS
mg L 1 %
0 1 60 70 80
Fig. 4-3, The depth distribution of selected parameters measured in triplicate sediment cores collected
in May 1989 from the center of Lake Apopka: a) alkaline phosphatase activity; b) porewater
soluble reactive phosphorus; c) volatile solids. Bars indicate 1 SE. No bar indicates
SE is smaller than symbol size.

DEPTH (cm)
NaOH extractable P
mg kg
0 400 800
Organic P
mg kg
700 1400 2100
Inorg P and TP
. -1
mg kg
700 1400 2100
Fig. 4-4. The depth distribution of selected parameters measured in triplicate sediment cores collected
in May 1989 from the center of Lake Apopka: a) NaOH extractable phosphorus; b) organic
phosphorus; c) total and inorganic phosphorus. Horizontal bars indicate 1 SE. No bar £
indicates SE is smaller than symbol size.

Table 4-2. Concentrations of parameters measured within the water column of cores collected in
September 1989, from the center of Lake Apopka (mean 1 SE) -
APA
Treat
ment
Time

TKN
TP
TSP
SRP
Total
Soluble
TSS
h
L'1
M9
L-1
nM
min'1
mg
l '1
my
L
Control
0
3.55

0.06
100

13
100

28
11

3
8.8
0.3
0.04

0.00
72

2
1
3.65

0.12
90

6
90

3
5

0
8.0
0.6
0.09

0.03
75

4
24
3.89

0.12
150

30
90

10
4

1
6.9
0.3
1.20

0.17
54

3
48
4.31

0.12
180

50
60

3
5

0
12.5
0.7
0.41

0.06
47

3
Resusp-
0
3.79

0.33
80

3
100

8

5
5.6
0.29

0.20
59
+
1
ended
1
96.00

5.65
5733

245
70

10
5

1
109.1
3.0
0.07

0.06
3236

38
24
5.44

0.12
160

23
80

3
4

0
6.1
0.2
0.55

0.08
64

1
48
4.33

0.55
130

3
70

3
5

1
14.5
0.8
0.15

0.04
70

8
* Time 0 represents samples taken prior to resuspension, 1 h represents end of period of suspension.
Only 1 replicate per measurement.

110
variation with no apparent change in concentration. The undisturbed
control sediment cores maintained constant nutrient concentrations
throughout the duration of the experiment.
Sediment. The APA in the control sediment cores decreased with
sediment depth. No distinct depth profile of APA was observed in the
cores where the top 10 cm of sediment was resuspended (Fig. 4-5),
although there was a tendency for the APA to be lower in the surface
sediment and increase with depth. There was no net change in porewater
SRP, TP, organic P and inorganic P concentrations in any of the cores.
Experiment 2
Water. The resuspension of different depths of surface
sediment into the overlying water column resulted in distinct
differences among measured parameters. Resuspension of the surface
10 cm resulted in TSS values of 3000 mg L'1 which decreased to 100 mg L'1
within 1 h. (Fig. 4-6a). The resuspension of 5 cm of sediment produced
TSS values around 1700 mg L'1 which decreased to 174 mg L'1 within 1 h.
This compares to the settling of the 2 cm depth increment which produced
a resuspended TSS of 400 mg L'1 and decreased more slowly to 140 mg L'1
within 1 h. As observed in experiment 1, increases in TP, TKN and TOC
corresponded to TSS increases (Fig. 4-6). The total APA also increased
with TSS, and declined as the suspended matter settled (Fig. 4-7).
Combining the data from both resuspension experiments, the
relationship between total APA and TSS measured within the water column
(Fig. 4-8) was best described by the following power equation:
Total APA = 0.831 x (TSS)0565; r2 = 0.91 n=32

DEPTH
111
0.0
2.5
E
5.0
7.5
10.0
l
4 5 6 7 8 9 10
ALKALINE PHOSPHATASE ACTIVITY
(/mol g d.w. 1 h 1)
Fig. 4-5. The depth distribution of alkaline phosphatase activity in
triplicate resuspended and undisturbed (control) sediment
cores collected in September 1989 from the center of Lake
Apopka. Horizontal bars indicate 1 SE. No bar indicates SE
is smaller than symbol size.

CONCENTRATION
112
en
E
Fig. 4-6. Concentrations of selected parameters measured in the
overlying water column following sediment resuspension of 0,
2, 5, and 10 cm surficial sediments: a) total suspended
solids; b) total organic carbon; c) total phosphorus;
d) total Kjeldahl nitrogen. Initial data point indicates
the conclusion of resuspension. Vertical bars indicate
1 SE. No bar indicates SE is smaller than symbol size.

TOTAL ALKALINE PHOSPHATASE ACTIVITY
113
75
(a)
50
25
-
\
k 2 cm
n
1 L_
1... zz
~*
TIME (h)
Fig. 4-7. The total alkaline phosphatase activity measured in the
overlying water column of triplicate sediment cores after
resuspension of surficial sediments: a) 2 cm resuspended;
b) 5 cm resuspended; c) 10 cm resuspended; d) control,
no resuspension. Initial data point indicates the conclusion
of resuspension. Vertical bars indicate 1 SE. No bar
indicates SE is smaller than symbol size.

TOTAL ALKALINE PHOSPHATASE ACTIVITY
114
(mg L 1)
Fig. 4-8. The relationship between alkaline phosphatase activity and
total suspended solids in the overlying water column of
sediment cores after resuspension of surficial sediments.

115
The soluble APA measured in the water column after resuspension of
2 cm of sediment exhibited the same trend as observed for total APA,
decreasing as the TSS decreased (Fig. 4-9). Soluble APA did not show
significant differences over time in the cores in which the surface 5
and 10 cm were resuspended.
Soluble reactive P data exhibit such variability that no
statistically significant response was observed. However, all cores
including undisturbed cores, exhibited a tendency to increase at time
12 h (Fig. 4-10). This would suggest that SRP was slowly desorbed from
the underlying sediment.
Sediment. Changes in total and porewater APA within the sediment
of control and disturbed cores were not significant (Table 4-3). The
highly variable porewater APA accounted for less than 1% of the total
activity.
Discussion
The physico-chemical parameters measured within the water column
of Lake Apopka showed no distinct stratification, however, DO, pH,
temperature and chlorophyll a tended to decrease with depth. The
relatively high concentrations of nutrients and chlorophyll a in the
water column are characteristic of hypereutrophic systems, although SRP
concentrations are very low. The APA is also representative of
productive systems (cf. Heath and Cooke 1975). The soluble APA
accounted for only 3% of the total APA, therefore, the majority of APA
within this system was associated with particulate matter. A possible
incomplete settling of particulate matter would explain the increase in

SOLUBLE ALKALINE PHOSPHATASE ACTIVITY
116
0.8
0.4
(c)

-
-
J
1
_l
1 1
'
1 0 cm
i i
Fig. 4-9. Soluble alkaline phosphatase activity measured in the
overlying water column of triplicate sediment cores after
resuspension of 0, 2, 5 and 10 cm surficial sediments:
a) 2 cm resuspended; b) 5 cm resuspended; c) 10 cm
resuspended; d) control, no resuspension. Initial data
point indicates the conclusion of resuspension. Absence of
vertical bar indicates symbol size is greater than 1 SE.

SOLUBLE REACTIVE PHOSPHORUS
117
J 1 I I I L
1 O cm
Fig. 4-10. Soluble reactive phosphorus concentrations measured in the
overlying water column of triplicate sediment cores after
resuspension of 0, 2, 5 and 10 cm surficial sediments:
a) 2 cm resuspended; b) 5 cm resuspended; c) 10 cm
resuspended; d) control, no resuspension. Initial data
point indicates the conclusion of resuspension. Vertical
bar indicates 1 SE. Absence of vertical bar indicates
symbol size is greater than SE.

118
Table 4-3. The distribution of alkaline phosphatase activity in
disturbed and undisturbed sediment cores collected from
the center of Lake Apopka in January 1990 (means 1 SE).
Alkaline phosphatase activity
Total Porewater
/xmol g dry wt. '1 h'1 /xmol L'1 h'1
Control
2 cm
14.0'
0.91
5 cm
15.3
0.22
10 cm
10.5
1.07
ResusDended
2 cm
17.8 0.2
0.45
0.14
5 cm
14.0 1.5
0.34
0.03
10 cm
9.5 0.5
1.27
0.38
Control sediment cores were not replicated.

119
APA at the sediment-water interface, while there was no apparent change
in soluble APA.
In the sediment, APA was shown to decrease with depth. Soluble
reactive P is a competitive inhibitor of APA (Coleman and Gettins 1983),
hence the increase of porewater SRP with sediment depth may partially
explain the decrease in APA, due to inhibition of the production of the
enzyme. Organic P and APA have been shown to be positively correlated
(Juma and Tabatabai 1978; Speir and Ross 1978). A high enzyme activity
may be maintained in the presence of competitive inhibitors by the
presence of increased substrate concentrations; however, the decrease in
organic and NaOH-extractable P with depth suggests a decrease in
substrate concentrations. Alkaline phosphatase activity was also found
to have a positive correlation with organic matter (Speir and Ross
1978), therefore the decrease in volatile solids (an indicator of
organic matter), may have contributed to the APA decrease. However, the
most probable cause for the reduced activity with depth is a decrease in
microbial biomass. Decreasing APA with sediment depth has been shown
to have a positive correlation with microbial biomass (Sayler et al.
1979; Ayyakkannu and Chandramohen 1971). Microbial biomass was not
measured in this study, however, a decrease with depth could be expected
in these sediments because of highly reduced (anaerobic) conditions at
lower depths in Lake Apopka sediments (Moore et al. 1991). Pul ford and
Tabatabai (1988) suggested that under anaerobic conditions, the higher
solubility of metals such as Fe and Mn results inhibition of APA (Juma
and Tabatabai 1978). Lake Apopka sediment is Ca dominated (Moore et al.

120
1991), thus, inhibition of APA under anaerobic conditions is probably
due to reduced microbial numbers and metabolism.
The sediment APA observed in this study is in the same range as
those reported for other freshwater systems (APA = 6-18 /imol
g dry wt.'1 h'1) with fine particulate organic matter (Sayler et al.
1979). The APA of marine sediments was much lower, i.e., 0.2-3.3 /imol g
dry wt.'1 h'1 (Ayyakkannu and Chandramohen 1971; Degobbis et al. 1984).
However, a comparison of the sediment APA values obtained in other
studies is difficult due to the lack of standardization in methodology
used. Other complications include the pretreatment of samples, e.g.
drying of the soil (Tabatabai and Bremner 1969) which may affect the APA
values of some soils (Skujins 1976; Speir and Ross 1978).
Short-term resuspension of surficial sediments increased TSS, TKN,
TP and TOC concentrations. This is expected because all these
parameters are interrelated. The concentrations of these species
decreased rapidly following the end of resuspension, as a result of
particle settling. However, the soluble fraction, i.e. SRP, did not
exhibit significant increases as observed previously (Pollman 1983;
Reddy and Graetz 1990) in Lake Apopka cores. The porewater SRP
concentrations of the cores used in these resuspension experiments was
very low (<5 /xg L'1), thus the resuspension of these sediments would not
result in an increase in the SRP concentration of the water column.
Although the TP concentration in the surficial sediments is high (1200
mg kg1), a major portion is organically bound and is not readily
available. The low porewater SRP concentrations suggest that the
surface sediments have a high P sorptive capacity. A high C/P ratio of

121
the surface sediments also suggests that microbial immobilization of
inorganic P occurs (Reddy and Graetz 1990).
The mechanism of P release from suspended particles will depend on
the rate of P desorption from the solid phase to the liquid phase and
the physico-chemical properties of the water column. Resuspension has
been shown to increase the biological breakdown of organic P (Pomeroy et
al. 1965). Resuspension of anaerobic sediments to the overlying
oxygenated water column will result in aeration of the sediments. This
may make the associated organic matter more susceptible to enzymatic
hydrolysis (Pulford and Tabatabai 1988). The pH of the surface sediment
was 7, compared to a water column pH of 8-9. Thus any SRP release into
the water column upon resuspension could be immediately precipitated as
calcium phosphates (Moore et al. 1991).
In shallow lakes sediment resuspension may play an important role
in P recycling (Ryding and Forsberg 1977). Due to the association of
APA with TSS, prolonged resuspension would result in higher APA levels
within the water column. As observed by Burns (1986), the attachment of
APA at a non-active site would result in increased longevity of the
enzyme within the aquatic system. If phosphomonoesters are present SRP
would be released. The insignificant, but apparent gradual increase in
SRP in all cores at 12 h, may be due to the enzymatic degradation of the
more easily hydrolyzed organic P compounds. Hence, APA may enhance the
ability of sediments and particulate matter to recycle P. To test the
relationship observed between TSS and total APA, seasonal data collected
in the field (chapter 2) was fitted to the equation A good fit was
observed when TSS were high (= 100 mg L"1) and chlorophyll a

122
concentrations were low. However, with normal TSS (= 70 mg L'1) and
high chlorophyll a, the equation underestimated the measured APA. This
suggests that the predictive capability of this equation is only valid
during periods of resuspension, when sediment particulate matter is the
dominant component of the TSS pool. After 48 h an increase in total APA
was observed in the water column of both control and resuspended cores,
this may be in response to APA production following P limitation of the
plankton population. These increases are apparent as the two points
above the regression line in Fig. 4-8. Consequently, during quiescent
periods other contributors to the TSS pool. e.g. phytoplankton, should
be included to predict APA.
Apart from the cores in which the surface 2 cm was resuspended,
soluble APA was not shown to increase due to resuspension. The sediment
porewater APA was greater than the water soluble APA; however, the
dilution effect due to resuspension brought the porewater concentration
to ambient levels. The APA attributed to porewater exhibited high
variability. This variability could be partially due to differences
between cores, and also the insensitivity of the method at such low APA.
The fluorescent technique used for lake water would be more sensitive,
however, different substrate specificities have been observed
(Pettersson and Jansson 1978). Even considering the variability in the
porewater APA, it was clearly demonstrated that >99% of the APA was
associated with the solids portion of the sediment. A recent study
demonstrated the association of APA with the larger particulate matter
(Rojo et al. 1990) and it has been suggested by Burns (1986) that the

123
covalent bonding of enzymes to organic matter would result in increased
stabilization of the enzyme.
Conclusions
Most studies investigating APA in aquatic systems have emphasized
the contribution of free-living bacteria, phytoplankton and zooplankton.
This study demonstrated the contribution of the sediment associated APA
to the total APA pool of the water column. Sediment resuspension
resulted in increased APA and TP in the overlying water column, and
hence there was an increase in potential organic P mineralization.
This increased APA remained high only while sediment particles were
still suspended in the water column and decreased upon settling of the
sediment. This indicates that no APA was released from the sediment
into the water column. The APA determined in both sediments and lake
water was mainly associated with particulate matter and thus may have
increased longevity. The high APA recorded both in the sediment and
water columns suggests that organic P mineralization, via APA, may play
a significant role in P cycling of Lake Apopka. The importance of this
process is dependent upon the concentration of organic substrates. The
forms of sediment organic P capable of acting as substrates needs to be
evaluated.

CHAPTER 5
THE EFFECT OF SEDIMENT AND WATER COLUMN ANOXIA ON
ORGANIC PHOSPHORUS MINERALIZATION
Introduction
Organic P constitutes the major component of total P in the
sediment and water column of lakes. When soluble inorganic P
concentrations within the water column are low plankton may produce
phosphatase enzymes, which hydrolyze organic P compounds with the
release of inorganic P (Kuenzler and Perras 1965; Reichardt 1971). As a
result the measurement of alkaline phosphatase activity (APA) has been
used as a tool to indicate P limitation and potential organic P
mineralization through enzymatic hydrolysis (Healey and Hendzel 1979b;
Gage and Gorham 1985).
Under well oxygenated conditions (aerobic), mineralization of
organic P is rapid. However, depletion of dissolved oxygen (DO)
concentrations can occur in the water column as a result of high
respiratory activity. Under reduced DO concentrations the rate of
enzymatic hydrolysis of organic P compounds could be reduced as the
metabolism of aerobic plankton is reduced. This may be of particular
importance at the sediment-water interface where DO concentrations are
often depleted. Oxygen concentrations in the water column may vary both
diurnally (Howeler 1972; Reddy 1981) and seasonally (Mortimer 1941). In
rare circumstances, metalimnetic DO depletion may occur with increasing
124

125
eutrophication (Shapiro 1960). More often, highly productive aquatic
systems exhibit clinograde DO distributions, resulting in the depletion
of DO with water depth in response to summer stratification (Miyake and
Saruhashi 1956; Wetzel 1983) and consumption during high respiratory
activity at the sediment-water interface (Charlton 1980; Bostrom et al.
1982).
Diffusion of DO from the water, bioturbation and sediment
resuspension can maintain relatively aerobic conditions at the sediment-
water interface. Under these conditions P release from sediments to
overlying water can be due to mineralization of organic P (Lee et al.
1977). Mineralization of organic P may be of particular importance in
sediments which possess abundant organic substrates, such as peat
sediments (Ayyakannu and Chandramohen 1971).
During stratification of the water column, the hypolimnion may
become anoxic, hence the surface sediment becomes anaerobic. A
significant consequence of anaerobic conditions at the sediment-water
interface is the increase in water soluble P in the overlying water
column (Holdren and Armstrong 1980; Ponnamperuma 1972; Mortimer 1941).
The release of P under these conditions is the result of the reduction
of ferric phosphates to the more soluble ferrous phosphates (Mortimer
1941). This flux of P from the sediment to the overlying water column
may have a significant impact upon APA in the water column. Inorganic P
is a competitive inhibitor of APA (Coleman and Gettins 1983), and
sedimentary release of P has been shown to inhibit APA within the
overlying water column (Pettersson 1980). Anaerobic conditions may also

126
result in the inhibition of organic P mineralization by APA in the
sediment (Pulford and Tabatabai 1988).
Regardless of the oxygen status of the surface sediments, redox
potential tends to decrease with sediment depth (Kobori and Taga 1979b;
Degobbis et al. 1984). Bacterial populations were also observed to
decrease with sediment depth, in response to redox potential changes
(Kobori and Taga 1979b). Bacterial numbers have been positively
correlated with APA in sediments (Ayyakannu and Chandramohen 1971),
hence APA also decreases with depth (Kobori and Taga 1979b). An
important effect of reduced Eh conditions may be the accumulation of
soluble organic P compounds due to incomplete mineralization. It is
apparent that oxygen concentrations both in the sediment and in the
water column may play a significant role in determining P
mineralization.
The objective of this study was to determine the effects of anoxia
upon organic P mineralization in the sediment and water column of a
highly productive lake. It is known that APA is a non-specific enzyme
capable of hydrolyzing numerous organic P compounds (Coleman and Gettins
1983; Cembella et al. 1984a). However, specific forms of sedimentary
organic P are mainly unknown; identification is based upon the
susceptibility to different extraction solutions (Bowman and Cole 1978;
Sommers et al. 1972). A second objective of this research was to
determine the forms of extractable P influenced by different redox
conditions, and their effect on the mineralization of organic P by APA.

127
Materials and Methods
Site Description
Lake Apopka is a 12,500 ha hypereutrophic lake, located in central
Florida, 28*37' N latitude, 81*37' W longitude. It has a mean depth of
2 m. Chlorophyll a values exceeding 100 /zg L'1 are frequently recorded
(chapter 2). Nutrient loading from the surrounding agricultural and
urban areas has resulted in the current hypereutrophic conditions in the
lake (USEPA 1979). The bottom sediments in the lake have an average of
30 cm unconsolidated flocculent layer at the surface, underlain by
consolidated flocculent material (Reddy and Graetz 1990).
Sampling Procedures
Water column. Water was collected on July 2 1990, 30 cm below the
water surface from the center of the lake in 15 and 30 L polycarbonate
containers. The water used for the first experiment was kept for less
than 24 h in the dark under ambient laboratory conditions prior to the
start of the experiment. The water used to dilute the sediment was
first coarse filtered and stored under ambient laboratory conditions in
polycarbonate containers until the start of the experiment.
Sediment. Grab samples of the surface 30 cm of sediment were
collected on 28 June 1990 from the center of the lake using an Eckman
dredge, and placed in polycarbonate containers, which were filled
completely to minimize air spaces. The samples were stored under
ambient laboratory conditions prior to use in the study.

