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Evaluation of Aquatic Plants for Phytoremediation of Eutrophic Stormwaters

Permanent Link: http://ufdc.ufl.edu/UFE0024791/00001

Material Information

Title: Evaluation of Aquatic Plants for Phytoremediation of Eutrophic Stormwaters
Physical Description: 1 online resource (127 p.)
Language: english
Creator: Lu, Qin
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: accumulation, eutrophication, metals, nitrogen, nutrients, ph, phosphorus, phytoremediation, removal, salinity, salvinia
Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: EVALUATION OF AQUATIC PLANTS FOR PHYTOREMEDIATION OF EUTROPHIC STORMWATERS By Qin Lu Water quality impairment by nutrient and metal enrichment from agricultural activities has been a concern worldwide. Phytoremediation technology using aquatic plants was evaluated for its efficacy in removing N, P, and metals from stormwater in detention ponds. Water lettuce (Pistia stratiotes) plants were grown in treatment plots in two stormwater detention ponds and water quality in both ponds was monitored. To better utilize water lettuce and investigate the possibility of a water lettuce-common salvinia (Salvinia minima) polyculture system, water lettuce and common salvinia were tested for their N and P requirements for normal growth with hydroponic studies conducted in a greenhouse. Water lettuce was also evaluated for its growth performance in water with different pH and salinity levels. Water quality in both ponds was improved by phytoremediation with water lettuce, as evidenced by decreased water turbidity, total solids, and nutrient concentrations. Water turbidity was decreased by more than 65%. Total solids decreased by about 20%. Ammonium-N and nitrate-N concentrations in the treatments plots were 31-72% lower than those in the control plots (without plant), and total Kjeldhal N was decreased by more than 20%. Reductions in ortho-P, total dissolved P, and total P concentrations in water were approximately 18-58% compared to the control plots. Annual removal of N and P from the water was 190 and 24.6 kg/ha, respectively in East Pond, and 329 and 34.1 kg/ha, respectively in West Pond by harvesting plant biomass. Compared to the control plots, Al, Fe, and Mn concentrations were reduced by an average of 20%, and K by 10% in the treatment plots. Calcium, Mg, and Na concentrations were also reduced by 5-10%. Metals were substantially accumulated in the roots of water lettuce. A larger proportion of Ca, Cd, Co, Fe, K, Mg, Mn, and Zn was attached to external root surfaces by adsorption or surface deposition while more Al, Cr, Cu, Ni, and Pb were absorbed and accumulated into the root. The critical N concentrations required for water lettuce and common salvinia to have net growth were 1.25 and 2.5 mg/L, respectively, and the critical P concentrations were 0.1 and 1 mg/L, respectively. Higher N and P requirements make common salvinia less desirable for a polyculture system with water lettuce. Water lettuce could tolerate the salinity level ( < 1766 micronS/cm) of freshwater but its biomass could be reduced by up to 30% by high salinity. This plant could not survive in brackish water with salinity > 6937 micronS/cm. We can also expect optimum performance from this plant in neutral and slightly alkaline water.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Qin Lu.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: He, Zhenli.
Local: Co-adviser: Graetz, Donald A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024791:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024791/00001

Material Information

Title: Evaluation of Aquatic Plants for Phytoremediation of Eutrophic Stormwaters
Physical Description: 1 online resource (127 p.)
Language: english
Creator: Lu, Qin
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: accumulation, eutrophication, metals, nitrogen, nutrients, ph, phosphorus, phytoremediation, removal, salinity, salvinia
Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: EVALUATION OF AQUATIC PLANTS FOR PHYTOREMEDIATION OF EUTROPHIC STORMWATERS By Qin Lu Water quality impairment by nutrient and metal enrichment from agricultural activities has been a concern worldwide. Phytoremediation technology using aquatic plants was evaluated for its efficacy in removing N, P, and metals from stormwater in detention ponds. Water lettuce (Pistia stratiotes) plants were grown in treatment plots in two stormwater detention ponds and water quality in both ponds was monitored. To better utilize water lettuce and investigate the possibility of a water lettuce-common salvinia (Salvinia minima) polyculture system, water lettuce and common salvinia were tested for their N and P requirements for normal growth with hydroponic studies conducted in a greenhouse. Water lettuce was also evaluated for its growth performance in water with different pH and salinity levels. Water quality in both ponds was improved by phytoremediation with water lettuce, as evidenced by decreased water turbidity, total solids, and nutrient concentrations. Water turbidity was decreased by more than 65%. Total solids decreased by about 20%. Ammonium-N and nitrate-N concentrations in the treatments plots were 31-72% lower than those in the control plots (without plant), and total Kjeldhal N was decreased by more than 20%. Reductions in ortho-P, total dissolved P, and total P concentrations in water were approximately 18-58% compared to the control plots. Annual removal of N and P from the water was 190 and 24.6 kg/ha, respectively in East Pond, and 329 and 34.1 kg/ha, respectively in West Pond by harvesting plant biomass. Compared to the control plots, Al, Fe, and Mn concentrations were reduced by an average of 20%, and K by 10% in the treatment plots. Calcium, Mg, and Na concentrations were also reduced by 5-10%. Metals were substantially accumulated in the roots of water lettuce. A larger proportion of Ca, Cd, Co, Fe, K, Mg, Mn, and Zn was attached to external root surfaces by adsorption or surface deposition while more Al, Cr, Cu, Ni, and Pb were absorbed and accumulated into the root. The critical N concentrations required for water lettuce and common salvinia to have net growth were 1.25 and 2.5 mg/L, respectively, and the critical P concentrations were 0.1 and 1 mg/L, respectively. Higher N and P requirements make common salvinia less desirable for a polyculture system with water lettuce. Water lettuce could tolerate the salinity level ( < 1766 micronS/cm) of freshwater but its biomass could be reduced by up to 30% by high salinity. This plant could not survive in brackish water with salinity > 6937 micronS/cm. We can also expect optimum performance from this plant in neutral and slightly alkaline water.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Qin Lu.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: He, Zhenli.
Local: Co-adviser: Graetz, Donald A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024791:00001


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EVALUATION OF AQUATIC PLANTS FOR PHYTOREMEDIATION OF EUTROPHIC
STORMWATERS



















By

QIN LU

















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


UNIVERSITY OF FLORIDA

2009

































O 2009 Qin Lu




































To my husband, Diangao, and my son, Xuanning










ACKNOWLEDGMENTS

First of all, I would like to express my deepest thanks to my advisor, Dr. Zhenli L. He, for

his encouragement, trust, and patience as my mentor. He has not only provided me professional

opportunities and offered me numerous insightful suggestions for my research, but also, as a role

model, he has shown me that hardworking and persistence as well as creativity and independent

thinking are ingredients of success. I am very grateful to my co-advisor, Dr. Donald A. Graetz.

He arranged for my airport pick up, social security number application, and first term

registration, all of which helped me adjust smoothly to a whole new environment and feel at

home. He has been giving invaluable suggestions and comments for my research. I would also

like to give my sincere thanks to Drs. Peter J. Stoffella, Yuncong Li and Samira Daroub for

serving on my advisory committee and making maj or contributions to my research. Special

thanks go to the late Dr. Dolen Morris, who had always been prompt in helping me improve my

writing. I profoundly appreciate South Florida Water Management District for funding the

research.

I thank Dr. Min Liu and Ms. Yu Wang, Lacey, Katrina, Leighton, and Brandon in Dr.

Graetz's lab and Sampson and many other friends in Gainesville for their help and friendship

which made my stay in Gainesville a pleasant one. I wish to thank the faculty, staff, and students

of the Soil and Water Science Department for their assistance and support.

Dr. Charles A. Powell of Indian River Research and Education Center at the University of

Florida is acknowledged for making his laboratory facilities available for my use. I wish to thank

all the faculty, staff, and students, especially Mrs. Youjian Lin, Hai Lu, Mrs. Cuifeng Hu, Mrs.

Maria Solis, Drs. Peter J. Van Blokland and Sandra B. Wilson, at Indian River Research and

Education Center of University of Florida. Their kindness and help in many ways made my stay

in Fort Pierce a memorable one.










I thank Dr. Xiaoe Yang for providing insight, expertise, and support. Special thanks go to

Drs. Guochao Chen, Jinyan Yang, Yuangen Yang, Frederico Vieira, Wenrong Chen, Yangbo

Wang, Mr. Douglas J. Banks, Mrs. Shaoqin Lu, and PhD students Jinghua Fan and Bruno Pereira

for providing assistance in laboratory analysis, expertise and laughter over the past three years.

Without their help, successful completion of my PhD study is impossible. I have always felt

fortunate to be part of Dr. He's group where I have learned, enjoyed and benefited from team

work.

I wish to express my appreciation to Dr. Xiaochang Wang for his continued interest in my

progress, encouragement and support.

I am very grateful to my parents, parents-in-law, and siblings for their love, support,

encouragement, and confidence in me, which have been the driving force for me to pursue my

dreams.

I am greatly indebted to my loving husband, Diangao, who has sacrificed so much to be

with me here in the United States and helped me in the field and in the lab. I thank my adorable

son, Xuanning, who has brought so much j oy and happiness into our life. They are the endless

source of strength I can always rely on.












TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. ...............4.....


LI ST OF T ABLE S ............ ...... .___ ...............9....


LIST OF FIGURES .............. ...............10....


AB S TRAC T ............._. .......... ..............._ 12...


CHAPTER


1 LITERATURE REVIEW ................. ...............14...............


Water Quality: A Worldwide Concern ..........._..._ ... .... .......... ...............14....
Phytoremediation of Contaminated Water Using Aquatic Plants .............. .....................17
Stormwater Treatment with Floating Aquatic Plants .............. ...............22....
Growth Factors of Aquatic Plants. ................ ................... .........................23
Research Objectives............... ...............2


2 NUTRIENT REMOVAL POTENTIAL OF WATER LETTUCE (PISTIA S7RA TIOTES
L.) FROM STORMWATER INT DETENTION SYSTEMS .............. .....................2

Introducti on ................. ...............27.................
Materials and Methods .............. ...............28....

Experimental Design .............. ...............28....
Chemical Analysis............... .. ...............3
Data Treatment and Data Analysis............... ...............32
Results and Discussion .............. .... .. ...............3
General Water Quality Improvement ................ ...............33................
Nitrogen and P Concentration Reduction............... ...............3
Nitrogen and P Removal Potential by Plant Uptake .............. ...............46....
Physiological Limits ................. ...............48.................
System Management .............. ...............49....
Conclusions............... ..............5


3 METAL REMOVAL POTENTIAL OF WATER LETTUCE (PISTIA STRA TIOTES L.)
FROM STORMWATER INT DETENTION SYSTEMS .............. ...............51....


Introducti on ................. ...............51.................
M materials and M ethods .............. ...............54....
Chemical Analy sis............... ...............55
Data Treatment ................. ...............55.......... ......
Re sults ................ ........ ............ .......... ..... ..........5
Metal Concentration Reduction in Water ................. ...............56........... ...
Metal Accumulation by Plant Root ................ ...............61........... ...












