Phosphorus cycling in a periphyton-dominated freshwater wetland


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Phosphorus cycling in a periphyton-dominated freshwater wetland
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x, 187 leaves : ill. ; 29 cm.
Scinto, Leonard Joseph, 1960-
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Subjects / Keywords:
Wetland ecology -- Florida -- Everglades   ( lcsh )
Freshwater ecology -- Florida -- Everglades   ( lcsh )
Periphyton -- Florida -- Everglades   ( lcsh )
Soil and Water Science thesis, Ph. D   ( lcsh )
Dissertations, Academic -- Soil and Water Science -- UF   ( lcsh )
bibliography   ( marcgt )
non-fiction   ( marcgt )


Thesis (Ph. D.)--University of Florida, 1997.
Includes bibliographical references (leaves 175-186).
Statement of Responsibility:
by Leonard Joseph Scinto.
General Note:
General Note:

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University of Florida
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Copyright 1997


Leonard Joseph Scinto

I dedicate this dissertation to the Mendicino and Scinto families.


Sincere gratitude goes to my advisor Dr. K.R. Reddy and to the members of my

committee; Drs. W.G. Harris, G.R. Best, E.J. Phlips, and P.S.C Rao. This work was

partially supported, both financially and with field assistance, by the South Florida Water

Management District. Dr. P.V. McCormick of SFWMD deserves special consideration. I

would also like to thank my friends in the Soil and Water Science Department of the

University of Florida, especially Ms. Y. Wang and Mr. M.M. Fisher, for their support.

I am deeply indebted to my bride, the former Ms. Kirsten Ilene Schneider, for her

affection, support, faith, and mostly patience.


ACKNOWLEDGMENTS .................................. ........ ....................... iv

A B ST R A C T ............................................................................ ......................... .......... viii


1 IN TRO D U CT IO N ..................................................................... ..............................1
Periphyton ........................................... ............................................................... 4
N eed for Research................................ .............................................................6
Site D escription.......................................................................... ........................7
O objectives ............................................................................... .................................9

WATER,AND VEGETATION ................... ........ .........................11
Introduction .................................... ....................................................................... 1
Materials and Methods........................................................... 15
Site D escription............................................ .............................................. 15
Water Sampling and Analysis................................ ............................ 17
Periphyton and Macrophyte Sampling and Analysis .............................................18
Soil Sampling and Analysis................................ ........................... 19
Fractionation of Soils and Benthic Periphyton ................................... ........... 20
Peat Accretion Rates ........................................ ................ ..........................23
Statistical Analysis....................................................................................24
Results and Discussion ............................................................................................24
Water...................... ......................................................................24
Periphyton .... .................. ..................................................................27
Saw grass ...................................................................... ............................ 30
- Phosphorus Forms in Soils and Benthic Periphyton......................................32
Long-term Peat and Nutrient Accretion Rates...............................................42
Conclusions.................. ........................................................................ 45

Introduction.................. .............................................................................46
Materials and Methods................... .......................................................48
Study Site .......................... .................................................................48
Characterization of Periphyton ............................................... .........................49
Development of Extraction Procedures ............... ............................51


Phosphorus Uptake by Periphyton: Laboratory Conditions.................................53
Phosphorus Uptake by Periphyton: Field Conditions ..........................................55
Calculation of Uptake Rates .......................................... ..............................56
Tracer Studies of Phosphorus Partitioning in Periphyton............................ ..58
Results........................................... ........... ............ ......................... 59
Characterization of Periphyton .................................. .........................59
Extraction Procedure................................................... .........................59
Phosphorus Uptake by Periphyton: Laboratory Conditions.................................61
Phosphorus Uptake by Periphyton: Field Conditions ..........................................70
Inorganic P uptake .................................. ... .........................70
Organic P uptake............................ ................... .........................74
Phosphorus Partitioning in Periphyton .......................... .............................74
Discussion...................................................................................................... 78
Characterization of Periphyton .................................. ........................78
Inorganic P Uptake......................................................................................80
Organic P Uptake.............................................................................................84
Partitioning of Phosphorus.........................................................................86
C onclusions............................... ................................................. ................................ 90

Introduction....................................................................... .....................................92
Materials and Methods.......................................................................................93
Site Description................... ..................................... .. ........................93
Field Experiments.....................................................................................94
Dissolved ions in porewater..................................................................94
In-situ phosphorus uptake................................................ ..........................95
Greenhouse and Laboratory Experiments .......................................................96
Diel ambient DRP flux .......................................................................96
Fate of water column P.........................................................................97
Statistical M ethods............. ..............................................................................99
R results ................................................... ..................................................... ....... .. 10
Physicochemical Properties of the Water Column ............................................100
Gradients in Dissolved P and other Ions.......................................................... 107
Phosphorus Removal from the Water Column.............................................. 116
In situ experiment ........................................................................... 116
Greenhouse experiment ................................................... ......................... 116
Fate of Phosphorus Under Light and Dark Conditions......................................121
D isscussion ................................ ...................................... ............................. 123
C onclusions............................. ............................................................ ......... .... 130

THE WATER COLUMN.......................................................................................132
Introduction.......................... ............................................... ........................ 132
Materials and Methods............................................... 135
Site D escription............................ .................................... ......... ...............135
Phosphorus Precipitation: Physicochemical Conditions..............................136

Laboratory reactors .......................................................................................136
Chemical Characteristics of Water, BP Interstitial Water, and Soil
Porew after ............................................. .............................................. 138
Porewater equilibrators .......................................... .......... ............................ 138
Oxygen and pH Profiles of Soil Columns with and without BP........................ 140
Analytical M ethods ........................................... ................ 140
M ineral Equilibria Calculations........................................ ............ 141
X-ray Diffraction of BP and Surficial Soils........................... .... .................142
Results and D discussion ........................................................................................... 142
--Phosphorus Solubility as Influenced by Fluctuating pH and CO2 .....................142
Chemical Characteristics of Water, BP Interstitial Water, and Soil
Porewater ................................................................................................. 152
Oxygen and pH Profiles of Soil Columns with and without BP..........................154
M ineral Equilibria ..........................................................................................154
Laboratory experiments .......................... ... .......................... 154
In situ soil porewater chemistry................................ ....... .................. 162
X-ray Diffraction of BP and Surficial Soils.................................................... 165
Conclusions.................... ......................................................................165

6 SUMMARY...................... ........ ...........................167

LITERATURE CITED ................................................................................................ 175

BIOGRAPHICAL SKETCH .................................................187

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



Leonard Joseph Scinto

May, 1997

Chairman: Dr. K.R. Reddy
Major Department: Soil and Water Science

Periphyton, the community of microorganisms growing on submerged substrates,

is a conspicuous feature of shallow, interior Everglades slough habitats. This research

was conducted to identify major pathways and storage involved in P cycling.

Specifically, this work centered on mechanisms functioning in unimpacted, periphyton

dominated areas of the northern Everglades (Water Conservation Area 2A). The field site

was dominated by calcareous blue-green algae (cyanobacteria).

Total P content was in the order of benthic (BP) > epiphytic (EP) = floating (FP)

periphyton, and was in the range of 130 390 mg kg-1. Calcium carbonate content

accounted for 20 50 % of periphyton dry weight. Total P in BP was approximately

equal to that in the surficial soil. Inorganic P (Pi), associated with Ca, was highest in the

surface 0-2 cm of soil and was directly related to of increased CaC03 deposition due to

calcification by BP. The presence of BP on the soil surface was shown to maintain higher

soil porewater concentrations of dissolved reactive P (DRP) and Ca2+ than when the BP

was removed.

Phosphorus uptake rates, measured in the laboratory, were 0.04 0.62 tmol P g-I

min-1 (dry weight periphyton) for EP and 0.02 0.2 gmol P g-I min-i for BP. Uptake

parameters for EP were; Va = 0.85 munol P g-I min-1, K = 9.9 pM, and for BP were;

Vm, = 0.10 rpmol P g-l min-1, K = 2.5 pM. Inorganic (Pi) and organic P (P,) uptake
rates by periphyton was higher under field conditions than under laboratory cultures.

Both biotic and abiotic processes were shown to regulate P uptake by periphyton, with

CaCO3 as a barrier between living periphyton and adjacent water column. Abiotic uptake

accounted for 10-30% of 32P activity in one hour and 3-8% after 12 hours, suggesting that

P initially associated with CaCO3 surfaces is biotically incorporated with time.

In situ P uptake was greater in cores with intact BP layers (+BP) than in cores

without BP (-BP). Under greenhouse conditions -BP uptake of P was initially as rapid as

+BP cores. With continued P loading -BP cores lost the ability to effectively remove

water column P. This suggests adsorption to soil mineral surfaces, or uptake by soil

microbes can rapidly assimilate P but have limited capacity for P removal. Partitioning
32P in intact cores (+BP) resulted in 14% abiotic uptake vs. 86 % biotically incorporated.

Laboratory studies were conducted to determine conditions necessary for CaCO3

precipitation and subsequent P coprecipitation. Phosphorus was removed from solution

when pH > 8.6 for prolonged periods. Phosphorus reduction was not observed when

solution pH varied between 7.0 8.8 on 12 h cycles. Mineral equilibria modeling, using

SOILCHEM, generally predicted hydroxyapatite as the stable mineral P form in field and

reactor solutions. X-ray diffraction analysis of dried BP, and peat soil from 0-2 cm and 2-

5 cm depths showed the presence of calcite but not of mineral Ca-P.

Periphyton activity controls short-term P retention via biotic uptake and creates

conditions, by influencing Ca2+ activity, that increases long-term, stable, abiotic P

retention. Loss of calcareous periphyton communities would decrease the Everglades

system capacity to maintain low P concentrations.


Phosphorus (P), often exists in low concentrations in the hydrosphere and

therefore is the component that commonly limits algal growth (Wetzel, 1983; Bostr6m et

al., 1982). Depending on the geochemical nature of the region, most uncontaminated

surface waters contain between 10 to 50 tg P L-1 (Wetzel, 1983). Therefore, additions of

P above that which a system has evolved to assimilate can have profound effects on its

structure and function. Phosphorus additions to shallow waterbodies, such as wetlands

and lakes, often lead to increased primary productivity due to increased algae growth. For

example, increased P loads to oligotrophic wetlands can lead to changes in the

community structure as was shown by Swift and Nicholas (1987) for the periphyton

community in the northern Everglades. In waters that received relatively high

concentrations of inorganic P and N, they observed algal communities dominated by the

blue-green algae (cyanobacteria) Microcoleus lyngbyaceus or the green alga Oedogonium

spp. The periphyton communities dominated by the calcareous blue-greens Schizothrix

calcicola (Ag.) Gom. and Scytonema hofmannii Ag. represented low nutrient, hard water

conditions (Swift and Nicholas, 1987).

Wetlands have traditionally been thought of as low-value wastelands since in their

natural state they cannot be used for most agricultural activities or urban development

(Lee et al., 1975). Historically, these areas have been "improved" by such "reclamation

projects" as drainage for agriculture or filling for urban development (Tiner, 1984).

There is little agreement as to the actual wetland acreage present at the time of this

country's settlement. One estimate claims there were approximately 87 million hectares

in the contiguous United States, of which an estimated 46% remains (Tiner, 1984).

The development of a global widespread environmental awareness in the last 20

years has led to an increased appreciation of the many benefits wetlands provide. The

remaining wetlands of the U.S. have become a rallying point for restoration, conservation,

and preservation (Kadlec and Knight, 1996). One of the most important, albeit least

understood, functions of wetlands is their ability to purify water (Hammer and Bastian,

1989). This ability is derived in part from wetlands being transitional areas, found at the

interface between terrestrial and aquatic systems (van der Valk et al., 1978; Tiner, 1984).

Wetlands, being complex hydrologic, chemical, and biochemical systems, can store

and/or transform environmentally damaging water-borne elements (Lee et al., 1975).

Phosphorus utilization in a wetland involves a complex biogeochemical cycle of

interacting pathways. Since many wetlands have evolved under a limited P supply and

because there is no atmospheric (gaseous) sink for P, its removal is difficult in wetland

systems. On an area basis, wetlands are generally not efficient in P removal (Kadlec and

Knight, 1996). An examination of the P cycle in a wetland system aids in understanding

the complex interactions of pathways involved.

The cycling of phosphorus within a shallow waterbody involves numerous

interactions between the P compartments of the water, biota, and soil (Fig. 1-1).

Processes controlling these interactions can be biological, as in algal uptake and release,

or physicochemical, such as adsorption/precipitation (Kadlec, 1987).

Phosphorus is present as inorganic (Pi) and organic (Po) forms in wetlands. These

P forms occur as both soluble and particulate forms. The relative P storage of the water is

small compared to that of the biota, and much smaller relative to the soil (Bostr6m et al.,

1982; Faulkner and Richardson, 1989). Dissolved inorganic P (DIP) is the P form that is

most biologically active and has the greatest impact on surface water quality (Bostr6m et

Fig. I -1. Phosphorus cycling in a periphyton dominated freshwater wetland.

al., 1982; Syers et al., 1973). Therefore, transformations between DIP in the water and

the other P components of the biota, water, and soils, and the mechanisms controlling

these transformations are of ecological interest.

Dissolved inorganic P is immobilized to particulate organic P (POP) through

biological uptake. Organic P, both dissolved (DOP) and POP, can account for a

substantial portion of the total P in wetlands, especially those with peat soils (Richardson

and Marshall, 1986). Phosphorus is bound in organic compounds as an ester (C-O-P)

(Golterman, 1973; Bostr6m et al., 1982), a form not used for autotrophic synthesis until it

undergoes hydrolysis. Hydrolysis is catalyzed by phosphotase enzymes produced by

bacteria and algae. Dissolved inorganic P can be retained as particulate inorganic P (PIP)

by a number of abiotic processes occurring in the water and soil including, adsorption and

precipitation. Dissolved inorganic P is released from PIP through desorption and

dissolution. These processes are dependent upon several environmental factors, the most

influential of which are pH, temperature, P content, concentration of other ions, redox

potential, and the type and amount of sorbing materials (Froelich, 1988; Jacobsen, 1978;

Shukla et al., 1971; Ballard and Fiskell, 1974; White and Taylor, 1977; Khalid et al.,

1977; Sharpley et al., 1981). Soluble P forms move between the water and soil

compartments by diffusion according to the concentration gradient. Particulate forms

settle from the water and become part of the soil matrix.


The term periphyton is originally of Russian origin and initially referred to

organisms growing on submerged, man-made objects. Eventually, it acquired a broader

meaning to include the attached microorganisms, both floral and faunal, growing on

natural and artificial substrates (Collins and Weber, 1978; Wetzel 1975; Browder et al.,

1994). Floating mats of algae are common in many shallow waterbodies and can also be

considered "periphytic", although various authors use numerous terms to differentiate

between true benthic periphyton (BP) and floating algal mats. Floating periphyton (FP)

has been shown to form from benthic algae that has risen in the water column because of

the entrappment of photosynthetically produced oxygen bubbles (Hillebrand, 1983;

Gleason and Spackman, 1974). There is considerable confusion in the nomenclature

relating to periphyton (for a review see Vymazal, 1995). Periphyton can be loosely

separated into types based on position in the water column although, as mentioned

earlier, transformation of types can occur. The three types of periphyton considered in

this research are: benthic periphyton, epiphytic periphyton (EP), and floating periphyton.

Benthic periphyton grows on the soil surface. Epiphytic periphyton grows on the

submerged stems of macrophytes, such as sawgrass (Cladium jamaicense Crantz.).

Floating periphyton is that material most associated with the surface of the floodwater

often in association with Utricularia spp.

My research is concerned with the function of periphyton or algal mats in shallow

water systems and is not specifically concerned with the taxonomic classification of the

multitude of autotrophic and heterotrophic organisms that compose the community.

Since the basic functions of nutrient uptake, photosynthesis, and respiration are common

to all, the term periphyton will be used here in a general sense.

Periphyton can be extremely important in some shallow waterbodies. The

periphytic algae may contribute up to 80% of the primary productivity and therefore

influence many of the biological and physicochemical aspects of these environments

(Hansson, 1992; Carlton and Wetzel, 1988). Periphyton has a significant influence on P

cycling. Periphyton can utilize both DIP and DOP (Bentzen et al., 1992) from floodwater

and the soil porewater depending upon the vertical position of the mat in the water

column (Hansson, 1989). Periphytic photosynthesis has been shown to produce marked

changes in the pH and oxygen content of surficial sediments (Carlton and Wetzel, 1988),

two of the more important regulators of P adsorption/desorption from sediments

(Bostrom et al., 1982). Periphyton is known to mediate the precipitation of CaCO3

(Gleason, 1972), which can interact with adsorbed P to form more crystalline forms such

as hydroxyapatite (Golterman, 1988).

Submerged and floating mats of periphytic algae are often conspicuous features of

the littoral zones of lakes, slow moving rivers and streams and other shallow waterbodies

(Wetzel, 1983; Swift and Nicholas, 1987). However, these mats have remained little

studied when compared to pelagic phytoplankton. This is partly due to the complex

nature of the periphyton communities and because of the more variable environmental

physicochemical and biotic parameters of shallow waterbodies than those of the open

water (Wetzel, 1983).

Need for Research

Agricultural runoff, alterations in the natural hydrology, and controlled release of

water from Lake Okeechobee are causing increased levels of nutrients, most notably P, to

enter into the Everglades ecosystem (Koch and Reddy, 1992; Reddy et al., 1993). This is

leading to increased eutrophication of the historically oligotrophic Everglades (Swift and

Nicholas, 1987).

Periphyton dominated "polishing" areas may be used as the final stage in a

wetland-utilizing process for the treatment of enriched water to achieve P concentrations

below those normally found in macrophyte wetlands. Since periphyton can affect the P

cycle in numerous ways, it becomes important to obtain a basic understanding of the

mechanisms, processes, and extent of these influences.

Site Description

The original Everglades covered an area of more than 10 000 km2 forming a

contiguous wetland system some 65 km wide and 160 km long, with water flowing from

Lake Okeechobee south to the mangrove estuaries of Florida Bay (Swift, 1981).

Considerable changes have occurred in the Everglades system over the last century. The

original Everglades has been fragmented into a series of hydrologic units composed of the

three Water Conservation Areas (WCA) and Everglades National Park. The WCAs

encompass almost 3500 km2, representing a little more than a third of the original

Everglades (Swift and Nicholas, 1987). Today, southward waterflow through the series

of WCAs is highly controlled by 2400 km of canals, 18 major pump stations, and

hundreds of water control structures (Reddy et al., 1993).