128
Experimental Design
The effect of dissolved oxygen on organic phosphorus
mineralization in the water column. Dissolved 02 minima in the water
column of lakes occur during the night hours when respiration exceeds
photosynthesis. To emulate this condition this experiment was conducted
in the dark, under ambient laboratory temperatures. Four hundred mL of
lake water were placed in each of six 500 mL erlenmeyer flasks. The
flasks were placed on stir plates and continuously stirred. Three
flasks were stoppered and maintained under anaerobic conditions (Fig. 5-
la), while three were maintained under aerobic conditions. One
replicate of each treatment had pH and DO probes in constant contact
with the water column; it was assumed that all three replicates would
respond similarly. Anaerobic conditions were obtained by closing the
flasks with rubber stoppers and purging with a mix of 330 ppm C02
balanced with N2 for approximately 1 1/2 h until the DO concentration
was <0.2 mg L'1. The pH and DO probes were tightly sealed in the
stoppers. A rubber septum was also included in the stopper arrangement
to allow sampling. At this point 30 mL water samples were removed by
syringe from all flasks. A time series of samples was subsequently
removed after 2, 4, 8 and 24 h. Samples were analyzed for total and
soluble APA, total soluble P (TSP), total soluble N (TSN), NH4-N, and
soluble reactive P (SRP). Initial chlorophyll a was also determined.
The effect of redox potential on organic phosphorus
mineralization in the sediment. Fresh sediment was diluted with
filtered lake water to obtain 10 g dry wt. L'1 of slurry, with a total
volume of 2.5 L and was placed in each of six 2.8 L flasks, which were

129
pH electrode
DO electrode
Purging gas
Sampling port
(a)
Lake water
Magnetic Stirring Plate
Fig. 5-1. Diagram illustrating the apparatus used to control dissolved
oxygen concentration and redox potential: a) anoxia control
in lake water; b) redox potential control of sediment
suspensions.

130
stoppered, placed on stir plates and attached to redox controllers (Cole
Palmer Model 5997-20) (Fig. 5-lb) modified from Patrick et al. (1973).
All flasks were purged with a mixture of 330 ppm C02 balanced with N2,
for 24 h. The flasks were then allowed to equilibrate for 1 month in
the dark under ambient laboratory conditions at the desired redox
potentials; -250, -100, 0, +100, +250 and +500 mV (actual potentials
measured upon sampling were; -242, -157, -2, +48, +338, +483 mV). After
equilibration, triplicate samples were withdrawn from the flasks and
analyzed for APA and fractionated for organic P using an adaptation of
the method of Sommers et al. (1972) (Fig. 5-2). Bicarbonate extractable
P was determined upon sediment samples after the removal of porewater
for pH determinations (Fig. 5-2). Total P and volatile solids were
measured on dried sediment samples.
Analytical Methods
Water. Alkaline phosphatase activity was determined
fluorometrically (Healey and Hendzel 1979a). One half mL of substrate,
3-o-methylfluorescein phosphate (Sigma Chemicals), at a concentration
determined to be non-limiting, in 0.1 M Tris buffer (Tham, Fisher
Scientific) pH 8.6, was added to 4.0 mL of lake water in a cuvette.
Anoxic water samples were injected into cuvettes which were sealed by a
rubber septum and had been purged with N2 and then evacuated. Both
total (whole lake water) and soluble (filtered through 0.45 im Gelman
membrane filter) APA were determined. The cuvettes were placed in a
water bath (25C). At timed intervals during a 20 min period the

131
ORGANIC PHOSPHORUS FRACTIONATION SCHEME
Fig. 5-2. The extraction scheme used to fractionate organic phosphorus
in sediment.

132
cuvettes were placed in the fluorometer and fluorescence was measured.
The enzyme activity was measured as an increase in fluorescence as the
substrate was enzymatically hydrolyzed to the fluorescent product.
Fluorescence units were converted to enzyme activity using a standard
calibration curve of 3-o-methylfluorescein (Sigma Chemicals). The
fluorescence was measured using a Turner fluorometer No. 110, equipped
with Turner lamp no. 110-853, in combination with 47 B primary and 2a-
12 secondary filters. Autoclaved lake water with substrate added was
used as a control.
Chlorophyll a was determined spectrophotometrically following
extraction with acetone and correction for pheophytin (APHA (1002-G),
1985). Total soluble P, TSN, NH4-N, and SRP were determined by standard
methods (APHA 1985).
Sediments. Alkaline phosphatase activity was determined by a
method adapted from Sayler et al. (1979). Sediment samples (1 g wet
sediment) were placed in centrifuge tubes. Three mL of 1 M Tris-Tris
HC1 buffer (pH 7.0) were added to each tube. The samples were sonicated
(Heatsystems Ultrasonics Model W-220F) for 45 sec at 30% relative
output, to release cell bound phosphatase. The samples were then
incubated at 25*C, with 1 mL p-nitrophenyl phosphate, at a concentration
of 50 mg mL'1 (Sigma Chemicals) for 1 hr. At the end of the incubation
3 mL of 1 M NaOH were added to the tubes to stop the reaction and
enhance p-nitrophenol color. To account for color interference controls
were obtained by incubating sediment without the substrate and
subsequently adding the substrate along with NaOH at the end of the
incubation. All samples were then centrifuged at 7000 rpm (7096 g) for

133
15 min. The liquid was removed and absorbance at 410 nm (Shimadzu Model
UV-160) measured. Concentrations were determined by calibration with a
standard curve of p-nitrophenol (Sigma Chemicals).
Porewater was extracted by centrifuging a subsample of sediment at
6000 rpm (5213 g) for 15 min. The porewater was immediately filtered
through 0.45 pm Gelman membrane filters. Total P, TSP and SRP were
measured using standard methods (APHA 1985). Porewater total organic P
(TOP) was defined as the difference between porewater TP and SRP.
Bicarbonate extractable P was determined after porewater extraction by
shaking the sediment with 0.5 M NaHC03 (1:50, on a dry weight basis) for
30 min. The extracted medium was then centrifuged at 6000 rpm (5213 g)
for 15 min and the supernatant filtered through Whatman 40 filter paper.
Total P and SRP were determined on the filtrate (APHA 1985). Labile
inorganic P was calculated as the sum of porewater SRP and HC03
extractable SRP. Labile organic P was defined as the difference between
total labile P and labile inorganic P.
Water content of the sediment was determined by drying a known
weight of wet sediment at 70*C to a constant weight. The dried sediment
was ground to a powder using a Spex 8000 grinding mill. Total P content
of the dried sediment was determined via ignition (Walker and Adams
1958). Volatile solids were reported as the loss in weight due to
ignition of dried sediment at 500*C for 2 h.
Statistical Analysis
Data were analyzed using the Statistical Analysis Systems (SAS
1985), Version 6.03 for personal computers. Data in the first

134
experiment were analyzed using repeated measures analysis which accounts
for the inherent within replicate correlation due to repeated sampling
of the same flasks. When data were unbalanced repeated measures
analysis could not be used, a split plot design, with time as the
subplot, was used to produce the analysis of variance and F test. Data
in both experiments were analyzed using Pearson correlation
coefficients.
Results
The Effect of Dissolved Oxygen on Organic Phosphorus Mineralization in
the Water Column
Chlorophyll a concentrations of the water samples collected in
July 1990 were extremely high (Table 5-1) and exceeded previous
measurements (chapter 2). Other parameters measured were within the
same range as previously recorded in Lake Apopka (chapter 2).
The response of the measured parameters over time was highly
varied under both aerobic and anaerobic conditions. During the 24 h
experimental period pH decreased from 8.2 to 7.5 and 8.0 under aerobic
and anaerobic conditions, respectively (data not shown). Total soluble
N exhibited significant differences over time (p=0.02). Under aerobic
conditions TSN increased within 2 h and subsequently declined over time
(Fig. 5-3a). Under anaerobic conditions the only significant difference
observed was the increase at 24 h versus initial concentrations. There
was an apparent decrease in NH4-N concentrations over time (Fig. 5-3b),
however, no statistically significant time or treatment effects were
determined for NH4-N. Both TSP and SRP concentrations also exhibited

135
Table 5-1. Concentrations of
Lake Apopka water
selected parameters measured in
in July 1990 (mean 1 SE).
Parameter
Concentration
Chlorophyll a (nq L'1)
220 4
Total APA (nM min'1)
21.9 0.1
Soluble APA (nM min'1)
0.82 0.4
TSP (nq L'1)
15 0
SRP (/xg L'1)
1 0
TSN (mg L'1)
2.48 0.0
NH4-N (mg L'1)
0.05 0

TSN (mg
136
TIME (h)
Fig. 5-3. Nutrient concentrations in Lake Apopka water
incubated in the dark under aerobic and anaerobic
conditions: a) total soluble nitrogen; b) NH4-N.
Vertical bars represent 1 SE. Absence of bar indicates SE
is smaller than the symbol size.

137
insignificant time and treatment responses (Fig. 5-4a and b), SRP
concentrations were below detection (< 1 ng L'1) for both treatments
after 24 h. A significant treatment response was observed for total APA.
(Fig 5-5a). Under anoxic conditions total APA remained constant over
the 24 h sampling period. Under aerobic conditions total APA increased
within 2 h and then remained constant until 24 h. At 24 h, the total
APA had increased from 27 to 43 nM min'1. Soluble APA accounted for 4%
of total APA (Table 5-1). No change in soluble APA was observed in
either treatment (Fig. 5-5b).
Significant correlations among the water chemistry parameters
measured were observed. A significant inverse relationship between
NH4-N and TSP was determined (r=-0.79), and significant positive
correlations between SRP and TSN (r=0.67) and NH4-N (r=0.70) were
observed.
The Effect of Redox Potential on Organic Phosphorus Mineralization in
the Sediment
The initial pH of the sediment prior to the month long incubation
was 7.12. Following equilibration at different Eh levels slight changes
in pH values were recorded (Table 5-2). Redox values were expressed as
pE + pH to account for the variability in pH among the reaction vessels
(pH range 6.35 to 7.15). pE was calculated as;
pE = Eh / 59
A significant negative relationship was observed between APA and
pE + pH (Fig. 5-6a). Conversely, a significant positive relationship
between pE + pH and porewater TOP was recorded (Fig. 5-6b). pE + pH
accounted for 77% of the variability in APA and 73% of porewater TOP

138
O 5 10 15 20 25 30
TIME (h)
Fig. 5-4. Nutrient concentrations in Lake Apopka water
incubated in the dark under aerobic and anaerobic
conditions: a) total soluble phosphorus; b) soluble reactive
phosphorus. Vertical bars represent 1 SE. No vertical bar
indicates SE is smaller than the symbol size.

SOLUBLE APA TOTAL APA
139
TIME (h)
Fig. 5-5. Alkaline phosphatase activity in Lake Apopka water
incubated in the dark under aerobic and anaerobic
conditions: a) total alkaline phosphatase activity;
b)soluble alkaline phosphatase activity. Vertical bars
represent 1 SE. No vertical bar indicates SE is smaller
than the symbol size.

140
Table 5-2. Concentrations of selected parameters measured
on sediments incubated under six different redox levels for
one month (mean 1 SE)
Porewater Labile
Eh pH pE + pH APA ~SRP TOP- ~P,
Mmol
mV g dry wt.'1 mg P kg dry wt.'1
h'1
483
6.35
15
6.89

0.21
26

0.45
2.1

0.45
48

1
11

3
338
6.49
12
4.01

0.65
51

0.78
7.0

2.81
74

3
16
i
3
48
6.78
8
3.64
+
0.39
8
+
1.19
13.8

2.51
23

2
19

2
-2
6.55
7
4.23
+
0.31
58

0.90
8.3
+
3.15
75

0
10
+
2
-157
6.78
4
1.07

0.59
44
+
1.35
16.1

1.96
66

2
22

2
-242
7.15
3
0.71
+
0.24
111

1.80
18.3
+
0.55
144
+
2
19

1

141
pE + pH
Concentration of selected parameters measured in sediments
incubated under six different redox levels for one month:
a) alkaline phosphatase activity; b) porewater total organic
phosphorus.
Fig. 5-6.

142
variability. Non linear redox effects were observed for other P
compounds. A significant increase in porewater SRP and labile inorganic
P were observed at a pE + pH value of 3 (Table 5-2). In contrast FA-TP
decreased at a pE + pH of 3. The HA-TP fraction tended to increase
under pE + pH values of 7. However, the majority of P forms measured
were not significantly affected by redox potential.
Volatile solids were 66% in both aerobic and anaerobic treatments.
The sediment TP as determined by ignition was 1047 ng g dry wt.'1. On
average the total recoverable P determined by the summation of HC1-TP,
NaOH-TP and porewater TP accounted for 74% of TP as determined by
ignition (Table 5-3). Acid hydrolyzable-TP extracted the largest
percentage of P (41%), followed by alkali extractable P (28%). Labile
inorganic and organic P were a small component of the TP, contributing
6.8 (2-14) and 1.4 (1-2)%, respectively. Readily available, i.e.
porewater SRP and TP, also contributed less than 20% of total P.
Porewater TP accounted for 5.8 (2-13)% of the total P pool.
Total and inorganic P extracted with HC1 were the same, indicating
that no organic P was extracted. Porewater TSP and SRP were also
equivalent suggesting that no porewater organic P was in the soluble
phase; TOP represented particulate organic P.
Significant correlations between the parameters measured were
observed (Table 5-4). Alkaline phosphatase activity had a high positive
correlation with pE + pH (r=0.90) while porewater TOP was strongly
inversely related to pE + pH (r0.92). Alkaline phosphatase activity
was also highly negatively correlated with labile organic P (r=-0.81)
and HA-TP (r=-0.74), while porewater TOP was highly positively related

143
Table 5-3. Concentrations of selected parameters measured
on sediments incubated under six different redox levels for
one month (mean + 1 SE).
NaOH-TP
Eh pH pE + pH HC1-TP FA-TP H^P
mV mgPkg dry wt.'1
483 6.35 15 490 13 171 5 132 3
338 6.49 12 393 26 162 4 134 1
48 6.78 8 431+4 180 1 134 0
-2 6.55 7 414 20 146 8 126 3
-157 6.78 4 421 15 150 15 167 2
-242 7.15 3 434+3 115 2 148 3

Table 5-4. Selected correlation coefficients between phosphorus compounds and
alkaline phosphatase activity measured on sediments incubated for
one month under six different redox levels.
pE + pH
APA
LabP;
LabP0
PW-SRP
PW-TP
PW-TOP
FA-TP
FA-P0 NaOH-TP
APA
0.90*
LabP¡
NS
NS
LabP0
NS -
0.81
NS
PW-SRP
NS
NS
1.00
NS
PW-TP
NS
NS
0.99
NS
0.99
PW-TOP
-0.92
-0.95
NS
0.84
NS
NS
FA-TP
NS
NS
-0.96
NS
-0.97
-0.98
NS
FA-P0
NS
NS
-0.92
NS
-0.94
-0.95
NS
0.98
NaOH-TP
NS
NS
-0.94
NS
-0.96
-0.94
NS
0.95
0.97
HA-P0
NS
-0.74
NS
0.81
NS
NS
NS
NS
NS NS

Unless otherwise indicated all correlations are significant at a=0.05
Significant at a=0.09

145
to labile organic P (Table 5-4). A strong inverse relationship between
APA and porewater TOP was observed (r=-0.95).
Other significant inverse correlations include porewater TP and
SRP with FA-P and NaOH-TP. Labile inorganic P was also highly inversely
correlated to these P forms. No relationship was observed between
HC1-TP and any other parameters measured.
Discussion
The Effect of Dissolved Oxygen on Organic Phosphorus Mineralization in
the Water Column
Phosphorus concentrations in lake water incubated in the dark for
24 h, did not show significant changes under either anaerobic
(<0.2 mg L'1) or aerobic (6 mg L'1) conditions. However, decreases in
TSN concentrations suggested that mineralization of organic N occurred,
but no increase in NH4-N was observed. This may be due to rapid
nitrification under aerobic conditions (Reddy and Graetz 1981) or loss
through volatilization (Stratton 1968, 1969). Initial TSN:TSP exceeded
200:1 and would suggest that the system may be P limited.
Under conditions of inorganic P limitation, phytoplankton may
produce APA which hydrolyzes organic P compounds with the release of
inorganic P (Kuenzler 1965). Specific APA (APA/chlorophyll a) ratios
were determined to be an effective means of determining inorganic P
limitation of Lake Apopka plankton (chapter 3). Under SRP limiting
conditions, APA and specific APA increase (Kuenzler and Perras 1965;
Heath and Cooke 1975; chapter 3). Initial and final specific APA ratios
under aerobic conditions were 0.1 and 0.2 nmol APA ng chlorophyll a1

146
min1, suggesting that the plankton population was not P limited
(Pettersson 1980; chapter 3). However, total APA increased from 22 to
43 nM min'1. This increase could be a demonstration of either increased
bacterial reproduction or alternatively, a response to decreased
internal P supply within the plankton (Fitzgerald and Nelson 1966;
Pettersson 1980; chapter 3). Internal P reserves, which include small
chain polyphosphates (Elgavish and Elgavish 1980) are broken down in
response to P demand (Rhee 1972, 1973, 1974, chapter 3). Once internal
P concentrations reach a certain critical level, APA is produced (Taft
et al. 1977; Sproule and Kalff 1978; Chrst and Overbeck 1987).
Under natural conditions and in continuous culture, P
concentrations may be low but they are continuously replenished. In
batch culture, a one time P input can result in rapid depletion of SRP,
hence internal P sources may be required for metabolism (Rhee 1972).
Once this P pool has been reduced to a critical level APA is produced
(Chrst and Overbeck 1987). This would explain the 24 h lag time
observed prior to the increase in APA observed under aerobic conditions.
In contrast, under anaerobic conditions, metabolism of aerobes is
reduced and eventually inhibited completely without the return of DO.
The dominant form of phytoplankton in Lake Apopka are the blue-
greens, Lyngbya sp. and Microcystis sp. (Shannon and Brezonik 1972;
Stites, D. L., unpublished data, St. John's River Water Management
District, Palatka, FL.). Under anaerobic conditions, only anaerobes
and facultative anaerobes are active; however, research has demonstrated
that after acclimatization cyanobacteria grow well under anaerobic
conditions with H2S acting as an electron donor in the photolysis of

147
water during photosynthesis (Stewart and Pearson 1970). The production
of APA is dependent upon the P stress within system (Fuhs et al. 1972;
Kuenzler and Perras 1965). Because of the low P requirements of
anaerobic bacteria, APA inhibition may occur at lower SRP levels than
under aerobic conditions, where P requirements of aerobic bacteria are
high. Enzyme activity did not cease under anaerobic conditions
suggesting that the enzyme itself was not inhibited by anaerobic
conditions. Although total APA under aerobic conditions differed from
total APA under anaerobic conditions within 2 h, no significant increase
in APA occurred until 24 h, this suggests that less severe low DO will
not affect APA organic P mineralization. Depth profiles of APA with
varying DO concentrations were shown to correlate with chlorophyll a and
bacterial counts rather than DO concentrations (Jones 1972a),
consequently metalimnetic 02 depletion is unlikely to affect APA.
However, in waters which become stratified the anoxic conditions at the
sediment-water interface may result in decreased APA after an extended
time period. This may be due to decreased production by anaerobes or as
a response to competitive inhibition by SRP released from sediments
(Pettersson 1980). Lake Apopka is a frequently mixed system under which
extended periods of 02 depletion are not likely to occur. Diel changes
in 02 will be too brief (< 8 h) to affect organic P mineralization.
No change in soluble APA was observed under either aerobic or
anaerobic conditions. Soluble APA has been shown to be a result of cell
lysis (Berman 1970) as well as excretions by zooplankton, bacteria and
phytoplankton (Wynne 1981; Pettersson 1980). It is thought that
significant cell death and lysis did not occur during the period of

148
anaerobiosis because increased nutrient concentrations and soluble APA
were not observed.
The Effect of Redox Potential on Organic Phosphorus Mineralization in
the Sediment
A significant effect of increasing anaerobiosis is the change from
a predominately aerobic microbial population to a smaller anaerobic
population. Under anaerobic conditions organic material accumulates due
to reduced mineralization rates. Microbial biomass is highly correlated
with APA (Sayler et al. 1979; Ayyakannu and Chandramohen 1971), and APA
was significantly inhibited by the decrease in redox potential. Hence
organic P mineralization will decrease under reduced conditions. In
general, redox potential did not have a significant effect on the
various organic P pool sizes. However, Lake Apopka sediments are poorly
poised (poorly redox buffered) and the low concentrations of electron
acceptors may reduce the rate of mineralization. Only APA and porewater
TOP were significantly correlated with pE + pH. More resistant P forms
may require longer incubation times to show a response to redox
potential.
Significantly lower porewater SRP and labile inorganic P
concentrations were observed at Eh 48 mV, than either Eh 338 or -2 mV.
This may be a problem associated with the control of redox conditions.
Redox levels may need to be established by utilizing specific electron
acceptors, rather than fluctuating the air input.
Redox potential tends to decrease with sediment depth (Kobori and
Taga 1979b; DeGobbis et al. 1984), while porewater SRP has often been
shown to increase as a function of depth (Mortimer 1941; chapter 4).