Metal Distribution in Plant ................. ...............61........... ...
Estimation of Annual Metal Removal ................ ...............62........... ...
Metal Uptake and Surface Adsorption .............. ...............63....
Metal Bio-concentrated by Plant ................. ...............64................
Discussion ................. ...............66.................
Conclusions............... ..............6


4 NITROGEN REQUIREMENT FOR WATER LETTUCE AND COMMON
S ALVINIA .................... ...............6


Introducti on ................. ...............69.................
M materials and M ethods .............. ...............70....

Experimental Design .............. ...............70....
Chemical Analysis............... ...............71
Statistical Analysis .............. ...............71....
Results and Discussion .................. .............. ... .. ..........7
Relationship between Plant Biomass Yield and N Concentration .............. ..................71
Relationship between Plant N and Solution N Concentration ................. ................ ..75
Plant Critical N Concentration .............. ...............78....
Conclusions............... ..............7


5 PHOSPHORUS REQUIREMENT FOR WATER LETTUCE AND common
S ALVINIA .................... ...............8


Introducti on ................. ...............8.. 0..............
Material s and Method s .............. ...............8 1....

Experimental Design .............. ...............8 1....
Chemical Analysis............... ...............82
Statistical Analysis .............. ...............82....
Results and Discussion .................. .............. .. ..... ... .......8
Relationship between Plant Biomass Yield and Solution P Concentration ....................82
Relationship between Plant P Concentration and Solution P Concentration ..................87
Plant Critical P Concentration ................ ...............90................
Conclusions............... ..............9


6 EFFECT OF SALINITY ON GROWTH OF WATER LETTUCE ................. ................. .92


Introducti on ................. ...............92.................
M materials and M ethods .............. ...............93....

Experimental Design .............. ...............93....
Chemical Analysis............... ...............94
Statistical Analysis .............. ...............95....
Results and Discussion .............. ........ ..... .... .... .......9
Plant Growth as Affected by a Salinity Gradient ................. ............... ......... ...95
Plant Biomass in Different Salinity .................. ... .......... ...............95.....
Plant Nutrient Status under Different Salinity Conditions ................ ......................98
Conclusions............... ..............10












7 EFFECT OF PH ON GROWTH OF WATER LETTUCE ................ ........................101


Introducti on ................. ...............101................
Materials and Methods .............. ...............102....

Experimental Design .............. ...............102....
Chemical Analysis............... ...............10
Statistical Analysis .............. ...............103....
Results and Discussion .................. ............ ...............103......
Plant Growth in Water at Different pH .............. ...............103....
Plant Biomass Yield at Different pH Treatments ................. ................ ......... .105
Plant Nutrition Status at Different pH Treatments ................ .......... ................1 05
Conclusions............... ..............10


8 SUMMARY AND CONCLUSIONS ................ ...............111...............


LIST OF REFERENCES ................. ...............115................


BIOGRAPHICAL SKETCH ................. ...............127......... ......










LIST OF TABLES


Table page

2-1 Water quality improvement in the treatment plots of the East and West Ponds. ...............36

2-2 Annual removal amounts of plant dry biomass, N, and P from the East and West
Ponds ................. ...............47.................

3-1 Annual metal removal rates by periodic harvesting of water lettuce. .............. .... ........._..65

4-1 Nutrient solution composition for N requirement study. ................. .................7

5-1 Nutrient solution composition for P requirement hydroponic study. ............. .................81

6-1 EC and ions contributing to water salinity in the waters of the East and West Ponds. .....92

6-2 Nutrient solution composition for the salinity tolerance study. .............. ....................93

7-1 Chemical composition of nutrient solution for pH effect study. ................ ..................102

7-2 Nutrient concentration and related properties of the nutrient solution at different pH
levels. ........._.__ _.... ____ ........_ .......110..











LIST OF FIGURES


FiMr page

2-1 Experimental set up in the West Pond and the East Pond. ............. ......................3

2-2 Total solid concentrations in the waters of the East and West Ponds. .............. ..... ........._.34

2-3 Turbidity in the East and West Ponds ................. ...............35..............

2-4 Water samples from treatment plot and control plot. ............. ...............37.....

2-5 Water EC in the East and West Ponds. .............. ...............38....

2-6 Water pH in the East and West Ponds. ............. ...............39.....

2-7 Nitrate-N in the waters of the East and West Ponds ................. ................. ..........40

2-8 Ammonium-N in the waters of the East and West Ponds ................. .......................41

2-9 Total Kj eldhal N in the waters of the East and West Ponds. .........._._.. .........___........42

2-10 Water PO4-P in the East and West Ponds. ............. ...............43.....

2-11 Total dissolved P in the waters of the East and West Ponds. ................ .....................44

2-12 Total P in the waters of the East and West Ponds ................. ...............45...........

2-13 Nitrogen concentrations in plant roots and shoots from the East and West Ponds............47

2-14 Phosphorus concentrations in plant roots and shoots from the East and West Ponds. ......48

3-1 Total dissolved metal concentrations in the treatment and control plots of the East
and West Ponds during 2005-2007 (n=122). ................ ...............56..............

3-2 Plant metal concentration factors (CFs) in the East and West Ponds ............... ...............61

3-3 Metal root/shoot ratio in concentration of the East and West Ponds. .............. .... ........._..62

3-4 Distribution of metals outside and inside of water lettuce root. ...........__.. ................ .64

3-5 Plant metal bio-concentration factors (BCFs) in the East and West Ponds. ......................66

4-1 The growth performance of water lettuce and common salvinia under different N
level s. ............. ...............73.....

4-2 Plant dry biomass yield at different N level treatments. .................. ................7

4-3 The shoot/root ratio of water lettuce dry biomass at different N levels. ................... .........74











4-4 Regression curve of plant dry biomass yield vs. solution N concentration. ................... ...74

4-5 Plant N concentration at different N level treatments ................. ................. ........ 75

4-6 Regression curve of plant N concentration vs. solution N concentration. ................... ......77

5-1 Growth performance of water lettuce and common salvinia under different P levels.......84

5-2 Plant dry biomass weights of different P level treatments ................. .......................84

5-3 Water lettuce shoot/root in dry biomass under different P level. ............. ....................85

5-4 Regression curves of plant dry biomass vs. solution P concentration.............. ..._............86

5-5 Plant P concentration in treatments with different solution P level .........._..... ..........._...88

5-6 Regression curve of plant P vs. solution P concentration. ................ ..................8

6-1 Growth performance of water lettuce in water with gradient salinity. ............. ..... ........._.94

6-2 Growth performance of water lettuce in water with gradient salinity. ............. ..... ........._.96

6-3 Plant dry biomass of water lettuce with different salinity treatments .............. ..............97

6-4 Plant nutrient concentrations with different salinity treatments. ............. ....................98

7-1 Growth of water lettuce under different pH treatments. ....._____ .... ... ._ ..............104

7-2 Dry biomass yield of water lettuce at different pH. ....._____ ... ......_ ...............106

7-3 Regression curve of water lettuce dry biomass vs. solution pH. ..........__.................106

7-4 Plant nutrient concentration of water lettuce at different pH treatments. ......................107









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

EVALUATION OF AQUATIC PLANTS FOR PHYTOREMEDIATION OF EUTROPHIC
STORMWATERS

By

Qin Lu

August 2009

Chair: Zhenli L. He
Cochair: Donald A. Graetz
Major: Soil and Water Science

Water quality impairment by nutrient and metal enrichment from agricultural activities has

been a concern worldwide. Phytoremediation technology using aquatic plants was evaluated for

its efficacy in removing N, P, and metals from stormwater in detention ponds. Water lettuce

(Pistia stratiotes) plants were grown in treatment plots in two stormwater detention ponds and

water quality in both ponds was monitored. To better utilize water lettuce and investigate the

possibility of a water lettuce-common salvinia (Salvinia minima) polyculture system, water

lettuce and common salvinia were tested for their N and P requirements for normal growth with

hydroponic studies conducted in a greenhouse. Water lettuce was also evaluated for its growth

performance in water with different pH and salinity levels.

Water quality in both ponds was improved by phytoremediation with water lettuce, as

evidenced by decreased turbidity, total solids, and nutrient concentrations. Turbidity was

decreased by more than 65%. Total solids decreased by about 20%. Ammonium-N and NO3-N

concentrations in the treatments plots were 31-72% lower than those in the control plots (without

plants), and total Kjeldhal N was decreased by more than 20%. Reductions in PO4-P, total

dissolved P, and total P concentrations in water were approximately 18-58% compared to the









control plots. Annual removal of N and P from the water was 190 and 25 kg ha- respectively in

the East Pond, and 329 and 34 kg ha- respectively in the West Pond by harvesting plant

biomass.

Compared to the control plots, Al, Fe, and Mn concentrations were reduced by an average

of 20%, and K by 10% in the treatment plots. Calcium, Mg, and Na concentrations were also

reduced by 5-10%. Metals were substantially accumulated in the roots of water lettuce. A larger

proportion of Ca, Cd, Co, Fe, K, Mg, Mn, and Zn was attached to external root surfaces by

adsorption or surface deposition while more Al, Cr, Cu, Ni, and Pb were absorbed and

accumulated into the root.

The critical N concentrations required for water lettuce and common salvinia to have net

growth in biomass were 1.25 and 2.5 mg L^1, respectively, and the critical P concentrations were

0.1 and 1 mg L^1, respectively. Higher N and P requirements make common salvinia less

desirable for a polyculture system with water lettuce.

Water lettuce could tolerate the salinity level (< 1766 CIS cm l) of freshwater but its

biomass could be reduced by up to 30% by high salinity (1766 CIS cm- ). This plant could not

survive in brackish water with salinity > 6937 CIS cm- We can also expect optimum

performance from this plant in neutral and slightly alkaline water.









CHAPTER 1
LITERATURE REVIEW

Water Quality: A Worldwide Concern

To meet the requirement of a burgeoning human population, fertilizers and chemicals have

been extensively used to boost crop production. Of the nitrogen (N) taken up by plants,

approximately 70% is provided by inorganic fertilizers (Singh and Verma, 2007). Nitrogen

loading to the land has doubled from the pre-industrial period (111 Tg yr ) to the present time

(223 Tg yr- ) due to anthropogenic activities (Green et al., 2004). Manures and biosolids are

usually applied based on crop N requirements, which provides phosphorus (P) in excess of crop

needs. Many fungicides contain heavy metals such as copper (Cu) and zinc (Zn). Repeated use of

the fungicides in citrus and vegetable crop production systems has resulted in accumulation of

Cu and Zn in the soils (Zhu and Alva, 1993).

Off-site migration of these nutrients and metals by runoff to surface water is a worldwide

concern because of the resulting degradation of the aquatic ecosystems and decreased water

availability.

Urbanization also contributes to the deterioration of the aquatic ecosystems by boosting

sediment loads because of decreased surface area available for absorption and infiltration of

rainwater and snow melt and by increasing heavy metal inputs from automobile usage. Fertilizers

and chemicals applied on urban/suburban lawns, gardens, and golf courses are also subj ect to

loss by surface runoff.