Water Conservation Area 2A (WCA-2A) is the smallest of the three WCAs (547

km2). It lies between WCA-1 and WCA-3A, and is heavily impacted by large quantities

of nutrient enriched water derived from the Everglades Agricultural Areas (EAA) (Swift

and Nicholas, 1987). The surface water quality (SWIM, 1992), nutrient gradient (Koch

and Reddy, 1992), and soil nutrient characteristics (DeBusk et al., 1994) of this area have

been recently studied. Water Conservation Area 2A has been shown to contain abundant

calcareous blue-green algal periphyton (Gleason and Spackman, 1974).

Previous research pertaining to the floating mats of periphytic algae in the WCAs

(Swift, 1981; Swift and Nicholas, 1987) focused on how environmental variables such as

light, hydroperiod, and water quality factors affect the growth and species composition of

the mats. The effects of periphyton on the physicochemical properties of the water

column and soils, especially in relation to the P cycle have had little study.

Water Conservation Area 2A (Fig. 1-2) receives nutrient enriched water via the

Hillsboro Canal (S-10C structure) causing a north to south P gradient (Koch and Reddy,

1992). In a recent study Rutchey and Vilcheck (1994) characterized about 12% of

Fig. 1-2. Map of Water Conservation Area 2A (WCA) showing field site 217.

WCA-2A as being periphyton dominated. However, field observations have shown

dense periphyton growth on the surface of peat and the stems of macrophytes in areas

these authors characterize as being dominated by sawgrass. The site used for this

research was in an area Rutchey and Vilcheck (1994) characterize as being sawgrass

dominated. The effects of the P gradient are evident in the periphyton species

composition, growth rate, and tissue nutrient concentrations. The dominant periphyton in

the enriched water near S-10C are pollutant tolerant species such as Microcoleus

lyngbyaceous and Oedogonium spp. The interior unimpacted sites are dominated by

calcareous blue-green cyanobacteria such as Schizothrix calcicola (Ag.) Gom. and

Scytonema hofmannii Ag., as well as hardwater diatoms. The growth rates of dominant

algal species were shown to be as much as 10 times higher at enriched sites (25 mg chl a
m-2 wk-1) than at the interior sites (0.1 2.3 mg chl a m-2 wk-l). The average periphyton

N to P ratios at enriched sites was shown to be approximately 9:1 while at the interior

sites this ratio was 107:1. The low surface water P content, periphyton tissue content, and

low algal growth rates suggest the interior of WCA-2A to be P limited (Swift and

Nicholas, 1987).


The overall objective of this research was to determine the influence of periphyton

on P retention in a freshwater wetland. It is hypothesized that periphyton aids in

maintaining low P concentration in the water through biotic and abiotic processes.

Specific objectives include:

Determine the quantity and form of P stored in three types of periphyton, water,

and soil. It is hypothesized that the quantity of P stored in periphyton depends on

periphytic type, and that periphytic CaCO3 production will increase the inorganic

P retention capacity of the peat soil.

Determine the P uptake kinetics of periphyton and partition uptake into biotic

and abiotic processes. Phosphorus uptake should be rapid in the P limited interior

of WCA-2A. Since periphyton contains both autotrophic and heterotrophic

organisms both Pi and Po should be utilized. It is also hypothesized that the

CaCO3 matrix of the periphyton will adsorb Pi thus providing abiotic uptake.

Determine the influence of BP on P flux between soil porewater and the

overlying floodwater. It is hypothesized that the activity of BP reduces P flux

from soil porewater at ambient concentrations. Under enriched conditions,

demand for P by BP should result in faster uptake as compared to periphyton-less

soil. Phosphorus uptake by BP, being a function of autotrophic and heterotrophic

organisms, should occur under light and dark conditions.

Determine the physicochemical conditions in the water column which lead to P

retention in stable mineral forms. It is hypothesized that high pH conditions

causes the precipitation of CaCO3 and coprecipitation of P and that high CO2

causes dissolution of the solids.

Assess in situ ion activities and physicochemical conditions to predict stable

mineral forms.

Data were generated from field, laboratory, and greenhouse experiments. Chapter

2 provides a characterization of the system. Phosphorus uptake kinetics using laboratory

and in situ batch incubations are presented in Chapter 3. Phosphorus flux between soil

porewater and floodwater was studied in intact cores in the field and greenhouse, and

these data are discussed in Chapter 4. Chapter 5 presents a laboratory reactor study using

WCA-2A-217 water incubated at varying pH to determine precipitation processes and

presents results of geochemical modeling for laboratory and in situ conditions. Finally,

Chapter 6 is a summary synthesizing the significance of the results to P retention in the




Phosphorus in wetlands exists in a state of dynamic equilibrium, being distributed

between numerous compartments including periphyton, vegetation, water, and soil. The

system is complex due to the number of storage compartments, each having different

uptake and release mechanisms (Howard-Williams, 1985; Kadlec and Knight, 1996;

Richardson and Marshall, 1986). The various compartments contain inorganic P (Pi) and

organic P (Po) in dissolved and particulate forms. The availability of P for biotic

utilization depends on the compartment and form of P contained. The dissolved

inorganic P (DIP) is the form that is most biologically active and therefore is in greatest

demand. The biological productivity of many aquatic systems is dependent upon how

quickly this form of P can be cycled through the system (Wetzel, 1983). Other forms of

P, especially recalcitrant Po, tend to accumulate in soils and can account for a substantial

fraction of the total P (Koch and Reddy, 1992; Faulkner and Richardson, 1989).

The proportion of P in various compartments varies depending on the stability and

liability of stored P. The relative P storage in the water compartment is small compared to

that of biota and soil (Bostrdm et al., 1982; Faulkner and Richardson, 1989; Richardson

and Davis, 1987; Verhoeven, 1986). Phosphorus in the water compartment is affected by

numerous pathways and generally has a short residence time. Periphyton and

microorganisms are responsible for the initial uptake of DIP from the water column,

while vegetation provides short-term storage of P in the form of biomass (Correll et al.,

1975; Howard-Williams and Allanson, 1981; Richardson and Marshall 1986).

Periphyton removes DIP from the water column and converts it into particulate organic P

(POP) in the form of biomass. Periphyton is short-lived and can return the DIP removed

for cell growth back to the water column in the form of POP. In oligotrophic or P-limited

systems, P released from dead cells is rapidly recycled back into the periphyton such that

the P stored in the periphyton layers is maintained at a semi-constant content (Wetzel,

1993). Emergent macrophytes and other aquatic vegetation tend to store P on a seasonal

basis, releasing large quantities of stored nutrients at senescence. The soil represents the

largest storage compartment and generally retains P on longer time scales than the other

storage. Dissolved P is labile and readily moves between the soil interstitial water and

the water column by diffusion according to the concentration gradient (Reddy et al.,


Phosphorus, as compared to other biotically essential elements, often exists in low

concentrations in the hydrosphere and therefore limits primary productivity (Wetzel,

1983; Bostrom et al., 1982). Depending on the geochemical nature of the region most

natural surface waters contain between 10 and 50 pg P L-1 (Wetzel, 1983). The size of

the water storage is dependent upon the water depth and P concentration. Interactions of

water-borne P with the uptake mechanisms of periphyton and soil depend largely upon

residence time of water over a given surface area of wetland. Emergent macrophytes

influence water storage by slowing flow thus increasing the residence time and allowing

greater removal of floodwater P (Howard-Williams, 1985).

Periphyton can be defined as the attached microorganisms, both floral and faunal,

growing on submerged natural or artificial substrates (Collins and Weber, 1978; Wetzel,

1983). Periphyton can be loosely separated into types based on position in the water

column although transformation of types occur. The water column position determines

the sources of nutrients available to the periphyton. Benthic periphyton (BP) is composed

of the microcommunities of autotrophic and heterotrophic microorganisms associated

with the flooded soil surface. Benthic periphyton often coats the soil surface with a loose

blanket of material or with a cohesive mat. Benthic periphyton has been shown to obtain

P from the overlying water column and from the soil interstitial water (Hansson, 1989).

This type of periphyton is also associated with the detrital layer of the wetland.

Particulate matter settling from the water column passes into the BP layer. Epiphytic

periphyton (EP) grows on the submerged surfaces of emergent macrophytes such as

sawgrass (Cladiumjamaicense Crantz.). The EP receives most of its P supply from the

water column. Small amounts of P from living macrophyte exudates can augment

floodwater P, but this amounts to a small fraction of periphytic P (Riber et al, 1983;

Carignan and Kalff, 1982). Upon senescence a substantial amount of macrophyte P can

be released. The proximity of EP aids in securing the P before it is diluted into the

surrounding water column (Wetzel, 1983). Floating periphyton (FP) receives P solely

from the water column.

The soil compartment contains the largest storage of P in wetlands. Almost all of

the P stored in the soil is held in mineral inorganic forms or in resistant organic forms and

is not readily available for biotic use. For example, Verhoeven (1986) found only 0.2 -

3.0% of the TP in minerotrophic peat mires was in an available form. Forms of P present

in soils and sediments have been characterized by their differential solubilities in

chemical extractants. Procedures for chemical fractionation of soil P are developed with

the intention of sequentially removing discrete pools of P. Generally, the biologically

available P is removed first, followed by labile Pi and Po, and then more stable forms of

inorganic and organic P. The terminology used to describe the various extracted P pools

is mostly operational (Psenner and Pucsko, 1988). Numerous extractants have been used

to remove P from mineral soils (Chang and Jackson, 1957; Hedley et al., 1982; Hedley

and Stewart, 1982), organic soils (Walbridge and Vitousek, 1987), and freshwater (Olila

et al., 1995; Williams et al., 1971; Frink, 1969; Syers et al., 1973) and marine (Hieltjes

and Lijklema, 1980; van Eck, 1982) soils/sediments. Several extraction procedures have

been recently applied to peat soils of the Florida Everglades (Koch and Reddy, 1992;

DeBusk et al., 1994; Qualls and Richardson, 1995). The extraction scheme presented by

Hedley et al. (1982) separates the P fractions into i) readily biologically available, ii)

labile Pi and Po sorbed on the soil surface, iii) labile Po, vi) chemisorbed Fe and Al-P, v)

internal soil particulate Pi, vi) apatite-type minerals, and vii) resistant P forms.

Modifications of this procedure have been used on Everglades peat soils (Quails and

Richardson, 1995).

Long-term storage of P in a wetland is limited. Leaf litter, decaying vegetation

and periphyton generally decompose to stable residuals in time spans of less than 24

months, depending upon numerous factors including microbial activity and temperature.

The residual material is a small portion of the original biomass. Therefore standing

biomass cannot be counted as a long-term, sustainable P removal mechanism

(Richardson, 1985). Soil retention of P is limited by the sorption capacity of the soil

unless new sites are formed. The soil compartment saturates unless there is an input of

new soil materials (Richardson, 1985). The two possible mechanisms for sustainable P

retention are the accretion of biomass residuals and the formation or accumulation of

mineral forms (Richardson, 1985). Peat soils can accumulate P in long-term storage by

the accretion of organic matter (Reddy et al., 1993; Richardson and Craft, 1993). In

systems with high calcium activity, photosynthesis induced pH changes can cause the

precipitation of CaCO3 and the coprecipitation of P (Otsuki and Wetzel, 1972) which is

subsequently deposited on the soil surface. The Ca-P complex is eventually incorporated

into the soil as shown by the linear relationship between long-term Ca and P

accumulation in the Everglades system (Reddy et al., 1993).

Peat soils are the largest reservoir of nutrients, including P, in the Everglades

system. The accretion of peat has been shown to regulate the long-term P storage of the

Everglades (Reddy et al., 1993; Craft and Richardson, 1993). The accretion of peat soils

is dependent upon a number of environmental factors including hydrology and nutrient

regimes. Peat accretion rates and corresponding nutrient accumulation rates have been

estimated in wetland systems using 137Cs as a marker (DeLaune et al., 1978). This

method was recently used in several studies of peat accretion in the Water Conservation

Areas (WCAs) of the northern Everglades (Craft and Richardson, 1993; Reddy et al.,


The objective of this study was to describe the major P storage in an oligotrophic,

periphyton dominated, wetland with peat soils. An area sparsely vegetated with sawgrass

of the northern Everglades will serve as an example. Knowing the sizes and forms of

stored P allows inferences to be made as to the potential mobility of stored P, estimates of

P accumulation rates, and identification of the mechanisms controlling long-term P


Materials and Methods

Site Description

Soil, water, periphyton, and vegetation characterized in this study were collected

from an interior site, designated 217, in Water Conservation Area 2A (WCA-2A-217)

(260 17.17' N, 80 24.73' W 30 m) of the Florida Everglades. The hydrology of WCA-

2A is intensively managed to provide water supply during the dry season and to provide

water storage during the rainy season. This area receives nutrient laden water across its

northern border via the Hillsboro Canal (Fig. 2-1). Steady external nutrient loading has

resulted in a P concentration gradient in the soil and water column with higher P

concentrations near inflow control structures and background P contents at interior sites

(SWIM, 1992; Koch and Reddy, 1992). The interior site of WCA-2A-217 is

characterized by peat soils, patchy sawgrass stands, and the presence of calcareous

Fig. 2-1. Map of Water Conservation Area 2A (WCA) showing field site 217.

periphyton; covering most of the peat, coating the submerged portions of older stems of

sawgrass, and occasionally forming floating mats on the water surface (Swift and

Nicholas, 1987).

Water Sampling and Analysis

Bulk water samples were collected from the surface floodwater on April 10, 1995.

The water depth averaged 0.6 m on the sampling date. Field-filtered and unfiltered

samples were collected near the water surface in the proximity of mats of FP. Water

samples were stored in polyethylene bottles and submerged in ice water until returned to

the laboratory. Samples were stored at 40 C until analyzed. Unfiltered water samples

were analyzed for pH, electrical conductivity (EC) (Method 2510, APHA, 1992),

alkalinity (Method 2320, APHA, 1992), and TP after persulfate oxidation (Method 4500-

P-B, APHA, 1992). Field-filtered (0.2 pm membrane filters; Gelman Supor, Gelman,

Inc.) water samples were split before analysis. One subsample was analyzed

colorimetrically for dissolved reactive P (DRP) (Ascorbic Acid Method 4500-P-E,

APHA, 1992) on an AutoAnalyzer II (Technicon Corp.). The other subsample was

digested and analyzed colorimetrically for total dissolved P (TDP). Total C (TC), total

organic C (TOC), and total inorganic C (TIC) were analyzed on unfiltered samples on a

DC-190 High-Temperature TOC Analyzer (Rosemount Analytical Inc. Dohrmann

Division, Santa Clara, CA). Filtered water samples were analyzed for the cations: Ca,

Mg, K, Zn, Na, Cl, Si, Cu, Mn, Al, and Fe using Inductively Coupled Argon Plasma
Spectrometry (ICP) (Method 3120B, APHA, 1992). Sulfate-S and NO3-N were analyzed

by ion chromatography on filtered samples using a Dionex 4500i (Dionex Corp.
Sunnyvale, CA.)(Method 4110, APHA, 1992).

Periphvton and Macrophvte Sampling and Analysis

Benthic periphyton was sampled on August 12, 1993 and on April 10 and 21,

1995. Benthic periphyton was collected by removing the material trapped in a 14.5 cm

I.D. acrylic core. Three replicate samples were collected during the 1993 sampling while

four replicates were collected in 1995. In April 1995, BP was collected in an area

sparsely populated with sawgrass. This BP was termed BPsawgs. Additionally, BP

samples were also collected from an open water area (slough) in the same vicinity. These

samples were designated BPslough. Epiphytic periphyton was collected by clipping

sawgrass at the floodwater/benthic periphyton interface and at the air/water interface.

The macrophyte and EP were returned to the laboratory where they were separated by

gently scraping the EP with a scalpel. Four clusters of heavily colonized plants were

sampled to provide replication. The separated sawgrass stems were then sectioned into

below water living and dead material, and above water live material. Floating periphyton

was collected with a swimming pool net from four floating mats.

Periphyton and sawgrass materials were oven-dried at 700 C to constant weight

for the determination of water content. Some of this material was then combusted in a

muffle furnace at 4500 C for 12 h to determine ash content. This modification of standard

practice (5500 C for 4 h) was done because of the dolomitic nature of the CaCO3

suspected in the dried material which begins to degrade at temperatures in the 5500 C

range. The objective was to remove all organic matter while leaving mineral carbon

forms intact. Recoveries of standard materials, which included CaCO3, dolomite, and

estuarine sediment, showed the modified ashing method to yield results within 10% for

the two methods. Oven-dried subsamples were ground and digested by perchloric acid

(Method 4500-P-B, APHA, 1992) for colorimetric analysis of total P. This solution was

analyzed for cations (Ca, Mg, Fe, and Al) by Flame Atomic Absorption (FAA) (Method

3111, APHA, 1992). Total C (TC) and total N (TN) were determined on 1 to 5 mg finely

ground (#100 mesh) oven-dried subsamples and analyzed using a Carlo-Erba NA- 1500

CNS Analyzer (Haak-Buchler Instruments, Saddlebrook, New Jersey) (Nelson and

Sommers, 1982). Total inorganic C (TIC) was determined on ashed material (as above

using the CNS analyzer). Total organic C was determined by difference. The CaCO3

content was calculated assuming that all TIC came from CaCO3. Multiplying TIC by

8.33 (mole percentage of C in CaCO3) yields an estimate of CaCO3 content.

Benthic periphyton (air dried, ground (#100 mesh)) samples were extracted with

1.0 M HCI in a 1:50 BP to solution ratio. After a 3 h equilibration under continuous

shaking, samples were centrifuged (6000 rpm, 15 min) and the supernatant filtered

through 0.45 pm membrane filters. Filtered solutions were analyzed colorimetrically for

DRP and by Flame Atomic Absorption for extracted cations, as described above.