149
Soluble reactive P concentrations measured at -242 mV were similar to
SRP concentrations measured in the 20-40 cm depth of cores collected in
January 1990 from the same site in Lake Apopka (chapter 4) indicating
that porewater SRP increased in response to reduced redox potential. In
Lake Apopka P fluxes were determined to be mainly a function of
decomposition at the sediment-water interface (Moore et al. 1991).
Aerobic conditions may also result in SRP release. Sediments may
be aerated through wind induced resuspension or by diffusion of 02 from
the hypolimnion. Aerobic release of P from sediments into the overlying
water is a function of the mineralization of organic P compounds (Lee et
al. 1977). Greater than 90% of organic P extracted from an upland soil
was shown to be in the form of phosphomonoesters (Condron et al 1985).
Thus a large portion of organic P may potentially be made available
through the action of phosphatase enzymes. Alkaline phosphatase
activity has been significantly correlated with SRP release in marine
sediments (Degobbis et al. 1985). In this study a significant inverse
correlation was observed between porewater TOP and sediment APA
(r=-0.95), hence high APA may result in the breakdown of porewater TOP,
although this may also indicate the inhibition of APA by porewater TOP.
Alkaline phosphatase activity was also highly inversely correlated with
labile organic P. Porewater TOP, which is already in the soluble
fraction of the sediment, may be more susceptible to enzyme hydrolysis
than labile organic P and hence will be hydrolyzed first. The porewater
TOP pool is subsequently replenished by the labile organic P pool of the
sediment. The solubility of the substrate is the factor limiting
organic P mineralization not the enzyme activity (Jackman and Black

150
1952). It was determined that the rate of phytate hydrolysis in
solution was over 71 times as important as the phytase activity; a
specific acid phosphatase in the limitation of hydrolysis (Jackman and
Black 1952).
The high volatile solids content of the sediment indicated the
high organic matter content. A significant portion of this organic
matter was highly resistant to hydrolysis, as apparent by the 26%
difference in TP determined by ignition and extraction. In this study,
APA was inversely correlated with HA organic P (r=-0.74), a resistant
form of P. This inverse relationship may be explained as the possible
binding of alkaline phosphatase to the humic substances which may
increase enzyme stability, thus resulting in some inhibition of APA
(Burns 1986; Kandeler 1990; Wetzel 1991). Organic P compounds may also
form complexes with humates (Stewart and Tiessen 1987; Brannon and
Sommers 1985). The two main chemical extractants, HC1 and NaOH, were
unable to extract organic P compounds incorporated into humic polymers
(Brannon and Sommers 1985). This chemically resistant organic P was
also resistant to enzymatic hydrolysis.
The high negative correlation between labile inorganic P and SRP
with FA-TP and NaOH-TP suggest that FA-TP, a component of NaOH-TP, may
be a significant contributor to the labile inorganic P pool through
enzymatic hydrolysis. It has been suggested that inositol phosphates
bound to FA are hydrolyzable by phytase (Herbes et al. 1975).

151
Conclusions
The results from this study show that short-term DO depletion will
not affect APA in the water column of Lake Apopka. However, extended
periods of anoxia (8 to 24 h) will result in APA inhibition and
decreased enzymatic breakdown of organic P. In the sediment, APA was
high under aerobic conditions and decreased with a decrease in Eh, hence
under anaerobic conditions the rate of organic P mineralization will be
slower. Inverse correlations between APA and porewater TOP and labile
organic P also suggest that these are susceptible to enzymatic
hydrolysis or may be inhibited by high concentrations of these
substrates. Based upon the resistant nature of HA-TP the inverse
relationship between HA and APA was attributed to the binding of the
enzyme in the formation of humic complexes which accumulate under
anaerobic conditions.

CHAPTER 6
ORGANIC PHOSPHORUS CYCLING IN LAKE APOPKA
Organic P plays a dominant role in the P cycle of aquatic systems
(Fig. 1-1). The bioavailability of organic P to plankton is regulated
by the activity of enzymes, types of substrates and associated physico
chemical factors in the system. The research presented in this
dissertation examined the specific organic P compartments (plankton,
water column and sediment) to evaluate the bioavailability of organic P
for plankton growth. Results obtained in this study are summarized in
the context of addressing key research issues raised in an attempt to
understand the P dynamics in Lake Apopka.
(1) How is the enzymatic hydrolysis of organic P affected by
other water chemistry parameters?
Both seasonal and spatial variability of total P (TP), total
soluble P (TSP) and alkaline phosphatase activity (APA), were observed
in the water column. Alkaline phosphatase activity, an indicator of
potential organic P hydrolysis, was dependent upon different water
chemistry parameters, both seasonally and spatially. The majority of
APA was associated with particulate matter, and soluble APA averaged
only 3% of total APA, therefore particulate interactions are a key
component of organic P cycling in Lake Apopka. The attachment of
enzymes to surfaces may increase enzyme longevity, but may also reduce
enzyme activity, either directly through sorption at the active site or
152

153
indirectly as a result of steric hindrance. Evidence of this was
observed by the inverse relationship between APA and total organic C
(TOC).
Alkaline phosphatase activity was higher in the pelagic zone
(sites 2-8), which was more productive and nutrient rich than the spring
(site 1). The mean annual total APA in the water column was 18 nM
min'1, under non-limiting substrate concentrations; this indicates a
potential inorganic P release rate of 0.6 ng P L'1 min'1. Natural
substrate concentrations are usually lower than those used in the APA
assay, thus in situ release rates will be slower.
(2) Is Lake Apopka plankton APA inhibited by inorganic P and is
it produced in response to inorganic P limitation?
Over 90% of APA within the natural plankton population was
inhibited following the addition of 1000 fig L'1 inorganic P to the
growth medium. Addition of lower inorganic P concentrations
(10 to 100 fig L1) did not produce such complete inhibition, thus
ambient soluble reactive P (SRP) concentrations (< 10 fig L1) in the
lake water will not inhibit APA.
The uptake of added inorganic P was very rapid, indicating high P
demand. The inorganic P was immediately incorporated into the plankton
cells as surplus P, as determined by hot water extraction. In general,
surplus P was inversely related to APA, suggesting that internal
inorganic P levels controlled APA. Under field conditions this was
reflected by an inverse relationship between acid hydrolyzable P and
APA. In batch culture, both hot water extractable inorganic and organic
P were used to provide P for growth.

154
Increased APA was associated with increased plankton biomass;
however, the intensity of APA was dependent upon the severity of P
limitation. During severe inorganic P limitation Lake Apopka plankton
produced specific APA (total APA/chlorophyll a) values > 1 nmol APA
ng chlorophyll a'1 min'1. Ambient lake water specific APA was
< 0.3 nmol APA /xg chlorophyll a'1 min'1, suggesting that Lake Apopka
plankton were not severely P limited.
(3) What effect does sediment resuspension have upon organic P
mineralization rates?
Resuspension of surficial sediments resulted in immediate
increases in total suspended solids (TSS), total Kjeldahl N (TKN), TP
and APA within the overlying water column. These elevated
concentrations decreased rapidly upon particle settling, indicating they
remained associated with the sediment particles. During resuspension,
increased APA and TP concentrations within the overlying water column
may result in increased organic P mineralization. Exposure of anaerobic
sediments to the oxygenated water column during resuspension may also
increase the rate of organic P mineralization within the sediment
itself. Porewater organic P was inversely related to pE + pH, while APA
was positively related to pE + pH, indicating reduced mineralization
rates of organic P under anaerobic conditions. Alkaline phosphatase
enzymes have been shown to bind to humic acid complexes, the inverse
relationship between humic acid P (HA-TP) and APA indicates inhibition
in response to binding. The overall conclusion is that mineralization
rates may be increased as a result of sediment resuspension and
aeration, and are reduced under anaerobic conditions.

155
The results presented in this study demonstrate significant
potential for organic P mineralization in Lake Apopka. Substrates
hydrolyzable by APA are very labile, hence the rate of organic P
mineralization in Lake Apopka is limited by substrate concentrations
rather than enzyme activity. Further research should focus more on
specific organic P compounds within the sediment-water column and their
relative turnover rates. Studies should also include evaluating the
change in enzyme kinetics in response to different organic P compounds.
To encourage P limitation, emphasis should be placed upon a means to
inhibit organic P mineralization.

APPENDIX A
LORAN COORDINATES
Table A. Loran coordinates of sampling sites on Lake Apopka
Group repetition interval:7980, Southeast USA.
Site number
Time
Y
delay ns
Z
1
44526.6
62460.6
2
44524.2
62446.4
3
44553.9
62452.3
4
44577.2
62449.9
5
44539.7
62416.8
6
44497.1
62403.0
7
44488.8
62420.2
8
44530.0
62432.2
156

APPENDIX B
CONCENTRATIONS OF SELECTED WATER CHEMISTRY PARAMETERS
DETERMINED BIMONTHLY FROM APRIL 1989 THROUGH
FEBRUARY 1990, AT 8 SITES IN LAKE APOPKA
Table B-l. Temperature.
Date
Site
Mean
' SE
1
2
3
4
5
6
7
8
c
APR
24.3
27.4
27.4
26.7
26.3
26.1
28.1
23.6
26.5
0.21
JUN
24.2
32.5
30.4
28.5
30.0
29.8
28.1
29.8
29.9
0.20
AUG
24.0
28.7
28.0
27.8
29.5
29.8
29.2
28.5
28.8
0.11
OCT
22.7
21.1
19.9
19.8
19.4
19.9
20.0
19.4
19.9
0.08
DEC
20.5
17.6
16.5
15.8
16.3
15.7
15.0
15.5
16.1
0.12
FEB
22.3
20.1
19.8
20.6
20.8
20.4
21.3
20.0
20.0
0.07
Mean
SE
23.1
0.3
25.5
1.0
24.4
1.0
23.7
0.9
24.3
1.0
24.3
1.0
24.1
1.0
23.4
1.0
Means listed in
this
column
are
means
of sites 2-8.
157

158
Table B-2. Secchi depth transparency.
Date
Site
Mean" SE
1 2 3 4 5 6 7 8
m
APR
1.00
0.24
0.20
0.25
0.23
0.24
0.23
0.25
0.23
0.00
JUN
1.50
0.21
0.21
0.24
0.24
0.21
0.24
0.21
0.22
0.00
AUG
1.90
0.20
0.23
0.30
0.25
0.25
0.23
0.23
0.24
0.00
OCT
1.38
0.27
0.28
0.28
0.27
0.35
0.24
0.30
0.28
0.00
DEC
1.00
0.25
0.25
0.39
0.31
0.32
0.25
0.27
0.29
0.01
FEB
0.72
0.26
0.24
0.30
0.25
0.22
0.25
0.25
0.25
0.00
Mean
SE
1.36
0.06
0.23
0.00
0.23
0.01
0.29
0.01
0.26
0.01
0.27
0.01
0.24
0.00
0.25
0.01
Means listed in this column are means of sites 2-8.

159
Table B-3. Dissolved oxygen.
Date
Site
Mean'
SE
1
2
3
4
5
6
7
8
1
my l
APR
4.5
12.7
13.0
11.2
12.3
12.8
13.0
10.7
12.2
0.13
JUN
2.4
7.3
10.5
3.7
5.2
8.0
6.3
9.8
7.3
0.35
AUG
2.5
8.7
9.3
5.8
10.2
11.0
10.8
9.6
9.3
0.25
OCT
4.2
9.7
8.6
8.9
9.1
9.6
10.6
10.2
9.5
0.10
DEC
5.3
9.5
9.0
9.9
9.1
9.9
10.1
9.3
9.5
0.06
FEB
5.5
7.7
10.2
10.0
9.6
10.8
12.1
9.0
9.9
0.20
Mean
3.8
9.6
10.1
7.9
9.2
10.3
10.2
9.9
SE
0.2
0.3
0.3
0.5
0.4
0.3
0.4
0.1
Means listed in this column are means of sites 2-8.

160
Table B-4. pH.
Date
Site
Mean'
SE
1
2
3
4
5
6
7
8
APR
8.2
9.4
9.3
9.1
9.3
9.4
9.4
9.2
9.3*
0.0
JUN
8.1
8.8
9.0
8.1
8.8
9.0
8.5
9.1
8.6
0.1
AUG
8.1
8.9
9.1
7.8
8.9
9.2
9.3
8.9
8.5
0.1
OCT
8.2
8.7
8.9
8.7
9.0
9.2
9.1
9.0
8.9
0.0
DEC
8.2
9.0
8.8
9.1
8.9
8.9
9.0
9.0
8.9
0.0
FEB
8.1
8.5
8.7
8.7
8.2
8.5
8.8
8.5
8.5
0.0
Mean
8.2
8.8
8.9
8.3
8.7
8.9
8.9
8.9
SE
0.0
0.0
0.0
0.1
0.0
0.0
0.1
0.0
Means listed in this column are means of sites 2-8.
Means were calculated following the conversion of pH to hydrogen
ion concentrations. Standard errors were calculated without prior
conversion of pH values.

161
Table B-5. Total solids.
Date
Site
Mean*
SE
1
2
3
4
5
6
7
8
mg
r1 ---
L
APR
ND*
ND
ND
ND
ND
ND
ND
ND
JUNE
148
349
373
337
411
411
300
404
369
6
AUG
156
309
317
328
334
335
323
350
328
2
OCT
202
387
396
385
402
395
402
411
397
1
DEC
229
369
369
389
370
373
393
396
380
2
FEB
271
531
499
524
511
459
496
508
504
3
Mean
201
389
391
393
406
395
383
414
SE
10
17
13
16
13
9
15
12
Means listed
in this
column
are
means
of sites 2-8.
* ND indicates not determined.

162
Table B-6. Total suspended solids.
Date
Site
Mean"
SE
1
2
3
4
5
6
7
8
1
mg l
APR
ND
4 ND
ND
ND
ND
ND
ND
ND
JUN
ND
ND
ND
ND
ND
ND
ND
ND
AUG
6
59
66
42
49
69
72
73
62
2
OCT
5
54
67
52
71
60
64
75
63
1
DEC
15
61
69
48
67
60
58
65
61
1
FEB
17
77
85
109
99
89
92
103
94
2
Mean
11
63
72
63
72
70
72
79
SE
2
2
2
8
5
3
4
4
Means
listed
in this
column
are
means
of sites 2-8.
4 ND indicates not determined.

163
Table B-7. Chlorophyll a
Date
Site
Mean'
SE
1
2
3
4
5
6
7
8
-1
/*y *-
APR
33
61
70
68
75
71
52
98
71
2
JUN
24
80
79
88
84
92
55
67
78
2
AUG
24
131
142
87
156
95
158
127
128
4
OCT
16
92
96
75
101
73
85
99
89
2
DEC
26
102
94
60
68
71
79
74
78
2
FEB
7
39
33
49
48
51
40
45
44
1
Mean
22
84
86
71
89
75
78
85
SE
1
5
6
3
6
3
7
5
Means
listed
in this
column
are
means
of sites 2
-8.

164
Table B-8. Total organic carbon.
Date
Site
Mean*
SE
1
2
3
4
5
6
7
8
mg L'1
APR
ND*
ND
ND
ND
ND
ND
ND
ND
JUN
0.3
30.6
29.9
29.8
31.6
31.5
24.3
32.2
30.0
0.38
AUG
2.4
32.5
34.0
40.5
32.4
31.7
33.1
34.0
34.0
0.43
OCT
1.2
28.5
29.8
30.5
29.0
32.9
28.3
29.2
29.7
0.22
DEC
9.1
30.1
28.6
29.4
31.1
32.9
27.8
31.9
30.3
0.26
FEB
10.6
35.2
36.9
33.7
44.5
32.6
34.0
39.2
36.6
0.59
Mean
SE
4.7
0.95
31.4
0.51
31.8
0.69
32.8
0.93
33.7
1.23
32.3
0.13
29.5
0.81
33.3
0.75
Means listed in this column are means of sites 2-8.
* ND indicates not determined.

165
Table B-9. Total Kjeldahl nitrogen.
Date
Site
Mean
SE
1
2
3
4
5
6
7
8
i
my l
APR
2.57
5.06
6.12
5.38
5.71
5.03
5.18
8.11
5.80
0.16
JUN
0.18
4.71
4.72
4.62
4.78
5.33
3.58
4.48
4.60
0.07
AUG
1.30
5.28
6.45
4.86
5.56
5.53
5.66
5.25
5.51
0.07
OCT
0.61
2.08
3.82
3.64
4.68
3.14
4.08
4.28
3.67
0.12
DEC
2.30
4.19
4.72
3.81
4.13
4.36
4.59
4.45
4.32
0.04
FEB
1.87
4.04
4.12
4.85
5.30
4.58
5.06
4.00
4.56
0.08
Mean
1.47
4.23
4.99
4.53
5.03
4.66
4.69
5.09
SE
0.16
0.19
0.18
0.11
0.10
0.14
0.13
0.26
Means listed in this column are means of sites 2-8.

166
Table B-10. Total and soluble alkaline phosphatase activity.
Date
Site
Mean" SE
1 2 3 4 5 6 7 8
nM min'1
Total
APR
5.9
12.2
12.8
22.0
18.7
20.1
20.7
22.8 18.5
0.61
JUN
3.8
11.8
11.2
44.7
29.9
30.6
20.8
19.2 24.0
1.70
AUG
3.7
10.2
10.6
10.3
6.4
17.8
20.8
11.6 12.5
0.71
OCT
5.7
27.0
28.2
18.2
19.6
21.9
25.4
24.9 23.6
0.54
DEC
7.7
23.8
24.6
19.8
18.8
17.9
20.3
17.4 20.4
0.40
FEB
5.6
9.6
10.3
11.6
9.5
10.6
10.9
8.9 10.2
0.13
Mean
5.4
15.8
16.3
21.1
17.2
19.8
19.8
17.5
SE
0.25
1.26
1.33
2.07
1.39
1.08
0.80
1.04
Soluble
APR 0.16
0.51
0.49
0.34
0.29
0.67
0.37
0.30 0.42
0.02
JUN
0.07
0.03
0.05
0.93
0.01
0.07
0.10
0.20 0.20
0.05
AUG
0.31
0.10
0.54
0.60
0.35
0.16
0.55
0.64 0.42
0.03
OCT
0.74
1.29
1.19
0.64
0.48
0.80
0.29
0.27 0.71
0.06
DEC
0.65
0.38
0.62
0.45
1.02
0.68
0.58
1.34 0.72
0.05
FEB
0.39
0.56
0.33
0.43
0.11
0.12
0.03
0.09 0.24
0.03
Mean
0.39
0.48
0.53
0.57
0.38
0.42
0.32
0.47
SE
0.04
0.08
0.06
0.04
0.06
0.06
0.04
0.08
Means listed in this column are means of sites 2-8.

167
Table B-ll. Phosphorus.
Date
Mean* SE
1
2
3
4
5
6
7
8
l
IP
APR
110
140
170
180
200
200
180
260
190
5
JUN
30
150
140
190
150
170
260
140
171
6
AUG
30
140
90
240
240
130
120
230
170
9
OCT
300
520
440
350
200
400
270
400
369
15
DEC
20
100
120
110
130
120
150
150
126
3
FEB
70
190
200
250
280
230
240
240
233
4
Mean
93
207
193
220
200
208
203
237
SE
18
26
21
13
9
17
10
16
ISP
APR
130
110
70
70
90
80
50
40
73
3
JUN
80
40
70
70
80
60
30
40
56
3
AUG
10
10
10
10
10
10
10
10
10
0
OCT
120
170
70
130
60
140
160
60
113
7
DEC
10
10
10
20
20
10
10
20
14
1
FEB
30
40
40
40
40
20
20
20
31
2
Mean
63
63
45
57
50
53
47
32
SE
9
11
5
7
5
9
10
3
SRP
APR
1
3
3
5
3
5
2
3
3
0
JUN
2
3
4
4
5
3
3
5
4
0
AUG
4
7
5
4
5
4
4
5
5
0
OCT
1
1
1
1
1
1
1
1
1
0
DEC
1
1
1
1
1
1
1
1
1
0
FEB
0
3
0
1
1
1
2
2
1
0
Mean
2
3
2
3
3
3
2
3
SE
0
0
0
0
0
0
0
0
Means listed in this column are means of sites 2-8.