Runoff from agricultural fields or urban area carries inorganic nutrients (Caccia and Boyer,

2005). In Europe, 65% of the Atlantic coast shows varying degrees of eutrophication (Diaz and

Rosenberg, 2008), and 55% of river stations had annual average dissolved P concentrations in

excess of 50 Clg P L^1 over the period 1992-1996 (Crouzet et al., 1999). Taking agricultural land









out of production brought both loads and concentrations of soluble reactive P and dissolved

inorganic N down by about 90% in a first-order agricultural stream in a small rural watershed,

Germany (Chambers et al., 2006). This strongly shows how much agriculture contributes to

increased nutrient inputs into the waterways.

Measurements in a lake (0.08-2.29 mg P L total P (TP) and 3-15 mg N L total Kjeldhal

N (TKN)) and upstream to the lake (0.6-3.8 mg P L^1 and 10-22 mg N L^1 respectively) indicated

eutrophication of lakes by receiving nutrient-rich surface runoff from urbanized areas of Central

Africa (Kemka et al., 2006). Approximately 10% of New Zealand's shallow lakes were classified

as eutrophic (> 50 Clg TP L^1) (Cameron et al., 2002).

An agricultural non-point source pollution survey in 18 townships in Fujian Province,

China revealed that N and P were the primary contaminants in the drainage area and that farm

nutrient loss, aquaculture, livestock and bird feces and urine were the largest three pollution

sources (Huang et al., 2008). In another province of south China, Guangdong, where fertilizers

are heavily applied in the orchards of its hilly and mountainous area, 90.5% of the runoff water

samples from the orchards in Dongyuan County had a total N (TN) concentration higher than

0.35 mg L^1 and 54.2% had a TP concentration higher than 0.1 mg L^1 (Zeng et al., 2008).

According to Diaz and Rosenberg (2008), 78% of the continental US coastal area show

varying degrees of eutrophication. An estimate of 45% of US waterways has impaired water

quality due to nutrient enrichment according to the US Environmental Protection Agency

(CEEP, 2001).

An average NO3-N concentration of 6.6 mg L^1 in surface runoff resulted from corn

production was measured in Lake Bloomington watershed, Illinois in a nine-year (1993 -2002)

and 36-site monitoring study (Smiciklas et al., 2008). Both N and P concentrations above the









eutrophic level in the receiving water bodies were observed by Yu et al. (2008) in a watershed

associated with sugarcane production in Louisiana. Lake Apopka in Florida was made

hypereutrophic by P loading from floodplain farms (Coveney et al., 2002).

An important source of heavy metals is highway runoff, especially in large cities such as

Guangzhou in south China (Gan et al., 2008). Highway runoff on the island of Crete, Greece

showed two-year (2005-2007 ) mean concentrations of Cu, Ni, Pb and Zn to be 56, 1 14, 49 and

250 Clg L^1, respectively (Terzakis et al., 2008). Copper was found to be the dominant metal in

the surface runoff from a suburban parking lot near Portland, Oregon (Mesuere and Fish, 1989).

Five times background levels of Cr, Cu, Ni, Pb and Zn concentrations were found in the

sediment of River Murray, Australia (Thoms, 2007). Higher metal concentrations in the river,

lake, or coastal sediments were often associated with increased agricultural and urban

development, accompanying with more anthropogenic activities (Amin et al., 2009).

Water quality throughout south Florida has been a maj or concern for many years. Nutrient

enrichment has been considered to impact ecological functions of the Everglades National Park,

Lake Okeechobee, and Indian River Lagoon (Capece et al., 2007; Ritter et al., 2007). Results

from recent monitoring study in Indian River Lagoon (IRL) by He et al. (2006b) indicate that

more than 50% of the surface runoff water samples contained TN of 1 to 5 mg L^1 and TP above

1.0 mg L^1. Mean concentrations of TN and TP in the runoff were 4. 1 and 1.6 mg L^1,

respectively, which are much greater than the USEPA critical levels for surface water (1.5 mg L-

Sfor total N and 0.1 mg L^1 for total P) (U. S. Environmental Protection Agency., 1976). The

intricate network of Canals C-23, C-24, and C-44, that drain the surrounding urban and

agricultural lands in the St. Lucie Basin and are connected to the IRL, are estimated to

collectively deliver at least 8.6xl05 kg of N, 9. 1 x10' kg of P, and 3.6xl0s kg of suspended solids









to the estuary annually (Graves and Strom, 1992). Overall IRL total N load is proj ected (year

2010) to increase by 32% (Woodward-Clyde Consultants, 1994).

Repeated use of the fungicides in citrus and vegetable crop production systems has resulted

in accumulation of Cu and Zn in the sediments of the St. Lucie Estuary (Haunert, 1988; He et al.,

2003). High concentrations of Cu and Zn were measured in storm runoff water from these

production systems (He et al., 2006a; Zhang et al., 2003).

Phytoremediation of Contaminated Water Using Aquatic Plants

Excessive nutrients (N and P) in surface runoff cause eutrophication in the receiving water,

such as lakes and estuaries, and lead to algal blooms and changes in species composition. The

increased metals in the receiving water are toxic to the living communities in the aquatic

ecosystem, and also cause health problems in human. The aquatic ecosystems are degraded by

the increased nutrients and metals, water quality is impaired, and water availability is decreased.

Actions are needed to remediate such polluted systems or to treat the surface runoff before

it gets into the receiving water. Unlike point source water pollution, which is localized and easier

to monitor and control (Smith et al., 1999), non-point source pollution is of a diffuse nature.

Conventional remediation methods suitable for point source pollution may not be desirable or

cost-effective when applied to non-point source pollution because of the relatively low pollutant

concentrations and large source area.

In addition to development of best management practices (BMPs) to reduce losses of

nutrients (N, P) and transport of contaminants (heavy metals and pesticides) from land to water,

constructed wetlands such as stormwater treatment areas (STAs), water detention systems, and

retention ponds have been increasingly built in South Florida to clean eutrophic water from

agriculture or urban areas before they are discharged to surface water systems such as Indian

River Lagoon. The functions of these systems are to settle down suspended solids and reduce









concentrations of dissolved nutrients and contaminants in water where aquatic plants can play an

important role.

Phytoremediation has been increasingly used to clean up contaminated soil and water

systems because of its lower costs and fewer negative effects than physical or chemical

engineering approaches (Ignjatovic and Marjanovic, 1985; Prasad and Freitas, 2003; Reddy and

DeBusk, 1986). The principles of phytoremediation system to clean up stormwater include: 1)

identification and implementation of efficient aquatic plant systems; 2) uptake of dissolved

nutrients including N and P and metals by the growing plants, and the plants creating a favorable

environment for a variety of complex chemical, biological and physical processes that contribute

to the removal and degradation of nutrients (Billore et al., 1998; Gumbricht, 1993); and 3)

harvest and beneficial use of the plant biomass produced from the remediation system.

Because of their fast growth rates, simple growth requirements, and ability to accumulate

biogenic elements and toxic substances, aquatic plants are utilized for nutrient and metal removal

from water. Since the first recognition of their value in water quality improvement in the 1960s

and the 1970s (Sheffield, 1967; Steward, 1970; Wooten and Dodd, 1976), aquatic plants have

been widely used to treat wastewaters or increasingly used to remediate eutrophic waters in

forms of constructed wetlands or retention ponds. This is a low-cost treatment with low land

requirements, which is attractive to urban areas with high land prices.

Aquatic plants are grouped into submerged, emergent, and floating/floating-leaved aquatic

plants according to their leaf s relation with water. Among the submerged aquatic plants,

coontail (Ceratophyllum demersum L.), hydrilla (Hydrilla verticillata), southern naiad (Naja~s

guadahepensis) are the most investigated (Badr and Fawzy, 2008; Bunluesin et al., 2004). Cattail

(Typha latifolia), bull rsh (Scirpus lacustris), and common reed (Phragmites australis) are the









most planted emergent plants in constructed wetlands to remove nutrients such as N and P

(Manab Das and Maiti, 2008). Among the floating/floating-leaved aquatic plants, water hyacinth

(Eichhornia crassipes), water lettuce (Pistia stratiotes), duckweed (Lemna spp. and Spirodela

polyrrhiza W. Koch), pennywort (Hydrocotyle umbellata), and common salvinia (Salvinia

minima baker) are the best candidates (John et al., 2008; Maine et al., 2004; Mishra et al., 2008;

Sanchez-Galvan et al., 2008). With regard to the uptake capacity of aquatic plants, and

subsequently the amount of nutrients or contaminants that can be removed when the biomass is

harvested, floating plants (especially large-leaved species) are in the lead, followed by emergent

species and then submerged species. Approximately 350 kg P and 2000 kg N ha-l yr- were

removed by large-leaved floating plants such as water hyacinths, whereas the capacity of

submerged macrophytes was lower (<100 kg P and 700 kg N ha-l yr- ) (Brix, 1997). Growing in

waters with similar P concentrations, water hyacinth had an average P concentration almost

twice that of hydrilla, hornwort, pondweed, eelgrass, or naiad, showing a much greater ability for

P scavenging (Easley and Shirley, 1974). Emergent macrophytes are mostly in the range of 30 to

150 kg P ha-l yr- and 200 to 2500 kg N ha-l yr- (Brix, 1994; Gumbricht, 1993).

Impressive removal rates of inorganic N (NO3-N, NH4-N, and total N) and P (PO4-P and

total P) have been reported from all kinds of phytoremediation systems using aquatic plants

especially when invasive floating aquatic plants such as water hyacinth were utilized in nutrient-

or metal-rich wastewaters. A wide range of nutrient reduction in wastewaters containing water

hyacinth has been reported. For inorganic N, Reddy et al. (1982) reported a reduction of about

80%, while Sheffield (1967) observed a 94% reduction. For ortho-P, a 40-55% reduction was

reported by Sheffield (1967). For total P, Reddy et al. (1982) measured about 32% reduction,

while Ornes and Sutton (1975) achieved a much higher removal rate of 80% in their treatment









pond. In a pilot scale study using a series of six tanks with water hyacinth for wastewater

treatment, the mean decrease in total N and total P in the effluent as it flowed the six tank series

was 27.6% and 4.48%, respectively (Bramwell and Devi Prasad, 1995). A pond containing water

hyacinth, with an air stripping unit and a flocculation and settling unit, was reported to remove

>99% ortho-P, 99% nitrate-N, and >99% ammonia-N (Sheffield, 1967). Plant uptake contributes

a large proportion to the N and P removal for very high uptake rates have been reported, for

instance, 1980 kg N and 322 kg P hal yl by Boyd (1970), 2500 kg N and 700 kg P hal yl by

Rogers and Davis (1972), and up to 53 50 kg N ha-l y^l and 1260 kg P hal yl by Reddy and

Tucker (1983).

Although at a lower rate compared to such large-leaved floating species as water hyacinth,

small-leaved floating species such as duckweed can also remove a considerable amount of

nutrients and have been utilized in remediation of wastewaters. Small tank polycultures of

duckweed species (Lemna minor and Spirodela polyrhiza~) were found to remove 404 mg N m-2

day-l (1460 kg N ha-l yr- ) and 84 mg P m-2 day-l (307 kg P ha-l yr- ) from dairy barn wastewater

(Whitehead et al., 1987). Phosphorus removal rates of 60-92.2% were achieved in a wastewater

system utilizing Lemna gibba (Hammouda et al., 1995). Two species of Azolla (Azolla

filiculoides and Azolla pinnate) removed N from mixed waste water resulting in more than 50%

decrease in concentration (Elsharawy et al., 2004).