Separate subsamples of field moist BP were extracted with 0.5 M NaHC03 at a

solid to solution ratio of 1:100. The suspensions were equilibrated for 16 h under

continuous shaking, then centrifuged at 7000 rpm for 15 min. Supernatant solution was

filtered through 0.45 pm membrane filter (Gelman Supor, Gelman, Inc.) and analyzed

colorimetrically for DRP. A second subsample of field moist was treated with 1 ml of

CHC13, vortexed, and allowed to evaporate overnight. This was followed by extraction

with 0.5 M NaHCO3, as described above. Extracted solutions were analyzed for DRP and

TP The difference between CHCI3/NaHCO3-TP and NaHCO3-P, was considered labile

Po. When this extraction was used on agricultural soils, the additional P extracted after

CHC13 treatment was shown to originate from lysed microbial cells (Hedley and Stewart,


Soil Samoling and Analysis

Soil samples were collected by slowly driving a 10 cm I.D., thin-walled aluminum

tube, with a sharpened edge, into the peat soil. On August 12, 1993, four replicate soil

cores were collected to a depth of 20 cm and were sectioned into 10 depth increments

(cm): 0-1, 1-2, 2-3, 3-4, 4-5, 5-7, 7-9, 9-12, 12-15, 15-20. On April 10, 1995, four

replicate cores were collected to a depth of 5 cm and sectioned into 0-2 and 2-5 cm layers.

Soil pH was determined by taking a 5 g homogenized subsample of each field

moist soil sample and mixing with 5 g distilled, deionized water (DDI-H20) to achieve a

1:1 soil slurry. The pH was obtained using a combination glass electrode and a standard

pH meter. A known mass subsample of the homogenized soil was dried to constant

weight at 700 C and bulk density was determined (g dry weight soil cm-3). The procedure

used follows that of Blake and Hartge (1986), with the exception that soils were dried at

700 C instead of at 1050 C. The alteration of standard method was done considering the

organic nature of these soils. Subsamples of air dried, ground (#100 mesh), and weighed

(approximately 0.2 g) soil was digested with perchloric acid and analyzed for TP as

above. Dried, ground (#100 mesh) soil was extracted with 1 M HCI in a 1:50 soil to

solution ratio and analyzed for Pi and selected cations, as previously described. Dried soil

samples were used to determine the TC, TIC, TOC, and CaCO3 content following the

procedures above.

Fractionation of Soils and Benthic Periphyton

The soil and BP samples, collected in 1993, were fractionated to determine

discrete pools of P using a modification of the extraction scheme as presented in Hedley

et al. (1982). The procedure used (Fig. 2-2) modified the original scheme by omitting

the initial removal of exchangeable P with anion exchange resin. The sonication and

second 0.1 M NaOH extraction were also omitted from our scheme. This fraction is

meant to represent P held at the internal surfaces of soil aggregates (Hedley et al., 1982).

Residual P was not determined by digestion of sequentially extracted soil, but rather as


+ 0.5 M NaHCO3 to maintain 1:100
soil:extractant ratio (25g)
16h shake, centrifuge, filter


0.2g dried ground soil
Perchloric digestion

+ Iml CHCI3, vortex
evaporate overnight
+ 0.5 M NaHCO3 to maintain 1:100
soil:extractant ratio (25g)
16h shake, centrifuge, filter
Persulfate digestion Sml subsample

Perchloric digestion 5ml subsample

and TP by digestion

Fig. 2-2. Diagrammatic representation of phosphorus extraction procedure.

the difference between the sum of the sequentially extracted P and TP by perchloric acid

digestion. Additionally, the 0.1 M NaOH extract was digested for TP.

Field moist soils were simultaneously extracted with 0.5 M NaHCO3 and 0.5 M

NaHCO3 following CHC13 treatment. Phosphorus extracted with NaHCO3 was

considered labile Pi and represents plant available P (Olsen and Sommers, 1982).

Phosphorus extracted with NaHCO3 after CHC13 treatment is composed of the labile P

from above, surface sorbed Po, and a portion of the P released from lysed microbial cells

(Hedley et al., 1982). Hedley and Stewart (1982) found that digested extracts of

CHCI3/NaHCO3 minus the P recovered solely from NaHCO3 extraction represented

about 40% of the microbial P they added to their soil. I consider the P release by

CHCl3/NaHCO3 minus the labile Pi to represent labile P,. The residual soil from the

labile Po extraction above was treated with 0.1 M NaOH to obtain a soil to solution ratio

of 1:100. Soil suspensions were equilibrated for 16 h under continuous shaking, then

centrifuged at 7000 rpm for 15 min. Supernatant solution was filtered and analyzed for

DRP as above. The NaOH-extractable P, as analyzed colorimetrically for DRP, is

considered to be Fe/Al-bound Pi (Ryden et al., 1977; van Eck, 1982). Hydrolyzable Po

was obtained by digestion of a subsample of NaOH extracted P (yielding TP) minus the

Fe/Al-bound Pi. This fraction is considered to be moderately resistant organic P

associated with humic and fulvic acids (Olila et al., 1995). Phosphorus extracted with 1

M HCI represents Ca-bound Pi (Williams et al., 1971). This P is associated with apatite

or apatite-like minerals, essentially as calcium phosphate. Phosphorus can also be present

in amorphous and transitional forms such as monocalcium, dicalcium, and octacalcium

phosphates. Unless soil conditions become acidic this fraction of P is not readily

solubilized (van Eck, 1982). Residual P (as the difference between perchloric acid TP

and sum of extracted forms) is the P remaining in soil at the end of the sequential

extraction and represents resistant organic and mineral P forms.

Peat Accretion Rates

Four replicate soil cores for 137Cs analysis were collected in the same manner as

cores used in soil fractionation. These cores were sectioned by 1 cm increments to a total

depth of 20 cm. Soil sections were air dried, weighed, and ground to pass a 1 mm sieve.

Vertical peat accretion rates were estimated by measuring the '37Cs distribution in the

soil profile (DeLaune et al., 1978). The 137Cs activity in each section was determined by

gamma counting oven-dried soils with a germanium detector and multichannel analyzer.
137Cesium entered the atmosphere as a by-product of aboveground thermonuclear testing.

This 137Cs was then deposited on the soil surface as fallout. The first noticeable levels of

137Cs appeared in 1954: the maximum level corresponds to the soil surface of 1964

(Pennington et al., 1973). Therefore 137Cs provides a 1964 time signature in the peat

soil. The accumulation of peat above the 1964 '37Cs peak can be considered peat

deposited in the past 29 years. From this information, peat accretion and the

accumulation rates of P and selected chemical components were calculated. Peat

accretion rate (PAR) was calculated as

PAR = 137Cs peak depth (mm) / Number of years since 1964 [2-1]

where PAR = peat accretion rate (mm yr1) (converted from cm depth increments), and

Number of years since 1964 = 29. Accumulation rates (AR) (mg m-2 yr1) for P and other

components were calculated by

AR= [(IC pd 1 cm) / n 104 for mg m-2 yr- or 10 for g m-2 yr- [2-2]

where AR = accumulation rate in mg m-2 yrI or g m-2 yr1, C = concentration (mg kg-i),

pd = bulk density for each 1 cm depth increment (g cm-3), n = number of years since 1964

(n = 29 yr), and 104 or 10 to convert to m2 basis.

Statistical Analysis

Differences in chemical composition of periphyton types (Table 2-2) and sawgrass
(Table 2-3) were analyzed statistically using a one-way analysis of variance. Where

appropriate, the level of significance (p-value) is designated (p-value; *** = 0.001, ** =
0.01, = 0.05, ns > 0.05). Sample means determined to be significantly different (a =

0.05) were evaluated by Tukey's Studentized Range Test. Values given in tables

followed by similar letters are not significantly different. Correlation analysis was

performed on physicochemical characteristics of periphyton and soil samples collected on

August 12, 1993. The correlation coefficients (r) and level of significance of the

correlation (p-value) are given for important relationships. All statistics were performed

on SAS Proprietary Software Release 6.08 (SAS Institute Inc. 1989, Cary NC.)

Results and Discussion


Surface water of the interior WCA-2A site was characterized by relatively high

electrical conductivity, a basic pH, and high alkalinity (Table 2-1). The major anions

were Cl and HCO- Major cations included Na, Ca, and Mg. These results generally

agree with those of Swift and Nicholas (1987) measured at this site between 1977 and

1983, except that the pH measured in our study was almost one unit higher, and SO4

about half their average. Swift and Nicholas (1987) suggest the highly mineralized nature

is due to the influx of high ionic strength water from agricultural production in the EAA.

Total inorganic C and TOC each represent about 50 % of the TC in the water column.

Swift and Nicholas (1987) describe the interior of WCA-2A as being a
minerotrophic peatland. Minerotrophic peatlands develop in areas that receive mineral

enriched water inputs by draining alkaline soils or that overlie mineral soils. The bedrock

Table 2-1. Chemical composition of WCA-2A-217 surface water collected on
April 10, 1995, (n=5 unless noted then n=4).

Parameter Units Mean SE

pH (mean and range)
Electrical Conductivity
Alkalinity (CaCO3 to pH=4.5)
Dissolved Reactive P
Total Dissolved P
Total P
Total C
Total Inorganic C
Total Organic C
Sulfate S
Cu, Mn, Al, and Fe
Nitrate N

mS m-1
mg L-1
gg L-1
gg L-1
gg L-1
mg L-'
mg L-1
mg L-1
mg L-1
mg L-
mg L-
mg L-1
mg L-1
mg L-
mg L-
mg L-1
mg L-1

7.93 (7.85 8.20)
83.2 0.7
230 2
9.4 0.1
10.7 2.1t
10.8 0.7
94.2 1.5
45.5 0.5
48.7 1.9
18.0 0.2
64.6 0.5
32.1 0.1
0.3 0.1
106 0.2
164 1
7.6 0.1

underlying the peat soil at WCA-2A-217 is calcareous limestone of the Ft. Thompson

formation (Gleason, 1972). Characteristics of minerotrophic peatlands are alkaline

conditions, hard water, bicarbonate as a dominant anion, high Ca concentrations, neutral

to basic pH, high EC, and low concentrations of inorganic nutrients (P) (Moore and

Bellamy, 1974).

Dissolved reactive P was 10 pg L-1 and accounted for nearly 90 % of TP. The

ratio of DRP to TP is expected as the hydrolysis of organic P compounds, represented by

ATP, is known to be rapid (see Chapter 3). The TP values obtained agree with those of

Swift and Nicholas (1987), but they obtained DRP concentrations < 2 pg L-' or < 20% of

their TP was DRP. Quails and Richardson (1995) found DRP (SRP) to comprise nearly

80% of TP at their unenriched sites in WCA-2A; however, their TP averaged 36 pg L- or

about 3 times the TP concentrations in our study. Concentrations of water P forms in the

Quails and Richardson (1995) study are averages of samples collected from several

transects, some of which are in closer proximity to drainage canals than was the site used

in this study.

Total P content in the water column is a function of P inputs: through

precipitation, by the influx of P from upstream sources, and by the cycling of P between P

containing compartments at the site. Although WCA-2A receives an estimated 5.4 x 104

kg P yr1 (South Florida Water Management District, 1991) as a function of the pumping

of nutrient laden water from the Everglades Agricultural Area (EAA), this P has been

shown to be removed from the water column upstream of WCA-2A-217 and does not

affect TP concentration at this site (Koch and Reddy, 1992). If TP concentrations are

considered to be relatively constant (as based on similarity in TP of Swift and Nicholas,

1987 study and results of this study), then the P cycle may be described as closed to

outside inputs. In a steady-state system, where nutrient inputs from outside the system

are minor compared to consumption rates, nutrient regeneration from within the system

becomes more important to overall system productivity (Howard-Williams, 1985).

Nutrient recycling rates are a function of the proportion of available nutrient to

unavailable form. Dissolved reactive P is the most biologically available form; therefore,

in a system with rapid recycling of nutrients it would be expected that mechanisms (e.g.

alkaline-phosphotase production) would be developed to increase the proportion of DRP

to TP. The high (90%) proportion of TP that was DRP from this analysis suggests

nutrient recycling at WCA-2A-217.


In WCA-2A-217 BP was associated with the peat substrate under sparsely

vegetated sawgrass (BPsawgra and BP collected in August 1993 (BP93)), and in open

sloughs (BPslough), EP was growing on sawgrass stems, and FP was present at various

positions in the water column, usually near the water surface. Benthic periphyton

consisted of loose accumulations of photosynthetic, mineral, and detrital organic

materials. Although stratification within the BP layers undoubtedly occurred (see pH

profiles Chapter 5) the BP at this site is best described as an integrated periphyton type

rather than a biofilm mat according to the descriptions given by Wetzel (1993). The

growth of EP was much greater on submerged, dead sawgrass stems than on living stems.

Similarly, Browder (1981) found preferential colonization on dead macrophyte stems.

Floating periphyton occasionally colonized Utricularia spp. When this occurred, the

Utricularia spp. was often completely covered and accounted for a very small portion of

the floating material.

Several similarities exist between BPswgrs collected in 1995 and the BP

collected in 1993, reflecting the stable nature of the interior of the northern Everglades.

Samples collected were from the same general area. The TP contents generally agreed,

with BPsawgss being higher but not statistically different from BP93 (Table 2-2). Water

Table 2-2. Characterization of benthic (BP), epiphytic (EP), and floating (FP) periphyton collected at WCA-2A-217 in August, 1993
(BP93) and April, 1995. In April 1995, BP was collected from a location sparsely vegetated with sawgrass (BPsawgrss) and from an open
slough (BPslough) in the same vicinity. The BPslough values are the averages of one sample collected April 10, 1995 and three samples
collected April 21,1995 (n = 4, unless noted t then n = 3, if noted t then only April 10 BPslough sample analyzed). Levels of
significantly different sample means are given by p-values (***= 0.001, **= 0.01, *= 0.05, ns > 0.05). Values followed by similar letters
were not significantly different.

August, 1993 April, 1995
Periphyton Type Units p-value BP93 BPsawgrass BPough EP FP
(dry wt.
Parameter basis) Mean SE Mean SE Mean SE Mean SE Mean SE

Total P mg kg- *** 330 5a 385 13a 385 71a 157 0 IOb 132 10b
Total Ca gkg1 *** NA 260 19b 542 61a 412 14a 502 17a
Total Mg g kg-l ** NA 7.17 0.41b 9.96 0.84a 7.69 0.37b 8.81 0.08ab
Total AI g kg *** NA 1.19 0.07a 1.01 0.18a 0.16 0.02b 0.24 0.03b
Total Fe gkg-1 *** NA 1.72 0.19a 1.05 0.19b 0.40 0.02c 0.26 0.02c
Water Content % *** 93.6 0.3ta 93.5 0.3a 95.8 t 0.5b 97.1 0.2b 96.9 0.2b
Ash Content % ** 33.4 2.1b 32.6 2.1b 61.1 10.4a 50.8 2.2ab 53.4 0.8ab
Total N g kg- *** NA 25.4 0.6a 22.1 0.7b 12.0 0.5c 10.9 0.5
Total C g kg *** 321 38ab 349 7a 279 6ab 251 4bc 220 1.8c
TC:TN ** NA 13.7 0.2b 12.6 0.1b 21.0 0.5a 20.3 0.7a
Total Inorganic C g kg1 ** 26.0 2.2b 23.6 2.4b 48.9 9.5a 38.6 2.8ab 38.8 1.3ab
Total Organic C g kg- *** 295 37ab 325 10a 230 12bc 212 7c 182 3c
CaCO3 gkg1 ** 217 19b 197 20b 408 78a 321 23ab 323 i lab
Bulk Density gem-3 *** 0.011 <0.001a 0.037 0.002b 0.029tb NA NA
Labile Pi mg kg *** 1.8 0.6tb 13.0 NA NA NA
Labile Po mg kg- *** 38 Itb 115 7a NA NA NA
I M HCI extractable Pi mg kg- ns 130 2 172 3 168 22 NA NA
I M HCI extractable Ca g kg *** 113 4a 176 8b 289tc NA NA
I M HCI extractable Mg gkg-1 ns 4 0.1 4.2 0.1 4.3t NA NA
I M HCI extractable Al g kg- ns 0.31 0.09 0.25 0.02 0.30t NA NA
I M HCI extractable Fe g kg- *** 0.34 0.02a 0.23 0.01b 0.20$c NA = not available

content, ash content, TC, TOC, TIC, CaCO3 content, and 1 M HCI extractable Mg, and

Al were all virtually the same for the BPwgrass and BP93. The dissimilarities between

BPsawgrass and BP93 appear in the bulk densities and in the concentrations of labile Pi,
labile Po, and HCI extractable Pi and Ca.

For the periphyton collected in 1995 the BP contained over twice as much TP than

did EP, or FP (Table 2-2). Total P content was in the order BPsawg = BPsough > EP =

FP. Similarly, TC and TN followed these trends, except that BPwgras had higher

concentrations than all other types. Total C in BPslough, EP, and FP were similar while

that of BPswgra was generally higher. The increase in TC of BPsawgrass was the result of

higher TOC concentration and not TIC. Both BP types had about twice the TN content of

EP and FP, resulting in C:N of half that of EP and FP. The higher TC and TN contents

reflect the increased supply of organic C and N available to BP from decaying sawgrass

litter. The ash content, total Ca, and CaC03 content were greater in the BPslough samples.

The total Ca contents of all periphyton forms were extremely high with total Ca

accounting for around 50% of the total dry mass of periphyton. If this Ca was associated

only with CaCO3, then the CaC03 contents would account for 65%, 136%, 103%, and

126% of the total dry weight of BPawgrass, BPlough, EP, and FP,

respectively. Ash content was > 50% for all periphyton types except BPsawgras. Total

inorganic C was 18% of the TC in FP, 15% in EP, 18% in BPslough, and 7% in BPwgrass.

Concentrations of Fe and Al were low in all periphyton types, especially EP and FP. The

Mg concentrations were similar for all periphyton types and followed the same order as

total Ca (BPslough > FP > EP > BPsawgrass).


The most dramatic difference between live and dead portions of sawgrass was that

of TP content (Table 2-3). Live sawgrass contained 5 7 times more P than dead stems.

There was not a corresponding decrease in TN or TC between submerged live and dead

stems. The TC:TN was about 130 in both cases. This suggests that upon senescence a

considerable portion of the plant P was either lost to the water column through leaching-

or incorporated into the EP. There was no evidence for a similar transfer of TC or TN.