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413. In A. L. Page, R. H. Miller, and D. R. Keeney [eds.j, Methods
of soil analysis, Part 2: Chemical and microbiological properties.
1982 ASA, SSSA. Madison, WI.

179
Weimer, W. C., and D. E. Armstrong. 1979. Naturally occurring organic
phosphorus compounds in aquatic plants. Environ. Sci. Technol.
13:826-829.
Wetzel, R. G. 1983. Limnology. 2nd. Edn. Saunders College,
Philadelphia.
Wetzel, R. G. 1991. Extracellular enzymatic interactions: storage,
redistribution, and interspecific communication, p. 6-28. In
R. J. Chrdst [ed.], Microbial enzymes in aquatic environments.
Science Tech. Publ.
Wolanski, E., T. Asaeda, and J. Imberger. 1989. Mixing across a
lutocline. Limnol. Oceanogr. 34:931-938.
Wynne, D. 1977. Alterations in activity of phosphatases during the
Peridinium bloom in Lake Kinneret. Physiol. Plant. 40:219-224.
Wynne, D. 1981. The role of phosphatases in the metabolism of
Peridinium cinctum, from Lake Kinneret. Hydrobiol. 83:93-99.
Wynne, D., and T. Berman. 1980. Hot water extractable phosphorus- an
indicator of nutritional status of Peridinium cinctum (Dinophyceae)
from Lake Kinneret (Israel)?. J. Phycol. 16:40-46.
Wynne, D., and M. Gophen. 1981. Phosphatase activity in freshwater
zooplankton. OIKOS 37:369-376.
Young, T. C., J. V. DePinto, S. C. Martin, and J. S. Bonner. 1985.
Algal available particulate phosphorus in the Great Lakes basin. J.
Great Lakes Res. 11:434-446.

BIOGRAPHICAL SKETCH
Susan Newman was born 26 June 1963 in Portsmouth, England. She
graduated with a BSc. Honors degree in management and chemical sciences,
from the University of Manchester Institute of Science and Technology,
in June 1984. Following a 6 month appointment at the Weed Research
Organization (the now defunct WRO), Susan determined it was time to
leave the cloudy skies of England. With emotions split between
excitement and trepidation, Susan began a Master of Science degree in
the Agronomy Department at the University of Florida in January 1985.
With an interest in wetland soils dating back to the making of mud pies
during her childhood, and unable to resist the opportunities of the
Sunshine State, she began a Ph.D. program in the Soil Science Department
at the University of Florida in 1987. The first course she completed
within this department was openwater scuba diving, however, following
that the more traditional courses were taken. Upon completion of her
Ph.D. Susan will continue her aquatic/wetland science interests by
obtaining a research position to investigate the nutrient dynamics in
wetlands.
180

I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
-K K sue _
Konda R. Reddy, Chair
Professor of Soil Science
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Joseph J. Delfino
Professor of Environmental
Engineering Sciences
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Donald A. Graetz
Professor of Soil Science
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Edward J,
Assistant^ofes^or of
Forest Resources and
Conservation

This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
May 1991
cu>k X.
ollege of
culture
Dean, Graduate School

This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
May 1991
cu>k X.
ollege of
culture
Dean, Graduate School



110
variation with no apparent change in concentration. The undisturbed
control sediment cores maintained constant nutrient concentrations
throughout the duration of the experiment.
Sediment. The APA in the control sediment cores decreased with
sediment depth. No distinct depth profile of APA was observed in the
cores where the top 10 cm of sediment was resuspended (Fig. 4-5),
although there was a tendency for the APA to be lower in the surface
sediment and increase with depth. There was no net change in porewater
SRP, TP, organic P and inorganic P concentrations in any of the cores.
Experiment 2
Water. The resuspension of different depths of surface
sediment into the overlying water column resulted in distinct
differences among measured parameters. Resuspension of the surface
10 cm resulted in TSS values of 3000 mg L'1 which decreased to 100 mg L'1
within 1 h. (Fig. 4-6a). The resuspension of 5 cm of sediment produced
TSS values around 1700 mg L'1 which decreased to 174 mg L'1 within 1 h.
This compares to the settling of the 2 cm depth increment which produced
a resuspended TSS of 400 mg L'1 and decreased more slowly to 140 mg L'1
within 1 h. As observed in experiment 1, increases in TP, TKN and TOC
corresponded to TSS increases (Fig. 4-6). The total APA also increased
with TSS, and declined as the suspended matter settled (Fig. 4-7).
Combining the data from both resuspension experiments, the
relationship between total APA and TSS measured within the water column
(Fig. 4-8) was best described by the following power equation:
Total APA = 0.831 x (TSS)0565; r2 = 0.91 n=32


REFERENCE LIST
Aaronson, S., and N. J. Patni. 1976. The role of surface and
extracellular phosphatases in the phosphorus requirement of
Oochromonas. Limnol. Oceanogr. 21:838-845-
Abbott, W. 1957. Unusual phosphorus source for plankton algae.
Ecol.38:152.
American Public Health Association (APHA). 1985. Standard methods for
the examination of water and wastewater. 16th edn. American Public
Health Association, Washington, D. C.
Ayyakkannu, K., and D. Chadramohen. 1971. Occurrence and distribution
of phosphate solubilizing bacteria and phosphatase activity in marine
sediments and Porto Novo. Mar. Biol. 11:201-205.
Baligar, V. C., R. J. Wright, and M. D. Smedley. 1988. Acid phosphatase
activity in soils of the Appalachian Region. Soil Sci. Soc. Am. J.
52:1612-1616.
Berman, T. 1970. Alkaline phosphatases and phosphorus availability in
Lake Kinneret. Limnol. Oceanogr. 15:663-674.
Bieleski, R. L. 1974. Development of an externally located alkaline
phosphatase as a response to phosphorus deficiency, p. 165-170. In.
R.L. Bieleski, A.R. Ferguson, and M.M. Cresswell, [eds.], Mechanisms
of regulation of plant growth. Bulletin 12, The Royal Society of
New Zealand, Wellington.
Boavida, M. J., and R. T. Heath. 1988. Is alkaline phosphatase always
important in phosphate regeneration? Arch. Hydrobiol. 111:507-518.
Bostrom, B., M. Jansson, and C. Forsberg. 1982. Phosphorus release
from lake sediments. Arch. Hydrobiol. Beih. 18:5-59.
Bowman, R. A., and C.V. Cole. 1978. An exploratory method for
fractionation of organic phosphorus from grassland soils. Soil Sci.
125:95-101.
Bradford, M. E., and R. H. Peters. 1987. The relationship between
chemically analyzed phosphorus fractions and bioavailable phosphorus.
Limnol. Oceanogr. 32:1124-1137.
168


169
Brannon, C. A., and L. E. Sommers. 1985. Stability and mineralization
of organic phosphorus incorporated into model humic polymers. Soil
Biol. Biochem. 17:221-227.
Burns, R. G. 1986. Interaction of enzymes with soil mineral and
organic colloids, p. 429-451. In P.M. Huang and M. Schnitzer [eds.],
Interactions of Soil Minerals with Natural Organics and Microbes.
SSSA Spec. Publ. no. 17.
Canfield, D. E. Jr., 1981. Chemical and trophic state characteristics
of Florida lakes in relation to regional geology. Final Report
submitted to Project Officer, Cooperative Fish and Wildlife Unit,
University of Florida, Gainesville, FL.
Cembella, A. 0., N. J. Antia, and P. J. Harrison. 1984a. The
utilization of inorganic and organic phosphorus compounds as
nutrients by eukaryotic microalgae: a multidisciplinary perspective:
Part 1. CRC Crit. Rev. Microbiol. 10:317-391.
Cembella, A. 0., N. J. Antia, and P. J. Harrison. 1984b. The
utilization of inorganic and organic phosphorus compounds as
nutrients by eukaryotic microalgae: a multidisciplinary perspective:
Part 2. CRC Crit. Rev. Microbiol. 11:13-81.
Charlton, M. N. 1980. Hypolimnion 02 consumption in lakes: discussion
of productivity and morphometry effects. Can. J. Fish. Aquat. Sci.
37:1531-1539.
Chiaudani, G., and M. Vighi. 1982. Multistep approach to
identification of limiting nutrients in northern Adriatic eutrophied
coastal waters. Water Res. 16:1161-1166.
Chrst, R. J., and J. Overbeck. 1987. Kinetics of alkaline phosphatase
activity and phosphorus availability for phytoplankton and
bacterioplankton in Lake PluBsee (north German eutrophic lake).
Microb. Ecol. 13:229-248.
Chrst, R. J., U. Mnster, H. Rai, D. Albrecht, P. K. Witzel, and J.
Overbeck. 1989. Photosynthetic production and exoenzymatic
degradation of organic matter in the euphotic zone of a eutrophic
lake. J. Plank. Res. 11:223-242.
Chrst, R. J., W. Siuda, D. Albrecht, and J. Overbeck. 1986. A method
for determining enzymatically hydrolyzable phosphate (EHP) in natural
waters. Limnol. Oceanogr. 31:662-667.
Coleman, J. E., and P. Gettins. 1983. Alkaline phosphatase, solution,
structure, and mechanism, p.381-452. In A. Meister [ed.j, Advances
in Enzymology Vol. 55. Wiley-Interscience, New York.


51
been reported (Fitzgerald and Nelson 1966; Chrst and Overbeck 1987).
Inorganic Pisa competitive inhibitor of APA (Coleman and Gettins 1983;
Moore 1969; Reid and Wilson 1971). Upon replenishment of external
inorganic P concentrations enzyme activity is inhibited (Lien and
Knutsen 1973; Torriani 1960; Perry 1972) and surplus P accumulates (Rhee
1973). Surplus P is measured as hot water extractable P (HEP), and in
conjunction with APA has been shown to accurately assess P demand in
some lakes (Sproule and Kalff 1978; Pettersson 1980) but not in others
(Wynne and Berman 1980). Wynne and Berman (1980) observed that the HEP
concentration in Lake Kinneret, Israel, remained stable throughout the
year even under conditions of P stress and concluded that HEP was a
metabolic intermediate rather than a form of P storage.
Lake Apopka is a hypereutrophic lake with soluble reactive P (SRP)
concentrations frequently < 1 ng L'1. In contrast, concentrations of
total soluble P (TSP) and APA are high (chapter 2). The objectives of
this study were to determine whether high APA in Lake Apopka was due to
high demand for P or P limitation, and to evaluate the N and P
requirements of native plankton.
Materials and Methods
Site Description
Lake Apopka is a 12,500 ha lake located in central Florida (28*
37' N. latitude, 81* 37' W. longitude). It has a mean depth of
2 m. It has been proposed that the nutrient loading from the
surrounding agricultural and urban areas has precipitated the current
hypereutrophic conditions in the lake (USEPA 1979).


CHLOROPHYLL a (fig
69
Fig. 3-5. Time courses of chlorophyll a concentrations following
nutrient enrichment of natural plankton populations
collected in August 1990. 0N,0P=no nutrient addition;
400N,0P=400 ng N L\ 0N,40P=40 ng P L\ 400N,40P=400 ¡tq
N L'1 and 40 /xg P L'1, and 800N,80P=800 /xg N L1 and
80 ng P L'1: a) plankton grown at 29*C; b) plankton grown at
19#C. Vertical bars indicate 1 SE. No vertical bar
indicates SE is smaller than symbol size.


17
percent of organic P in seawater (Kobori and Taga 1979a) and 32% of
organic P in freshwater (Hino 1988) were hydrolyzed by phosphatase
enzymes. Organic P of algal origin is particularly sensitive to
hydrolysis; 74% of algal extracted P was hydrolyzed by APA, while 80% of
organisms involved in the decomposition of plankton produced
phosphatases (Halemejko and Chrst 1984). In eutrophic situations
TP and organic P concentrations can be high (Jones 1979b), resulting in
higher APA levels than in lower trophic states (Jones 1979b; Pick 1987).
Alkaline phosphatase activity may be a significant mechanism of
satisfying high P demand in eutrophic situations, as well as a means of
overcoming P limitation in nutrient poor environments. The intensity of
APA is subject to the physico-chemical conditions in the environment.
Enzyme activity is pH dependent and can respond negatively or positively
to pH fluctuations (Torriani 1960). Dissolved oxygen (DO) and
temperature also influence microbial enzyme activity and metabolism,
thus they may directly or indirectly affect APA (Garen and Levinthal
1960). These environmental effects demonstrate the potential for
seasonal and diel fluctuations in APA.
This chapter examines the impact of seasonality on APA in one of
Florida's largest hypereutrophic lakes, Lake Apopka. Despite low SRP
concentrations < 1 /ig L'\ chlorophyll a concentrations are
regularly > 100 ng L'1 (Canfield 1981; Reddy and Graetz 1990). High
standing crops may be maintained through the rapid recycling of SRP or
by obtaining P from other sources. Total soluble P (TSP) concentrations
of 255 ng P L'1 have been recorded in Lake Apopka (Reddy and Graetz
1990). Total soluble P may represent bioavailable P (Bradford and


132
cuvettes were placed in the fluorometer and fluorescence was measured.
The enzyme activity was measured as an increase in fluorescence as the
substrate was enzymatically hydrolyzed to the fluorescent product.
Fluorescence units were converted to enzyme activity using a standard
calibration curve of 3-o-methylfluorescein (Sigma Chemicals). The
fluorescence was measured using a Turner fluorometer No. 110, equipped
with Turner lamp no. 110-853, in combination with 47 B primary and 2a-
12 secondary filters. Autoclaved lake water with substrate added was
used as a control.
Chlorophyll a was determined spectrophotometrically following
extraction with acetone and correction for pheophytin (APHA (1002-G),
1985). Total soluble P, TSN, NH4-N, and SRP were determined by standard
methods (APHA 1985).
Sediments. Alkaline phosphatase activity was determined by a
method adapted from Sayler et al. (1979). Sediment samples (1 g wet
sediment) were placed in centrifuge tubes. Three mL of 1 M Tris-Tris
HC1 buffer (pH 7.0) were added to each tube. The samples were sonicated
(Heatsystems Ultrasonics Model W-220F) for 45 sec at 30% relative
output, to release cell bound phosphatase. The samples were then
incubated at 25*C, with 1 mL p-nitrophenyl phosphate, at a concentration
of 50 mg mL'1 (Sigma Chemicals) for 1 hr. At the end of the incubation
3 mL of 1 M NaOH were added to the tubes to stop the reaction and
enhance p-nitrophenol color. To account for color interference controls
were obtained by incubating sediment without the substrate and
subsequently adding the substrate along with NaOH at the end of the
incubation. All samples were then centrifuged at 7000 rpm (7096 g) for


138
O 5 10 15 20 25 30
TIME (h)
Fig. 5-4. Nutrient concentrations in Lake Apopka water
incubated in the dark under aerobic and anaerobic
conditions: a) total soluble phosphorus; b) soluble reactive
phosphorus. Vertical bars represent 1 SE. No vertical bar
indicates SE is smaller than the symbol size.


158
Table B-2. Secchi depth transparency.
Date
Site
Mean" SE
1 2 3 4 5 6 7 8
m
APR
1.00
0.24
0.20
0.25
0.23
0.24
0.23
0.25
0.23
0.00
JUN
1.50
0.21
0.21
0.24
0.24
0.21
0.24
0.21
0.22
0.00
AUG
1.90
0.20
0.23
0.30
0.25
0.25
0.23
0.23
0.24
0.00
OCT
1.38
0.27
0.28
0.28
0.27
0.35
0.24
0.30
0.28
0.00
DEC
1.00
0.25
0.25
0.39
0.31
0.32
0.25
0.27
0.29
0.01
FEB
0.72
0.26
0.24
0.30
0.25
0.22
0.25
0.25
0.25
0.00
Mean
SE
1.36
0.06
0.23
0.00
0.23
0.01
0.29
0.01
0.26
0.01
0.27
0.01
0.24
0.00
0.25
0.01
Means listed in this column are means of sites 2-8.


BIOAVAILABILITY OF ORGANIC PHOSPHORUS
IN A SHALLOW HYPEREUTROPHIC LAKE
By
SUSAN NEWMAN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1991


48
EHP. No EHP was found in Lake Apopka during the October fractionation
experiment. However, this does not reflect the absence of these
substrates. In some lakes the release rate of P from EHP satisfies the
P uptake rate (Chrst and Overbeck 1987) while in others a large
discrepancy exists between these two rates (Heath 1986; Boavida and
Heath 1988). Low EHP concentrations have been attributed to the rapid
hydrolysis of this fraction (Berman 1970; Taft et al. 1977).
Alternatively, this may reflect methodological problems; 1) the method
to measure EHP requires the addition of extracted APA from Escherichia
coli to filtered lake water. This enzyme was not adapted to this system
and consequently may not be as efficient or may require a longer
incubation time, 2) filtered lake water does not represent the entire P
pool available, as particulate organic P, a large portion of TP, may
also be susceptible to enzymatic hydrolysis (Jansson 1977), 3) enzymes
added from E. coli were more inhibited by inorganic P additions than
natural enzyme populations (Chrst et al. 1986). The first and third
problems were resolved by measuring the increase in SRP in filtered
water without the addition of the enzyme (Chrst et al. 1986). However,
in a lake which has high particulate APA, this would not be a true
representation of the potential APA.
Conclusions
Seasonal and spatial differences in water chemistry were observed.
In general, seasonal variability was greater than spatial variability.
The system was highly productive as evidenced by chlorophyll a
concentrations > 150 /g L'1, and an annual mean of 81 /ig L'1. Annual


TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
LIST OF TABLES v
LIST OF FIGURES viii
ABSTRACT xii
CHAPTERS
1 INTRODUCTION 1
Statement of the Problem 1
Need for Research 2
Organic Phosphorus Mineralization 3
Alkaline Phosphatase Activity in the Water Column 8
Alkaline Phosphatase Activity in the Sediment 10
Objectives 12
Dissertation Format 14
2 SEASONAL VARIABILITY IN ALKALINE PHOSPHATASE ACTIVITY IN A
SHALLOW HYPEREUTROPHIC LAKE 16
Introduction 16
Materials and Methods 18
Results 23
Discussion 39
Conclusions 48
3 RESPONSE OF NATURAL PLANKTON POPULATIONS
TO NUTRIENT ENRICHMENT 50
Introduction 50
Materials and Methods 51
Results 60
Discussion 86
Conclusions 94
iii


94
appeared to be a reliable indicator of the nutritional status of the
plankton.
Conclusions
Results from this study show that APA of Lake Apopka plankton is
inhibited by high concentrations of inorganic P. Hence, ambient lake
water SRP concentrations (<10 ng L'1) will not be sufficient to inhibit
APA. Both P limitation and co-limitation of N and P were observed.
During conditions of inorganic P limitation, plankton P uptake
demand was very high as shown by the rapid uptake of SRP. Uptake rates
as high as 1.5 /xg L'1 min'1 were reported. The first step in the
metabolism of the added P was the accumulation of internal P as
identified by hot water extraction. After the apparent removal of all
SRP from the growth medium, plankton utilized P from the HEP-SRP and
HEP-TSP pools for growth. Studies assessing the importance of surplus P
frequently only determine HEP-SRP, however, this study highlighted the
need to measure both HEP-TSP and HEP-SRP.
Alkaline phosphatase activity tended to increase with growth of
the plankton, however, the intensity of APA was dependent upon the
conditions of P limitation. Severe P limitation was indicated by
specific APA values > 1 nmol APA /zg chlorophyll a'1 min'1. Specific APA
values < 1 nmol APA ng chlorophyll a'1 min'1 may indicate slight P
limitation, but APA is associated with numerous organisms in this
eutrophic system, which change both spatially and temporally, hence P
limitation should be confirmed by nutrient enrichment bioassays.