According to Ruan et al. (2006), polluted river water was efficiently treated by pilot-scale

constructed wetland systems planted with emergent aquatic plants, Typha latifolia and Scirpus

lacustris, with mean NH4-N removal rates of over 85%. Wetlands with emergent macrophytes

were reported to remove P at rates from 0.4 to 4.0 g m-2 -1l, with more eutrophic systems

achieving higher removal rate (Mitsch, 1992).









Tatrai et al. (2005) observed an increase in transparency and a decrease in the

concentrations of P simultaneously with increased presence of submerged macrophytes in the

lake.

Aquatic plants also demonstrate tremendous potential in metal accumulation and removal

from the surrounding waters. Free water surface and subsurface flow pilot-size wetlands were

constructed to treat highway runoff with metal removal rates of 47%, 23%, 33%, and 61% for

Cu, Ni, Pb and Zn, respectively, with their respective two-year mean concentrations of 56, 114,

49 and 250 Clg L^1 (Terzakis et al., 2008). Azolla filiculoides removed 91.0, 41.5, 82.5, 37.7, 12.1,

46.7 and 67.2% of the initial Fe, Zn, Cu, Mn, Co, Cd and Ni, respectively from mixture of waste

waters, while Azolla pinnata removed 92.7, 83.0, 59.1i, 65.1, 95.0, 90.0 and 73.1%, respectively

(Elsharawy et al., 2004). Although all three plants, water lettuce (Pistia stratiotes L.), duckweed

(Spirodela polyrrhiza W. Koch), and water hyacinth (Eichhornia cra~ssipes) demonstrated high

removal rates of Fe, Zn, Cu, Cr, and Cd (>90%) without reduction in growth, water hyacinth

were the most efficient followed by water lettuce and duckweed (Mishra and Tripathi, 2008).

Many researchers have reported that high heavy metal concentrations (Cu, Cd, Mn, Pb, Hg, etc.)

were measured in the tissues of aquatic plant growing in waters with elevated metal

concentrations and no toxic effects or reduction in plant growth were observed (Badr and Fawzy,

2008; Mishra et al., 2008; Okafor and Nwaj ei, 2007).

Common duckweed and water hyacinth have been reported to be the top species as Cd

accumulators (Wang et al., 2002; Zayed et al., 1998; Zhu et al., 1999). Both Salvinia herzogii

and Pistia stratiotes efficiently removed Cr from water at the concentrations of 1, 2, 4, and 6 mg

Cr L^1 (Maine et al., 2004). Lead concentrations in plant tissue (mg kg- ) were found to be 1621

and 1327 times those in the external solution (mg L^1) for C. demersum and C. caroliniana,










respectively (Fonkou et al., 2005). Salvinia minima has been reported as a hyperaccumulator of

Cd (Olguin et al., 2002) and Pb (Olguin et al., 2005) with bioconcentration factors (metal

concentration in plant tissue over that in external solution) of approximately 3000 for both heavy

metals.

Stormwater Treatment with Floating Aquatic Plants

To enhance the performance of stormwater detention ponds, aquatic plants are often

planted. Biomass production, growth rate, and easiness of management and harvest are the

considerations that should be taken into in selecting aquatic plants.

Floating aquatic plants can grow in a vertical as well as horizontal direction, thereby

increasing the photosynthetic surface area. In addition, unlike submerged species, they

photosynthesize in an aerial environment where CO2 is not a constraining factor and water

supply is abundant. All these factors together make floating aquatic plants, especially large-

leaved species, one of the earth' s most productive communities. Their annual primary production

was estimated to be up to 85 Mt in dry matter per hectare in subtropical and tropical regions

(Westlake, 1963). Floating plants are more favorable in terms of energy and machinery use in

management. In addition, harvesting floating plants causes minimal disturbance to the system,

thus reducing sediment re-suspension.

Water hyacinth is a free-floating vascular aquatic plant found throughout the tropical and

subtropical regions of the world (Holm et al., 1969). It extracts nutrients from the water through

a system of fine, feathery roots. Water hyacinth is one of the earliest and most widely used

floating aquatic plants with extensive publications on its biomass production, growth rate,

nutrient uptake dynamic and ability. According to Knipling et al. (1970), the harvesting of one

acre of water hyacinths would remove 170 kg of N and 60 kg of P from Lake Alice in

Gainesville, Florida.










Compared to water hyacinth, the other large-leaved free-floating aquatic plant, water

lettuce, has a lower nutrient uptake capacity and lower nutrient concentration. For example, the

N, P and ash contents of biomass were about 1.5 times higher in water hyacinths than in water

lettuce (Aoi and Hayashi, 1996). However, when considering management, the smaller biomass

of water lettuce renders an easier removal of biomass from water bodies.

Biomass yields of small-leaved floating plants such as Salvinia, Lemna, and Azolla are

significantly lower than those of large-leaved species, which makes these plants unsuitable for

monoculture systems. But they were reported to have high P removal capacity (Sutton and

Ornes, 1975) and low light requirements (Wedge and Burris, 1982). Reddy and DeBusk (1985)

suggested they be integrated into treatment systems based on large-leaved species to improve

overall nutrient removal efficiency.

Growth Factors of Aquatic Plants

For a phytoremediation system to work efficiently, optimal plant growth is the key. Many

environmental factors can influence plant growth and its performance, such as temperature,

nutrient concentration, pH, solar radiation, and salinity of the water. The weight and size of

aquatic plants are a function of these factors. For example, growth of water hyacinth plants

cultured in nutrient solution were significantly influenced by the seasonal changes in temperature

and solar radiation, shorter time was required to reach maximum biomass yield in summer with

high growth rate (Reddy et al., 1983). If maximum growth is obtained, one hectare of water

hyacinths could remove about 2500 kg N yr- (Rogers and Davis, 1972) and as high as 7629 kg

N ha-l yr- was reported by Reddy and Tucker (1983) for water hyacinth cultured in a nutrient

solution.

Although large-leaved floating plants such as water hyacinth and water lettuce can produce

high biomass and remove large amounts of nutrients and metals, they may not be suitable for









temperate or frigid areas due to their sensitivity to cool temperature which significantly affects

their performance (Clough et al., 1987). Instead, duckweed or azolla could be a better choice

because of their tolerance to colder weather (Reddy et al., 1983). This also explains why

pennywort removed 20% more N and 30% more P from primary domestic effluent than water

hyacinth during the winter in central Florida (Clough et al., 1987).

Nutrient availability affects the growth and performance of aquatic plants. Within the

studied nutrient concentration ranges, mean number of ramets, mean height and total biomass of

water hyacinth significantly increased with increasing nutrient level (Zhao et al., 2006). A 200-

fold difference in dry weight of water lettuce was reported by Aoi and Hayashi (1996) between

cultivated in rain water and treated sewage water. Similar to terrestrial species, aquatic plants

respond positively to nutrient concentration increases up to a certain point followed by no further

response or a negative response. Five and a half mg N per liter and 1.06 mg P L^1 were such

points reported for water hyacinth growth (Reddy et al., 1989; Reddy et al., 1990), while 20 mg

N L^1 and 2 mg P L^1 were found for Salvinia molesta (Cary and Weerts, 1984). Not only nutrient

concentration itself, but also ratios between different nutrients play an important role in plant

growth. It was reported that the highest production of water hyacinth occurs when the N:P ratio

in the water was close to 3.6 (Reddy and Tucker, 1983).

Stormwater varies in salinity which may have significant effects on aquatic plants' growth

and performance. Utilization of such invasive aquatic plants as water hyacinth and water lettuce

has its advantages as discussed above and its concern of plant escape from the detention systems

into the lagoons or estuaries. Knowledge on salinity tolerance of candidate plants) can help

better utilize the plants) without bringing disaster. Salt concentrations of 1660 and 2500 mg kg-l









(equivalent to 2683 and 4040 CIS cm l) were reported to have toxic effects on water lettuce and

water hyacinth, respectively (Haller et al., 1974).

pH plays a role in plant growth directly by hydrogen ion (H ) injury at low pH and

indirectly by affecting availability and toxicity of mineral elements(Pessarakli, 1999). Generally,

plant grows best in the pH range of 5.5-7.0. Optimum pH ranges 6.5-7.5 and 5.8-6.0 were

reported for water hyacinth (El-Gendy et al., 2004; Hao and Shen, 2006). Macroalga Chlorella

sorokmniana grew best at pH 7-8 (Moronta et al., 2006).

Research Objectives

Taking their high biomass production and easiness in management into consideration, free

floating aquatic plants were chosen for my dissertation studies. Compared to water hyacinth,

water lettuce has been overlooked with little investigation. Compared to large-leaved floating

plants, small-leaved floating plants such as azolla (Azolla filiculoides and Azolla pinnata),

duckweed (Spirodela polyrrhiza W. Koch), and common salvinia (Salvinia minima) produce

much less biomass, which is a disadvantage for application to phytoremediation (Reddy and

Bagnall, 1981; Reddy, 1984). But it was also shown that these small-leaved floating plants have

a narrower N/P ratio indicating they are efficient in removing P (Reddy and DeBusk, 1985). It

was suggested that small-leaved floating plants can be included in polyculture systems with

large-leaved plants (Reddy and DeBusk, 1985). Among the small-leaved aquatic plants, common

salvinia has been shown to produce dry biomass twice that of duckweed when cultured in

nutrient solution (Olguin et al., 2002) and outcompete duckweed for growth surface in a mixed

culture (Olguin et al., 2007). Common salvinia was also reported to be capable of removing over

70% of NH4-N and PO4-P from coffee processing effluent (Olguin et al., 2003). It was of our

interest to compare water lettuce and common salvinia in terms of their nutrient uptake ability










and determine the possibility of include common salvinia in a polyculture system with water

lettuce.

It is critical to select appropriate plants for water treatment taking into account the

characteristics of the water to be remediated. The overall objective of this study was to evaluate

water lettuce's nutrient and metal removal potential in stormwater detention ponds and its

growth response to environmental factors. Specific obj ectives addressed in this dissertation

include :

* Evaluation of water lettuce for its potential in N and P removal from stormwater;

* Investigation of water lettuce regarding its metal accumulation ability and mechanism, and
metal distribution in the plant;

* Determining N requirements of water lettuce and common salvinia for both net and
maximum growth;

* Determining P requirement of water lettuce and common salvinia for both net and
maximum growth;

* Assessing the effects of salinity on the growth of water lettuce;

* Assessing the effects of pH on the growth of water lettuce.









CHAPTER 2
NUTRIENT REMOVAL POTENTIAL OF WATER LETTUCE (PISTIA S7RATIOTES L.)
FROM STORMWATER IN DETENTION SYSTEMS

Introduction

Chemical fertilizers have been playing a very important role in agricultural production in

the modern society. Because of crops' quick response to chemical fertilizers, to many farmers,

fertilizer application seems to be the only guarantee of high crop yield. But the ever increasing

use of fertilizer results in significant build-up of nutrients, such as nitrogen (N) and phosphorus

(P), in the soils (Smith et al., 2007). These nutrients are subj ect to loss to surface and ground

water. Water quality is impaired and water availability is reduced because of accelerated

eutrophication (Carpenter et al., 1998).