Davis (1990, as reported in Table 14-2 of Kadlec and Knight, 1996) reported P contents

of 400 mg kg-' in live material and 200 mg kg-' in dead sawgrass and litter material from

the oligotrophic Everglades areas he studied. The TP concentrations in the live sawgrass

analyzed in this study were about 1/2 and the dead sawgrass contained about 1/7 those

reported by Davis (1990). Soluble substances are rapidly leached from senescent

macrophytes. Leaching was shown to cause the loss of 50% of the P content in Typha

lattifolia L. in the first three weeks following senescence (Kulshreshtha and Gopal 1982).

Nitrogen content did not behave in this manner; little change in N content was observed

between live and dead plant material (Kulshreshtha and Gopal 1982). It should be noted

that the water content of submerged dead sawgrass was much greater than that of living

stems. The implications of this are that much larger surface areas of

dead material contributed to the analyzed concentrations of nutrients. Epiphytic

periphyton growing on the surface of dead stems would therefore be in contact with

macrophyte material that contains proportionately lower concentrations of nutrients. The

availability of macrophytically derived nutrients to EP was not determined. However, the

TP content of EP was not statistically greater than FP, suggesting that macrophyte P was

not a significant, additional P source. Total inorganic C and subsequently CaCO3 contents

of the dead material was greater than that of the live material. The accumulation of EP on

the dead stems may lead to calcification in the decaying vegetation.

Table 2-3. Characterization of sawgrass (Cladiumjamaicense Crantz.) stems from
within (live and dead) and above water surface collected from WCA-2A-217 on April 10,
1995. Below water stems were substrate for epiphytic periphyton (n = 4 unless noted t
then n = 3). Levels of significantly different sample means are given by p-values
(***=0.001, **= 0.01, *= 0.05, ns > 0.05). Values followed by similar letters are not
significantly different.

Below Water Above Water
Units p-value Live Dead Live
Parameter (dry) Mean SE Mean SE Mean SE

Total P mgkg- *** 165 20a 28 5 197 15a
Total Ca g kg-' ns 24.4 5.5 38.3 5.1 38.9 2.5
Total Mg g kg- ** 0.02 0.02b 1.42 :0.28a 1.10 0.22a
Total Al gkg-1 ns 0.10 0.03 0.11 0.01 0.06 0.02
Total Fe gkg- *** <0.003b 0.12 0.02a 0.07 0.01a
Water content % *** 74.1 1.9a 98.3 0.3b 60.0 2.0C
Ash % ** 2.67 0.23a 3.57 0.58ab 4.78 0.26b
Total N g kg-' ** 3.53 0.25ta 3.68 0.68ta 5.71 t 0.06b
Total C g kgl ** 455 3ta 448 t5ab 442 lb
TC1TN ** 130 11 130 23 77 1
Total Inorg. C gkg-1 *** 0.8 0.3ta 68.0 5.8tb 9.5 2.1a
Total Org. C g kg- *** 454 2ta 380 10tb 432 3a
CaCO3 g kg-' *** 6.7 3ta 566 42tb 79.0 17a

Phosphorus Forms in Soils and Benthic Perihvton

Soil and BP collected in August 1993 were characterized for P forms and selected

cations associated with P retention. Total P in BP equaled that in the surface layer of the

soil column (Fig. 2-3g.) (Table 2-4). Total P remained constant to a depth of 4-5 cm then

decreased to approximately one-half that of the surface at 15-20cm depth (Fig. 2-3g).

Concentrations of all soil P forms generally decreased exponentially with depth. Non-

linear regressio I

fractions than did lower soil lay (a = 0.05, r2 ranged 0.53 0.98). This trend was most

pronounced for Ca-Pi, Fe/Al-Pi, labile Po, residual Po, and TP and was less distinct for

labile Pi, and hydrolyzable Po (Fig. 2-3a-g.). The parameters labile Po (r = 0.57***),

Fe/Al Pi (r = 0.57***), Ca-P, (r = 0.56***), residual Po (r = 0.64***) and TP (r =

0.88***) were correlated with depth suggesting higher concentrations at the soil surface.

Depth of the BP layer at the time of sampling was 7 cm. Depth values for the BP layer

are represented at +3 cm (Fig. 2-3). Of the extracted P forms, labile Po (r = 0.78***),

Fe/Al-Pi (r = 0.66***), and Ca-Pi (r = 0.54***) were most highly correlated to TP.

Labile P forms (Pi and Po) comprised 12 -18 % of TP (Fig 2-4). Being labile,

these forms of P can have the greatest effect on surface water quality by being those

most biologically available. Labile Pi accounts for a smaller percentage of TP in the BP

than in all soil layers (Fig. 2-4). On a mass basis, labile Pi in BP is also smaller than in all

soil layers, even when soil TP drops to half that of the BP (Fig. 2-3a). This agrees with

the P limited condition ofWCA-2A-217. Any readily available Pi is rapidly incorporated

into the periphyton biomass (see Chapter 3). Similarly, the labile Po of the BP is lower

than surficial soils on a mass basis (Fig. 2-3b), but this trend is not as distinct as that of

labile P,. Benthic periphyton was also shown to rapidly incorporate organic P, such as

adenosine triphosphate (ATP) (see Chapter 3).

-5 I--


-15- LablePi LabilePo F/Al-Pi
-20 Hyd able Po

0 5 10 15 0 20 40 60 0 20 40 60 0 10 20 30 40
5e tf
Soil So-l Sol so
-5- ResidualP ,


-15- Ca-Pi TotalP

-20 .... .
0 20 40 60 80 0 50 100 150200250 150 250 350

Phosphorus, mg kg1
Fig. 2-3. Distribution of P forms extracted from WCA-2A-217 benthic periphyton and soil collected in August, 1993. Depth of BP,
averaging 7 cm is graphicaly represented by point at +3 cm. Data are means SE of 4 replicate soil cores and 3 BP samples.

Table 2-4. Characterization of WCA-2A-217 benthic periohvton (BP) and soil collected Aueust 12. 1993. (n = 4. if noted t then n = 3).

Depth Increment Units BP 0-lcm 1-2cm 2-3cm 3-4cm 4-5cm
Mean SE
Total P mg kg- 330 5 334 6 325 7 323 5 322 6 315 18
I M HCI extractable Pi mgkg.t 127.9 2.3t 81.9 4.5 73.2 1.0 70.6 1.0 70.7 2.4 69.2 2.0
I M HCI extractable Ca g kg-' 112.9 4.0 48.8 0.9 42.9 2.0 39.5 1.7 37.5 1.3 35.7 0.9
I M HCI extractable Mg gkg' 4.0 0.1 3.4 0.0 3.2 0.1 3.3 0.1 3.1 0.1 3.2 0.1
I M HCI extractable Al mg kg-' 305 91 630 20 598 53 553 90 510 78 475 76
I M HCI extractable Fe mg kg-1 335 23 223 20 208 30 175 13 185 16 180 16
pH (mean) 7.72 7.64 7.45 7.44 7.34
pH (range) 7.64-7.85 7.49-7.75 7.29-7.73 7.25-7.62 7.21-7.57
Bulk Density gcm-3 0.011 <0.001 0.112 0.005 0.110 0.014 0.093 0.019 0.089 0.014 0.093 0.014
Water Content % 93.6 0.3t 86.6 1.1 87.3 1.8 87.9 1.9 88.7 1.3 89.2 0.9
Ash % 33.4 2.1 15.6 0.6 13.7 0.6 12.4 1.9 11.8 0.8 11.1 0.8
Total C g kg1 321 38 415 6 426 8 435 8t 441 8 447 6t
Total Inorganic C g kg-' 26.0 2.2 6.8 0.3 5.6 0.6 4.9 0.5 4.6 0.4 4.3 0.3
Total Organic C g kg-! 295 37 408 6 421 8 431 8t 436 8 443 6t
CaCO3 g kg- 216.8 18.7 56.4 2.3 46.8 5.0 40.7 4.3 38.3 t 3.1 35.9 2.7

Depth Increment 5-7cm 7-9cm 9-12cm 12-15cm 15-20cm

Total P mg kg- 293 20 263 16 237 19 203 6 18011
I M HCI extractable Pi mg kg- 61.2 8.3 49.1 2.5 51.85.1 40.5 1.9 35.1 1.3
I M HCI extractable Ca g kg-' 33.8 2.6 30.8 3.0 33.6 0.7 32.9 0.4 31.6 0.8
I M HCI extractable Mg g kg-l 3.0 0.1 2.8 0.3 2.9 0.1 2.9 0.1 2.7 0.1
I M HCI extractable AI mgkg-l 465 99 375 89 483 60 548 51 575 29
I M HCI extractable Fe mg kg- 160 14 178 43 213 22 230 18 230 19
pH (mean) 7.22 7.22 7.15 7.39 7.27
pH (range) 6.96-7.40 7.07-7.43 6.88-7.39 7.29-7.44 7.00-7.55
Bulk Density g cm-3 0.071 0.011 0.077 0.007 0.075 0.006 0.083 0.004 0.084 0.003
Soil Water % 89.6 0.7 90.1 0.5 90.4 0.3 90.2 0.1 90.4 0.3
Ash % 10.7 0.8 9.9 0.5 9.4 0.2 9.3 0.3 9.0 0.2
Total C gkg-1 427 17 457 5 467 5 467 10 456 7
Total Inorganic C g kg-1 4.1 0.3 4.2 0.3 4.3 0.4 4.1 0.3 4.1 0.2
Total Organic C g kg-1 424 17 457 5 463 5 463 10 452 8
CaCO, g kg-1 34.5 2.7 35.3 2.5 35.6 3.0 34.2 2.3 34.0 1.7

Benthic Periphyton

Soil, 4-5 cm Depth


Soil, 0-1 cm Depth


* Labile Pi
SLabile Po
* Hydrolyzable Po
[E Ca-Pi
O Residual P

TP =334 6mg kg'

TP=315 18mgkg'

Soil, 15 -20 cm Depth


TP = 180 11 lmgkg-'

Fig. 2-4. Average relative extracted P form as a percentage of total P (TP) in benthic periphyton (BP) and selected soil depths. Total
P content by perchloric acid digestion is shown under diagrams.

The Fe/Al-bound Pi makes up 15 % of TP in the surface 0-1 cm of the soil and

decreased depth to 4% of TP. Hydrolyzable Po maintains a consistent percentage of TP,

accounting for about 7 8 % in all depths (Fig. 2-4). However, the extracted amounts of

this fraction were highly variable (Fig. 2-3d). The Fe/Al-bound Pi fraction may be

overestimated. The NaOH extractant used to separate this fraction solubilizes humic and

fulvic acids and therefore may be releasing some organically bound P. Further evidence

to suspect overestimation of Fe/Al-P, is that the extracted solutions analyzed were highly

colored, and the 1 M HCl-extractable concentrations of Fe and Al were relatively low

compared to the amount of Pi. This portion of the sequential extraction was suspect in

other studies (Koch and Reddy, 1992). Calcium bound Pi accounted for between 4 and

11 % of TP. There is a very distinct enrichment of this fraction in the surface 0-2 cm of

soil depth (Fig. 2-3e).

The residual P accounted for the largest portion of P in all samples (Figs. 2-3f and

2-4). Residual P as a percentage of total extractable P was similar in the BP and the 15 -

20 cm soil depths (Fig. 2-4). For this organic soil, the residual P is probably composed

mostly of recalcitrant organic material and not resistant mineral forms. The residual P,

also known as recalcitrant organic P, was found to be the fraction in largest proportion in

other studies on Everglades peat soil (Reddy et al., 1997; Quails and Richardson, 1995).

The decrease in residual P between the BP layer and that of the surficial soils and a

corresponding increase in the inorganic fractions (labile Pi, Fe/Al- Pi, Ca-Pi) is evidence

of increased mineralization in surficial soils. Increased biological activity in this layer

probably increased mineralization of decomposing BP. The residual P fraction increased

as a proportion of TP after the first few surficial depths because the remaining material

becomes increasingly resistant to microbial degradation. Phosphorus mineralization in

peat soils of fens in the Netherlands was shown to be faster at a soil depth of 10 cm than

at 25 cm (Verhoeven et al., 1990). Verhoeven and coworkers (1990) believed the

increased mineralization was due to the effects of temperature and/or redox conditions on

microbial metabolic activity but also suggest that a change in substrate quality with depth

could have been partially responsible.

In addition to the sequential P fractionation, soils and BP samples were analyzed

for several physicochemical parameters (Table 2-4). Soil bulk density ranged 0.07 0.11

g cm-3 and generally decreased with depth, although the correlation was weak (r = 0.35*).
The bulk density of these soils were lower than those of the Netherlands minerotrophic

fens, 0.07 0.25 g cm-3 (Verhoeven, 1986), but agrees well with those from several other

locations in the Everglades (Koch and Reddy, 1992; Craft and Richardson, 1993) The pH

of the soils averaged slightly above neutral with the highest recorded pH (7.85) being in

the 0 1 cm depth increment and the lowest (6.88) being at 9-12 cm. Ash (r = 0.68***).

CaCO3 (r = 0.57***), and TIC (r = 0.57***) contents were correlated to depth. The

CaCO3 content of the BP averaged almost 22% (dry wt. basis). High CaCO3 content are

expected in the calcareous cyanobacteria dominated BP of WCA-2A-217 (Swift and

Nicholas, 1987). The CaCO3 content of the BP during this sampling was lower than was

seen at this site during the April 1995 sampling (Table 2-2). Conversely, there was an

inverse relationship between TC and TOC with proximity to the soil surface. Total C and

TOC concentrations increased with depth in the soil column.

Phosphorus and selected cation concentrations were analyzed after extraction of

air dried, ground soil samples with 1 M HC1. The Pi extracted with 1 M HCI also

estimates total soil Pi (TP,). The TP, contents were slightly higher than that estimated by

sum of the sequential extraction. Both methods showed similar trends with depth.

Recently, Reddy et al., (1997) analyzed TPi by sequential and 1 M HCI extraction for 210

soil samples collected from a large area of the Everglades. They showed that the

empirical relationship between the two estimates closely agreed. The TPi was strongly

correlated to HCI extracted Ca (r = 0.87***), and Mg (r = 0.79***), was weakly

correlated to extracted Fe (r = 0.44***), and was not correlated to extracted Al (r = -

0.24ns). Total Pi was strongly correlated with depth (r = 0.87***). Extracted cations

were generally correlated with ash content except for Al. The concentrations of extracted

Ca in the soil column were 55 85 times higher than extracted Al and between 130 and

230 times greater than extracted Fe. Extracted Mg averaged 7 and 15 times greater than

soil Al and Fe, respectively (Table 2-4). The extractable Ca was correlated with depth (r

= 0.64***) showing an accumulation of Ca in the soil surface. Phosphorus completed

with cations in the soil is dominated by the Ca/Mg cations, not Al/Fe. This is consistent

with the alkaline nature of the site. Aluminum and Fe tend to dominate P complexation

in acid soils. The relatively low concentrations of Fe in WCA-2A-217 soil suggests that

P retention would not be strongly influenced by redox conditions.

A value termed the inorganic P index (IPI) was developed to describe changes in

the proportion of total P that was composed of by Pi. The IPI was determined by

summing the inorganic P forms and expressing the total as a ratio of TP

IPI = (labile Pi + Fe/Al-Pi + Ca-Pi) / TP [2-3]

The IPI for WCA-2A-217 soils was larger in the surface 0 4 cm (Fig 2-5) and

decreased exponentially with depth. A nonlinear regression analysis of the relationship of

IPI to depth showed IPI to be statistically higher in the surface soil than with depth (a =

0.05). The equation

IPI = 0.216 e-0.335 x + 0.130 [2-4]

where x = soil depth, best described the trend (r2 = 0.64). The relative contribution of Po

forms to TP content increased with depth. This, coupled with increased levels of CaCO3,

HCl-extractable Pi, Ca, and Mg in the soil surface suggests accumulation of Pi in the

surface soils. It is hypothesized that accumulation of Pi forms in the surface soil is due to

the decomposition of organic materials, which results in the release of labile Po that is
then mineralized to Pi. Some of the Pi is then recycled back into the biota while a

portion is precipitated with Ca, coprecipitated with CaCO3, or otherwise absorbed to

Inorganic Phosphorus Index (IPI)

0 0.1 0.2 0.3 0.4 0.5
5i .

BP 1993
Si U 1964 n

5 -5-




Fig. 2-5. Inorganic phosphorus index (IPI) with depth in the soil column. Inorganic P
index is defined as the ratio of all extracted inorganic P forms to total P. IPI = (labile Pi +
Fe/Al-Pi + Ca-Pi) / (TP). Soil/benthic periphyton (BP) interface is shown for 1993 by
solid line and by dashed line for soil/BP interface position in 1964 as determined by '37Cs

inorganic solid phases. The exact mechanism, and the relative proportions of Pi being

recycled versus precipitated on a short-term basis are currently unknown (Reddy et al.,


Data from the sequential extraction of the 1993 soils were averaged over the 0-2

and 2-5 cm increments so that comparisons could be made between soil chemistry of

August 1993 and April 1995. Statistical comparisons were made for each of the two

depth increments. Significant comparisons are denoted by p-values (Table 2-5).

Although not statistically different the bulk density of the 0-2 cm increment was higher in

the 1993 soils than in the 1995 soils. For the 2-5 cm increment, the bulk densities are the

same; therefore, concentration data are directly comparable. Concentrations of P in 1993

surface soils were slightly lower than in 1995 when normalized for bulk densities. There

was generally little difference in labile P concentrations in the 2-5 cm soils from the two

sampling dates; however, the TP concentrations in the 2 5 cm soil was higher

in 1995 than in 1993. The TP data for the 1995 sampling was more variable than that of

the 1993 samples. Although statistically significant, the differences in TP concentration

may be due as much to spatial variation as to temporal P changes. The 1 M HCI extracted

Ca, and ash contents of the 1993 soils were significantly lower than those of 1995. This

indicates a lower degree of calcification in the 1993 soils. Conversely, this difference

was not seen in the CaCO3 or TIC contents, as expected if there was a difference in

mineralogical components of the soils. Differences in the pH of the soils between

sampling times did not manifest in changes in the CaCO3 contents suggesting that the

CaCO3 is stable at pH slightly lower than neutral. Overall, the soil samples collected 21

months apart were not appreciably different, attesting to the stable nature of the interior

sites of WCA-2A.

Table 2-5. Characterization of WCA-2A-217 surface soils collected August 1993 and
April 1995 (n = 4). Statistical comparisons were made between sampling times for soil
parameters of each depth increment. (p-values: *** = 0.001,** = 0.01, = 0.05, ns >
Aueust 1993 April 1995
Soil Depth 0-2cm 2-5cm 0- 0-2cm 2-5cm 2-
Parameter Units Mean SE Mean SE pval. Mean SE Mean SE pval.
(dry wt.)