LIST OF TABLES
Table page
2-1. Means of selected weather data measured at the central
station (mean 1 SE) 24
2-2. Correlation coefficients for chlorophyll and alkaline
phosphatase activity measured bimonthly at 7 sites in
Lake Apopka (significant at a=0.05, n=7) 32
2-3. Correlation coefficients between alkaline phosphatase
activity and selected parameters measured bimonthly at
8 sites in Lake Apopka (significant at a=0.05, n=6) 33
2-4. Correlation coefficients of selected water chemistry
parameters determined at 7 sites in October 1989
(significant at a=0.05, n=7) 42
3-1. Nutrient additions made to diluted lake water collected
in November 1989 54
3-2. Nutrient additions made to diluted lake water collected
in April and August 1990 56
3-3. Initial concentrations of selected parameters measured in
diluted lake water prior to nutrient addition
(triplicate samples) in November 1989 (mean 1 SE) 61
3-4. Chlorophyll a and specific alkaline phosphatase activity
measured in natural plankton populations collected in
November 1989, 72 h after receiving nitrogen and
phosphorus additions 62
3-5. Initial concentrations of selected parameters measured
in diluted lake water prior to nutrient addition
(triplicate samples) (mean 1 SE) 66
3-6. Phosphorus uptake rates for natural plankton populations
collected in August 1990, 30 min after receiving nitrogen
and phosphorus additions (mean 1 SE) 74
3-7. Hot water extractable phosphorus concentrations of composite
lake water samples collected in April 1990, 216 h after
nutrient additions 78
v


I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
-K K sue _
Konda R. Reddy, Chair
Professor of Soil Science
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Joseph J. Delfino
Professor of Environmental
Engineering Sciences
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Donald A. Graetz
Professor of Soil Science
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Edward J,
Assistant^ofes^or of
Forest Resources and
Conservation


52
Sampling Procedures
Water samples were collected 30 cm below the water surface from
the west side of the lake on November 16 1989, and from the east side on
April 18 and August 21 1990 (Fig. 3-1). Water was stored in
polycarbonate and polyethylene carboys in the dark and at ambient
laboratory temperature, for no more than 24 h prior to the start of the
experiments.
Experimental Design
The effect of inorganic phosphorus concentrations upon alkaline
phosphatase activity
Lake water collected in November 1989 was diluted 2:1 (260 mL
unfiltered:140 mL filtered) with filtered lake water (0.45 /im), to
reduce the chlorophyll a concentration. Four hundred mL were placed in
each of 15 wide mouth 500 mL erlenmeyer flasks. The experimental design
was completely randomized with 5 treatments and 3 replicates. The water
was spiked with nutrient additions (Table 3-1). Nitrogen was added at
an N:P ratio of 10:1 to avoid N limitation. The flasks were capped with
cotton wool and placed on magnetic stir plates, under a black plastic
enclosure in the greenhouse. Temperature within the enclosure was
maintained using window air conditioning units and fans (mean 1SE,25*C
+ 0.21). Light was supplied at 200 /xmol photons m'2 s'1 using cool-white
fluorescent lamps. The light:dark schedule was 16:8. The flasks were
shaken and aliquots were withdrawn by syringe from treatments 1 and 2 at
0, 24, 72 and 96 h. Aliquots were removed from remaining treatments at
24, 72, and 168 h. Additional sampling times of 268 and 312 h were
included for cultures which received 1000 nq L'1. Samples requiring


TSN (mg
136
TIME (h)
Fig. 5-3. Nutrient concentrations in Lake Apopka water
incubated in the dark under aerobic and anaerobic
conditions: a) total soluble nitrogen; b) NH4-N.
Vertical bars represent 1 SE. Absence of bar indicates SE
is smaller than the symbol size.


58
cuvettes were placed in the fluorometer and fluorescence was measured.
The enzyme activity was measured as an increase in fluorescence as the
substrate was enzymatically hydrolyzed to the fluorescent product.
Fluorescence units were converted to enzyme activity using a standard
calibration curve of 3-o-methylfluorescein (Sigma Chemicals). The
fluorescence was measured using a Sequoia Turner fluorometer Model 110,
equipped with Turner lamp no. 110-853, in combination with 47 B
excitation and 2a-12 emission filters. Autoclaved lake water with
substrate added was used as a control.
Chlorophyll a was determined by measuring in vivo fluorescence,
using a Turner Design Model 10 fluorometer equipped with a Turner lamp
no. 110-853, in combination with 5-60 excitation and 2-64 emission
filters. A Sequoia Turner fluorometer Model 110 equipped with the same
light source and filters was used to measure chlorophyll a fluorescence
in the first experiment. Pheophytin a fluoresces at the same wavelength
as chlorophyll a, so chlorophyll a concentrations are uncorrected for
pheophytin. The calculation of chlorophyll a was based on equations by
Lorenzen (1967);
ABS x (vol. extracted (mL)) x (unit of measure factor) x 1
89 (vol. filtered (mL)) pathlength
(cm)
ABS = absorbance at 664 nm
89 = absorption coefficient for chlorophyll a in 90% acetone
Unit of measure factor = 108 for ng L'1.


TOTAL ALKALINE PHOSPHATASE ACTIVITY
113
75
(a)
50
25
-
\
k 2 cm
n
1 L_
1... zz
~*
TIME (h)
Fig. 4-7. The total alkaline phosphatase activity measured in the
overlying water column of triplicate sediment cores after
resuspension of surficial sediments: a) 2 cm resuspended;
b) 5 cm resuspended; c) 10 cm resuspended; d) control,
no resuspension. Initial data point indicates the conclusion
of resuspension. Vertical bars indicate 1 SE. No bar
indicates SE is smaller than symbol size.


42
Table 2-4. Correlation coefficients of selected water chemistry
parameters determined at 7 sites in October 1989
(significant at a=0.05, n=7).
TP
TRP
TAH
TOP
TSP
SAH
SOP TSUSP
SUSAHP
CHL
TOP
O
o
*
SAH
0.92
SOP
TSUSP
SUSRP
SUSAHP
0.95
0.78
0.99
0.94
1.00
0.86
SUSOP
TAPA
CHL
TOC
0.97
-0.80
0.96
-0.66*
-0.73
0.99
-0.84
-0.68
TSS
-0.78
0.74
0.73
Blank space indicates not significant at a=0.05.
* Significant at or=0.10.


DEPTH
111
0.0
2.5
E
5.0
7.5
10.0
l
4 5 6 7 8 9 10
ALKALINE PHOSPHATASE ACTIVITY
(/mol g d.w. 1 h 1)
Fig. 4-5. The depth distribution of alkaline phosphatase activity in
triplicate resuspended and undisturbed (control) sediment
cores collected in September 1989 from the center of Lake
Apopka. Horizontal bars indicate 1 SE. No bar indicates SE
is smaller than symbol size.


161
Table B-5. Total solids.
Date
Site
Mean*
SE
1
2
3
4
5
6
7
8
mg
r1 ---
L
APR
ND*
ND
ND
ND
ND
ND
ND
ND
JUNE
148
349
373
337
411
411
300
404
369
6
AUG
156
309
317
328
334
335
323
350
328
2
OCT
202
387
396
385
402
395
402
411
397
1
DEC
229
369
369
389
370
373
393
396
380
2
FEB
271
531
499
524
511
459
496
508
504
3
Mean
201
389
391
393
406
395
383
414
SE
10
17
13
16
13
9
15
12
Means listed
in this
column
are
means
of sites 2-8.
* ND indicates not determined.


TOTAL P CONCENTRATION
38
Fig. 2-9. Distribution of phosphorus compounds determined in whole lake
water at 8 sites in October 1989. TP=total phosphorus, T0P=
total organic phosphorus, TAH=total acid hydrolyzable
phosphorus, TRP=total reactive phosphorus. Vertical bars
represent 1 SE.


179
Weimer, W. C., and D. E. Armstrong. 1979. Naturally occurring organic
phosphorus compounds in aquatic plants. Environ. Sci. Technol.
13:826-829.
Wetzel, R. G. 1983. Limnology. 2nd. Edn. Saunders College,
Philadelphia.
Wetzel, R. G. 1991. Extracellular enzymatic interactions: storage,
redistribution, and interspecific communication, p. 6-28. In
R. J. Chrdst [ed.], Microbial enzymes in aquatic environments.
Science Tech. Publ.
Wolanski, E., T. Asaeda, and J. Imberger. 1989. Mixing across a
lutocline. Limnol. Oceanogr. 34:931-938.
Wynne, D. 1977. Alterations in activity of phosphatases during the
Peridinium bloom in Lake Kinneret. Physiol. Plant. 40:219-224.
Wynne, D. 1981. The role of phosphatases in the metabolism of
Peridinium cinctum, from Lake Kinneret. Hydrobiol. 83:93-99.
Wynne, D., and T. Berman. 1980. Hot water extractable phosphorus- an
indicator of nutritional status of Peridinium cinctum (Dinophyceae)
from Lake Kinneret (Israel)?. J. Phycol. 16:40-46.
Wynne, D., and M. Gophen. 1981. Phosphatase activity in freshwater
zooplankton. OIKOS 37:369-376.
Young, T. C., J. V. DePinto, S. C. Martin, and J. S. Bonner. 1985.
Algal available particulate phosphorus in the Great Lakes basin. J.
Great Lakes Res. 11:434-446.


DEPTH (cm)
APA
. J 1 u~1
/imol g d.w. h
0 2 4 6 8 10
SRP VOLATILE SOLIDS
mg L 1 %
0 1 60 70 80
Fig. 4-3, The depth distribution of selected parameters measured in triplicate sediment cores collected
in May 1989 from the center of Lake Apopka: a) alkaline phosphatase activity; b) porewater
soluble reactive phosphorus; c) volatile solids. Bars indicate 1 SE. No bar indicates
SE is smaller than symbol size.


173
Jansson, M., H. Olsson, and K. Pettersson. 1988. Phosphatases; origin,
characteristics and function in lakes. Hydrobiol. 170:157-175.
Jones, J. G. 1972a. Studies on freshwater bacteria: association with
algae and alkaline phosphatase activity. J. Ecol. 60:59-75.
Jones, J. G. 1972b. Studies on freshwater micro-organisms: phosphatase
activity in lakes of differing degrees of eutrophication. J. Ecol.
60:777-791.
Juma, N. G., and M. A. Tabatabai. 1978. Distribution of
phosphomonoesterases in soils. Soil Sci. 126:101-108.
Kandeler, E. 1990. Characterization of free and dissolved phosphatases
in soils. Biol. Frtil. Soils 9:199-202.
Klotz, R. L. 1985a. Factors controlling phosphorus limitation in
stream sediments. Limnol. Oceanogr. 30:543-553.
Klotz, R. L. 1985b. Influence of light on the alkaline phosphatase
activity of Selenastrum capriconutum (Chlorophyceae) in streams.
Can. J. Fish. Aquat. Sci. 42:384-388.
Kobori, H., and N. Taga. 1979a. Phosphatase activity and its role in
the mineralization of organic phosphorus in coastal seawater. J.
Exp. Mar. Biol. Ecol. 36:23-39.
Kobori, H., and N. Taga. 1979b. Occurrence and distribution of
phosphatase in neritic and oceanic sediments. Deep Sea Res. 26:799-
808.
Krausse, G. L., and J. Sheets. 1980. Nutrient chemistry laboratory
notes: hot water extractable and particulate phosphorus. Great Lakes
Res. Div., Univ. of Michigan. Inhouse note.
Kuenzler, E. J. 1965. Glucose-6-phosphate utilization by marine algae.
J. Phycol. 1:156-164.
Kuenzler, E. J., and J. P. Perras. 1965. Phosphatases of marine algae.
Biol. Bull. 128:271-284.
Lean, D. R. S., and E. White. 1983. Chemical and radiotracer
measurements of phosphorus uptake by lake plankton. Can. J. Fish.
Aquat. Sci. 40:147-155.
Lee, G. F. 1970. Factors affecting the transfer of materials between
water and sediments. Univ. of Wisconsin. Eutrophication Information
Program, Literature Review No. 1. Madison, Wisconsin.
Lee, G. F., W. C. Sonzogni, and R. D. Spear. 1977. Significance of
oxic vs anoxic conditions for Lake Mendota sediment phosphorus


119
APA at the sediment-water interface, while there was no apparent change
in soluble APA.
In the sediment, APA was shown to decrease with depth. Soluble
reactive P is a competitive inhibitor of APA (Coleman and Gettins 1983),
hence the increase of porewater SRP with sediment depth may partially
explain the decrease in APA, due to inhibition of the production of the
enzyme. Organic P and APA have been shown to be positively correlated
(Juma and Tabatabai 1978; Speir and Ross 1978). A high enzyme activity
may be maintained in the presence of competitive inhibitors by the
presence of increased substrate concentrations; however, the decrease in
organic and NaOH-extractable P with depth suggests a decrease in
substrate concentrations. Alkaline phosphatase activity was also found
to have a positive correlation with organic matter (Speir and Ross
1978), therefore the decrease in volatile solids (an indicator of
organic matter), may have contributed to the APA decrease. However, the
most probable cause for the reduced activity with depth is a decrease in
microbial biomass. Decreasing APA with sediment depth has been shown
to have a positive correlation with microbial biomass (Sayler et al.
1979; Ayyakkannu and Chandramohen 1971). Microbial biomass was not
measured in this study, however, a decrease with depth could be expected
in these sediments because of highly reduced (anaerobic) conditions at
lower depths in Lake Apopka sediments (Moore et al. 1991). Pul ford and
Tabatabai (1988) suggested that under anaerobic conditions, the higher
solubility of metals such as Fe and Mn results inhibition of APA (Juma
and Tabatabai 1978). Lake Apopka sediment is Ca dominated (Moore et al.


SOLUBLE APA TOTAL APA
139
TIME (h)
Fig. 5-5. Alkaline phosphatase activity in Lake Apopka water
incubated in the dark under aerobic and anaerobic
conditions: a) total alkaline phosphatase activity;
b)soluble alkaline phosphatase activity. Vertical bars
represent 1 SE. No vertical bar indicates SE is smaller
than the symbol size.


CHAPTER 6
ORGANIC PHOSPHORUS CYCLING IN LAKE APOPKA
Organic P plays a dominant role in the P cycle of aquatic systems
(Fig. 1-1). The bioavailability of organic P to plankton is regulated
by the activity of enzymes, types of substrates and associated physico
chemical factors in the system. The research presented in this
dissertation examined the specific organic P compartments (plankton,
water column and sediment) to evaluate the bioavailability of organic P
for plankton growth. Results obtained in this study are summarized in
the context of addressing key research issues raised in an attempt to
understand the P dynamics in Lake Apopka.
(1) How is the enzymatic hydrolysis of organic P affected by
other water chemistry parameters?
Both seasonal and spatial variability of total P (TP), total
soluble P (TSP) and alkaline phosphatase activity (APA), were observed
in the water column. Alkaline phosphatase activity, an indicator of
potential organic P hydrolysis, was dependent upon different water
chemistry parameters, both seasonally and spatially. The majority of
APA was associated with particulate matter, and soluble APA averaged
only 3% of total APA, therefore particulate interactions are a key
component of organic P cycling in Lake Apopka. The attachment of
enzymes to surfaces may increase enzyme longevity, but may also reduce
enzyme activity, either directly through sorption at the active site or
152


18
Peters 1987), hence organic P compounds in this pool may potentially be
hydrolyzed by APA and release inorganic P. With the exception of
extensive research by Berman and colleagues on Lake Kinneret, Israel
(Berman 1970; Wynne and Berman 1980), the majority of studies
investigating APA have been conducted in cold temperate zones. Warmer
climates with mild winters which result in extended periods of
productivity, may result in increased P demand. More studies in warmer
climates are necessary.
The primary objective of this study was to examine the seasonal,
spatial and diel changes in APA to determine whether it represents P
limitation or high P demand. This would also determine what effect the
water chemistry has upon APA. Zooplankton, phytoplankton and
bacterioplankton may all contribute to the total APA pool (Jansson 1976;
Jones 1979a; Kuenzler and Perras 1965; Wynne and Gophen 1981). A second
objective was to estimate the relative importance of these contributors
based on filter size fractionation. Total soluble P may be used as an
indicator of bioavailable P; however, a third objective was to determine
the relationship between APA and other components of the TP pool.
Materials and Methods
Site Description
Lake Apopka is a 12,500 ha, located in central Florida, 28* 37' N
latitude, 81* 37' W longitude (Fig. 2-1). It has a mean depth of 2 m.
Water influxes to the lake include Apopka Springs and backpumping from
surrounding agricultural land. Outflow is northward through the Apopka-


167
Table B-ll. Phosphorus.
Date
Mean* SE
1
2
3
4
5
6
7
8
l
IP
APR
110
140
170
180
200
200
180
260
190
5
JUN
30
150
140
190
150
170
260
140
171
6
AUG
30
140
90
240
240
130
120
230
170
9
OCT
300
520
440
350
200
400
270
400
369
15
DEC
20
100
120
110
130
120
150
150
126
3
FEB
70
190
200
250
280
230
240
240
233
4
Mean
93
207
193
220
200
208
203
237
SE
18
26
21
13
9
17
10
16
ISP
APR
130
110
70
70
90
80
50
40
73
3
JUN
80
40
70
70
80
60
30
40
56
3
AUG
10
10
10
10
10
10
10
10
10
0
OCT
120
170
70
130
60
140
160
60
113
7
DEC
10
10
10
20
20
10
10
20
14
1
FEB
30
40
40
40
40
20
20
20
31
2
Mean
63
63
45
57
50
53
47
32
SE
9
11
5
7
5
9
10
3
SRP
APR
1
3
3
5
3
5
2
3
3
0
JUN
2
3
4
4
5
3
3
5
4
0
AUG
4
7
5
4
5
4
4
5
5
0
OCT
1
1
1
1
1
1
1
1
1
0
DEC
1
1
1
1
1
1
1
1
1
0
FEB
0
3
0
1
1
1
2
2
1
0
Mean
2
3
2
3
3
3
2
3
SE
0
0
0
0
0
0
0
0
Means listed in this column are means of sites 2-8.


SRP (/g
71
l
90
80
70
60
50
40
30
20
10
0
0 48 96 144 192
TIME (h)
Fig. 3-6. Time courses of soluble reactive phosphorus concentrations
following nutrient enrichment of natural plankton
populations collected in April 1990. ON,OP=no nutrient
addition; 400N,0P=400 ng N L'1; 0N,40P=40 /xg P L*1;
400N,40P=400 ng N L1 and 40 ng P L*\ and 800N,80P=
800 ng N L'1 and 80 ig P L'1. Vertical bars indicate 1 SE.
No vertical bar indicates SE is smaller than symbol size.


60
Statistical Methods
Data were analyzed using SAS (Statistical analysis systems)
version 6. Balanced data (equal number of observations for each
treatment) were analyzed using the repeated measures procedure which
accounts for the within replicate correlation over time, due to repeated
sampling from the same flasks. Unbalanced data (unequal number of
observations per treatment) were analyzed using a split plot design with
time as the subplot.
Results
The Effect of Inorganic Phosphorus Concentrations upon Alkaline
Phosphatase Activity
The majority of N, P and APA were associated with particulate
matter (Table 3-3). Due to chlorophyll a analysis problems arising from
fluorometer calibration, only chlorophyll a data from 0 and 72 h are
presented and used for statistical analysis (Table 3-4). Increased
chlorophyll a concentrations were observed at 72 h in all cultures
except those which received no nutrient addition. Increased growth was
associated with higher nutrient additions, a 69% increase in chlorophyll
a was observed in treatment 5 (N=2500 P=1000) cultures. Chlorophyll a
increases of 33% for treatment 4 (N=1000 P=100) and 11 % for treatments
2 (N=500 P=0) and 3 (N=0 P=10) cultures were observed. The opposite was
observed for both total and soluble APA (Fig. 3-2a and 3-2b). Total and
soluble APA decreased significantly over time in cultures receiving the
highest P additions (100 and 1000 ng L'1). No decrease in total APA was
observed for treatments 1 (N=0 P=0) and 3 (N=0 P=10), but soluble APA


CHAPTER 5
THE EFFECT OF SEDIMENT AND WATER COLUMN ANOXIA ON
ORGANIC PHOSPHORUS MINERALIZATION
Introduction
Organic P constitutes the major component of total P in the
sediment and water column of lakes. When soluble inorganic P
concentrations within the water column are low plankton may produce
phosphatase enzymes, which hydrolyze organic P compounds with the
release of inorganic P (Kuenzler and Perras 1965; Reichardt 1971). As a
result the measurement of alkaline phosphatase activity (APA) has been
used as a tool to indicate P limitation and potential organic P
mineralization through enzymatic hydrolysis (Healey and Hendzel 1979b;
Gage and Gorham 1985).
Under well oxygenated conditions (aerobic), mineralization of
organic P is rapid. However, depletion of dissolved oxygen (DO)
concentrations can occur in the water column as a result of high
respiratory activity. Under reduced DO concentrations the rate of
enzymatic hydrolysis of organic P compounds could be reduced as the
metabolism of aerobic plankton is reduced. This may be of particular
importance at the sediment-water interface where DO concentrations are
often depleted. Oxygen concentrations in the water column may vary both
diurnally (Howeler 1972; Reddy 1981) and seasonally (Mortimer 1941). In
rare circumstances, metalimnetic DO depletion may occur with increasing
124


21
phytoplankton and bacteria, water samples collected from sites 1, 4 and
8 received further filtration. Subsamples of water were filtered
through 150, 8, 2.5, 0.45 and 0.2 /im filters. Water from these size
fractions was analyzed for chlorophyll a and determined to represent 80,
9, 3, 1 and 0% chlorophyll a distribution. To minimize the filtration
time, the filtration was not performed sequentially. Alkaline
phosphatase activity and TP were determined on all samples.
Dissolved oxygen and temperature (YSI, model 58), and pH (Orion,
model SA 230) were recorded at 0.3 m. Light penetration was estimated
by measuring Secchi disk transparency. Samples were collected at
approximately the same time at each sampling period to minimize the
effects of diel variation.
Piel sampling. Diel studies were conducted on March 21 1989 and
February 6 1990. In March 1989 DO and pH of the water were measured
from a pontoon boat which was anchored at the central station (site 8)
for 24 h. In February 1990, pH and DO measurements were determined by
SJRWMD personnel. Water samples at both time periods were collected
using an automatic sampler, and kept cool until returned to the
laboratory for analysis.
Fractionation of lake water phosphorus. To elucidate the
relationship between P and APA in the water column, the P forms were
separated analytically using discrete extraction procedures. In October
1989, water samples from all 8 sites were partitioned into total and
soluble; reactive P, acid hydrolyzable and organic P (APHA 1985).
Enzyme hydrolyzable P (EHP) was also determined.