Estuaries are among the most biologically productive ecosystems in the world. The St.

Lucie Estuary (SLE), rich in habitats and species, is one of the largest and most ecologically

diverse estuaries located on the central east coast of Florida and a maj or tributary to the Indian

River Lagoon (IRL). Surrounded by a rapidly growing human population, its health has been a

concern for years due to growing pressures from anthropogenic sources of nutrients and

pollutants (Chamberlain and Hayward, 1996; Phlips et al., 2002). Results from recent monitoring

study in IRL by He et al. (2006b) indicate that more than 50% of the surface runoff water

samples contained TN of 1 to 5 mg L^1 and TP above 1.0 mg L^1. Mean concentrations of TN and

TP in the runoff were 4. 1 and 1.6 mg L^1, respectively, which are much greater than the USEPA

critical levels for surface water (1.5 mg L^1 for total N and 0.1 mg L^1 for total P) (U. S.

Environmental Protection Agency., 1976). The intricate network of Canals C-23, C-24, and C-

44, that drain the surrounding urban and agricultural lands in the St. Lucie Basin and are

connected to the IRL, are estimated to collectively deliver at least 8.6xl05 kg of N, 9. 1 x10' kg of

P, and 3.6xl0s kg of suspended solids to the estuary annually (Graves and Strom, 1992).









Best management practices (BMPs) have been implemented to reduce N and P export from

urban area and agricultural field and approximately 10-15% reduction may be realized based on

our previous BMPs proj ect (He et al., 2005). This reduction is still far below the goals (30-70%

reduction in N and P) established in the Surface Water Improvement and Management Plan

(SWIM Plan) (SFWMD and SJRWMD, 1994) for the St. Lucie Estuary watershed. The

stormwater needs to be further treated before it is dischargeable to the St. Lucie Estuary.

Large constructed wetlands or stormwater treatment areas have been operating since early

1990's to filter nutrients in eutrophic stormwater from Everglades Agricultural Area (EAA)

before they are drained into water conservation area in the Everglades National Park. Stormwater

detention systems are to be constructed in the Indian River area for cleaning up nutrients and

pollutants in stormwater from agriculture and urban area. Key to the performance of the

constructed wetlands including STAs, water detention systems, and retention ponds is the

establishment and sustainability of desired vegetation communities.

The primary obj ectives of this study were to evaluate the effectiveness of water lettuce

(Pistia stratiotes L.) in removing nutrients including N and P from stormwater in the constructed

water detention systems and to quantify the potential of this plant in improving stormwater

quality in detention pond system.

Materials and Methods

Experimental Design

Two stormwater detention ponds (called the West Pond and the East Pond), located to the

west and east side, respectively, of the University of Florida, Indian River Research and

Education Center (IRREC) Facility in Fort Pierce, were selected for this study (Figure 2-1). The

East Pond has an area of approximately 2500 m2 and the West pond, approximately 5000 m2.

The West Pond receives stormwater from IRREC teaching gardens. The land surrounding the









East Pond was used for citrus production but has been left fallow since the occurrence of canker

five years ago. Besides receiving stormwater from the fallow land, the East Pond also receives

stormwater from the ditch along Kings Highway.

For each pond there were two plots, i.e. the control (without plants) and the treatment plot

(with plants), which were separated from each other and from the rest part of the pond by a soft

wall made of weather-resistant plastic material, which allows only water and dissolved ions to

pass through. The bottom of the soft wall was inserted into the sediment by an impregnated

stainless iron chain and its top was floated on water surface by means of a wrapped foam bump.

The height of the soft wall is equal to the maximum water depth (2 m and 3 m in the East and

West Pond, respectively) of the plot site when the pond is full of water. Therefore, the height of

soft wall can change according to water level. Each plot had an area of 72 m2 (12 m x 6 m).

Water lettuce (Pistia Stratiotes) was selected for this study because of its high yield

potential and high uptake capacity for nutrients. Due to low levels of N and P in the two ponds at

the time of proj ect implementation, known amounts of N and P were spiked in both plots before

water lettuce was planted into the treatment plots. Water lettuce was transplanted in the treatment

plot of each pond on August 22, 2005, and was maintained to cover three-fourth of water surface

of the plot. Known amounts of N and P were also spiked on January 27 and September 5, 2006

because of low N and P concentrations in both ponds without receiving stormwater for a certain

period. For each spiking, the same amounts of the same nutrient (N or P) were added into both

the control and treatment plots for each pond.

Sampling of water and plant from both plots began after the full establishment of the plants

in the treatment plots, which was approximately two months after the experimental set up.

























































Figure 2-1. Experimental set up in the West Pond and the East Pond.







30









Water samples were collected weekly from the control and the treatment plots for two and

a half years (September 2005-March 2008) and analyzed for water quality parameters, including

total P (TP) and total Kj eldahl N (TKN), NO3-N, NH4-N, ortho-P, pH, EC, turbidity, and total

solids (TS).

Water lettuce was sampled monthly from the treatment plots. After being rinsed

thoroughly with deionized water to remove adhering materials and blotted dry, root and shoot

were separated and their fresh weights were recorded. Plant samples were oven-dried at 700C for

three days and then pulverized to <1 mm with a 4-Canister Ball Mill (Kleco Model 4200, Kinetic

Laboratory Equipment Company, Visalia, CA) prior to analysis for N and P concentrations in

both root and shoot.

Besides monthly sampling, plants were also periodically harvested to maintain three-fourth

coverage of the water surface of the treatment plot. For each harvest, the total fresh weight of

plant was recorded, plant moisture and nutrient (N and P) concentrations of plant root and shoot

were determined, and total amount of dry biomass yield was calculated. Total amount of

nutrients removed from the water by the harvested plant biomass was quantified as the sum of

the amounts in both root and shoot. The amount of nutrients (N, P) in root or shoot was the

product of root or shoot dry biomass yield and the nutrient concentration in that plant part.

Chemical Analysis

Prior to filtration, pH and EC of the water samples were determined using a

pH/ion/conductivity meter (pH/Conductivity Meter, Model 220, Denver Instrument, Denver,

CO) following EPA method 150.1 and 120.1, respectively. Turbidity was measured using a

turbidity meter (DRT-100B, HF Scientifie Inc., Fort Myers, FL) on the unfiltered water sample.

Total solids in unHiltered water samples were measured using a gravimetric method at 105oC

(EPA 160.3). Total P in the unHiltered water sample was determined by the molybdenum-blue









method after digestion with acidified ammonium persulfate (EPA method 365.1). As dissolved N

and P were of primary interest in the phytoremediation study, sub-samples of the water were

filtered through a Whatman 42 fi1ter paper for TKN measurement, in which the fi1trate was

digested with acidified cupric sulfate and potassium sulfate and NH4-N concentration in the

digested solution was determined following EPA method 351.3 using an N/P Discrete Analyser

(Easychem Plus, Systea Scientific, LLC, Illinois, USA). Portions of the sub-samples were further

filtered through a 0.45 Clm membrane filter for the measurement ofNH4-N, NO3-N, total

dissolved P (TDP), and PO4-P. Concentrations of NO3-N and PO4-P were measured within 48 h

after sample collection using an ion chromatography (DX 500; Dionex Corporation, Sunnyvale,

CA) following EPA method 300.0. Concentration of NH4-N in water samples was determined

using an N/P Discrete Analyser (Easychem Plus, Systea Scientific, LLC, 1L) following EPA

method 351.3. Total dissolved P in water was determined using inductively coupled plasma

optical emission spectrometry (ICP-OES, Ultima, JY Horiba Inc. Edison, NJ) following EPA

method 200.7.

Plant N concentration was determined using a CN analyzer (vario Max CN, Elementar

Analysensystem GmbH, Hanau, Germany). Subsamples (each 0.400 g) of plant material were

digested with 5 mL of concentrated HNO3 in digestion tube using a block digestion system (AIM

500-C, A.I. Scientific Inc., Australia), and P concentration in the digested solution was

determined using the ICP-OES.

Data Treatment and Data Analysis

As very high concentrations of N and P were measured in the water after fertilizer spikes,

data from the month following each nutrient spike were discarded and not included in graph or

data analysis.









At times when N or P concentration in water was so low that it was below detection limit,

half of its method detection limit (MDL) was used in graph or in calculation (USEPA, 2006).

Differences between control and treatment were tested using the MEANS procedure in

SAS software (SAS Institute, 2001). All statistical analysis tests were performed using a

significance level of 5%.

Results and Discussion

General Water Quality Improvement

Total solids and turbidity in the waters of both plots varied seasonally: increasing during

the rainy season from late May to mid-November and decreasing during the dry season from

mid-November to late May with values in the rainy season being several times higher than those

in the dry season (Figures 2-2 and 2-3). The increase in these parameters during the rainy season

was likely due to the input of stormwater, which carried soil particles and solutes, including

nutrients.

The growth of water lettuce improved water quality by significantly decreasing TS and

turbidity in water of the treatment plots (Table 2-1). Total solids in the water column was

decreased by an average of approximately 20% in the treatment plots, as compared with that in

the control plots (Figure 2-2) due to better particle sedimentation in the plant-growing plots (Brix,

1997). In addition, the presence of plants decreased water disturbance by wind, thus reducing

sediment resuspension. On average, water turbidity was reduced by approximately 65% in

treatment plot as compared to the control in both ponds (Figure 2-3). Water lettuce growth

blocks available sunlight for algae and phytoplankton growth, which, together with

sedimentation, contributes to clearer water (Figure 2-4). The much larger decrease in turbidity

than TS indicated that algae and phytoplankton contributed a high proportion to water turbidity

while minimal to TS due to their negligible biomass weight.


















1.6 *East Control
o East Treatment*


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~o West Treatment
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0.2

S0200 0~ -









Sampling date



Figure 2-2. Total solid concentrations in the waters of the East and West Ponds.


















*East Control
120 o East Treatment *

100-







60 -





20k~ -E.~L






in iSampling date






1 10

*West Control
12 West Treatment

100-


80 -1


60-

S40-



20










Sampling date



Figure 2-3. Turbidity in the East and West Ponds.










Table 2-1. Water quality improvement in the treatment plots of the East and West Ponds (time period: 9/13/2005-3/28/2008a), n=122).
Total dissolved Total Kjeldhal
Location Treatment Turbidity Total Solid PO4-PP Total P NO3-N NH4-N

NTU g L' ------------------------------------------------- mg L' -----------------------------------------------
East Pond Control 20.5119.7ab) 0.41310.295a 0.13210.597a 0.12310.215a 0.410+1.056a 0.01510.025a 0.099 0.138a 1.78 0.72a
Remediation 6.8013.91b 0.33010.238b 0.05510.108b 0.08710.144b 0.22910.441b 0.00710.009b 0.069 0.126b 1.4010.74b
Reduction 67% 20% 58% 29% 44% 52% 31% 21%
West Pond Control 21.4116.2a 0.26010.211a 0.22010.369a 0.27710.485a 0.45810.687a 0.02210.046a 0.080 0.132a 1.0510.63a
Remediation 7.2514.90b 0.21210.175b 0.15710.331b 0.22810.478b 0.32010.533b 0.00610.005b 0.044 0.044b 0.75+0.44b
Reduction 66% 18% 29% 18% 30% 72% 45% 29%
a) Not include data from Jan. 25 to Feb. 27 and from Sept. 8 to Oct. 8 of 2006, each of which was about one month after fertilizer spike.
b) Data shown are mean + standard deviation; different letters following the numbers denote significant difference between the means
of control and treatment.
