Labile Pi
Labile Po
Total P
Total Ca
Total Mg
Total Al
Total Fe
pH (mean)
pH (range)
Bulk Density
Total N
Total C
Total in. C
Total org. C
NA = not availal

mg kg-1
mg kg-

7.6 0.8 9.4 0.9 ** 13.0 0.5 16.2 3.5 ns
47.5 2.2 46.1 3.8 104.8 20.1 77.5 15.0 ns
298 10 253 8 427 45 403 46 *

mgkg1 78 3 701 ns 148 32 124 33 ns
gkg' 45.9 1.5 37.6 0.8 78.1 2.4 62.9 2.0 ***
g kg-1 3.29 0.06 3.22 0.06 ** 3.48 0.03 3.30 0.06 ns
gkg-1 0.61 0.03 0.51 0.04 ns 0.48 0.05 0.52 0.05 ns
gkg-1 0.22 0.02 0.180.01 ns 0.200.02 0.19 0.01 ns
gkg-1 NA 117 4 934
gkg-1 NA 5.47 0.26 5.48 0.26
gkg- NA 2.120.11 2.090.17
gkg- NA 1.72 0.11 1.58 0.16
7.7 7.4 ** 7.0 6.9 **
7.5-7.9 7.2-7.7 6.7-7.3 6.7-7.1
gem-3 0.11 0.01 0.09 0.01 ns 0.08 0.01 0.090.01 ns

% 86.9 1.0 88.60.8 91.20.5 90.40.9 ns
% 14.70.5 11.7 0.5 17.5 0.5 14.7 0.5
gkg-1 NA 31.4 0.8 33.0 0.7 ns
gkg-1 421 5 442 4 ns 430 4 455 6 ns
NA 13.7 0.4 13.8 0.5
gkg-1 6.2 0.4 4.6 0.2 ns 4.9 0.6 5.5 1.1 ns
gkg-1 4145 437 4 ns 425 4 449 6 ns
gkg-1 51.6 3.1 38.3 1.9 ns 40.5 5.2 45.7 9.2 ns

Long-term Peat and Nutrient Accretion Rates

The peak 137Cs concentration occurred at 2 cm soil depth (Fig. 2-6) which

represents a peat accretion rate of 0.68 mm yr1. This level of peat accretion was lower

than the 4 11.3 mm yr-1 accretion rates found by Reddy et al., (1993) for a transect along

a nutrient gradient in WCA-2A. Craft and Richardson (1993) found accretion rates in

unenriched WCA-2A to average 1.6 mm yr-1. Several factors influence the peat accretion

rates including hydrology and the level of nutrients. For instance, Reddy et al., (1993)
------- -----
found decreasing peat accretion rates with increasing distance from nutrient sources in

WCA-2A. Hydrology affects peat accretion by decreasing the oxidation of organic matter

under increased water depth and duration. The water depth at WCA-2A-217 periodically

drops below the soil surface (SFWMD, data not shown)gaowing nxihti" f th prt

layers. Accumulation rates of P, C, and selected cations since 1964 were calculated for 4

replicate cores (Table 2-6). The accretion rates of organic P

forms: labile Po, Hydrolyzable Po, and residual Po, are about 2.5 times higher than that of

the inorganic P forms; Labile Pi, Fe/Al-bound Pi, and Ca-bound Pi. Organic P forms

accounted for 71%, and inorganic P forms made up 29% of the TP accumulated since

1964. Accumulation rates are dependent upon two factors: accretion of peat and

concentration of materials in the peat (Qualls and Richardson, 1995).

Estimated accretion rates and the standing TP content in BP biomass at the time of

sampling were used to calculate the proportion of BP-TP incorporated into the surface

soil. On an areal basis, the BP contained 254 mg m-2 and the TP accumulation in the

surface 2 cm of soil was 25.2 mg m-2 yr- over the past 29 yrs. Dividing accumulated TP

by that in the standing BP results in approximately 10% of the TP in the BP accumulating

in the surface soil. These calculations assume a constant standing stock of BP, constant

TP content in the BP, and the same accumulation rate in each of the past 29 yrs. Under

the caveat of these assumptions, it is hypothesized that 10% of the P content of BP is

8-9 3
9-10 .. 1

20 40

100 120 0 20 40 60

Fig. 2-6. Depth distribution of 137Cs in four replicate soil cores of WCA-2A-217
collected in August 1993. Maximum 137Cs accumulation denotes soil surface in 1964.
Maximum generally occurs within 2 cm of soil surface.

. ni I i

80 100 120



Table 2-6. Peat accretion rate and accumulation rate of P forms and selected
parameters since 1964. Based on depth to 137Cs peak at 2 cm in soil profiles of four
replicate cores.

Accumulation Rate Accumulation as
Parameter (Mean SE, n = 4) Percentage of Total P

Peat 0.7 mm yr1

mg m-2 yr1
Labile Pi 0.6 0.1 2.3 0.4
Labile P, 3.6 0.3 14.4 0.5
Fe/Al-bound P, 3.4 0.4 13.9 1.9
Hydrolyzable Po 1.9 0.5 7.5 1.6
Ca-bound Pi 3.3 0.6 13.0 2.2
Residual P 12.3 0.8 48.9 2.6
Total P 25.2 1.7
HC1 Extractable
Pi 5.9 0.4 23.5 0.7
Mg 251 12
Al 47.5 5.0
Fe 16.6 2.0

g m-2 yr1
Ca 3.5 0.1
Total Carbon 32.1 1.5
Total Inorganic C 0.5 0.0
Total Organic C 31.6 1.5
CaCO, 3.9 0.1

added to the soil each year; 90% is either recycled through the BP or exported

downstream. Similarly, estimates of TC accretion result in 13% of the TC in the BP

being incorporated into the soil.


The interior of WCA-2A, as exemplified by site 217, can be characterized as an

oligotrophic, minerotrophic peatland. The TP content of the water was around 10 pg L-1

with DRP accounting for 90% of TP. The DRP:TP is evidence of a P-limited system

with mechanisms developed to maximize P availability. Calcareous cyanobacterial

periphyton exists as floating, epiphytic, and benthic forms. Phosphorus contents of these

types are in the order; BP > EP = FP. The higher TP contents of the BP reflect the water

column position. Forming a layer between the floodwater and the soil surface the BP

receives detrital materials from the water column. Epiphytic periphyton preferentially

colonized dead sawgrass stems. Sawgrass stems probably provide only physical support

as the P concentration in the dead material was very low. However, the exchange of

nutrients between the macrophyte and EP was not studied.

The peat soil contained TP concentrations around 330 mg kg-1 in the surface and

about half that at 15-20 cm. Similarly, the concentrations of all P forms generally

decreased exponentially with depth. Residual P, considered to be recalcitrant organic P,

accounted for the largest fraction of TP. Inorganic P was completed with Ca and Mg

reflecting the alkaline nature of the site. The inorganic P index (IPI) showed

proportionally higher Pi at the soil surface than at depth, suggesting increased

complexation with Ca or other inorganic solid phases at the soil surface.

Peat accretion, based on 137Cs dating, is slow, accounting for up to 0.7 mm yr1.

Estimates of BP-TP entering the soil are about 10% yr1. Stage recording shows periodic

drying of this area, thus allowing oxidation. This reduces net accretion.



Phosphorus is an essential nutrient that frequently limits the productivity of

freshwater ecosystems. The cycling of phosphorus (P) in wetlands involves numerous

interactions between the P compartments of the water, biota, and soil. Processes

controlling these interactions can be biological, as in uptake and release by periphyton

and vegetation or physicochemical, such as adsorption/precipitation. Periphyton refers to

the communities of attached microorganisms, both floral and faunal, that grow on

submerged surfaces (Wetzel, 1983). Periphyton, often a conspicuous feature of shallow

water bodies, may contribute up to 80% of the primary productivity to an aquatic system

and can therefore influence many of the biological and physicochemical aspects of these

environments (Hansson, 1992; Carlton and Wetzel, 1988).

Periphyton covers a large extent of the oligotrophic Everglades Marsh. It is

believed that the activity of calcareous periphyton has had a primary role in the biogenic

formation of the calcitic mud found in the southern Everglades (Gleason, 1972). The

calcitic nature of the surface soil and periphyton plays a significant role in the long-term P

storage (Reddy et al., 1993). The uptake of P by periphyton is dependent upon P

concentration in the water column, antecedent P content in the periphyton tissue, the

forms of P available, and the growth stage and thickness of the mat (Sand-Jensen, 1983;

Homer et al., 1983; Cotner and Wetzel, 1992). Several studies suggest that algal tissue P

content in the range of 1.1 2.8 mg g- (dry wt.) is necessary for normal growth (Miller,

1983; Riber et al., 1983; Swift and Nicholas, 1987). However, Grimshaw et al. (1993)

observed that the P content of periphytic material in oligotrophic areas of the Everglades

Water Conservation Areas (WCA) was <0.1 mg g-1. Generally, as dissolved reactive P

(DRP) in water increases, so does the cellular P content of the periphyton (Swift and

Nicholas, 1987; Adey et al., 1993). Many species of algae have been found to accumulate

P at concentrations much higher than needed for normal growth. This "luxury uptake"

has been shown in numerous studies with enriched waters (Miiller, 1983; Riber et al.,

1983; Swift and Nicholas, 1987; Cotner and Wetzel, 1992). In a recent study, Adey and

coworkers (1993) found that the P content in periphyton, cultured in agricultural drainage

water, increased to a saturation level of approximately 3.5 4.2 mg g-1 (dry wt.) at

solution P concentrations as low as 25 jig L-1. However, it should be noted that P

saturation is dictated by P loading (mass of P applied per unit biomass or area) not by

concentration alone.

In addition to the uptake of inorganic P (Pi), numerous species of algae are able to

utilize organic P (Po) (Cotner and Wetzel, 1992; Bentzen et al., 1992). A number of

factors, including C:P ratio of organic matter undergoing decomposition and rate of

phosphatase enzyme production, are important in regulating organic P mineralization

(Golterman, 1973; Syers et al., 1973; Newman and Reddy, 1993). Bentzen and

coworkers (1992) found a significant effect of dissolved Pi concentration on Po uptake by

limnetic plankton, but concluded that dissolved Po was also actively utilized when

demand was high.

The calcareous cyanobacteria that dominate the periphyton in wetlands such as the

interior of WCA-2A are known to cause the precipitation of CaCO3 (Gleason, 1972).

This CaCO3 is commonly seen as sheaths, stalks, or other encrustations. It is

hypothesized that P can be completed with this CaCO3 leading to abiotic removal of P

from the water column. Recently, Browder et al. (1994) posed the premise that P

adsorption to CaCO3 may be controlling watercolumn P levels. In this context,

periphyton removal of water column P column would then be a function of both abiotic

and biotic processes.

Previous research on the periphyton in the WCAs (Swift, 1981; Swift and

Nicholas, 1987) focused on how environmental variables such as light, hydroperiod, and

water quality factors affect the growth and species composition of the mats. However,

limited information is available on the influence of periphytic activity on the fate of P in

the water column.

The objectives of this study were to determine the Pi and Po uptake kinetics of

periphyton, compare Pi uptake kinetics between morphologically different periphyton

types, and to assess the relative magnitude of biotic and abiotic uptake mechanisms.

Phosphorus uptake was determined in a series of laboratory and field studies using a

solution depletion technique (Reuter et al., 1986). Partitioning of periphyton incorporated

P by biotic and abiotic mechanisms was evaluated using 32P and a simple extraction


Materials and Methods

Study Site

The original Everglades has been fragmented into a series of hydrologic units

consisting of the Everglades Agricultural Area (EAA), three Water Conservation Areas

(WCA) and Everglades National Park (ENP). The three WCAs encompass almost 3500
km2, representing a little more than one third of the original Everglades (Swift and

Nicholas, 1987). Water Conservation Area 2 is the smallest of the three WCAs (547

km2), lies between WCA-1 and WCA-3A, and is heavily impacted by large quantities of

nutrient enriched water derived from the Everglades Agricultural Areas (EAA) (Swift and

Nicholas, 1987). The surface water quality (SWIM, 1992), nutrient gradient (Koch and

Reddy, 1992), and soil nutrient characteristics (DeBusk et al., 1994) of this area have

been recently studied. The interior unimpacted sites of WCA-2A are dominated by

calcareous blue-green algae (cyanobacteria), such as Schizothrix calcicola (Ag.) Gom.

and Scytonema hofmannii Ag., as well as hardwater diatoms (Swift and Nicholas, 1987).

The average periphyton N to P ratio in the impacted sites was 9:1 while at the interior

sites this ratio was 107:1, suggesting that the interior of WCA-2A is P limited (Swift and

Nicholas, 1987).

Characterization of Periphvton

Periphyton used in laboratory studies was collected from the field site station 217

(Fig. 3-1; 260 17.17' N, 800 24.73' W 30 m) on October 18, 1994 for initial studies, then

again on June 8, 1995 for 32p laboratory studies. Benthic periphyton (BP) was collected

by gently running a swimming pool screen of 1 mm mesh size along the soil surface.

Epiphytic periphyton (EP) was obtained by clipping senescent stems of sawgrass

(Cladiwnjamaicense Crantz.) just above the soil surface. Periphyton, forming a nearly

continuous coating on soils and sawgrass and extending into the water as floating

material, was collected, gently homogenized, and termed mixed periphyton (MP). Upon

return to the laboratory EP was removed from the stems by scrapping with a scalpel.

Periphyton was placed in plastic trays filled with bulk surface water, also field collected,

and stored with occasional stirring in a light booth that provided 10 W m-2 on a 16 h

light/8 h dark cycle. Subsamples were removed as needed for experiments.

Portions of the bulk periphyton material were oven dried at 700 C to constant
weight. A known mass of dry periphyton was combusted in a muffle furnace at 4500 C

for 12 h to determine ash content. Subsamples were digested with perchloric acid (4500-

P-B, APHA, 1992) and the digestate was analyzed colorimetrically for total P (TP)

(Method 4500-P-F, APHA, 1992). Total C (TC) and N (TN) were determined on oven-

Fig. 3-1. Map of Water Conservation Area 2A (WCA) showing field site 217.

dried samples using a Carlo-Erba NA-1500 CNS Analyzer (Haak-Buchler Instruments,

Saddlebrook, New Jersey) (Nelson and Sommers, 1982). Total inorganic C (TIC) was

determined on ashed material, as above, using the CNS analyzer. Total organic C (TOC)

was determined by difference (Browder et al., 1982).

In addition, BP collected in 4 replicate bulk samples on June 8, 1995 was also

characterized for bulk density and CaCO3-associated P (CaCO3-P). Bulk density was

determined by collecting a known volume of BP in an acrylic cylinder. Measurements

were made to insure negligible compaction and to provide depths of the BP layers. The

CaCO3-P of fresh BP samples was extracted in a 1:100 solid to solution ratio with 0.01 M

HCI for 2 h (see following section). Extractant solution was filtered (0.2 Jim, Gelman

Supor, Gelman Inc., or Cameo acetate membrane, MSI Inc.) and analyzed

colorimetrically for DRP on an AutoAnalyzer II (Technicon Corp.) (Method 4500-P-F,

APHA, 1992).

Water samples were collected and filtered (0.2 mni) in the field on June 7 and 8,

1995. Samples were analyzed for DRP and digested by persulfate oxidation (Method

4500-P-B, APHA, 1992) and analyzed for total dissolved P (TDP) (Method 4500-P-F,

APHA, 1992).

Development of Extraction Procedures

It was hypothesized that the P abiotically associated with periphyton would be

bound to CaCO3 (CaCO3-P). Preliminary analysis of dried, ground BP resulted in a

CaCO3 content of about 50%. Removing the CaCO3 by dissolution would cause the

release of this P. An optimal extractant would remove CaCO3-P but would not remove

appreciable amounts of biotically bound organic P. Assuming the extractant removes all

CaCO3-P and that extracted solution TDP = DRP then either no biotic P was extracted or

any biotic Po extracted was completely hydrolyzed. A series of preliminary experiments

were conducted using varying molarities of HCI, solid:solution ratios, and extractant

times. Fresh BP was gently homogenized and mixed with the acid extractant. Samples

were collected at selected times, 0.2 ntm filtered, and analyzed colorimetrically for DRP.

A portion of this subsample was digested by persulfate oxidation and analyzed

colorimetrically for TDP. The results of these tests showed that 0.01 M HCI in an

approximately 1:100 fresh weight BP to solution ratio provided highest extracted P

concentrations yet maintained the relationship TDP = DRP. At weaker molarities very

little P was released, and at higher molarities TDP > DRP.

In the first of the experiments presented which demonstrate the applicability of

this method, duplicate fresh BP samples were extracted with 0.01 M HCI (1:100).

Solution samples were collected at 2, 5, 10, 30, 60, and 120 minutes, 0.2 pm filtered and

analyzed colorimetrically for DRP. An aliquot of this sample was analyzed for TDP.

Differences between the DRP and TDP concentrations in the extract were analyzed for

significance by t-tests. In the course of continuing experimentation, additional BP

samples were collected and subjected to a 2 h extraction with 0.01 M HCL These data

were also used to substantiate the method.

The pH of the BP containing extracting solution was generally 2.5 3.0. To

determine if this level of acidity could hydrolyze soluble organic P, selected inorganic

and organic P compounds were added to a known amount of CaCO3 and subsequently

extracted with 0.01 M HC1. Reagent grade KH2PO4 was the source of Pi. Both glucose

6-phosphate (G6P) and ATP were used as Po. Additions of these compounds were

combined with a mass of CaCO3 typically found in periphyton, about 0.5g g-1 dry weight,

and allowed to equilibrate in a dessicator for about 16 h before extraction. All P

containing species were added in amounts to increase the P content per volume of

extractant solution by 0.9 pM. The 0.01 M HCI was added to these samples and gently

stirred periodically for the next 2 h. The pH was monitored during the extraction.

Samples were collected at 10, 30, 60, and 120 minutes. Samples were 0.2 Pim filtered and

analyzed colorimetrically for DRP or were digested and analyzed for TDP.