TSP
75
TIME (h)
Fig. 3-8. Time courses of nutrient concentrations following nutrient
enrichment of natural plankton populations collected in
April 1990. 0N,0P=no nutrient addition; 400N,0P=400 ng
N L1; 0N,40P=40 ng P L'1; 400N,40P=400 ng N L'1 and 40 ng
P L'1, and 800N,80P=800 ng N L'1 and 80 ng P L'1: a) total
soluble phosphorus; b) [N03 + N02]-N; c) NH4-N. Vertical
bars indicate 1 SE. No vertical bar indicates SE is smaller
than symbol size.


SOLUBLE REACTIVE PHOSPHORUS
117
J 1 I I I L
1 O cm
Fig. 4-10. Soluble reactive phosphorus concentrations measured in the
overlying water column of triplicate sediment cores after
resuspension of 0, 2, 5 and 10 cm surficial sediments:
a) 2 cm resuspended; b) 5 cm resuspended; c) 10 cm
resuspended; d) control, no resuspension. Initial data
point indicates the conclusion of resuspension. Vertical
bar indicates 1 SE. Absence of vertical bar indicates
symbol size is greater than SE.


fiage
4-3. The depth distribution of selected parameters measured in
triplicate sediment cores collected in Masy 1989 from
the center of Lake Apopka 107
4-4. The depth distribution of selected parameters measured in
triplicate sediment cores collected in May 1989 from
the center of Lake Apopka 108
4-5. The depth distribution of alkaline phosphatase activity in
triplicate resuspended and undisturbed (control) sediment
cores collected in September 1989 from Lake Apopka Ill
4-6. Concentrations of selected parameters measured in the
overlying water column following sediment resuspension of
0, 2, 5, and 10 cm surficial sediments 112
4-7. The total alkaline phosphatase activity measured in the
overlying water column following sediment resuspension of
0, 2, 5, and 10 cm surficial sediments 113
4-8. The relationship between alkaline phosphatase activity and
total suspended solids in the overlying water column of
triplicate sediment cores after resuspension of surficial
sediments 114
4-9. Soluble alkaline phosphatase activity measured in the
overlying water column following sediment resuspension of
0, 2, 5, and 10 cm surficial sediments 116
4-10. Soluble reactive phosphorus concentrations measured in the
overlying water column following sediment resuspension of
0, 2, 5, and 10 cm surficial sediments 117
5-1. Diagram illustrating the apparatus used to control dissolved
oxygen concentration and redox potential 129
5-2. The sediment extraction scheme used to fractionate organic
phosphorus in sediment 131
5-3. Nutrient concentrations in Lake Apopka water incubated
in the dark under aerobic and anaerobic conditions 136
5-4. Nutrient concentrations in Lake Apopka water incubated
in the dark under aerobic and anaerobic conditions 138
5-5. Alkaline phosphatase activity in Lake Apopka water
incubated in the dark under aerobic and anaerobic
conditions 139
x


104
W-220F) for 45 s at 30% relative output, to release cell bound
phosphatase. The samples were then incubated with 1 mL of 50 mg mL 1 of
p-nitrophenyl phosphate, (Sigma Chemicals) at 25* C for 1 h. After
1 h, 3 mL of 1 M NaOH were added to the tubes to stop the reaction and
enhance p-nitrophenol color. Controls to account for substrate color
and color release during solubilization of organic matter by the NaOH,
were obtained by incubating sediment without the substrate and
subsequently adding the substrate along with NaOH at the end of the
incubation. All samples were then centrifuged at 7000 rpm (7096 g) for
15 min. The liquid was removed and absorbance at 410 nm (Shimadzu Model
UV-160) measured. Concentrations were determined by calibration with a
standard curve of p-nitrophenol (Sigma Chemicals).
Porewater was extracted by centrifuging the sediment subsample
under anaerobic conditions at 5000 rpm (3620 g) for 15 min. The
porewater was immediately filtered through 0.45 pm Gelman membrane
filters. Soluble reactive P was measured using standard methods (APHA
1985). Sodium hydroxide extractable P was obtained by shaking 5 g of
wet sediment with 20 mL of 0.1 M NaOH for 16 h. Total P and SRP of
filtered extracts were then measured using standard methods described
for water.
Water content was determined by drying a known weight of wet
sediment at 70*C to a constant weight. The dried sediment was ground to
pass through a 20 mesh screen, using a Spex 8000 grinding mill.
Volatile solids were reported as the loss in weight due to ignition of
dried sediment at 500*C for 2 h. Total and organic P content of the
dried sediment was determined via ignition (Walker and Adams 1958).


115
The soluble APA measured in the water column after resuspension of
2 cm of sediment exhibited the same trend as observed for total APA,
decreasing as the TSS decreased (Fig. 4-9). Soluble APA did not show
significant differences over time in the cores in which the surface 5
and 10 cm were resuspended.
Soluble reactive P data exhibit such variability that no
statistically significant response was observed. However, all cores
including undisturbed cores, exhibited a tendency to increase at time
12 h (Fig. 4-10). This would suggest that SRP was slowly desorbed from
the underlying sediment.
Sediment. Changes in total and porewater APA within the sediment
of control and disturbed cores were not significant (Table 4-3). The
highly variable porewater APA accounted for less than 1% of the total
activity.
Discussion
The physico-chemical parameters measured within the water column
of Lake Apopka showed no distinct stratification, however, DO, pH,
temperature and chlorophyll a tended to decrease with depth. The
relatively high concentrations of nutrients and chlorophyll a in the
water column are characteristic of hypereutrophic systems, although SRP
concentrations are very low. The APA is also representative of
productive systems (cf. Heath and Cooke 1975). The soluble APA
accounted for only 3% of the total APA, therefore, the majority of APA
within this system was associated with particulate matter. A possible
incomplete settling of particulate matter would explain the increase in


122
concentrations were low. However, with normal TSS (= 70 mg L'1) and
high chlorophyll a, the equation underestimated the measured APA. This
suggests that the predictive capability of this equation is only valid
during periods of resuspension, when sediment particulate matter is the
dominant component of the TSS pool. After 48 h an increase in total APA
was observed in the water column of both control and resuspended cores,
this may be in response to APA production following P limitation of the
plankton population. These increases are apparent as the two points
above the regression line in Fig. 4-8. Consequently, during quiescent
periods other contributors to the TSS pool. e.g. phytoplankton, should
be included to predict APA.
Apart from the cores in which the surface 2 cm was resuspended,
soluble APA was not shown to increase due to resuspension. The sediment
porewater APA was greater than the water soluble APA; however, the
dilution effect due to resuspension brought the porewater concentration
to ambient levels. The APA attributed to porewater exhibited high
variability. This variability could be partially due to differences
between cores, and also the insensitivity of the method at such low APA.
The fluorescent technique used for lake water would be more sensitive,
however, different substrate specificities have been observed
(Pettersson and Jansson 1978). Even considering the variability in the
porewater APA, it was clearly demonstrated that >99% of the APA was
associated with the solids portion of the sediment. A recent study
demonstrated the association of APA with the larger particulate matter
(Rojo et al. 1990) and it has been suggested by Burns (1986) that the


102
were sectioned into 2.5 cm intervals, placed in 125 mL centrifuge tubes,
immediately purged with N2 and stored at 4*C until analysis. The
sediments were analyzed for total APA, TP, organic P and porewater SRP.
Experiment 2. A second experiment was conducted in January 1990
to further evaluate the effect of varying depths of bottom sediment
resuspension upon APA and associated parameters within the water column.
Twelve sediment cores were collected on January 23 1990, to a depth of
20 cm. The overlying water was siphoned off to leave a 45 cm water
column. There were three treatments; the top 2, 5 or 10 cm of
triplicate cores were resuspended for 15 min using the procedures
described in experiment 1. There were 3 replicates for each treatment
and 1 undisturbed core to serve as a control. At predetermined
intervals, 70 mL water samples were withdrawn. The amount of resettling
was measured and the water withdrawn from the midpoint of the exposed
water. Alkaline phosphatase activity, TP, TKN, SRP, TSS and total
organic carbon (TOC) of the water column were determined. At the end of
the sampling period, the surface 2, 5 and 10 cm sediment fractions
representing the respective treatments were sectioned in resuspended and
control cores, and analyzed as described above. In addition, APA was
determined in the porewater using the method described for total
sediment APA, to determine whether the activity was predominantly
particulate or soluble.
Analytical Methods
Water. Alkaline phosphatase activity was determined
fluorometrically (Healey and Hendzel 1979a). One half mL of substrate,


HEP-TSP (fig
79
120
80
40
TIME (h)
Fig. 3-10. Time courses of hot water extractable total soluble
phosphorus following nutrient enrichment of natural plankton
populations collected in August 1990. 0N,0P=no nutrient
addition;400N,0P=400 pg N L ; 0N,40P=40 /xg P L'1;
400N,40P=400 M9 N L'1 and 40 jig P L'1, and 800N,80P=800 ng
N L'1 and 80 /xg P L'1: a) plankton grown at 29C; b)
plankton grown at 19C. Vertical bars indicate 1 SE. No
vertical bar indicates SE is smaller than symbol size.


120
1991), thus, inhibition of APA under anaerobic conditions is probably
due to reduced microbial numbers and metabolism.
The sediment APA observed in this study is in the same range as
those reported for other freshwater systems (APA = 6-18 /imol
g dry wt.'1 h'1) with fine particulate organic matter (Sayler et al.
1979). The APA of marine sediments was much lower, i.e., 0.2-3.3 /imol g
dry wt.'1 h'1 (Ayyakkannu and Chandramohen 1971; Degobbis et al. 1984).
However, a comparison of the sediment APA values obtained in other
studies is difficult due to the lack of standardization in methodology
used. Other complications include the pretreatment of samples, e.g.
drying of the soil (Tabatabai and Bremner 1969) which may affect the APA
values of some soils (Skujins 1976; Speir and Ross 1978).
Short-term resuspension of surficial sediments increased TSS, TKN,
TP and TOC concentrations. This is expected because all these
parameters are interrelated. The concentrations of these species
decreased rapidly following the end of resuspension, as a result of
particle settling. However, the soluble fraction, i.e. SRP, did not
exhibit significant increases as observed previously (Pollman 1983;
Reddy and Graetz 1990) in Lake Apopka cores. The porewater SRP
concentrations of the cores used in these resuspension experiments was
very low (<5 /xg L'1), thus the resuspension of these sediments would not
result in an increase in the SRP concentration of the water column.
Although the TP concentration in the surficial sediments is high (1200
mg kg1), a major portion is organically bound and is not readily
available. The low porewater SRP concentrations suggest that the
surface sediments have a high P sorptive capacity. A high C/P ratio of


22
Analytical Methods
Alkaline phosphatase activity was determined fluorometrically
(Healey and Hendzel 1979). One half mL of substrate,
3-o-methylfluorescein phosphate (Sigma Chemicals), at a concentration
determined to be non-limiting, in 0.1 M Tris buffer (Tham, Fisher
Scientific) pH 8.6, was added to 4.0 mL of lake water in a cuvette.
Both total (whole lake water) and soluble (filtered through 0.45 nm
Gelman membrane filter) APA were determined. The cuvettes were placed
in a water bath (25*C). At timed intervals during a 20 min period the
cuvettes were placed in the fluorometer and the fluorescence measured.
The enzyme activity was measured as an increase in fluorescence as the
substrate was enzymatically hydrolyzed to the fluorescent product.
Fluorescence units were converted to enzyme activity using a standard
calibration curve of 3-o-methylfluorescein (Sigma Chemicals). The
fluorescence was measured using a Turner fluorometer No. 110, equipped
with Turner lamp no. 110-853, in combination with 47 B primary and 2a-
12 secondary filters. Autoclaved lake water with substrate added was
used as a control.
Chlorophyll a was determined spectrophotometrically following
extraction with acetone (APHA (1002-G), 1985). Total organic C was
measured using an 0. I. Corporation Model 524C TOC analyzer, following
oxidation by potassium persulfate. Total P, TSP, and TKN were
determined following Kjeldahl digestion. Soluble reactive P, TSP and TP
were analyzed via ascorbic acid using standard methods (APHA 1985).
Total solids and TSS were determined by standard methods (APHA 1985).


99
Field sampling procedures
Water. Water samples were collected on May 23 1989, from the
center of the lake (Fig. 4-1.) using an alpha sampler (Wildco), at
depths 0, 0.5, 1 and 1.5 m below the surface. Water was stored in 1 L
polyethylene bottles kept on ice until return to the laboratory.
Dissolved oxygen (DO) and temperature (YSI, Model 58) and pH (Orion,
Model SA 230) were recorded with depth. Light penetration was estimated
by measuring the Secchi disk transparency. Within 24 h of return to the
laboratory, samples were analyzed for total and soluble APA. Other
parameters measured were total Kjeldahl N (TKN), total P (TP), SRP, and
chlorophyll a.
Sediment. Three intact sediment cores were collected from the
deck of a boat in May 1989 from the center of the lake using a 6 cm
(I.D.) x 1 m (length) Plexiglass-PVC sediment core sampler (Reddy and
Graetz, 1990). The cores were taken to a depth of 40 cm, capped and
brought to the laboratory for sectioning. The cores were sectioned at
0-2, 2-5, 5-10, 10-20 and 20-40 cm intervals, placed in 125 mL
centrifuge tubes, immediately purged with N2 and stored at 4C until
analysis. Preliminary studies demonstrated that sediments could be held
for at least 3 weeks at 4'C with no change in APA. Alkaline phosphatase
activity, porewater SRP, and water content were determined on wet
sediment. NaOH-extractable P, an indication of bioavailable inorganic P
and labile organic P (Dorich et al. 1985; Young et al. 1985) was also
measured. Total P, organic P and volatile solids content of dried
sediment were determined.


m3
3-2. Time courses of alkaline phosphatase activity following
nutrient enrichment of natural plankton populations
collected in November 1989 63
3-3. Time courses of nutrient concentrations following
nutrient enrichment of natural plankton populations
collected in November 1989 65
3-4. Time courses of chlorophyll a concentrations following
nutrient enrichment of natural plankton populations
collected in April 1990 68
3-5. Time courses of chlorophyll a concentrations following
nutrient enrichment of natural plankton populations
collected in August 1990 69
3-6. Time courses of soluble reactive phosphorus concentrations
following nutrient enrichment of natural plankton
populations collected in April 1990 71
3-7. Time courses of soluble reactive phosphorus concentrations
following nutrient enrichment of natural plankton
populations collected in August 1990 73
3-8. Time courses of nutrient concentrations following nutrient
enrichment of natural plankton populations collected in
April 1990 75
3-9. Time courses of nutrient concentrations following nutrient
enrichment of natural plankton populations collected in
August 1990 grown at 29C 76
3-10. Time courses of hot water extractable total soluble
phosphorus following nutrient enrichment of natural plankton
populations collected in August 1990 79
3-11. Time courses of hot water extractable soluble reactive
phosphorus following nutrient enrichment of natural plankton
populations collected in August 1990 82
3-12. Time courses of alkaline phosphatase activity following
nutrient enrichment of natural plankton populations
collected in April 1990 84
3-13. Time courses of alkaline phosphatase activity following
nutrient enrichment of natural plankton populations
collected in August 1990 85
4-1. Map showing the location of Lake Apopka 98
4-2. Diagram of the sediment resuspension device 101
ix


CHAPTER 4
THE EFFECT OF SEDIMENT RESUSPENSION ON ALKALINE PHOSPHATASE ACTIVITY
Introduction
In many lakes, exchange of P between the sediment and the water
column is dependent upon diffusion related processes (Stumm and Leckie
1971; Tessenow 1972). However, in shallow lakes, P exchange also occurs
due to sediment resuspension during wind events which increase the
interaction between the sediment and the overlying water column. It has
been estimated that the upper 10 cm of sediment is actively involved in
exchange reactions as a result of resuspension with the overlying water
column (Tessenow 1972; Schindler et al. 1977). However, the amount of
sediment that will mix with the water column is a function of the shear
stress and sediment type (Lee, 1970).
The immediate result of sediment resuspension is the increase in
suspended solids concentration in the water column. The suspended
sediment has been shown to provide 28-41% algal available P (Dorich et
al. 1985), as well as physically transporting soluble P to the water
(Ryding and Forsberg 1977). Sediment resuspension also results in
increased exchange of soluble reactive P (SRP) from the sediment to the
water column (Holdren and Armstrong 1980; Pollman 1983). The increase
in exchange has been associated with biological activity (Pomeroy et al.
1965). Conversely, SRP may be removed from the water column as a result
95


page
B-l. Temperature 157
B-2. Secchi depth transparency 158

B-3. Dissolved oxygen 159
B-4. pH 160
B-5. Total solids 161
B-6. Total suspended solids 162
B-7. Chlorophyll a 163
B-8. Total organic carbon 164
B-9. Total Kjeldahl nitrogen 165
B-10. Total and soluble alkaline phosphatase activity 166
B-l 1. Phosphorus 167
vii


TSP
76
l
200
^ 150
_i
2* 100
50
0
0.9
i
cn
E
0.6
0.3
0.0
0.3
0.2
X cn
z E
w0.1
0.0
0 24 48 72 96
TIME (h)
Fig. 3-9. Time courses of nutrient concentrations following nutrient
enrichment of natural plankton populations collected in
August 1990 grown at 29C. ON,OP=no nutrient addition;
400N,0P=400 fig N L*1; 0N,40P=40 fig P L'1; 400N,40P=400 ¡iq
N L1 and 40 fig P L'\ and 800N,80P=800 fig N L'1 and 80 fig
P L'1: a) total soluble phosphorus; b) [N03 + N02]-N;
c) NH4-N. Vertical bars indicate 1 SE. No vertical bar
indicates SE is smaller than symbol size.


84
I
TIME (h)
Fig. 3-12. Time courses of alkaline phosphatase activity following
nutrient enrichment of natural plankton populations
collected in April 1990. 0N,0P=no nutrient addition;
400N,0P=400 nq N L'1; 0N,40P=40 ng P L1; 400N,40P=400 nq
N L'1 and 40 ng P L'1, and 800N,80P=800 nq N L'1 and 80 nq
P L'1: a) total alkaline phosphatase activity; b) specific
alkaline phosphatase activity. Vertical bars indicate 1 SE.
No vertical bar indicates SE is smaller than symbol size.


PHOSPHORUS (/zg L ) APA (nM min
27
Fig. 2-
30
APR JUN AUG OCT DEC FEB
1989-90
3. Seasonal variability of selected parameters determined
bimonthly at 7 sites in Lake Apopka: a) alkaline phosphatase
activity; b) phosphorus. Vertical bars represent 1 SE.