Figure 2-4. Water samples from treatment plot and control plot.

Water lettuce growth decreased water EC in the treatment plot of both ponds (Figure 2-5),

due to salt removal from the waters by plant uptake or root adsorption. Compared to the West

Pond, the EC of water from the East Pond was higher (close to 2000 CIS cml in some seasons)

with large fluctuations. The reason could be due to the fact that besides receiving stormwater

from the fallow land, the East Pond also received stormwater from the ditch along Kings

Highway and runoff from roads with heavy traffic was reported to be enriched with salts

including heavy metals (Gan et al., 2008; Terzakis et al., 2008).

High pH (8.5-9.7) was measured from April to June in the control plots (Figure 2-6).

Water sampling was performed at approximately 2 pm when solar radiation was strong and

temperature was high. Therefore, the increase in pH in the control plots compared to the

treatment plots might result from the photosynthetic activity of periphyton and phytoplankton

communities or algae which depleted dissolved CO2 fTOm the water and raised water pH (Reddy



































































































Figure 2-5. Water EC in the East and West Ponds.


Sampling date


*West Control
o West Treatment

















~~ ~Fo ~P8b~m~g~O


and DeLaune, 2008). A pH value as high as 9.5 in the afternoon was documented in an aquatic


system containing algae by Reddy and Patrick (1984).


East Control
o East Treatment










o

99

0rbr~'


-

-

-

-

-

-

-

-


10 -
0-

10 -
0-

10 -
10-

10 -
10-

10 -

10 -
0-


2200

2000

1800

1600

1400

oE1200

S1000

800

600










200

200










180

160

140

oE120

100

80

60

40

20


cl cl


Sampling date
















East -Control
o8 East Treatment
9-9

8* *
o+o




6- Go o







100



8~ o. .













Sampling date




Nitrogen~~~ andt P onenrain edcto

Changes~~~~~~~~~~ ofNH4-N NO-,TN P4P D, n Pi atmerorthpeido


September~~~ 200 to Mac 08aesoni iue2- oFgr -2 hi vrg










concentrations were calculated and shown in Table 2-1. Like TS and turbidity, nutrient

concentrations in the waters showed seasonal changes during the year, which were affected by

external inputs from stormwater. Higher NH4-N concentration than NO3-N in both ponds may

indicate atmospheric input of NH4-N (Pauziah Hanum et al., 2009).

Although there are many reports showing that aquatic plants, such as Salvinia molest and

Elodea densa, preferred NH4-N to NO3-N (Reddy et al., 1987; Shimada et al., 1988) and

theoretically NH4+ uptake is energetically more efficient than that ofNO3-, reduction rate of

NH4-N (31 and 45% in the East and West Pond, respectively) was smaller than that of NO3-N

(52 and 72% in the East and West Pond, respectively) in both ponds. Besides plant uptake,

denitrification may also contribute to the decreased NO3-N concentration in the treatment plots

as a more anaerobic condition (dissolved oxygen < 1.5 and 0.7 mg L^1 in the East and West

Pond, respectively) at water surface was created by the growing plants. Other anaerobic micro-

sites may also contribute to NO3-N removal through denitrification (Gumbricht, 1993; Reddy,

1983).



Es Wst Cotro
S5-yena et02 -O Ws Treatment
020.1 020-1 *
015- 015-






ooSaplm date Sapn date





Figure 2-7. Nitrate-N in the waters of the East and West Ponds.


















*East Control
o East -Treatment









~ *
e

6. ** o'
i~Qc~p* *Lf~~ei


West Control
o West Treatment











8


1.0 -


0.8


oo0.6


0.4


0.2


0.0 -


Sampling date


CO CO
O O O
O O O


4~4
o o


1.2 -


1.0 -


0.8 -





0.4 -


0.2 -
-.


Sampling date


g g


Figure 2-8. Ammonium-N in the waters of the East and West Ponds.


W 0
















*East Control *
5 -1 1 East Treatment



o o

3- O



0- W o




Sampling dat


West Control
5 -; ~~ Q Wet retmn





o o





O 0~ O




Sampling date



Figue 29. otalKiedha N n th waersof te Est nd est- Ponds.


















*East Control
o East Treatment









*'


*cm,
nmnannmn mmranrlann~lgO


o o o o o o o o o

Sampling date



Figure 2-10. Water PO4-P in the East and West Ponds.


2.0 -



1.5



S1.0 -



0.5 -

0 -


(D (D
O O O
O O O
e~e
o o

Sampling date


2.5



2.0



1.5







0.-


0.0


CO CO
O O O
O O O


B~e


*West Control
~o West Treatment














o oo aI-IIIIIIII


















3.0 *East Control
o East Treatment

2.5


S2.0-


1.5-
1.o


S0.5



-0.0







Sampling date







*West Control
3.0-
~o West Treatment




S2.0 -




1 .50. -














Sampling date




Figure 2-11i. Total dissolved P in the waters of the East and West Ponds.

















*East Control
4 -1 1 East Treatment


oo 'o

3-8












Sampling date







4 o o Wes Tramn














Sampling date





Figure~ ~ ~ ~ ~~~~~~Wet 2-2 otlPintewteso teEs adWstPns










Inorganic P (PO4-P) removal (58 and 29% in the East and West Pond, respectively) was as

efficient as inorganic N (NH4-N and NO3-N) in the remediation plots of both ponds (Table 2-1).

Sheffield (Sheffield, 1967) measured a much higher reduction rate (94%) in inorganic N than

ortho-P (40-55%) in a water hyacinth system. Total P had a higher reduction than total dissolved

P (Table 2-1), which indicates that the role aquatic plants play in such a remediation system is far

more than uptake. More importantly, the aquatic plants play a crucial role by providing

additional surface and favorable environment in the root zone for microorganisms to grow and

involve in a variety of complex chemical, biological and physical processes, such as nitrification,

that contribute to the removal and degradation of nutrients, which was considered the most

important functions of aquatic plants (Brix, 1997). A higher removal rate in total P than in

dissolved total P may result from the additional sedimentation effect of plant growth on

particulate P.

Nitrogen and P Removal Potential by Plant Uptake

Nitrogen and P concentrations in the plant were averaged 17 and 3 g kg- respectively,

with N concentration being higher in root than shoot (Figure 2-13) but only a minimal difference

in P concentration between root and shoot (Figure 2-14). Nitrogen and P concentration typically

averaging 15-40 g N and 4-10 g P kg-l for such large-leaved floating plants as water lettuce and

water hyacinth (Eichhornia cra~ssipes) (Aoi and Hayashi, 1996).

Annual removal of N and P by water lettuce were 190 and 25 kg ha- respectively in the

East Pond, and 329 and 34 kg ha- respectively in the West Ponds, with dry matter being

approximately 9 (the East Pond) and 15 Mg ha-l (the West Pond). Research has also been

conducted on another invasive, large-leaf floating aquatic plant, water hyacinth (Eichhornia

cra;ssipes). Very high uptake rates have been reported, for instance, 1980 kg N and 322 kg P hal











yr- by Boyd (1970), 2500 kg N and 700 kg P ha-l yrl by Rogers and Davis (1972), and up to

5350 kg N ha-l yr- and 1260 kg P ha-l yr- by Reddy and Tucker (1983). Reasons behind this big

difference in nutrient uptake rate between this study and those in the literature include: 1) water


hyacinth has a higher nutrient uptake and biomass yield potential than water lettuce, 2) previous

studies were conducted using nutrient solution with nutrient concentrations much higher than

those in the stormwater detention ponds, thus resulting in higher removal rates, and 3) the high


reported values were based on short-term experiments and extrapolated to one year, which often

overestimates the nutrient uptake rate of the plant. As a 1.5-fold difference was reported by Aoi

and Hayashi (Aoi and Hayashi, 1996), the much lower nutrient uptake values from this study

also indicated that the water lettuce in the stormwater detention ponds was far from reaching its

maximum nutrient uptake potential.

Table 2-2. Annual removal amounts of plant dry biomass, N, and P from the East and West
Ponds.
Location Dybiomass N P
Mg ha ------------------ kg ha-' -------------------
East Pond 9 190 25
West Pond 15 329 34



East -Rootl west -Root
So East -Shoot o West -Shoot
40-, 40-

S30- 30-Oo

S20 oe **2
o o 000 oo oo a oa*
lo-0 10 o ag *0





Samplmg date Samphng date



Figure 2-13. Nitrogen concentrations in plant roots and shoots from the East and West Ponds.
















S *East -Root
o East -Shoot




8 o
'fo oo


4 o ao *

o o
oo o
O







Sampling date




*West Root
~o West Shoot

7-1 *

6- o


e- o
.o oe
S4- O O


3-~ O 0
2- O **




8 8 8 8 8 8 8



Sampling date


Figure 2-14. Phosphorus concentrations in plant roots and shoots from the East and West Ponds.


Physiological Limits


Plant growth is influenced by many environmental factors such as solar radiation and


temperature, and thus nutrient removal efficiency, as reflected in both nutrient concentrations in









the plant and biomass yield of water lettuce, showed strong seasonal dependence (Figures 2-13

and 2-14). This seasonal variation in plant growth and thus nutrient removal capacity was also

discussed by Reddy and Sutton (1984). They stated that in Florida, 50% of the annual biomass

yield was produced from May to August and 34% from March to April and from September to

October.

The West Pond worked better than the East Pond in removing N and P from the waters

(Table 2-2) by producing a much higher biomass, which could be related to the differences in

total dissolved organic carbon (averages of 30 and 12 mg L^1 in the East and West Ponds,

respectively) and EC of the waters (Figure 2-5, 180-2000 and 100-400 CIS cm-l in the East and

West Ponds, respectively) between the two ponds. It was reported that an EC of 2683 CIS cml

was toxic to water lettuce (Haller et al., 1974). High EC in the East Pond negatively affected

water lettuce's growth, leading to less nutrient removal from the water.

System Management

For efficient water treatment, some aquatic macrophyte biomass must be removed from

water bodies to keep an optimum plant density (0.2-0.7 kg dry biomass m-2 was suggested by

Reddy and DeBusk, 1984). If not harvested, the vast maj ority of the nutrients that have been

incorporated into the plant tissue would be returned to the water by the decomposition processes

(Brix, 1997). It was shown that more intensive management with more frequent and timely

harvest of plant biomass usually leads to a higher nutrient removal rate (DeBusk and Reddy,

1991). In Florida during the wet season when temperature is also favorable for water lettuce

growth, plants should be harvested every other week to maintain about three-forth coverage of

the water surface (DeBusk and Reddy, 1991).

Harvested plant biomass, rich in nutrients and organic matter, can be used as a soil

amendment, processed into livestock feed, or converted to methane (Reddy and Sutton, 1984).









Conclusions

Water lettuce worked well in such low nutrient systems as stormwater detention ponds.