Benthic periphyton was examined under a scanning electron microscope (SEM) to

confirm the removal of CaCO3 after acid extraction. One subsample of BP was extracted

with 0.01 M HCI as above; the other was not acid treated. Samples were fixed in Trumps

solution, washed in phosphate buffer saline, dehydrated by an ethanol series and dried via

hexamethyldisilazane (HMDS) (Nation, 1983). After evaporation to dryness in a

dessicator, the samples were attached to carbon coated posts with graphite glue. The

posts were then sputter coated with gold and observed on a field emission SEM (S-4000,

Hitachi, Tokyo, Japan).

Phosphorus Uptake by Periphvton: Laboratory Conditions.

On October 20, 1994, laboratory experiments began with the incubation of
approximately 2 g periphyton with 200 ml bulk water (obtained from the field site) to

achieve a nominal ratio of 1:100 freshweight to solution. Each of the three types of

periphyton: EP, MP, and BP, were gently homogenized before incubation. Inorganic P as

KH2PO4 was added in 2 ml aliquots of 10 mg L-1 stock solution to bring the initial

concentration to 100 g L-' above ambient (Co = 4.7 -6.0 pM, normalized for periphyton

dry weight). Triplicate vessels of each periphytic type and a control without added

periphyton were incubated under the light booth with occasional stirring. Temperature

was approximately 270 C, and light intensity was 10 W m-2. Solution samples (10 ml)

were collected at 0, 5, 15, 30, and 45 min after spiking, filtered through 0.2 pm, and

analyzed colorimetrically for DRP by standard methods (see above) on an Alpkem Rapid

Flow Analyzer (Perstorp Analytical, Wilsonville, OR). Each sample withdrawal reduced

the solution volume originally at 200 ml by 10 ml; therefore, calculations of uptake were

corrected for decreasing sample volume. Concentration of DRP in solution was

normalized for solution volume and periphyton oven dry weight.

Between October 24 and 26, 1994, a second experiment was conducted with three

types of periphyton where each type was incubated for a short time period (maximum

incubation period of 90 min) at 15 initial DRP concentrations. Initial DRP, as KH2PO4,

concentrations ranged from ambient (0.19 pM) to 62.2 plM. Experimental protocol and

sample handling was done as above, however, each reaction vessel was sampled only

once after a predetermined time. Sampling periods were based on the linear portions of

DRP depletion from previous experiments.

On November 23 and 24, 1994, a third experiment was conducted to determine P
uptake by two types of periphyton (EP and BP) at several initial DRP concentrations in

the range of ambient (0.23 pM) to about 14.5 pM. These experiments were conducted to

determine the uptake of Pi by periphyton at numerous initial concentrations in a more

narrow concentration range than was provided in previous experiments. Solution samples

were collected with time at 10, 60, 120, 240, and 480 min after DRP addition.

Experimental methods and analysis were similar to those previously described.

The concentration of all solution samples were corrected for matrix effect,

probably Si, which caused slightly elevated levels of DRP when measured

colorimetrically on an Alpkem Rapid Flow Analyzer with a 3 cm flowcell. The

correction factor was obtained by making additions of standards to previously analyzed

samples, analyzing the spiked samples, and back calculating the concentration in the

original sample. The calculated concentration was subtracted from the initial analyzed

concentration and the difference was obtained. This difference was found to be 0.27

0.01 pM (n = 8). Spiked recoveries of samples analyzed by a Technicon AutoAnalyzer H

showed no matrix effect; thus, in subsequent experiments this instrument was used for

DRP analysis.

Phosphorus Uptake by Periphyton: Field Conditions

On June 8, 1995, a field experiment was conducted to determine the uptake of Pi

(as KH2PO4) by BP. Bulk surface water was collected in large polycarbonate carboys in

the vicinity of WCA-2A-217 on each day of field experimentation. Water depth was 42.3

0.3 cm (mean SE, n=4) and BP depth was 12.8 0.5 cm (n=4). Bulk density of the

BP layer was 0.015 0.001 g cm-3. A known amount of surface water (900 ml) was

added to 946 ml polypropylene mason jars that were fit into floating racks. Styrofoam

floats were glued to acrylic sheets to provide flotation and stability. The reaction vessels

were open to the atmosphere with the liquid portion almost completely submerged in the

water column to help maintain ambient conditions. Approximately 9 g of homogenized

BP was added to reaction vessels (jars) to provide a nominal 1:100 fresh-weight BP to

water ratio. The water was spiked to achieve five initial concentrations. There were four

replications of each concentration. Initial concentrations were ambient (0.19 pM), 1.5,

2.7, 5.6, and 10.6 pM DRP. Samples were collected with time at 5, 15, 30, 45, and 60

minutes after P addition. The water samples (20 ml) were filtered (0.2 pm), stored in ice

water, and transported to the laboratory. At the end of the last sampling, the BP in each

of the reaction vessels was filtered by gravity onto Whatman #41 filter paper (Whatman,

Inc.) and dried at 70 C to constant weight (about 72 h). Since solution volume was

reduced by 20 ml with each sample, solution mass P was adjusted accordingly.

Phosphorus removed from solution was normalized per gram dry weight of BP.

To determine of hydrolysis and uptake of Po by BP, an experiment was conducted

on June 7, 1995. Experimental methods were similar to those described for the Pi uptake

study. Each vessel was spiked with a known volume, (up to 4 ml) of solution containing

1.5 mM P as adenosine triphosphate (ATP), to obtain TDP concentrations of ambient

(0.9 AM), 3.3, 5.5, and 10.1 pM. Each treatment was replicated four times. Water

samples were collected from each vessel before P addition and at 15, 30, 60, 120, and 240

minutes after spiking. Samples were then filtered and analyzed for DRP and for TDP

(APHA, 1992). Benthic periphyton was collected after the uptake study and dried as

above. Phosphorus in solution was determined colorimetrically on an AutoAnalyzer II

(Technicon Corp.) by standard methods (APHA, 1992).

An unfiltered water control (without BP), spiked to ambient + 3.23 pM P, was

included in both experiments. Hydrolysis rates in a periphyton-less water column could

be determined by this control during the Po experiments, and a decrease in the DRP of the

control would aid in quantifying the magnitude of chemical precipitation for both


Calculation of Uptake Rates

The solution DRP concentrations at each sampling time (t) were normalized for

solution volume and dry weight periphyton. Dissolved reactive P remaining in solution

was plotted against sampling time to obtain depletion curves. Phosphorus uptake was

described by two methods:(i) initial uptake rate in the linear portion of depletion curves,

and (ii) first-order kinetics for the determination of uptake rate constants.

Initial uptake rate calculations made over a range of initial P concentrations, were

used to obtain the parameters needed for the Michaelis-Menten equation:

v=(Va C)/(Km +C) [3-1]

where v = P uptake rate by periphyton (pmol g-1 min-'), Vma is the maximum P uptake

rate by periphyton (jimol g-I min-1), Km is the concentration of P at one-half Vma (pM),

and C is the P concentration in solution (pM). All uptake rates were calculated on

periphyton dry weight basis.

Maximum uptake rates occur when the P concentration available to periphyton is
non-limiting. At these concentrations C >> Km, and equation 3-1 reduces to

v = Va [3-2]

where initial uptake rate equals the maximum uptake rate. Initial uptake rates were

calculated from the linear portion of the P depletion curve, which occurred anywhere

between 5 and 90 minutes (time in minutes between t = 0 and t = tx) after spiking

depending on initial P concentration and experimental conditions. Over this range the

velocity is constant and independent of C. The initial uptake rates were used to develop

Lineweaver-Burk plots. The Lineweaver-Burk plot is a double reciprocal plot which

transforms the typical Michaelis-Menten equation into a linear form

1/v = (1/Vmax) + (Km/Vmax)(l/C) [3-3]

In the Lneweaver-Burk plot, the reciprocal of the solution concentration (1/C) at

any sampling time is the x-axis. The y-axis is obtained by the reciprocal of the uptake

rate at that time (1/ v). These data were then plotted and a linear regression was fit. The

Michaelis-Menten parameter of V, is taken as the reciprocal of the y-intercept, Km is

obtained by multiplication of the slope by Vm.

When C
equation [3-1] can be modified to

v = kC [3-4]

where k = Va/Km. These equations were used to describe v at any given concentration

above ambient along the depletion curve. The first-order rate constant (k) describes a

linear relationship for v vs. C at C<
Overall P depletion during an experimental period was best described by first-

order kinetics. The following integrated form of equation [3-4] was used to obtain P

uptake coefficients

C = Co e-kt [3-5]

where C is P concentration at any time, Co is the initial solution P concentration, and k is

the first order rate constant.

Tracer Studies of Phosphorus Partitioning in Periphvton

Benthic periphyton and surface water collected in bulk from the field site on June

8, 1995, were used in the following laboratory studies to determine the partitioning of

added 32P into biotic and abiotic components of BP. These experiments were conducted

on June 13 and 14, 1995.

Benthic periphyton of known fresh weight (0.5 g) was added to 50 ml (four

replications per treatment) of 0.2 ipm filtered water to maintain about a 1:100 fresh-

weight BP to water ratio. Depending on the experiment, either 32P as H3PO4 in 0.02 M

HCI, or as 5'-[y-32P]ATP (DuPont NEN) were added. The inorganic 32P was carrier-free.

The stock [32P]ATP had an activity of 111 TBq mM-1. Additions of 0.2-0.7 kBq were

made to each vessel. Unlabeled ATP or KH2PO4 was also added to manipulate the 31P

concentration of several treatments.-The 31P was either maintained at ambient ( Pi = 0.13

pM, P0 = 0.32 pM) concentrations or was spiked to 3.2 pM above ambient (Pi = 3.4 pM,

Po = 3.6 pM). One set of vessels were incubated in the light booth (10 W m-2), while

another set was incubated in the dark. The duration of the incubations was 1 and 12 h for

the vessels which received inorganic P and 4 and 12 h for the organic P incubations.

Replicated distilled water blanks were also spiked with 32P and were sampled

periodically to provide control activities. After timed incubation, vessel contents were

0.2 pm filtered and 100 L of the filtrate was added to 10 ml of scintillation cocktail

(Scinti-Verse L Fisher Scientific). Radioactivity was determined with a Beckman liquid

scintillation counter (LSC) (LS3801, Beckman Instruments Inc.). The BP and the

membrane filter were placed into 50 ml of 0.01 M HCI. The BP was washed from the

filter by gentle spraying of the dilute acid. The BP in dilute acid was stirred occasionally

for the next 2 h. After this dilute acid extraction, the solution was again filtered (0.2 jnm)

and a 100 pl subsample was added to 10 ml scintillation cocktail and analyzed by LSC.

The initial activity was partitioned into water activity, abiotic periphyton activity, and

biotic periphyton activity. Activity remaining in solution after the uptake period was

considered water activity. Initial activity minus water activity was the total activity in BP.

Total BP activity was further separated into the activity extracted with 0.01 M HCI, the

abiotic activity, and the biotic activity. Biotic activity was calculated by difference.


Characterization of Periphvton

Total P contents of the periphyton types were in the order BP > MP > EP (Table

3-1). The chemical contents of each periphyton type remained essentially the same from

time of collection until experimentation. This suggests that culturing under lights for 5

weeks did not appreciably change antecedent chemical conditions of the periphyton. The

TP of the BP was similar for all dates except the June 13 14, 1995, laboratory studies

where the TP was generally about 20% lower than that used at other times. Chemical

composition of MP is approximately equidistant between that of EP and BP, suggesting

the continuity of periphyton types. Ash contents are of the order EP > MP > BP

indicating that EP is more encrusted with CaCO3 than the other forms. Total C and TOC

increased with decreasing ash content. Total N:TP ratio was between 69 106, with

higher values for EP than for MP or BP. Total N increased proportionally to an increase

in TOC content and was between 8 11% of TOC for all samples. Total P increased with

increasing TC and TOC and inversely to TIC and ash content.

Extraction Procedure

Total dissolved P and DRP concentrations extracted from BP with 0.01 M HCI
were not significantly different when statistically analyzed by a t-test (p = 0.05). The 0.01

M HC1 extractant completely solubilized all added KH2PO4 as evidenced by >100%

Table 3-1. Selected chemical characteristics of periphyton used in the uptake studies.

Experiment Date, Total Total Total Total Total Ash
Periphyton Type P C inorganic organic N Content
mg kg-1 g kg-
October 20, 1994
EP 104 218 105 113 11 619
MP 189 247 104 143 13 541
BP 289 298 96 202 20 434

October 24-26, 1994
EP 104 223 105 118 11 603
MP 214 270 101 169 16 506
BP 284 318 95 222 21 429

November 23-24, 1994
EP 109 221 103 118 11 611
BP 273 295 95 200 20 445

June 7-8, 1995
BP 272 282 96 187 23 472

June 13-14, 1995
BP (P) 233 276 96 180 23 471
BP (P,) 222 268 96 173 21 473

recovery in DRP analysis of the CaCO3 + KH2PO4 treatment and in accounting for 50 -

60% DRP recoveries when added to CaC03 and the organic P forms (Table 3-2). If all Pi

were extracted without any hydrolysis of the organic forms, then expected recoveries

during DRP analysis would be 50%. The low recoveries (<12%) of DRP in the CaCO3 +

Po treatments suggests that organic P was not effectively hydrolyzed by the extractant.

The organic P forms were recovered after digestion and analyzed as TDP. The -

CaCO3 control (no added P) shows that the CaCO3 in the mixtures added small amounts

of P to the extracted solutions. Total dissolved P in extracted solutions showed

recoveries between 85 115%.

Visual inspection of BP tissue under SEM shows that filaments of the untreated

periphyton are encrusted with CaC03 spicules (Fig. 3-2). These spicules were absent in

periphyton tissue treated with 0.01 M HC1. It is hypothesized that this CaCO3 abiotically

binds the P removed during the dilute acid extraction. The filaments remained intact after

acid extraction, suggesting that the method does not cause extreme cell damage and

subsequent release of cellular constituents.

Phosphorus Uptake by Periphvton: Laboratory Conditions.

The first experiment compared P uptake by three types of periphyton in solution

with Co = 4.7 6.0 pM P. Dissolved reactive P depletion from solution shows that P

uptake by EP was more rapid than MP and BP (Fig. 3-3). Benthic periphyton had the

slowest uptake. During the first 45 minutes of the incubation period, DRP in the solution

containing EP returned to ambient levels (0.28 0.02 pM P, mean SE, n = 3). During

this time period, the DRP levels in solution containing MP were 1.5 0.2 pM, and 2.5

0.1 pM for BP containing solutions.

The initial uptake rates were calculated from the linear portion of the depletion

curves. When using the solution depletion technique for a specific nutrient, the nutrient

Table 3-2. Phosphorus recovered after a 2 h extraction of reagent grade chemicals with 0.01 M HCI in a nominal 1:100
solid:solution ratio. Extracted solutions were analyzed colorimetrically for dissolved reactive P (DRP) and digested by persulfate
oxidation and analyzed for total dissolved P (TDP).

Expected TDP Extracted P Extracted P Percent of Percent of
if all added P recovered recovered Total added Total added
released and during DRP during TDP P recovered P recovered
hydrolyzed analysis analysis during DRP during TDP
Pure Compounds Extracted during extraction analysis analysis
-------------- pM -------------- -------- % --------

CaCO3 0.0 0.05 0.006 0.18 0.010
CaCO3 + KH2PO4 0.92 1.05 0.013 1.06 0.074 114 115
CaCO3 + Glucose 6-phosphate 1.85 0.07 0.003 0.84 0.090 8 91
CaCO3 +KH2PO4 + Glucose 6-phosphate 1.85 1.02 0.010 1.56 0.061 55 85
CaCO3 +Adenosine triphosphate 0.92 0.11 0.013 0.87 0.055 11 94
CaCO3 +KH2PO4 + Adenosine triphosphate 0.92 1.08 0.003 1.65 0.094 59 89

-1 ^1'

Fig. 3-2. Scanning electron micrograph of typical filament from benthic periphyton (BP)
showing CaC03 encrustation (upper) and a typical filament in BP after 0.01 M HC1
extraction (lower). Note lack of encrustation after 2 h treatment with dilute acid.

0 5 10 15 20 25 30 35 40 45 50
Time, minutes

Fig. 3-3. Depletion of solution dissolved reactive P (DRP) concentration, normalized for
solution volume and periphyton dry weight, during a laboratory experiment conducted
October 20, 1994. Curves describing first-order kinetics were fit to the depletion
data.Fig. 3-3.

should not become limiting (Reuter et al., 1986). With few exceptions, the solution P

content in reaction vessels was not reduced by > 50% during any of the initial uptake rate

experiments. Depletion during the linear phase was usually around 30% of the initial


Initial uptake rates for three periphyton types were; 0.127 0.010 (imol DRP g-i

min-1 for EP, compared to 0.106 0.042 imuol DRP g-' min-1 for MP, and 0.072 0.007

plmol DRP g-1 min-' for BP. Dissolved reactive P depletion from solution was best

described by first-order kinetics with rate constants in the order EP > MP > BP (Fig 3-3).

Phosphorus uptake by EP did not fit first-order kinetics as well as did MP or BP. Uptake

by EP can accurately be described by a 2-phase kinetic model with the initial uptake rate,

given above, describing uptake in the first 5 minutes, and a slower uptake of

approximately 0.077 pmol DRP g-1 min-1 for the next 25 minutes.

In the next experiment, uptake rates by three periphyton types were determined for

one time step at 15 initial concentrations (Co = 0.2 62.2 pM P). Results showed a wide

range in P uptake rates: EP = 0.039 0.623 pmol P g-1 min-', MP = 0.028 0.397 imol P

g-1 min- and BP = 0.017 0.195 -pmol P g-1 min-1 At ambient levels (about 0.22 pM

P) the change in solution concentration was minimal and was within the analytical

variability for DRP. Thus, uptake was not determined at ambient concentration. For all

periphyton types initial uptake rate was dependent on the initial P in solution. In all cases

initial uptake was in the order EP > MP > BP, with EP generally being about 2-3 times

higher than the other two types.

Initial uptake rates were used to generate Lineweaver-Burk plots to obtain an

estimate of Michaelis-Menten uptake parameters. Maximum predicted uptake rate (Vm)

and the Michaelis constants (Ki) were in the order EP > MP > BP (Table 3-3). Using

resultant Vma and Km values, initial uptake rate data were fit to predicted Michaelis-

Menten plots (Fig. 3-4). There was generally good agreement between measured and

predicted values. Phosphorus uptake by EP followed Michaelis-Menten

Table 3-3. Michaelis-Menton uptake parameters for P uptake by periphyton.