93
by normalizing the data to ATP, and thus express the indicator relative
to living biomass (Healey and Hendzel 1980).
Although the focus of this research has been on P limitation, Lake
Apopka has been found to often be N limited or co-limited by both N and
P (Aldridge, F. J., unpublished data, Department of Fisheries and
Aquaculture, University of Florida, Gainesville, FL.). The uptake of
N03-N, shown to be the preferred form of N for Lake Apopka phytoplankton
(Aldridge, F. J., unpublished data), gives an insight into the N
requirements of the system. In April, a situation of co-limitation,
[N03 + N02]-N concentrations were shown to decrease over time, and NH4-N
concentrations tended to increase over time, suggesting that
mineralization of organic N occurred. In August, when plankton were
established as being slightly P limited, no decrease in [N03 + N02]-N
concentrations were observed.
The limitation of the analyses used in this study is that APA and
HEP concentrations do not differentiate between P limitation and co
limitation. Nitrogen limitation parameters, such as ammonium
enhancement, should also be determined to assess the N requirements of
the plankton and thus enable the assessment of N limitation in
conjunction with P limitation (Vincent et al. 1984).
The continued measurement of HEP-SRP as a P limitation indicator
has been questioned based on its highly dynamic nature (Wynne and Berman
1980; Cembella 1984b). Under ambient conditions such rapid
accumulation of HEP-SRP may not occur (Wynne and Berman 1980). Others
have shown that HEP-SRP is a good indicator of the nutritional status of
plankton (Pettersson 1980; Sproule and Kalff 1978). In this study it


CHAPTER 1
INTRODUCTION
Eutrophication may be defined as the nutrient and/or organic
matter enrichment that produces high biological productivity (Likens
1972). This process is often accelerated by man, through allochthonous
loading to the system from surface runoff, agricultural drainage and
wastewater effluent.
Eutrophication of our waterbodies has recently become a major
concern due to the ever increasing need for resource conservation.
Consequently, efforts are now being made to further understand the
process of eutrophication and to identify key management strategies to
abate this process.
Statement of the Problem
Lake Apopka is a 12,500 ha lake located in central Florida. It
has a mean water depth of 2 m, overlying highly flocculent organic
sediments (Reddy and Graetz 1990). Historically, the lake had clear
water, submersed macrophytes and supported substantial sport fish
populations. However, the physico-chemical properties of the lake have
been altered through nutrient enrichment following the construction of
the Apopka-Beauclair canal, discharge of sewage to the lake, and back
pumping from the surrounding muck farms (USEPA 1979). Following the
1947 hurricane, the submerged vegetation was uprooted, and the first
1


129
pH electrode
DO electrode
Purging gas
Sampling port
(a)
Lake water
Magnetic Stirring Plate
Fig. 5-1. Diagram illustrating the apparatus used to control dissolved
oxygen concentration and redox potential: a) anoxia control
in lake water; b) redox potential control of sediment
suspensions.


178
Stratton, F. E. 1968. Ammonia nitrogen losses from streams. J.
Sanitary Eng. Div. Proc. Am. Soc. Civ. Eng. 94:1085-1092.
Stratton, F. E. 1969. Nitrogen losses from alkaline water
impoundments. J. Sanitary Eng. Div. Proc. Am. Soc. Civ. Eng.
95:223-231.
Strickland, J. D. H., and T. R. Parsons. 1968. A practical handbook of
seawater analysis. Bull. 167 Fish. Res. Bd. Can.
Stumm, W., and J. 0. Leckie. 1970. Phosphate exchange with sediments;
its role in the productivity of surface waters. Fifth Int. Water
Poln. Res. Conf. 111:2611-2616.
Tabatabai, M. A., and J. M. Bremner. 1969. Use of p-nitrophenyl
phosphate for assay of soil phosphatase activity. Soil Biol.
Biochem. 1:301-307.
Taft, J. L., M. E. Loftus, and W. R. Taylor. 1977. Phosphate uptake
from phosphomonoesters by phytoplankton in the Chesapeake Bay.
Limnol. Oceanogr. 22:1012-1021.
Tarapchak, S. J., S. M. Bigelow, and C. Rubitschum. 1982. Soluble
reactive phosphorus measurements in Lake Michigan: filtration
artifacts. J. Great Lakes Res. 8:550-557.
Tessenow, U. 1972. Losungs-, Diffusions- und Sorptionsprozesse in der
Oberschicht von Seesedimenten. 1. Ein Langzeitexperiment unter
aseroben und anaeroben Bedingungen in Fleibgleichgewicht. Arch.
Hydrobiol. Suppl. 38:353-398.
Torriani, A. 1960. Influence of inorganic phosphate in the formation
of phosphatases by Escherichia coli. Biochim. Biophys. Acta 38:460-
469.
U. S. Environmental Protection Agency (USEPA). 1979. Environmental
impact statement. Lake Apopka restoration project. Lake and Orange
Counties, Florida. (EPA 904/0-8-79-043). U. S. Environmental
Protection Agency, EMSL, Cincinnati, OH.
Vincent, W. F., W. Wurstbaugh, C. L. Vincent and P. J. Richerson. 1984.
Seasonal dynamics of nutrient limitation in a tropical high-altitude
lake (Lake Titicaca, Peru-Bolivia): Application of physiological
bioassays. Limnol. Oceanogr. 29:540-552.
Walker, T. W., and A. F. R. Adams 1958. Organic phosphorus, p. 411-
413. In A. L. Page, R. H. Miller, and D. R. Keeney [eds.j, Methods
of soil analysis, Part 2: Chemical and microbiological properties.
1982 ASA, SSSA. Madison, WI.


121
the surface sediments also suggests that microbial immobilization of
inorganic P occurs (Reddy and Graetz 1990).
The mechanism of P release from suspended particles will depend on
the rate of P desorption from the solid phase to the liquid phase and
the physico-chemical properties of the water column. Resuspension has
been shown to increase the biological breakdown of organic P (Pomeroy et
al. 1965). Resuspension of anaerobic sediments to the overlying
oxygenated water column will result in aeration of the sediments. This
may make the associated organic matter more susceptible to enzymatic
hydrolysis (Pulford and Tabatabai 1988). The pH of the surface sediment
was 7, compared to a water column pH of 8-9. Thus any SRP release into
the water column upon resuspension could be immediately precipitated as
calcium phosphates (Moore et al. 1991).
In shallow lakes sediment resuspension may play an important role
in P recycling (Ryding and Forsberg 1977). Due to the association of
APA with TSS, prolonged resuspension would result in higher APA levels
within the water column. As observed by Burns (1986), the attachment of
APA at a non-active site would result in increased longevity of the
enzyme within the aquatic system. If phosphomonoesters are present SRP
would be released. The insignificant, but apparent gradual increase in
SRP in all cores at 12 h, may be due to the enzymatic degradation of the
more easily hydrolyzed organic P compounds. Hence, APA may enhance the
ability of sediments and particulate matter to recycle P. To test the
relationship observed between TSS and total APA, seasonal data collected
in the field (chapter 2) was fitted to the equation A good fit was
observed when TSS were high (= 100 mg L"1) and chlorophyll a


4 THE EFFECT OF SEDIMENT RESUSPENSION ON ALKALINE PHOSPHATASE
ACTIVITY 95
Introduction 95
Materials and Methods 97
Results 105
Discussion 115
Conclusions 123
5 THE EFFECT OF SEDIMENT AND WATER COLUMN ANOXIA ON
ORGANIC PHOSPHORUS MINERALIZATION 124
Introduction 124
Materials and Methods 127
Results 134
Discussion 145
Conclusions 151
6 ORGANIC PHOSPHORUS CYCLING IN LAKE APOPKA 152
APPENDICES
A LORAN COORDINATES 156
B CONCENTRATIONS OF SELECTED WATER CHEMISTRY
PARAMETERS DETERMINED BIMONTHLY FROM
APRIL 1989 THROUGH FEBRUARY 1990,
AT 8 SITES IN LAKE APOPKA 157
REFERENCE LIST 168
BIOGRAPHICAL SKETCH 180
iv


8
The intensity of APA is dependent on the severity of P limitation.
As much as 6% of the total protein produced under P limiting conditions
may be attributed to APA (Garen and Levinthal 1960). The increase of
APA in response to inorganic P deficiency has resulted in the use of APA
as a tool to assess the P limitation of plankton.
Alkaline Phosphatase Activity in the Water Column
Inverse relationships between APA and SRP have been reported in
many species of plankton (Healey 1973; Olsson 1990; Pettersson 1980;
Pettersson et al. 1990). Under low SRP concentrations APA is
derepressed, and upon replenishment of external inorganic P, APA is
inhibited. In some situations no significant relationship is observed
between APA and SRP (Berman 1970; Taft et al. 1977). It has been
suggested that under these circumstances high concentrations of soluble
organic P counteract the inhibition caused by SRP by stimulating
induction of APA (Kuenzler 1965; Cembella et al. 1984a). Alternatively,
where no correlation exists, APA may reflect P demand rather than P
limitation (Taft et al. 1977).
In combination with the depletion of external concentrations of
inorganic P, P limitation in plankton is also demonstrated by reduced
internal P concentrations (Chrst and Overbeck 1987; Rhee 1973).
Inverse relationships between APA and surplus P have been recorded (Lien
and Knutsen 1973; Rhee 1973). Once internal P concentrations have been
reduced below critical levels, APA is produced (Chrst and Overbeck
1987; Fuhs et al. 1972). Alkaline phosphatase activities have been


103
3-o-methylfluorescein phosphate (Sigma Chemicals), at a concentration
determined to be non-limiting, in 0.1 M Tris buffer (Tham, Fisher
Scientific) pH 8.6, was added to 4.0 mL of lake water in a cuvette.
Both total (whole lake water) and soluble (filtered through 0.45 nm
Gel man membrane filter) APA were determined. The cuvettes were placed
in a water bath (25*C). At timed intervals during a 20 min period the
cuvettes were placed in the fluorometer and the fluorescence measured.
The enzyme activity was measured as an increase in fluorescence as the
substrate was enzymatically hydrolyzed to the fluorescent product.
Fluorescence units were converted to enzyme activity using a standard
calibration curve of 3-o-methylfluorescein (Sigma Chemicals). The
fluorescence was measured using a Turner fluorometer No. 110, equipped
with Turner lamp no. 110-853, in combination with 47 B primary and 2a-12
secondary filters. Autoclaved lake water with substrate added was used
as a control.
Chlorophyll a was determined spectrophotometrically following
extraction with acetone and correction for pheophytin (APHA (1002-G),
1985). Total P, TSP, TKN, TOC, SRP and TSS were determined by standard
methods (APHA 1985).
Sediments. Alkaline phosphatase activity was determined by a
method adapted from Sayler et al. (1979). Sediment samples (1 g wet
sediment) or 1 mL of porewater, were placed in centrifuge tubes.
Three mL of 1 M Tris-Tris HC1 buffer (pH 7.6) were added to each tube.
The average pH found in Lake Apopka sediments was approximately 7.6.
The samples were sonicated (Heatsystems Ultrasonics Model


143
Table 5-3. Concentrations of selected parameters measured
on sediments incubated under six different redox levels for
one month (mean + 1 SE).
NaOH-TP
Eh pH pE + pH HC1-TP FA-TP H^P
mV mgPkg dry wt.'1
483 6.35 15 490 13 171 5 132 3
338 6.49 12 393 26 162 4 134 1
48 6.78 8 431+4 180 1 134 0
-2 6.55 7 414 20 146 8 126 3
-157 6.78 4 421 15 150 15 167 2
-242 7.15 3 434+3 115 2 148 3


131
ORGANIC PHOSPHORUS FRACTIONATION SCHEME
Fig. 5-2. The extraction scheme used to fractionate organic phosphorus
in sediment.


146
min1, suggesting that the plankton population was not P limited
(Pettersson 1980; chapter 3). However, total APA increased from 22 to
43 nM min'1. This increase could be a demonstration of either increased
bacterial reproduction or alternatively, a response to decreased
internal P supply within the plankton (Fitzgerald and Nelson 1966;
Pettersson 1980; chapter 3). Internal P reserves, which include small
chain polyphosphates (Elgavish and Elgavish 1980) are broken down in
response to P demand (Rhee 1972, 1973, 1974, chapter 3). Once internal
P concentrations reach a certain critical level, APA is produced (Taft
et al. 1977; Sproule and Kalff 1978; Chrst and Overbeck 1987).
Under natural conditions and in continuous culture, P
concentrations may be low but they are continuously replenished. In
batch culture, a one time P input can result in rapid depletion of SRP,
hence internal P sources may be required for metabolism (Rhee 1972).
Once this P pool has been reduced to a critical level APA is produced
(Chrst and Overbeck 1987). This would explain the 24 h lag time
observed prior to the increase in APA observed under aerobic conditions.
In contrast, under anaerobic conditions, metabolism of aerobes is
reduced and eventually inhibited completely without the return of DO.
The dominant form of phytoplankton in Lake Apopka are the blue-
greens, Lyngbya sp. and Microcystis sp. (Shannon and Brezonik 1972;
Stites, D. L., unpublished data, St. John's River Water Management
District, Palatka, FL.). Under anaerobic conditions, only anaerobes
and facultative anaerobes are active; however, research has demonstrated
that after acclimatization cyanobacteria grow well under anaerobic
conditions with H2S acting as an electron donor in the photolysis of


Sediment Suspension Device
Intact Sediment Core
Turbulence Generator
Fluid-Mud Mixture
(Lutocline)
Consolidated Sediment
(Gyttja)
Fig. 4-2. Diagram of the sediment resuspension device (Source: Reddy and Fisher 1990).


CONCENTRATION
26
en
E
A J A O D F
1989-90
O*
E
A J A 0 D F
1989-90
Fig. 2-2. Seasonal variability of selected parameters determined
bimonthly at 7 sites in Lake Apopka: a) total suspended
solids, data were not collected in April and June;
b) chlorophyll a; c) total organic carbon, data were not
collected in April; d) total Kjeldahl nitrogen. Vertical bars
represent 1 SE.


32
Table 2-2. Correlation coefficients for chlorophyll a and alkaline
phosphatase activity measured bimonthly at 7 sites in Lake
Apopka (significant at a=0.05, n=7).
Month
Correlations with
Chlorophyll a
Total APA
Soluble APA
April
TKN 0.88
TP 0.85
DO -0.74
temp -0.96
TP 0.75
SPP -0.67
NS*
June
TKN 0.92
TSP 0.76
TOC 0.68
SPP 0.78
soluble APA 0.74
DO -0.76
pH -0.79
August
NS
NS
NS
October
TOC -0.70
TSS 0.73
SPP -0.66
NS
TKN -0.75
TP 0.75
temp 0.71
TS -0.67
December
total APA 0.82
temp 0.68
secchi -0.80
TSP -0.86
SPP -0.86
TP 0.70
TSP 0.81
SPP 0.81
February
TP 0.71
TOC -0.69
NS
NS indicates not significant at a = 0.05.


HEP SRP (/g
82
Fig. 3-11. Time courses of hot water extractable soluble reactive
phosphorus following nutrient enrichment of natural plankton
populations collected in August 1990. 0N,0P=no nutrient
addition-,400N,0P=400 ^g N l/1; 0N,40P=40 /xg P L'1-,
400N,40P=400 ng N L1 and 40 /xg P L*1, and 800N,80P=800 /xg
N L'1 and 80 ng P L'1: a) plankton grown at 29C; b)
plankton grown at 19C. Vertical bars indicate 1 SE. No
vertical bar indicates SE is smaller than symbol size.


160
Table B-4. pH.
Date
Site
Mean'
SE
1
2
3
4
5
6
7
8
APR
8.2
9.4
9.3
9.1
9.3
9.4
9.4
9.2
9.3*
0.0
JUN
8.1
8.8
9.0
8.1
8.8
9.0
8.5
9.1
8.6
0.1
AUG
8.1
8.9
9.1
7.8
8.9
9.2
9.3
8.9
8.5
0.1
OCT
8.2
8.7
8.9
8.7
9.0
9.2
9.1
9.0
8.9
0.0
DEC
8.2
9.0
8.8
9.1
8.9
8.9
9.0
9.0
8.9
0.0
FEB
8.1
8.5
8.7
8.7
8.2
8.5
8.8
8.5
8.5
0.0
Mean
8.2
8.8
8.9
8.3
8.7
8.9
8.9
8.9
SE
0.0
0.0
0.0
0.1
0.0
0.0
0.1
0.0
Means listed in this column are means of sites 2-8.
Means were calculated following the conversion of pH to hydrogen
ion concentrations. Standard errors were calculated without prior
conversion of pH values.


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
BIOAVAILABILITY OF ORGANIC PHOSPHORUS
IN A SHALLOW HYPEREUTROPHIC LAKE
By
Susan Newman
May 1991
Chairman: K. R. Reddy
Major Department: Soil Science
Field and laboratory studies were conducted to determine the
importance of organic P mineralization in the sediment-water column of
Lake Apopka, a shallow hypereutrophic lake located in central Florida.
Alkaline phosphatase activity (APA) was used as a tool to indicate the
bioavailability of organic P to native plankton populations.
Spatial and temporal variability in total APA occurred in the
water column (range=4 to 45 nM min'1) in response to different water
chemistry characteristics. Nutrient enrichment studies demonstrated
that APA increased with plankton biomass and specific APA
(APA/chlorophyll a) values > 1 nmol APA ng chlorophyll a'1 min'1 occurred
during severe inorganic P limitation. In both the sediment and the
water column APA was mainly associated with particulate matter.
The APA of the plankton was inhibited by high inorganic P
concentrations. Phosphorus demand of the plankton was high, as
evidenced by the rapid uptake of added inorganic P. During the
xii


96
of sorption to the particulate material (Gchter and Mares 1985; Reddy
and Fisher 1990).
Resuspension results in increased aeration of the sediments.
Under aerobic conditions P release from sediments has been associated
with the decomposition of organic matter (Lee et al. 1977). If the
sediments are dominated by Fe, aeration results in increased
sedimentation of P (McQueen et al. 1986); however, in highly organic
sediments, biological processes catalyzed by numerous phosphatase
enzymes are likely to dominate (Ayyakkannu and Chandramohen 1971).
Organic P mineralization in sediments is regulated by the activity of
enzymes such as phosphatases, particularly alkaline phosphatase activity
(APA) in sediments at neutral and alkaline pH (Ayyakannu and
Chandramohen 1971; Kobori and Taga 1979b). A significant positive
correlation between SRP released and phosphatase activity in the water
column was observed during resuspension of marine sediments (Degobbis et
al. 1984). Thus the level of APA within the sediment will have an
effect on the APA subsequently resuspended in the overlying water
column.
Phosphatase activity decreases with depth of soils (Juma and
Tabatabai 1978; Speir and Ross 1978) and sediments (Degobbis et al.
1984; Kobori and Taga 1979b). This corresponds to a decrease in
microbial biomass, C, N and organic P with depth (Juma and Tabatabai
1978; Speir and Ross 1978, Baligar et al. 1988). Consequently the depth
of material resuspended is significant in influencing APA in the water
column.


Table 5-4. Selected correlation coefficients between phosphorus compounds and
alkaline phosphatase activity measured on sediments incubated for
one month under six different redox levels.
pE + pH
APA
LabP;
LabP0
PW-SRP
PW-TP
PW-TOP
FA-TP
FA-P0 NaOH-TP
APA
0.90*
LabP¡
NS
NS
LabP0
NS -
0.81
NS
PW-SRP
NS
NS
1.00
NS
PW-TP
NS
NS
0.99
NS
0.99
PW-TOP
-0.92
-0.95
NS
0.84
NS
NS
FA-TP
NS
NS
-0.96
NS
-0.97
-0.98
NS
FA-P0
NS
NS
-0.92
NS
-0.94
-0.95
NS
0.98
NaOH-TP
NS
NS
-0.94
NS
-0.96
-0.94
NS
0.95
0.97
HA-P0
NS
-0.74
NS
0.81
NS
NS
NS
NS
NS NS

Unless otherwise indicated all correlations are significant at a=0.05
Significant at a=0.09


170
Condron, L. M., K. M. Goh, and R. H. Newman. 1985. Nature and
distribution of soil phosphorus as revealed by a sequential
extraction method followed by 31P nuclear magnetic resonance
analysis. Soil Sci. 36:199-207.
Cotner, J. B., and R. T. Heath. 1988. Potential phosphate release from
phosphomonoesters by acid phosphatase in a bog lake. Arch.
Hydrobiol. 111:329-338.
Currie, D. J., E. Bentzen and J. Kalff. 1986. Does algal-bacteria
phosphorus partitioning vary among lakes? A comparative study of
ortho phosphate uptake and alkaline phophatase activity in
freshwater. Can. J. Fish. Aquat. Sci. 43:311-318.
Currie, D. J., and J. Kalff. 1984. A comparison of the abilities of
freshwater algae and bacteria to acquire and retain phosphorus.
Limnol. Oceanogr. 29:298-310.
Degobbis, D., E. Homme-Maslowska, A. A. Orio, R. Donazzolo, and B.
Pavoni. 1984. The role of alkaline phosphatase in the sediments of
Venice Lagoon on nutrient regeneration. Estuar. Coast. Shelf Sci.
22:425-437.
Dorich, R. A., D. W. Nelson, and L. E. Sommers. 1985. Estimating algal
available P in suspended sediments by chemical extraction. J.
Environ. Qual. 14:400-405.
Elgavish, A., and G. A. Elgavish. 1980. 31P-nmr differentiation
between intracellular phosphate pools in Cosmarium (Chlorophyta). J.
Phycol. 16:368-374.
Elser, J. J., and B. L. Kimmel. 1986. Alteration of phytoplankton
phosphorus status during enrichment experiments: implications for
interpreting nutrient enrichment bioassay results. Hydrobiol.
133:217-222.
Fitzgerald, G. P. 1969. Field and laboratory evaluations of bioassays
for nitrogen and phosphorus with algae and aquatic weeds. Limnol.
Oceanogr. 14:206-212.
Fitzgerald, G. P., and T. C. Nelson. 1966. Extractive analyses for
limiting or surplus phosphorus in algae. J. Phycol. 2:32-37.
Forsberg, C., and S. R. Ryding. 1980. Eutrophication parameters and
trophic state indices in 30 Swedish waste receiving lakes. Arch.
Hydrobiol. 89:189-207.
Francko, D. A. 1986. Epilimnetic phosphorus cycling: Influence of
humic materials and iron on coexisting major mechanisms. Can. J.
Fish. Aquat. Sci. 43:302-310.