Water quality in both ponds was improved, as evidenced by significant decreases in turbidity,

total solids and nutrient concentrations. Inorganic N (NH4-N and NO3-N) concentrations in

treatments plots were more than 30% lower than those in the control plots (without plant). TKN

was reduced by more than 20%. Reductions in PO4-P, TDP, and total P were approximately 18-

58%, as compared to the control plots. By periodic harvesting, water lettuce removed 190-329 kg

N ha-l yr- and 25-34 kg P ha-l yr- from the waters.









CHAPTER 3
METAL REMOVAL POTENTIAL OF WATER LETTUCE (PISTIA S7RA TIOTES L.) FROM
STORMWATER INT DETENTION SYSTEMS

Introduction

Intensive use of commercial fertilizers, liming materials and agro-chemicals in agriculture

has resulted in heavy metal accumulation in the soils. Significantly higher concentrations of

extractable Cu, Zn, Mn, Fe, Co, and Cr than those in forest soils (nonagricultural soils) were

measured in soils from eleven field sites (seven at commercial citrus groves and four at vegetable

production farms) in St. Lucie and Martin Counties, Florida (He et al., 2004). High

concentrations of Cu and Zn were measured in storm runoff water from these production systems

(He et al., 2006a; Zhang et al., 2003). Although median concentrations of Cd, Cu, Pb, and Zn in

Ten Mile Creek, a maj or tributary of the Indian River Lagoon (IRL), were below U. S. EPA

drinking water critical levels and the threshold levels recommended for aquatic organisms, their

individual pulse concentrations were above U.S. EPA recommended limits (Yang et al., 2008).

Accumulation of Zn and Cu in the sediments of the St. Lucie Estuary has also been reported

(Haunert, 1988; He et al., 2003).

There are extensive studies on metal accumulation by aquatic plants. The aquatic plants

include floating plants, such as Salvinia herzogii (Maine et al., 2004), water hyacinth

(Eichhornia cra~ssipes) (Mishra et al., 2008; Muramoto and Oki, 1983), duckweed (including

Lemna polyrrhiza L., Lemna minor, and Spirodela polyrrhiza W. Koch) (John et al., 2008;

Mishra and Tripathi, 2008), mosquito fern (Azolla pinnata R. Brown) (Mishra et al., 2008), and

water lettuce (Pistia stratiotes) (Maine et al., 2004; Mishra et al., 2008), emergent plants such as

common cattail (Typha latifolia) (Manab Das and Maiti, 2008), and submerged plants, such as

pondweed (Potttttttttttttttttamgton pectinatus or Potttttttttttttttttttaogeo crispus) (Badr and Fawzy, 2008; Mishra et

al., 2008), hydrilla (hydrilla verticillata) (Bunluesin et al., 2004; Mishra et al., 2008), and










coontail (Ceratophyllum demersum L.) (Badr and Fawzy, 2008; Bunluesin et al., 2004).

Interested metals accumulated by these aquatic plants were mainly micronutrients or heavy

metals, namely, Fe (Almeida et al., 2006; Manab Das and Maiti, 2008), Mn (Mishra et al., 2008;

Vardanyan and Ingole, 2006), Cu (Almeida et al., 2006; Badr and Fawzy, 2008), Ni (Manab Das

and Maiti, 2008; Vardanyan and Ingole, 2006), Co (Vardanyan and Ingole, 2006), Zn (Manab

Das and Maiti, 2008; Vardanyan and Ingole, 2006), Cd (Badr and Fawzy, 2008; Bunluesin et al.,

2004; Mishra et al., 2008), Hg (Mishra et al., 2008; Molisani et al., 2006), Cr (Almeida et al.,

2006; Mishra and Tripathi, 2008), Ti (Vardanyan and Ingole, 2006), Ba (Vardanyan and Ingole,

2006), and Pb (Almeida et al., 2006; Badr and Fawzy, 2008; John et al., 2008).

Most studies were conducted in laboratory or greenhouse settings using metal-enriched

nutrient solutions (Bunluesin et al., 2004; John et al., 2008; Maine et al., 2004; Mishra and

Tripathi, 2008). Results from these studies were usually very impressive with high metal uptake

or accumulation (>90%, Mishra and Tripathi, 2008). However, it may be entirely different when

these aquatic plants are applied to field condition such as lakes, reservoirs, and estuaries where

both metals and nutrients are of much lower concentrations and other environmental factors are

far less favorable. On the other hand, the performance of aquatic plants in natural water bodies is

more meaningful as degradation of natural aquatic ecosystem is a worldwide concern and yet

conventional physical or chemical treatments are not cost-effective due to the nature of non-point

source pollution.

Investigations have been conducted in natural water bodies such as lakes (Badr and Fawzy,

2008; Vardanyan and Ingole, 2006), reservoirs (Mishra et al., 2008; Molisani et al., 2006), and

estuaries (Almeida et al., 2006). But related information on man-made water bodies, stormwater

detention ponds, is minimal. Stormwater carries with it nutrients, heavy metals, and chemicals









from urban area and agricultural fields and may contribute to the degradations of aquatic

ecosystems (Casey et al., 2005; He et al., 2006b). Stormwater detention ponds are constructed to

collect and remediate eutrophic stormwater before it is discharged into water bodies such as

estuaries. Aquatic plants are useful in enhancing the water treatment performance of man-made

and natural wetland systems. Knowledge on metal removal potential is necessary for better use

of these plants for water quality improvement.

Because of the greater availability of soluble ferrous iron species in the anoxic conditions

(Ponnamperuma, 1972) and leakage of 02 frOm the roots of aquatic plants (Armstrong, 1979), Fe

tends to precipitate in the oxidized zone of root surface, forming Fe oxyhydroxides as coatings

on roots, which is often termed iron plaque and has been widely observed in aquatic plants and

terrestrial plants when subjected to flooding (Crowder and St-Cyr, 1991; Hansel et al., 2001;

Otte et al., 1989; Ye et al., 1997). Once formed, the large surface area of the iron plaque (which

is often in excess of 200 m2 -1l) prOVides a reactive substrate to sequester metals such as Zn, Cu,

and Ni (Otte et al., 1989; Taylor and Crowder, 1983b).

As the partitioning of metals on the root surface, within the root and shoot has an important

implication for predicting their potential bioavailability and/or movement upon changing

physicochemical conditions, it is of our interest to differentiate metal outside and within the plant.

In addition, such knowledge is necessary when making plant disposal decisions.

Among the methods used to extract the metals located on the external surfaces of the root,

the dithionite-citrate-bicarbonate (DCB) extraction has been shown to be the best for removing

all the external precipitate on root surface (McLaughlin et al., 1985; Taylor and Crowder,

1983a). This technique involves the use of sodium dithionite (Na2S204) aS a strong reducing

agent, sodium citrate (Na13C6H507r2H20) as a chelating agent to maintain the extracted metals in










solution and sodium bicarbonate (NaHCO3) aS a buffer. The DCB method is very efficient in

removing the iron oxyhydroxide coating without damaging root tissues (Bienfait et al., 1984;

Otte et al., 1989) or leaving considerable Fe on the surface of the washed roots as other methods

do. This method has been applied to rooted aquatic plants such as submerged and emergent

aquatic plants. No attempt has been made to apply this method to such free floating aquatic

plants as water lettuce. Also interests have been mainly on a few metals, namely Fe, Mn, Zn, and

Pb, on characterization of the iron plaque, and on the interactions between iron plaque and

metals. As DCB solution can remove not only Fe oxide and its associated metals but also metals

adsorbed on the surface, it can be used to quantify the amount of metals on the external surface

of root. In this study, we utilized the DCB method to differentiate metals outside from inside the

root, so that we have better understanding of mechanisms involved in the removal of metals by

aquatic plants.

Compared to heavy metals such as Cd, Cu, Zn, and Pb, non-heavy metals such as K, Ca,

Na, Mg, and Al are usually overlooked. Although they are not as deteriorating as heavy metals,

they also affect water quality and are factors in algal bloom. Also for recycling purpose, we need

to monitor these metals' concentrations in the plant. Therefore, the obj ective of this study was to

investigate the removal potential of both heavy metals (Fe, Mn, Zn, Cu, Cr, Ni, Pb, Cd, Co) and

non-heavy metals (K, Ca, Na, Mg, Al) by water lettuce in stormwater detention ponds and to

understand the mechanisms of metal removal by this plant.

Materials and Methods

Experimental design, weekly water sampling, monthly plant sampling, water and plant

sample preparation and processing are the same as described in the Materials and Methods

section of Chapter 2.









Chemical Analysis

For the measurement of total dissolved metal concentration, water samples were filtered

through a 0.45 Clm membrane filter and preserved at pH < 2.0 by adding concentrated HNO3

before analysis using inductively coupled plasma optical emission spectrometry (ICP-OES,

Ultima, JY Horiba Inc. Edison, NJ) following EPA method 200.7. Similar to N and P, total

amount of each metal removed from the water by the harvested plant biomass was the sum of

the amount in plant shoots and roots, which was calculated as the product of root/shoot dry

biomass yield and root/shoot metal concentration.

The plant samples (root or shoot) were oven-dried, pulverized and digested with

concentrated nitric acid, metal concentrations in the digested solution were determined using the

ICP-OES.

To differentiate metals that were absorbed into the interior of root from those attached to

the external surface of root, the DCB extraction technique was applied (McLaughlin et al., 1985;

Taylor and Crowder, 1983a). Briefly, twenty-five g of fresh roots were soaked in 450 mL of

DCB solution (containing 400 mL 0.3 mol L-1 sodium citrate, Na3C6H507r2H20, 50 mL 1.0 mol

L- sodium bicarbonate, NaHCO3, and 3 g sodium dithionite, Na2S204) at 60 oC for 20 min.

Then, the roots were removed, rinsed several times with deionized water and blotted dry before

they were oven dried, pulverized, and analyzed for metal absorbed by the root using the ICP-

OES. The DCB extract was filtered and analyzed for metal concentrations using the ICP-OES.

Metals dissolved in the DCB solution is considered as those attached to the external surfaces of

the roots by adsorption or surface deposition.

Data Treatment

When metal concentration was below detection limit, half of its method detection limit

(MDL) was used in graph or in calculation (USEPA, 2006).













Metal Concentration Reduction in Water

Figure 3-1 shows the total dissolved metal concentrations of water samples from both the

treatment and control plots of the East and West Ponds. Aluminum, Ca, Fe, K, Mg, and Na were

the main metals detected in the waters (about 0.2-50 mg L^)~. Copper, Mn, Ni, and Zn were of

very low concentrations. As Cd, Co, Cr, and Pb concentrations were mostly below MDLs, they

were not shown in Figure 3-1. The two ponds had similar concentrations in Al, Cu, Ni, and Zn,

while the concentrations of Ca, Fe, K, Mg, Mn, and Na in the East Pond were about two times

higher than those in the West Pond, which agreed with the EC measurement (see Chapter 2).



160

Ca
140-

120-

S100-

80-

$ 60-



40


East-control East-treatment West-control West-treatment
Plot


Figure 3-1. Total dissolved metal concentrations in the treatment and control plots of the East
and West Ponds during 2005-2007 (n=122). The middle line is the median value of
the data range. The error bars represent the 5 and 95 percentile of the data. The upper
value of the box is the 75 percentile and the lower value of the box is the 25
percentile. The dots are outliners.