Experiment Date and pM DRPt Vma Km r2
Periphyton Type range
Laboratory Conditions pmol g- min-i pM
October 24 -26, 1994.
EP 0.3- 62.2 0.852 9.9 0.96
MP 0.2-40.7 0.248 4.1 0.96
BP 0.2- 38.2 0.101 2.5 0.87

November 23-24, 1994.
EP 0.4-14.5 0.198 1.4 0.86
BP 0.6-7.7 0.038 1.1 0.94

In situ Conditions
June 8, 1995.
BP 1.5 -10.6 0.640 5.3 0.80

June 7. 1995. TDPt
BP 5.5- 10.1 0.661 21.4 0.99

t = pM total dissolved P (TDP) concentration for the June 7, 1995, Po uptake study.



0.6: Epiphytic

0.4 m


C 0.5
E 0.4- Mixed



0 5 10 15 20 25 30 35 40 45
Initial Concentration (Co), uM

Fig. 3-4. Plots of uptake rate (v) versus initial dissolved reactive P (DRP) concentration.
Based on initial uptake rates for laboratory experiments conducted October 24-26, 1994.
Lines represent theoretical Michaelis-Menton values.

kinetics to a greater extent than either MP or BP. The V, of the predicted Michaelis-

Menten kinetics tends to underestimate that attained for MP and BP at solution P

concentrations greater than about 15 pAM.

A third experiment was conducted using the periphyton collected on October 18,

1994, and cultured for a period of five weeks at ambient DRP levels in a laboratory light

booth (12 h light/dark cycle, light intensity = 10 W m-2). At initial P concentrations (Co

= 0.4 -14.5 pM), DRP levels in EP containing solution reached ambient levels (0.4 PM P)

within 120 minutes (Fig. 3-5). Depletion of DRP followed an exponential decrease,

being most rapid initially and slowing with time. Phosphorus depletion in BP containing

solutions was slower than EP. Like EP, P depletion in BP containing solutions was rapid

during early samplings (< 60 min), followed by exponential decrease with time. It took

up to 240 minutes to reach ambient P levels at Co <6.7 pM. The treatment with the

highest initial DRP concentration (7.7 pM) approached, but did not return to, ambient

concentrations within 480 minutes. As with the previous experiments, these data also

suggest that DRP uptake by EP to be greater than that of BP.

Time increments to determine initial uptake rates of the EP and BP curves ranged

from 10 60 min, and from 10 120 min, respectively. There was generally a trend that

uptake rate increases with initial concentration. Initial uptake rates for EP ranged from

0.051 0.176 pmol P g-1 min-1 over the initial concentration range of 1.1 14.5 iM. The

ranges of BP uptake rates were between 0.012 0.036 pmol P g-' min-1 Again, the

uptake rate increased slightly with increasing initial DRP concentration. The BP initial

uptake rates increased from the Co = 0.6 2.5 WM, and then tended to maintain around

0.032 nimol P g-1 min-l in the initial concentration range of 3.2- 7.7 pM. Michaelis-

Menten parameters were obtained from the initial rate data. The resultant Vm and Km

are presented in Table 3-3.

Time, minutes

Fig. 3-5. Depletion of solution dissolved reactive P (DRP) concentration, normalized for
solution volume and periphyton dry weight, during a laboratory experiment conducted
November 23, 1994. Samples were collected at time increments from 0 to 480 minutes.
Graph was truncated since all vessels returned to ambient concentrations (about 0.4 pM)
within 120 minutes.

The rapid depletion of solution DRP did not allow exponential curve fitting for

the EP treatments. Depletion of solution DRP was described only by the initial uptake

rates (Fig. 3-5). Curves resulting from the BP uptake experiment (Fig. 3-6) produced

first-order rate constants ranging from 0.006 0.02 min-1 (Table 3-4). The rate constants

were generally between 0.01 and 0.02 for initial concentrations Co = 0.60 4.0 pM. The

rate constants were lower at Co > 4.0 pM, with a slight tendency for the rate constants to

decrease with increasing initial concentration.

Phosphorus Uptake by Periphvton: Field Conditions

Inorganic P uptake

Rapid depletion of P was observed at Co > 1.5 pM. At ambient P levels (0.19 pM) no

detectable change in concentration was observed. Ambient concentrations of about 0.19

pM were reached within 30 minutes when the initial solution P was 1.5 0.1 and 2.7

0.1 pM (Fig. 3-7) (mean SE, n = 4). After 60 minutes, solution P concentrations for

treatments with Co = 5.6 0.1 and 10.6 0.2 pM were reduced to 0.7 0.1 and 1.7 0.1

pM respectively.

Initial uptake rates were determined by depletion of solution DRP in 5 min. Initial

uptake rates ranged from 0.077 0.007 to 0.429 0.036 pmol P g-' min-'. There was a

distinct trend of increasing uptake rate with increasing concentration. Michaelis-Menten

parameters show that the predicted Km value is several times higher than the ambient P

level (Table 3-3).

First-order rate constants were obtained from the depletion curves for treatments

with Co > 1.5 pM (Fig. 3-7 and Table 3-4). The first-order rate constants were in the

range of 0.03 -0.07 min-1, and showed a decreasing trend with an increase in DRP


0 50 100 150 200 250 300 350 400 450 500
Time, minutes

Fig. 3-6. Depletion of solution dissolved reactive P (DRP) concentration, normalized for
solution volume and periphyton dry weight, during a laboratory experiment conducted
November 24, 1994. Curves describing first-order kinetics were fit to the depletion data.

Table 3-4. Predicted initial P concentration, rate constant, and r2 for first-order
equations of the form C= Co e -kt fit to depletion curves.

Experiment Date,
Periphyton Type, and Predicted First-order
Initial Concentration Initial Rate
iM DRPt (mean SE) Concentration (Co) Constant (k). r

Laboratory Conditions pM min-1
October 20, 1994.

EP 6.0 0.2 4.9 0.0841 0.98
MP 4.8 0.1 4.6 0.0261 0.99
BP 4.7 0.2 4.6 0.0135 0.99

November 24, 1994. BP
0.6 0.6 0.0172 1.00
1.0 1.0 0.0203 1.00
1.7 1.7 0.0153 0.97
2.5 2.3 0.0096 0.96
3.2 3.0 0.0124 1.00
4.0 3.8 0.0120 1.00
4.9 4.8 0.0088 1.00
5.8 5.0 0.0057 0.98
6.7 6.3 0.0064 1.00
7.7 7.8 0.0056 1.00

In situ Conditions
June 8, 1995. BP
1.5 0.1 1.4 0.0529 0.94
2.7 0.1 2.8 0.0708 1.00
5.6 0.1 5.7 0.0323 0.97
10.6 0.2 9.9 0.0302 0.99

June 7. 1995. BP, TDP
3.3 0.2 ND
5.5 0.1 5.5 0.0318 1.00
10.1 0.2 10.9 0.0199 0.98

t = pM total dissolved P (TDP) concentration for the June 7, 1995 P, uptake study.

0 10 20 30 40 50 60 70
Time, minutes

Fig. 3-7. Depletion of solution dissolved reactive P (DRP) concentration, normalized for
solution volume and dry weight benthic periphyton (BP), for field experiment conducted
June 8, 1995. Curves describing first-order kinetics were fit to the depletion data.

Organic P uptake

The uptake of Po by BP was studied in situ on June 7, 1995. Solutions were

spiked with ATP at four initial concentrations (0.9 10.1 AM). Changes in P

concentrations of the water show that virtually all of the added ATP was hydrolyzed to

DRP within 15 min (Fig. 3-8). This occurred in all BP treatments and in a water-only

control (Co = 3.8 pM). Once hydrolyzed the P concentration in the BP-containing

treatments decreased rapidly, but the P in the control treatment remained constant.

Generally, P concentrations returned to ambient levels within 120 minutes for all BP-

containing treatments (Fig. 3-8).Initial uptake rates were determined at the 15 min time

step for all concentrations > ambient (0.9 pM). One replicate in the Co = 10.1 pM

treatment was omitted as an outlier, therefore n = 3, all other treatments n = 4. The initial

uptake rates varied between 0.099 0.024 pmol TDP g-1 min-I for Co = 3.3 pM to

0.203 0.016 pmol TDP g-1 min-1 for Co = 10.1 pM. Initial uptake rate increased with

increasing concentration. Michaelis-Menten uptake parameters were derived from these

results (Table 3-3). Exponential equations, fit to points with P concentration > than

ambient for the treatments with Co = 5.5 and 10.1 pM, produced first-order rate

constants of 0.03 and 0.02 min-' respectively (Table 3-4).

Phosphorus Partitioning in Periphvton

Benthic periphyton, brought from the field site on June 8, 1995, was subjected to

laboratory 32p uptake studies. A higher proportion of available P was taken into the

abiotic fraction at ambient P concentrations, Pi = 0.13 IM and Po = 0.32 pM, than at

spiked concentrations, Pi = 3.4 pM, Po = 3.7 pM. This result occurred irrespective of

irradiance, P-form, or incubation time (Fig. 3-9 and 3-10). At ambient concentrations,

>20% of 32pi activity was in the abiotic compartment after 1 h, compared to <6.5% at

spiked concentrations. In 12 h the ambient abiotic P pool had >8% 32Pi activity; whereas,

at Co = 3.4 pM <3% 32P, activity was in this fraction (Fig. 3-9). Similarly, at






- -2
o 12-

t 10-


Initial TDP =0.9 uM

0 50 100 150 200 250 0 50 100 150 200 250

Time, minutes

--- TDP -- DRP -r Po

-a- TDPw -- DRPw -- Pow

Fig. 3-8. Concentrations of total dissolved P (TDP), dissolved reactive P (DRP), and
organic P (Po) (by difference), normalized for volume and dry weight benthic periphyton
(BP) during field'Po uptake experiment. Unfiltered WCA-2A-217 water was spiked to
0.9 (ambient), 3.3, 5.5, and 10.1 pM TP with ATP. Unfiltered water controls (UFW)
devoid of BP were also run at Co = 3.8 pM (TDPw, DRPw, Pow). This study was
conducted on June 7, 1995. Points indicate mean solution concentration (SE, n=4).

Initial TDP = 3.3 uM


80- Pi Light




S- --- -

S I Water Activity e] Abiotic Activity O Biotic Activity

g 100
Pi Dark




0.1 uM 3.4 uM 0.1 uM 3.4 uM
--- 1 hour- 12 hour-

Fig. 3-9. Partitioning of added 32P between solution and periphyton. Benthic periphyton
(BP) was incubated in 0.2 pm filtered WCA-2A-217 water to which 32P was added as
carrier-free H3PO4 for either 1 or 12 h. Spiked treatments involved additions of 3.23 pM
31P as KH2PO4 to bring Co = 3.4 pM. Concurrent sample sets were incubated under light
and dark conditions. After the uptake experiments the BP was collected by filtration and
extracted with 0.01 M HCI for 2 h. The activity released during extraction is considered
that abiotically completed. The activity not extracted (by difference) represents biotic
incorporation. Bars represent mean SE, n = 4.


h. o

( 100







Water Activity O Abiotic Activity O Biotic activity

03 uM

3.5 uM

0.3 uM

----4 hour--

3.5 uM

---12 hour-

Fig. 3-10. Partitioning of added 32P between solution and periphyton. Benthic
periphyton (BP) was incubated in 0.2 pm filtered WCA-2A-217 water to which 32p was
added as adenosine 5'-(y-32P) triphosphate (3000 Ci mM-1) for either 4 or 12 h. Spiked
treatments also had additions of 3.23 pM 31p as ATP to bring Co = 3.4 pM. Concurrent
sample sets were incubated under light and dark conditions. After the uptake experiments
the BP was collected by filtration and extracted with 0.01 M HCI for 2 h. The activity
released during extraction is considered that abiotically completed. The activity not
extracted (by difference) represents biotic incorporation. Bars represent mean : SE, n =

Po Dark



ambient Po concentrations, approximately 16% of the 32po activity was in the abiotic pool

after 4 h compared to 5% at spiked concentrations. By 12 h, 9-12% of the added activity

was in the abiotic pool at Co = 0.3 pM TDP where about 3% was in this portion for Co =

3.5 pM TDP (Fig. 3-10). There was a shift in activity from the water and abiotic fractions

to the biotic fraction with time. At 1 h incubation around 70% of total added 32pi activity

for ambient Pi treatments and 36-55% of the activity at Co = 3.4 pM DRP was in the

biotic compartment. By 12 h, over 88% of total added activity was in the biotic portion

for all Pi treatments. The 32po fraction contained in the biotic compartment was 70 82

% of the total activity at 4 h incubation and increased to 86 97 % by 12 h (Fig. 3-10).

Irradiance had little effect on the partitioning of 32P activity in either Pi or Po treatments.


Characterization of Periphvton

Total P content of periphyton used in uptake experiments ranged 104 mg kg-I to

289 mg kg-' (dry wt). Other researchers have reported total P content in periphyton in

the range of 100 400 mg kg-' (Swift and Nicholas, 1987; Vymazal et al., 1994). The

slough site sampled by Vymazal et al. (1994) was located closer to a gated culvert than

was the WCA-2A-217 site from which the periphyton in the present experiments was

obtained. Canals have been shown to effect the nutrient concentration of the periphyton,

water, and soil such that concentrations generally decreased with increasing distance from

the inflow (SWIM, 1992; Swift and Nicholas, 1987; Koch and Reddy, 1992; DeBusk et

al., 1994). In fact, Vymazal et al. (1994) found TP contents in EP from a mixed

sawgrass/cattail community near a culvert of WCA-2B averaged 671 21 mg kg-'.

Vymazal et al.(1994) suggested the degree of calcification inversely affected the P
content of periphyton. In this study EP had the highest ash content, indicating the greatest

degree of calcification (Table 3-1). Ash content can generally be used as an estimation of

CaCO3 content (Browder et al., 1994). Epiphytic periphyton total P contents were about

40% that of BP but, when normalized for differences in organic matter content (TOC),

the TP content was about 70% that of BP.

The differences in P content of two periphytic forms may be due to the sources of

P available to them. Benthic periphyton has been shown to utilize P from both the water

column and the soil porewater (Hansson, 1989, 1992). The soil porewater at WCA-2A-

217 has been shown to have higher equilibrium DRP concentrations than the overlying

water (Koch and Reddy, 1992). Hansson (1992) observed that as the floodwater P

concentration increases, the sediment porewater P utilization decreases. Given very low

ambient floodwater DRP of the interior marsh (< 0.16 piM) it seems plausible that any P

source would be in demand. Epiphytic periphyton can receive P from the water column

and the exudation of P from the macrophytic tissue. The contribution of P from healthy

macrophytes was shown to be only 3 9% of the P in EP (Carignan and Kalff, 1982).

Epiphytic periphyton in the Everglades has been shown to preferentially colonize dead

macrophyte stems (Browder, 1981). Old or dead macrophyte tissues might release more

P than healthy ones, therefore adding available P to EP; however, Riber et al. (1983)

found that on a dry weight basis the EP grown on new Phragmites spp. stems had a

higher P content than that on older stems.

The TN content of periphyton was between 11 and 23 mg TN g-' (dry wt.). Like

P, TN content increased in the order BP > MP > EP. The TN content of BP was

consistent for all times. Lower TN values were reported for periphyton located in slough

sites (Vymazal et al., 1994). In another study, the TN content of the periphyton from

WCA-2A-217 was reported to be in the range of 9 36 mg g-1 (Swift and Nicholas

1987). The N:P ratio from 69 to 106:1, with the ratio generally higher in EP than in BP.

Earlier studies have reported a wide range in N/P ratios (53 205) of periphyton sampled

in WCA-2A (Swift and Nicholas, 1987; Vymazal et al., 1994). The high N/P ratio

suggests that periphyton is P limited.

Ash content ranged 429 619 g kg- (dry wt.) with highest values for EP followed

by MP > BP. The ash content in periphyton samples from the southern Everglades
represented 49-8 l%.of the total mass and varied little between seasons (Browder et al.,

1982). Vymazal et al. (1994) reported slough EP organic contents (as AFDM) as 318 -

525 g kg-' which corresponds to an ash content of 48 68%. Miiller (1983) reported ash

content of 33-73% for a Clodaphora community obtained from sandstone substrata

(epilithic) in a eutrophic lake in Sweden, with lowest values observed during a July

sampling event when the total Ca content was at a maximum. In our study, the ash

content was only slightly higher for BP during the summer sampling (June, 1995) than for

a fall sampling (October, 1994).

Inorganic P Uptake

Several studies have identified the effect of P loading on species compositional
changes (Swift and Nicholas, 1987; Radar and Richardson, 1992; Gleason and Spackman,

1974), on periphytic biomass production (Bothwell, 1989; Hansson, 1992; ), transfers

between substrate and periphyton (Carignan and Kalff, 1982; Riber et al., 1983; Hansson,

1989), and on P contents as affected by physical conditions (e.g. water velocity)(Horner

et al., 1983; Loch and John, 1979). A few studies have described P uptake by periphyton

on an area basis (Adey et al., 1993; House et al., 1995; Vymazal, 1988). Reuter et al.

(1986) determined the kinetics of N uptake by epilithic periphyton. However, to my
knowledge there are no reported studies on P uptake kinetics of periphyton.

The uptake estimates of Km and Vma, were obtained by using the P depletion
technique (Reuter et al., 1986). An advantage of this method is that measurements over a

full range of external concentrations can be made with the same sample (Drew et al.,

1984). The value of Km represents the affinity an organism or a community has for a

given substrate (Reuter, 1986). The lower the K,, i.e. the lower the P concentration

resulting in half the maximum uptake rate, the higher is the affinity of P. Uptake rates are

sensitive at concentrations lower than K, (Epstein, 1972). These values have been used

in algal competition studies as a measure of a species advantage for limited P (Grover,

1989; Bentzen and Taylor, 1991a). The maximum uptake velocity (Vmax) is a capacity

factor denoting the maximum uptake rate when all carrier sites are loaded (Epstein,

1972). A high capacity for nutrients, i.e. a large V,m, is considered advantageous in

nutrient rich environments in that it allows maximal exploitation of the available

substrate (Cotner and Wetzel, 1991).