CHAPTER 3
RESPONSE OF NATURAL PLANKTON POPULATIONS
TO NUTRIENT ENRICHMENT
Introduction
Soluble inorganic P is the main form of P utilized directly by
plankton. Phytoplankton growth rates close to maximal have been
determined in the apparent absence of inorganic P (Fuhs et al. 1972;
Smith and Kalff 1981). Methods used to measure soluble inorganic P are
for the most part limited in sensitivity (Rigler 1956; Tarapchak et al.
1982). This has resulted in the use of physiological indicators to
determine the nutritional status of plankton. Information is obtained
through a variety of methods including the determination of P uptake
rates (Lean and White 1983; Rigler 1956), the measurement of surplus P
concentrations (Fitzgerald and Nelson 1966; Rhee 1973) and the
determination of alkaline phosphatase activity (APA) (Berman 1970;
Kuenzler and Perras 1965). As external inorganic P concentrations
decline, plankton are able to utilize internal pools of surplus P to
maintain growth (Fitzgerald and Nelson 1966; Rhee 1972, 1973, 1974;
Wynne and Berman 1980). Once this internal source of P has been
reduced to a critical level some phytoplankton produce phosphatase
enzymes, which hydrolyze organic P compounds to inorganic P, to satisfy
nutritional demands (Chrst and Overbeck 1987; Kuenzler and Perras 1965;
Reichardt 1971). An inverse relationship between APA and surplus P has
50


78
Table 3-7. Hot water extractable phosphorus concentrations of composite
lake water samples collected in April 1990, 216 h after
nutrient additions. l=no nutrient addition, 2=400 nq N L'1,
3=40 nq P L'1, 4=400 ng N L1 and 40 ng P L'1,5=800 nq N L'1
and 80 nq P L1.
Treatment
Hot water extractable SRP
K L-1
1
3.0
2
2.8
3
7.2
4
6.8
5
18.4


172
Heath, R. T. 1986. Dissolved organic phosphorus compounds: do they
satisfy planktonic phosphate demand in summer ? Can. J. Fish. Aquat.
Sci. 43:343-350.
Heath, R. T., and G. D. Cooke. 1975. The significance of alkaline
phosphatase in a eutrophic lake. Verh. Int. Ver. Limnol. 19:293-304.
Herbes, S. E. 1974. Biological utilizability of dissolved organic
phosphorus in natural waters. Ph.D. Dissertation. Univ. of
Michigan. 236 pp.
Herbes, S. E., H. E. Allen, and K. H. Mancy. 1975. Enzymatic
characterization of soluble organic phosphorus in lake water.
Science 187:432-434.
Hio, S. 1988. Fluctuation of algal alkaline phosphatase activity and
the possible mechanisms of hydrolysis of dissolved organic phosphorus
in Lake Barato. Hydrobiol. 157:77-84.
Holdren, G. C. Jr., and D. E. Armstrong. 1980. Factors affecting
phosphorus release from intact lake sediment cores. Environ. Sci.
Technol. 14:79-87.
Howeler, R. H. 1972. The oxygen status of lake sediments. J. Environ.
Sci. Technol. 14:79-87.
Huber, A. L., J. 0. Gabrielson, and D. K. Kidby. 1985. Phosphatase
activities in the waters of a shallow estuary, western Australia.
Estuar. Coast. Shelf Sci. 21:567-576.
Huber, W. C., P. L. Brezonik, J. P. Heaney, R. E. Dickinson, S. D.
Preston, D. S. Dwornik and M. A. DeMaio. 1982. A classification of
Florida lakes. Final Report submitted to Florida Department of
Environmental Regulation, Tallahassee, FI.
Hutchinson, G. E., and V. T. Bowen. 1950. Limnological studies in
Conneticut. IX. A quantitative radiochemical study of the phosphorus
cycle in Linsley Pond. Ecol. 31:194-203.
Istvnovics, V., K. Pettersson, and D. Pierson. 1990. Partitioning of
phosphate uptake between different size groups of planktonic
microorganisms in Lake Erken. Verh. Int. Ver. Limnol. 24:231-235.
Jackman, R. H., and C. A. Black. 1952. Hydrolysis of phytate
phosphorus in soils. Soil Sci. 73:167-171.
Jansson, M. 1976. Phosphatases in lake water: characterization of
enzymes from phytoplankton and zooplankton by gel filtration.
Science 194:320-321.
Jansson, M. 1977. Enzymatic release from subarctic lakes in Northern
Sweden. Hydrobiol. 56:175-180.


DEPTH (cm)
NaOH extractable P
mg kg
0 400 800
Organic P
mg kg
700 1400 2100
Inorg P and TP
. -1
mg kg
700 1400 2100
Fig. 4-4. The depth distribution of selected parameters measured in triplicate sediment cores collected
in May 1989 from the center of Lake Apopka: a) NaOH extractable phosphorus; b) organic
phosphorus; c) total and inorganic phosphorus. Horizontal bars indicate 1 SE. No bar £
indicates SE is smaller than symbol size.


SUSPENDED P CONCENTRATION
41
SITE
Fig. 2-11. Distribution of suspended phosphorus compounds determined by
difference between whole and soluble P measured at 8 sites in
October 1989. TP=total phosphorus, T0P=total organic
phosphorus, TAH=total acid hydrolyzable phosphorus, TRP=total
reactive phosphorus. Vertical bars represent 1 SE.


28
were considerably lower and Secchi was significantly greater. Apopka
spring temperatures tend to be consistent throughout the year, only a
4*C fluctuation in water temperature was observed. Algal biomass was
significantly lower than observed in the rest of the lake. Annual
chlorophyll a concentrations averaged 21.8 /xg L'1 at site 1, while
values averaged 81 ng L1 at other sites.
The contribution of the various components in lake water to the
total APA pool was determined via filter size fractionation. The
distribution of APA followed that of chlorophyll a, with the majority of
the activity associated with the larger size fraction (Fig. 2-4), and
the distribution of APA within the different size fractions remained
constant throughout the year. The greatest amount of soluble APA
occurred in December. In general, a greater proportion of APA was
associated with 8 and 2.5 urn filtered samples in spring water at site 1,
than was observed in samples from sites 4 and 8. A similar distribution
was also determined for P (Fig. 2-5), although a greater proportion was
associated with the soluble fraction. Phosphorus concentrations peaked
in October at all three sites.
Relationships between Water Chemistry Data and Selected Environmental
Parameters
Seasonal patterns in PAR, wind speed and water temperature
collected at site 8 (lake center) were observed (Table 2-1). Water
temperature and PAR peaked in June and August. Wind speed measured 5 m
above the surface was generally greater than that measured 1 m above the
surface (Table 2-1). Total P was positively correlated with wind speed
observed 5 m above the water surface (r=0.88) and SRP was


VO
Fig. 2-1. Location of Lake Apopka and sampling sites.


10
The interpretation of APA as a measure of P limitation is
complicated by the uncertainty of the origin of APA. Bacteria,
phytoplankton and zooplankton are considered to be dominant contributors
to this pool, and it is suggested that APA of algal origin is the most
important in the epilimnion (Jansson et al. 1988). High levels of
soluble APA indicate filterable activity and may reflect bacterial
associated APA (Stewart and Wetzel 1982), zooplankton excretion (Jansson
1976; Wynne and Gophen 1981) and cell lysis (Berman 1970). In many
lakes APA was determined to be mainly associated with phytoplankton,
based on co-occurrence of APA and algal blooms (Heath and Cooke 1975),
and as shown by correlations with chlorophyll a (Jones 1972a; Matavulj
and Flint 1987; Siuda et al. 1982; Smith and Kalff 1981) and size
fractionation of phosphatase activity (Chrst et al. 1989; Jansson
1977). Alkaline phosphatase activity has also been attributed to
bacteria through correlations with bacterial numbers (Jones 1972a;
Kobori and Taga 1979a). In shallow lakes, a large portion of
particulate material may be sedimentary in origin. Concentrations of P
compounds and bacterial numbers may be higher in sediments. Interaction
between sediment and the overlying water column may significantly affect
the mineralization of organic P in the overlying water. Hence, wind
events in shallow lakes can significantly affect APA.
Alkaline Phosphatase Activity in the Sediment
In lake sediments as much as 70% of TP can be in organic form
(Weimer and Armstrong 1979). In highly organic sediments, the relative
abundance of organic substrates may result in enhanced breakdown of


83
Table 3-9. Specific hot water extractable phosphorus measured over time
in natural plankton populations collected in August 1990
after receiving nitrogen and phosphorus additions (mean
1 SE). l=no nutrient addition, 2=400 M9 N L1, 3=40 M9 P L'\
4=400 ng N L'1 and 40 M9 P L'1, 5=800 ng N L'1 and 80 ng P
L'1, and 6=no nutrient addition and 7=40 M9 P L'1 and
plankton grown at 19*C.
Time h
Treatment ~~0 2 24 48 96
/ig P M9 chlorophyll a'1
1
0.41 0.05 0.48

0.07
0.34

0.01
0.29

0.01
0.22

0.00
2
0.53

0.01
0.35

0.02
0.27
+
0.00
0.19

0.01
3
1.14
+
0.06
0.64

0.04
0.35

0.01
0.28

0.01
4
1.07
+
0.06
0.67

0.06
0.37
+
0.01
0.21

0.02
5
1.79

0.07
0.82

0.03
0.51

0.02
0.24

0.00
6
0.54
+
0.02
0.44

0.01
0.31

0.04
0.35

0.05
7
0.87
+
0.05
0.73

0.01
0.48

0.01
0.47
+
0.03


This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
May 1991
cu>k X.
ollege of
culture
Dean, Graduate School


45
Resuspension of sediment high in organic matter could bind enzymes
and/or release sediment bound APA to the overlying water column.
Organic inputs, living or dead should be considered when measuring APA
(Healey and Hendzel 1980).
Soluble APA is positively related to Secchi (r=0.87) suggesting
that there is a relationship between APA and water quality. But the low
proportion of APA observed in the soluble pool suggest that free
dissolved enzymes from cell lysis (Berman 1970) and enzymes excreted by
zooplankton (Wynne and Gophen 1981) were not as important as particulate
associated APA in the system. Organic phosphorus mineralization is
mainly be achieved by APA bound to particulate matter. Examining the
data from all sites (excluding site 1), APA was infrequently related to
chlorophyll a. The overall lack of correlation between APA and
chlorophyll a may be hidden due to the frequent mixing of the lake
water. The particulate nature of APA as determined by the size
fractionation scheme corresponds to the chlorophyll a distribution.
The correlation between APA and chlorophyll a leads to the expression of
APA as a ratio, i.e., APA/chlorophyll a (Pettersson 1980). This ratio
tends to increase with P limitation and decrease with trophic state
(Pick 1987). Combining data from numerous studies, Pettersson (1980)
determined that a ratio between 0.2 to 0.7 nmol APA ng chlorophyll a'1
min'1 could be used to indicate P limitation. When ambient lake
specific APA was consistently < 0.3 nmol APA /xg chlorophyll a'1 min'1
ratios greater than this were determined to indicate P limitation, i.e.,
elevated specific APA indicates P limitation (Istvnovics et al. 1990).
In this study a ratio of < 0.3 nmol APA nq chlorophyll a'1 min'1 was


87
Table 3-10. Specific alkaline phosphatase activity measured over time
in natural plankton populations collected in August 1990
after receiving nitrogen and phosphorus additions
(mean 1 SE). l=no nutrient addition, 2=400 ng N L'1, 3=40
ng P L'1, 4=400 /xg N L'1 and 40 ng P L'1, 5=800 ng N L'1 and
80 ng P L'\ and 6=no nutrient addition and 7=40 ng P L'1 and
plankton grown at 19C.
Time h
Treatment 0 24 48 96
nmol APA ng chlorophyll a'1 min
1
0.34 0.00
0.47

0.01
1.07
+
0.03
0.45

0.03
2
0.47

0.02
1.01
+
0.07
0.51

0.06
3
0.63

0.01
0.64

0.02
0.16

0.02
4
0.63

0.02
0.70

0.03
0.17

0.00
5
0.67
+
0.02
0.48
+
0.01
0.07

0.00
6
0.22
+
0.01
0.43

0.01
1.12

0.03
7
0.32
+
0.02
0.52

0.01
1.00

0.04


ACKNOWLEDGEMENTS
I would like to thank Dr. Reddy, my major advisor, whose help and
guidance made this undertaking an enjoyable learning experience. I
would also like to thank the members of my committee, who were always
willing to share their expertize. Appreciation is also expressed to Mr.
Rick Aldridge whose assistance and lively discussion enabled me to
conduct the nutrient enrichment experiments.
I would also like to thank my friends and colleagues in the Soil
Science Department, particularly those associated with the Wetland Soils
Laboratory, whose encouragement, assistance and cooperation made this
project flow more smoothly.
I would like to thank my parents, Joyce and Chris Newman, whose
love and support enabled me to complete my Ph.D. Without their
encouragement I would not have continued on to higher education.
Last but not least, I would like to thank Tom, whose love,
companionship and support boosted my morale numerous times throughout
this study.
ii
A


91
appears that HEP-TSP was utilized metabolically, and supports the
suggestion of Wynne and Berman (1980), that HEP-TSP can be utilized for
nutrition.
It was interesting to note that HEP-TSP concentrations increased
within 2 h in cultures which were not enriched with P. Phytoplankton
have been shown to grow even at SRP concentrations of < 3 /xg L'1
provided this concentration is maintained at the cell surface (Fuhs et
al. 1972). Hence, although SRP concentrations were low and did not
change, phytoplankton may have utilized P at the cell surface. In
conjunction with this, SRP concentrations were at the limit of detection
and not sensitive enough to record further decreases. Thus, it is
apparent that the monitoring of P limitation responses would provide a
more accurate assessment of the nutritional status of the plankton.
Alkaline phosphatase activity has been shown to increase with
eutrophication, in combination with increased plankton biomass (Gorham
and Gage 1985; Jones 1972b). Attempts to associate APA with specific
parameters has resulted in the normalization of APA to protein (Stevens
and Parr 1977), particulate organic matter (Gage and Gorham 1985) and
ATP (Healey and Hendzel 1980; Pettersson 1980). Specific APA (i.e.
normalized to chlorophyll a) has frequently been used as a measure of P
limitation and tends to decrease with trophic status (Pick 1987).
Specific APA values of 0.2-0.7 nmol APA /xg chlorophyll a'1 min'1 under
field conditions and values > 0.08 nmol APA /xg chlorophyll a'1 min'1
obtained in laboratory cultures have been suggested to indicate P
limitation (Pettersson 1980; Healey and Hendzel 1979a). In this study,
initial specific APA (0.3-0.4 nmol APA /xg chlorophyll a'1 min'1) would


80
concentrations in treatment 1 (N=0 P=0) cultures were lower than initial
concentrations while HEP-TSP concentrations in treatments 3 (N=0 P=40)
and 4 (N=400 P=40) were higher than initial concentrations. Normalizing
the data to chlorophyll a, a decrease in HEP-TSP was observed for all
treatments following the initial increase at 2 h (Table 3-8). The final
ratios were lower than the initial ratios.
The treatment by time response was different for HEP-SRP
(Fig. 3-11 a). The relative increases were greater. An increase in HEP-
SRP concentrations was observed after 48 h in cultures which received P,
except for treatment 4 (N=400 P=40). Hot water extractable P in
treatments 1 (N=0 P=0) and 2 (N=400 P=0) cultures remained constant. A
significantly lower increase was observed in treatment 7 (N=0 P=40,
temperature=19*C) (Fig. 3-llb a=0.06). Normalizing the data to
chlorophyll a resulted in a continuous decline in HEP-SRP. No increase
after 48 h was observed (Table 3-9). A significant correlation between
HEP-SRP and chlorophyll a was observed (r=0.56).
Alkaline phosphatase activity. Considerable differences in the
production of APA were observed between April and August (Fig. 3-12a and
13a). In both experiments, increases in APA were observed, however in
August this increase only lasted 48 h for all cultures grown at 29*C.
In April, APA in cultures receiving both N and P additions was inhibited
within 2 h, 28% and 11% inhibition for treatments 5 (N=800 P=80) and 4
(N=400 P=40), respectively. No inhibition was observed in treatments
receiving only a P addition. After initial inhibition, which lasted
24 h, APA increased (Fig. 3-12a). A significant interaction between N
and P was apparent at 48 h. Cultures receiving only P had a delayed


155
The results presented in this study demonstrate significant
potential for organic P mineralization in Lake Apopka. Substrates
hydrolyzable by APA are very labile, hence the rate of organic P
mineralization in Lake Apopka is limited by substrate concentrations
rather than enzyme activity. Further research should focus more on
specific organic P compounds within the sediment-water column and their
relative turnover rates. Studies should also include evaluating the
change in enzyme kinetics in response to different organic P compounds.
To encourage P limitation, emphasis should be placed upon a means to
inhibit organic P mineralization.


140
Table 5-2. Concentrations of selected parameters measured
on sediments incubated under six different redox levels for
one month (mean 1 SE)
Porewater Labile
Eh pH pE + pH APA ~SRP TOP- ~P,
Mmol
mV g dry wt.'1 mg P kg dry wt.'1
h'1
483
6.35
15
6.89

0.21
26

0.45
2.1

0.45
48

1
11

3
338
6.49
12
4.01

0.65
51

0.78
7.0

2.81
74

3
16
i
3
48
6.78
8
3.64
+
0.39
8
+
1.19
13.8

2.51
23

2
19

2
-2
6.55
7
4.23
+
0.31
58

0.90
8.3
+
3.15
75

0
10
+
2
-157
6.78
4
1.07

0.59
44
+
1.35
16.1

1.96
66

2
22

2
-242
7.15
3
0.71
+
0.24
111

1.80
18.3
+
0.55
144
+
2
19

1


125
eutrophication (Shapiro 1960). More often, highly productive aquatic
systems exhibit clinograde DO distributions, resulting in the depletion
of DO with water depth in response to summer stratification (Miyake and
Saruhashi 1956; Wetzel 1983) and consumption during high respiratory
activity at the sediment-water interface (Charlton 1980; Bostrom et al.
1982).
Diffusion of DO from the water, bioturbation and sediment
resuspension can maintain relatively aerobic conditions at the sediment-
water interface. Under these conditions P release from sediments to
overlying water can be due to mineralization of organic P (Lee et al.
1977). Mineralization of organic P may be of particular importance in
sediments which possess abundant organic substrates, such as peat
sediments (Ayyakannu and Chandramohen 1971).
During stratification of the water column, the hypolimnion may
become anoxic, hence the surface sediment becomes anaerobic. A
significant consequence of anaerobic conditions at the sediment-water
interface is the increase in water soluble P in the overlying water
column (Holdren and Armstrong 1980; Ponnamperuma 1972; Mortimer 1941).
The release of P under these conditions is the result of the reduction
of ferric phosphates to the more soluble ferrous phosphates (Mortimer
1941). This flux of P from the sediment to the overlying water column
may have a significant impact upon APA in the water column. Inorganic P
is a competitive inhibitor of APA (Coleman and Gettins 1983), and
sedimentary release of P has been shown to inhibit APA within the
overlying water column (Pettersson 1980). Anaerobic conditions may also