The half-saturation constants (Kn) predicted for Pi uptake varied between 1.1 pM

and 9.9 pM. The K, values obtained during the 1994 experiments suggest that BP has a

higher affinity for low DRP concentrations than does EP. A few studies have sought to

determine the uptake kinetics of filamentous algae. Borchardt et al (1994) reported Km

values for Spirogyrafluviatilis Hilse to be 0.4 1.5 pM P, slightly lower but of a similar

magnitude to those reported here. Tarutani and Yamamoto (1994) reported Km = 0.7 pM

for Pi uptake by the marine diatom Skeletonema costatum. Using whole water samples

from a Canadian shield lake, Lean and White (1983) obtained Km values for

phytoplankton ranging 2.4 9.5 pM P when Km was calculated from standard 31P

additions of up to 20 Ag L-' and 0.3 4.0 pM when calculated from 32P additions. Cotner

and Wetzel (1992) reported much lower Km values for whole water phytoplankton P,

uptake studies of between 0.05 to 0.19 pM.

The Km values obtained for periphyton are high, being 1 to 2 orders of magnitude
greater than the ambient DRP concentration typical of this wetland (< 0.16 pM). Reuter

et al. (1986) found Km values for nitrate and ammonium uptake by periphyton in Lake

Tahoe to also be 1-2 orders of magnitude greater than typical ambient concentrations.

Their results seemed to contradict those found for phytoplankton in the same setting

causing them to conclude that care must be taken when generalizing between

phytoplankton and periphyton communities, even in the same water body.

The calculated maximum uptake rates (Vm,) ranged 0.04 0.85 pmol g-1 min-1.

The highest reported value was for EP. The in situ Vm, for BP (June, 1995) approaches

this maximum. These data suggest a higher capacity of EP to take up P when P is present

in excessive concentrations and that Vm, is dependent on the physiological state of the

periphyton. However, under ambient condition, P uptake rate will not approach Vma, as

normal ambient DRP and TP concentrations are around 4 and 10 ig L-', respectively

(Swift and Nicholas, 1987).

The estimate of V,, predicted by fitting initial uptake rates at concentrations

from ambient to about 40 pM DRP (Fig. 3-4) show that at higher initial concentrations (>

ca. 10 pM) the predicted V, underestimates the experimentally determined uptake rate.

The reasons for which two seemingly different uptake rates would result as initial P

concentration increases is unknown. An analogy between P uptake by MP and BP and

that of P adsorption to CaCO3 may lead to speculation of possible mechanisms involved.

It has been reported that the retention of P to CaCO3 occurs initially by rapid filling of

monolayer sorption sites followed by slower rearrangement of phosphate ions in CaCO3

crystal growth (Berkheiser et al., 1980; Griffin and Jurniak, 1974). As the initial solution

P increases, precipitation of CaHPO4 may be favored (Berkheiser et al., 1980). A

possible mechanism to explain the Vx predictions (Fig. 3-4) is that biotic uptake and P

adsorption to CaCO3 occurs at all concentrations; however, as the initial P concentration

increases Ca-P precipitation reactions have a greater affect on P removal from solution.

Turnover times, calculated as the reciprocal of the first-order constant (k), ranged

14 180 min (Table 3-4). At DRP concentrations ca. 4.8 pM, turnover time was 12 min

for EP and 38 min for MP. The turnover time of BP during this experiment was 74 min.

Turnover times increased with increasing concentration. Lean and White (1983) found a

P turnover time in whole water phytoplankton studies to be 5.5 min at ambient P levels

(<1 gg L-1), but that the turnover time increased to 20 min with the addition of only 2 pg
P L-1. They suggested that at very low P concentrations rapid uptake and exchange of P

was primarily due to bacteria and microalgae; at higher concentrations uptake is by larger

algae or clumps of algal cells (Lean and White, 1983)

The rates of Pi uptake varied between the October and November 1994

experiments. Initial uptake rates compared for a given periphyton type at a similar initial

concentrations were generally in close agreement for the October 20, and October 24 -26,

1994 experiments (data not shown). However, incubation under artificial light (10 W m-
2) for a period of about 5 weeks affected the uptake ability of EP and BP. For both

periphyton types, uptake rates were about one-half of those measured for the October

experiments. The October uptake rates for BP ranged 0.033 0.084 jimol P g- min-,

while those of the November experiment were within the range of 0.018 0.036 imol P

g-1 min-. The uptake rates for BP at similar concentrations measured in the field during
June 1995 were 0.077 0.429 pimol P g-1 min-i. Similarly, the EP uptake rates at the

October sampling with initial concentrations of 4.6 12.6 pM were 0.162 0.348 Pmol

g-1 min-1, and those of November were 0.092 0.173 lmol g-1 min-1. The ability of

periphyton to maintain the uptake rates of the October experiments was reduced during

laboratory incubation. There is not a corresponding change in the chemical

characteristics studied (Table 3-1). For example, if the degree of calcification reduces the

ability of periphyton to obtain P, then a reduced ash content in the November periphyton

could explain the reduced uptake. Ash content, and presumably the degree of

calcification, were maintained during this period.

The mass of DRP removed by BP during the field study of June 1995 was greater
than that of either the October or November 1994 laboratory BP uptake studies. These

results suggest that seasonal variation in BP uptake ability, or in variations due to

experimental conditions (e.g. field vs. laboratory), or a combination of these factors,

influence P uptake rates.

The material used in laboratory experiments was collected on October 18, 1994,

when the water level at WCA-2A-217 was uncharacteristically high (about 1 meter). The

BP collected at this time showed visual symptoms of stress. The EP, being under

relatively less water depth due to its position in the water column showed no apparent

stress. Gleason and Spackman (1974) and Browder et al. (1994) suggested optimal

conditions for benthic calcareous periphyton mats in the Everglades develops in water

depths < 0.6 m. In deeper water, or where shading is excessive, the mats will degenerate

to "a crumbly mass or a thin coating of algae" and will begin to take on a deeper green

appearance (Gleason and Spackman, 1974). The water depth at WCA-2A-217 on June 7,

1995 was approximately 0.4 m. The benthic periphyton at this time had the normal

golden appearance. The bulk chemical parameters measured for periphyton used (Table

3-1) did not change appreciably with time for each type, but it is likely that the proportion

of biologically active portion is affected by storage time and seasonality.

Organic P Uptake

Phosphorus as ATP was used to determine the uptake rate of P0 by BP under in

situ conditions in June 1995. Bentzen and Taylor (1991b) found [32P]ATP (as a model

phosphomonoester, PME) was an effective substrate for tracing Po dynamics in

phytoplankton in a manner comparable to using 32PO43- to study P043- dynamics.

Added ATP had been rapidly hydrolyzed to DRP within 15 min. Since all ATP
was hydrolyzed by the first sampling, the actual hydrolysis rate could not be determined.

However, based on the maximum ATP addition of ca 7.0 pM P (217 [Lg L-') and a 15 min

time period, the calculated hydrolysis rates were > 0.47 pM (14.5 gg L-I min-1 ). The in

situ uptake studies showed similar maximum uptake rates for Pi and P0 (Pi Vm, = 0.64 jp

mol g-I min-, and 0.66 jimol g-' min-I for P,). Bentzen et al. (1992), found that PME

(ATP) and PO43- uptake by phytoplankton to be comparable, with P043- uptake being

slightly greater. The positive, significant relationship between these substrates led them

to conclude that similar forces are involved in Pi and P0 utilization (Bentzen et al., 1992)

As is the case with Pi uptake, there is virtually no research to date on the uptake

and utilization rates of P0 by periphyton. Numerous studies have been conducted on the

uptake of Po by plankton and, discussion of seemingly analogous mechanisms may

explain Po utilization by periphyton. Several studies have shown the utilization of P0 by

algal and bacterial components of plankton (Bentzen et al., 1992; Bentzen and Taylor,

1991b; Cotner and Wetzel, 1992; and Ammerman and Azam, 1985). Naturally occurring

Po, in the form of phosphomonoesters (PME) is not readily available for algal or bacterial

(and by analogy periphyton) uptake until it is converted to inorganic P.

Phosphomonoesters are hydrolyzed enzymatically to Pi by several phomonoesterases.

The most widely recognized phosmonoesterase in aquatic systems is the non-specific

alkaline phosphatase (AP) (Bentzen et al., 1992). However, Ammerman and Azam

(1985) reported that marine and freshwater bacterial plankton have a cell surface 5'

nucleotidase that rapidly hydrolyzes 5' nucleotides and regenerates Pi. They suggest that

this enzyme activity might also be a result of algae and cyanobacteria, but that 5'

nucleotidase has not yet been reported in photoautotrophs (Ammerman and Azam, 1985).

A number of factors influence the hydrolysis of PME including; the amount and

availability of P containing organic compounds, the overall P demand, the rate of

phosphatase enzyme production, and the amount and availability of Pi (Syers et al., 1973;

Ammerman and Azam, 1985; Cotner and Wetzel, 1991). Uptake of P from PME can

enter a cell directly if the ecto-phosphomonoesterase is closely associated to the P

transport mechanism (Lugtenberg, 1987 as cited in Bentzen et al. 1992) or indirectly if

hydrolyzed PO43- is released to solution or if some enzyme is free in solution (Bentzen et

al., 1992). Once hydrolyzed, the Pi is then available for uptake either by the organism

that hydrolyzed it or by other organisms. For instance, in California coastal water 85 90

% of the Pi hydrolyzed by bacterial 5' nucleotidase was released to the environment where

it could supply an estimated 50 100 % of the Pi consumption by phytoplankton

(Ammerman and Azam, 1985). Cotner and Wetzel (1991) isolated bacterial fractions

producing AP activity along a transect from the littoral to the pelagic zone of a lake they

studied and found the highest AP activity was in the bacterial isolate from the littoral

zone. WCA-2A-217 shares more similarities to a lake littoral zone than to the pelagic

waters. Inorganic Pi as P043- has been shown to inhibit the activity of

phosphomonoesterases such as AP (Bentzen et al., 1992; Cotner and Wetzel, 1991);

however, Ammerman and Azam (1985) found 5' nucleotidase to be relatively insensitive

to high concentrations of Pi. They observed no inhibition at Pi concentrations below 100

piM (Ammerman and Azam, 1985). The Pi concentrations at WCA-2A-217 never

approaches 100 pM; therefore, if this mechanism exists at WCA-2A-217, then the

enzyme should be active at all times. Benzten et al. (1992) and Cotner and Wetzel (1991)

also suggested that even with Pi inhibition, Po hydrolysis occurs when the P demand is


Orthophosphate is considered to be the P form preferentially utilized in aquatic

systems; however, the available fraction of Po, as exemplified by ATP, is also actively

utilized by Everglades BP. The rapid hydrolysis rates, the rapid uptake rates, and a

maximum uptake rate similar to that of Pi all indicate that P from PME is in high demand

at WCA-2A-217.

Partitionina of 32Phosphorus

Rates measured by solution depletion estimate P uptake by periphyton without

differentiating the mechanisms involved. We hypothesized that an abiotic interaction,

mediated by periphytic activity occurs between Ca2+, CaCO3, and P043-, and that this

interaction is at least partially responsible for removing P from solution. To test this

hypothesis, BP was incubated under laboratory conditions in solutions containing either

32pi as H3PO4 or [32P]ATP.

In short-term incubations (1 and 4 h for Pi and Po, respectively) and at ambient

concentrations, greater than 70% 32P activity was biotically incorporated (Fig. 3-9 and 3-

10). Ratios of biotically incorporated 32P activity were similar between light and dark


The activity associated with the abiotic compartment was greater for 32pi than for
[32P]ATP during the short-term incubations. This may be due to a shift from abiotic

compartment to biotic with time (4 h vs. 1 h) or may suggest a higher affinity for Po

uptake by biotic mechanisms. Similarly, abiotic mechanisms may be better able to

complex Pi than Po. As mentioned, Po is not readily taken up until it is hydrolyzed to Pi

by ecto-phosphatases. During this hydrolysis the majority of the [32P]ATP is associated

with cell surfaces, and if the ecto-phosphatases are close to the transport system, the
[32P]ATP would be incorporated into the biotic compartment before it has an opportunity

to associate with CaCO3. That [32P]ATP which appeared in the abiotic compartment was

possibly hydrolyzed by free enzymes in solution.

Periphyton at WCA-2A-217 is dominated by filamentous cyanobacteria and

hardwater diatoms (Swift and Nicholas, 1987). Everglades cyanobacteria have the ability

to precipitate CaCO3 (Gleason, 1972; Gleason and Spackman, 1974; Browder et al.,

1994). Several researchers have shown P to coprecipitate with CaCO3 in freshwater

during periods of high algal activity, or in water that mimics such activity (House et al.,

1995; Otsuki and Wetzel, 1972; Murphy et al., 1983). Diaz et al. (1994) observed rapid

precipitation of P as Ca-P in Everglades water when pH was manipulated to > 8.0. The

conditions that favor periphytic CaCO3 encrustation and subsequent P coprecipitation are

high calcium concentrations, high alkalinity, high pH, low partial pressure of CO-, high

levels of CaCO3 saturation, and high temperatures (Gleason and Spackman, 1974:

Browder et al., 1994; Otsuki and Wetzel, 1972; and House, 1990). Photosynthesis creates

microenvironments around cells with conditions that favor rapid calcite formation

(McConnaughey, 1991; Gleason and Spackman, 1974). Additionally, the algal surface

acts to catalyze the calcite precipitation by providing the initial nuclei for further crystal

growth (Mtiller, 1983; Murphy et al., 1983; Gleason and Spackman, 1974). The

concentration of available P is also important but mostly from its inhibitory effect on

calcite crystal growth. Depending on the level of supersaturation, solution P may stop

crystal growth completely (House, 1987, as cited in House 1990). The CaCO3-P

coprecipitation reaction occurs when solution P becomes adsorbed onto CaCO3 and

becomes incorporated as the crystal grows (Griffin and Juriak, 1974; House, 1990).

This process is more important for removing P rather than P becoming adsorbed on the

particulate CaCO3 after the formation of crystals (Otsuki and Wetzel, 1972)

Other researchers have stated that CaCO3-P coprecipitation is not responsible for

the removal of P. Based on the inverse relationship between Ca and P contents Vymazal

et al. (1994) promoted that P is bound to organic matter in periphyton rather than to

calcite precipitations. They suggested that the relationships between algal calcite

precipitation, P uptake, and calcium phosphate precipitation are unknown, but suggests

Ca3(P04)2 precipitation is not directly linked to algal calcite precipitation. They further

stated that calcareous periphyton does not function as a P storage mechanism as has been

suggested by Gleason (1974). They allowed the possibility of water column calcium P

precipitation, but that the direct Ca3(PO4)2 precipitation by periphyton is probably not the

mechanism. Recent analysis by X-ray diffraction of dried, ground BP, containing around

550 mg CaCO3 g-1, produced results very similar to that of pure CaCO3 with no mineral

forms (e.g. Ca3(P04)2 ) of calcium phosphates present (see Fig. 5-6). This does not

exclude the possibility that P coprecipitates with CaCO3 as an amorphous form, or that P

precipitated in a mineral form with Ca occurs in concentrations too low to appear in X-

ray diffraction. The inverse relationship between Ca and P contents can be explained by

the differences in the density of highly calcareous periphyton to that of less calcareous

periphyton. On a dry weight basis, the increased mass due to CaCO3 is going to dilute

the mass of all other elements.

The stability of P coprecipitated with CaC03 needs further study. Gleason (1972),

Gleason and Spackman (1974) and others described the diurnal fluctuations in

physicochemical parameters leading to CaCO3 precipitation in the water column over

periphyton communities. Generally, supersaturation and subsequent precipitation during

the day was at least partially balanced by release during night-time undersaturation

(Gleason and Spackman, 1974). Other factors, such as water depth (Gleason and

Spackman, 1974) and increased levels of P have been shown to reduce the levels of

calcification in periphyton. Vymazal et al. (1994) cited enrichment studies where the

addition of P caused a disintegration of the calcareous mat but the dominant

cyanobacteria remained. Several other researchers have shown a loss of the calcareous

periphyton and a replacement by other algae species, mostly green algae after prolonged

exposure to increased P nutrition (Swift and Nicholas, 1987; Steward, 1973; and Omes

and Steward, 1973 as cited in Vymazal et al., 1994). The fate of the P within the

periphytic matrix is unknown.

I propose that P uptake by periphyton involves a number of simultaneous

reactions. Phosphorus can move directly to cell surfaces where it is taken up biotically

or, in the case of Po, is first hydrolyzed and then taken up. Inorganic P is rapidly, but

weakly completed to the surface of CaCO3 crystals. Much of this P eventually moves

into the biotic compartment within a few hours. Some surface adsorbed P may become

incorporated into more stable Ca-P if the adsorption occurs during active CaCO3 crystal



The evidence presented here indicates that the periphyton of the interior of WCA-

2A-217 exists in a condition of P limitation. Parameters describing the P uptake by

periphyton were obtained by the solution depletion technique for the linear portion of the

depletion curves. Rate processes that are a function of substrate concentration, as were

ours, are never truly linear, yet appear that way when uptake is relatively slow. Results

showed no change in P concentration in ambient treatments; therefore, P uptake was only

determined after enrichment. At ambient concentrations, the periphyton is in steady-state

with the environment; and therefore, net community influx equals efflux. It is difficult to

chemically determine differences in influx/efflux at ambient levels without the use of

radioisotopes. The uptake rates after enrichment should not be interpreted as a measure

of long-term uptake potential, but are useful in quantifying uptake rates for intermittent

pulses of P enriched water and to determine the maximum P uptake rates (Vm,) and half-

saturation constants (K,). Ambient DRP levels in the interior of WCA-2A are always

below KI: and therefore, uptake rates are always < Vm. The periphyton of the interior

of WCA-2A was shown to have rapid uptake rates and high capacities to assimilate P.

The CaCO3 P coprecipitation mechanism as represented by abiotic uptake did

not remove a substantial percentage of added P during the short-term enrichment

experiments. The precipitation of CaC03 by periphyton and the interaction of CaCO3

with P could add P retention capacity to peat soils of WCA-2A-217, leading to long-term

storage. The P within the periphyton will cycle internally. The abiotic P is also available

to biotic consumption. Although the CaCO3 P coprecipitation did not prove to be an

appreciable P removal mechanism the fact that this mechanism exists deserves further

attention in the hopes of developing processes that increase P retention. Mat forming

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