Results








































East-control East-treatment West-control West-treatment

Plot





Mg









- atcnrlEs-ramn etcnrlWs-ramn
*lo


-N










*atcnrlEs-ramn etcnrlWs-ramn
Pl*


20 -



15 -


o



c 0-














O

O 0












200




150


100




5 0


Figure 3-1.Continued.


." *

g 3














1.0

1.4 -.A

1.2-

1.0-

S0.8 :

S0.6

0.4-

0.2

0.0 -~g


East-control East-treatment West-control West-treatment

Plot




1.8 -*F

1.6-

1.4-

S1.2-



S0.8-

0 0.6-


0.4





East-control East-treatment West-control West-treatment

Plot


Figure 3-1.Continued.
















Cu


























I, i






East-control East-treatment West-control West-treatment

Plot


0.06


0.05


0.04


0.03


0.02


0.01


0.00








0.14

0.12-

0.10-

0.08 -

0.06-

0.04-

0.02 -

0.00-


Figure 3-1.Continued.














Ni
















East-control East-treatment West-control West-treatment
Plot



Zn


East-control East-treatment West-control
Plot


West-treatment


Figure 3-1. Continued.

Compared to the control plots, Fe, Mn, and Al concentrations in water were reduced by an

average of more than 20% by planting water lettuce. Potassium was reduced by more than 10%

in the treatment plots. Calcium, Mg, and Na concentration reduction in the water was close to

10% in the East Pond and about 5% in the West Pond as compared to the control plots.










Metal Accumulation by Plant Root

Figure 3-2 shows the metal concentration factor (CF) of water lettuce, which was

calculated as the ratio of metal concentration in plant root regardless of mechanisms (mg kg- )

over that in the surrounding water (mg L^)~. All the investigated metals (Al, Ca, Cd, Co, Cr, Cu,

Fe, K, Mg, Mn, Na, Ni, Pb, and Zn) had a CF higher than 102, with Al, Cd, Co, Cr, Fe, Mn, and

Pb having a CF higher than 104. The CF values of the 14 metals changed in the following order

for the East Pond: Cr >Mn >Co >Pb >Fe >Zn >Cd >Al >Ni >Cu >K >Ca >Mg >Na. For

the West Pond the order was: Cr > Fe > Mn > Co > Al > Pb > Cd > Ni > K > Zn > Cu > Mg >

Ca >Na.



S106

.fC- I West Pond

10 -



S104





F410 -


Al Ca Cd Co Cr Cu Fe K Mg Mn Na Ni Pb Zn
Element

Figure 3-2. Plant metal concentration factors (CFs, metal concentration, mg kg- in root divided
by metal concentration in the surrounding water, mg L^1) in the East and West Ponds.

Metal Distribution in Plant

Most of the metals investigated were not effectively transported to shoot from root, with a

root/shoot (R/S) ratio in metal concentration higher than 1 (Figure 3-3). Of the 14 metals, only

Ca had an R/S ratio less than 1, which means higher Ca concentrations were in the shoot than in










the root. Potassium, Mg, and Na had an R/S ratio close to 1. For Cr, Cu, Fe, and Ni, more than

80% of their accumulation occurred in the root, with an R/S ratio close to or higher than 6. This

was most prominent in the case of Fe with an R/S ratio higher than 17. Much higher

concentrations of the above four elements in the root than in the shoot were also observed by

Jayaweera et al. (2008) and many other researchers (Maine et al., 2004; Manab Das and Maiti,

2008; Qian et al., 1999). Some physiological barriers were believed to play a role in preventing

their transport to the aerial tissues (Zhu et al., 1999), which is one of the mechanisms protecting

the aerial part (where photosynthesis takes place) from being damaged by excessive metals (Fe,

Cu, Ni, and Cr). Although Fe, Cu, and Ni are essential for plant growth, when at high

concentrations, they are toxic to plant. For heavy metals which are not essential and toxic to

plant such as Cd, Co, and Pb, they were only detected in the root of water lettuce.




HEM East Pond
[ West Pond
a 20











Ca K Mg Na AI Cd Co Cr Cu Fe Mn Ni Pb Zn
Metal


Figure 3-3. Metal root/shoot ratio in concentration of the East and West Ponds.

Estimation of Annual Metal Removal

Periodic harvesting of water lettuce plant is necessary not only for maintaining an optimum

growth density, but also for effective removal of nutrients (N and P) and metals from the waters,









otherwise the nutrients and metals would be released back into the water system after the plant

died and decomposed. Harvesting was mainly conducted in the summer when both temperature

and rainfall were high and the plant growth rate was the highest during a year. Water lettuce

removed a considerable amount of macroelements such as Ca, K, and Mg, and a sizable amount

of microelements such as Fe and Mn from the stormwater (Table 3-1). High metal concentrations

in the roots of water lettuce have been reported elsewhere (103 8 mg Cu kg-l (Qian et al., 1999),

9.43 mg Co kg- 27.07 mg Pb kg- 107.32 mg Cr kg-l (Vardanyan and Ingole, 2006)). In

comparison, water lettuce in the two ponds were far from reaching its potential in removing trace

metals, especially for Cd, Co, Ni, and Pb because of their low concentrations in the waters. For

both dry matter and in most cases, individual elements, the West Pond's annual removal rate was

twice that of the East Pond. The higher rate in the West Pond was related to higher biomass yield

due to more favorable conditions, as high total organic carbon and EC in the East Pond might

have negatively affected the growth of water lettuce (see Chapter 2 for discussion).

Metal Uptake and Surface Adsorption

According to their distribution between outside and inside the root (Figure 3-4), the 12

metals (as Na is a component of the DCB solution and the highly mobile nature of K in plant,

these two elements are excluded) can be grouped into 2 categories: 1) higher proportion was

located on the external surfaces of the root: Ca, Cd, Co, Fe, Mg, Mn, and Zn, and 2) higher

proportion was located inside the root: Al, Cr, Cu, Ni, and Pb. Many studies have been

conducted on elements such as Fe, Mn, Cd, Pb, Cu, and Zn (Hansel et al., 2001; Vesk et al.,

1999). The distribution patterns of Fe, Mn, and Zn agree with those from St-Cyr and Campbell's

research (St-Cyr and Campbell, 1996). As a plant essential nutrient, Ni was found mainly inside

the root (> 90%). Although Cr is a non-essential element, more than 90% of the plant

accumulated Cr had made its way into the root. This part of Cr could have been strongly bound











by the cell wall to prevent possible damage to the plant (Maine et al., 2004). Magnesium was

equally distributed outside and inside the root. About 80% of the Fe was located on the external

surface of the root as the main component of the iron plaque (St-Cyr and Campbell, 1996).



140
MM Outside the root
12 Inside the root

100 ---

80-

S 60-

40-




Al Ca Cd Co Cr Cu Fe Mg Mn Ni Pb Zn
Metal


Figure 3-4. Distribution of metals outside and inside of water lettuce root.

Metal Bio-concentrated by Plant

As a portion of the metals taken up by plant from water was actually located on the


external surfaces of the roots by adsorption or deposition instead of being absorbed into the


plant, the CFs previously calculated based on the total amount of metal removed by plant may

not accurately indicate the bio-accumulation capacity of a plant for certain metals. Therefore, it

is necessary to make some corrections. Another index, bio-concentration factor (BCF), the ratio


of metal concentration within plant root (mg kg- ) over that in the surrounding water (mg L^)~,

which can more accurately reflect the plant's uptake potential, was calculated (Figure 3-5). For


metals such as Cd, Fe, and Mn with a large proportion being adsorbed on the external surfaces of

the roots, their BCF value was much smaller than the respective CF value. For metals like Cr and


Ni with a large proportion being absorbed into the roots, the difference between their BCF and

CF value was small.










Table 3-1. Annual metal removal rates by periodic harvesting of water lettuce.

Location Dry Al Ca Fe K Mg Mn Na Zn Cd Co Cr Cu Ni Pb
matter
--------------------------------- kg ha-' ------------------------------------ --------------------- g ha ----------
East Pond 10455 16 357 29 344 70 5.3 138 1.3 4.0 4.9 92 107 31 51
West Pond 26005 55 546 57 853 134 5.3 370 1.2 11 10 189 336 52 110
















S10 -




10 -





~j 102
Al Ca Cd Co Cr Cu Fe Mg Mn Ni Pb Zn
Element


Figure 3-5. Plant metal bio-concentration factors (BCFs), the ratio of metal concentration within
plant root (mg kg- ) over that in the surrounding water (mg L^)~, in the East and West
Ponds.

Discussion

Planting water lettuce in the stormwater detention ponds not only improved water quality

by decreasing turbidity, total solids and nutrients (N and P) in the water as shown in Chapter 2,

but also by removing metals (Figure 3-1). Better metal removal performance by aquatic plants

was reported by many researchers with removal rates close to or higher than 90% (Mishra and

Tripathi, 2008; Mungur et al., 1997). But the high removal rates were usually associated with

laboratory or greenhouse experiments which provided more favorable environmental conditions

for plant growth in terms of light, temperature, and nutrient concentrations. In addition, high

spiked metal concentrations in the water were used in those studies (Ingole and Bhole, 2003;

Maine et al., 2001). High metal removal rates are also common when aquatic plants were applied

to the remediation of wastewater which usually contains high concentrations of metals (Kao et

al., 2001).










The plant is one of the sinks for metals in water column. As the metals except Ca were not

effectively transported to shoot from root (Figure 3-3), the root is the important final destination

for the metals. High concentrations of such metals as Cd, Co, Cr, and Pb in the plant can pose a

hazard to the plant. Fortunately, only a portion of the total metal located in root can made its way

into the root while the remainder stayed on the external surface of the root, completed or

adsorbed. This was confirmed qualitatively by Hansel et al. (2001) applying X-ray microprobe

and X-ray fluorescence microtomography to freeze-dried root cross-sectional slice and

quantitatively by the DCB extraction in this study (Figure 3-4).

A plant is commonly defined as a hyperaccumulator of a metal if the CF of that metal is

over 103 (Bunluesin et al., 2004). According to this definition, water lettuce can be considered a

hyperaccumulator of such trace metals as Cr, Cu, Fe, Mn, Ni, Pb, and Zn. But when we talk

about hyperaccumulation, we tend to emphasize the amount of metals accumulated within the

plant by absorption. Therefore, the BCF, which excludes the portion of metals on the external

surfaces of the roots, is a more appropriate index than CF for the differentiation of

hyperaccumulation, accumulation or non-accumulation plants for metals. Based on the BCF

index of 103 as the criterion, we found that water lettuce is a hyperaccumulator for Cr, Cu, Fe,

Mn, Ni, Pb, and Zn. Many reported BCFs are actually CFs without excluding that portion of

metals on the external surfaces of the roots (Bunluesin et al., 2004; Zayed et al., 1998), although

this may not change the conclusion regarding a hyperaccumulator for certain metals, as is the

case in this study. However, it is important that a BCF is used for differentiating a

hyperaccumulator from a regular plant based on plant physiology principle. In addition, this

differentiation help understand the mechanisms of metal accumulation and detoxification by

plants.










Conclusions

Growth of water lettuce reduced metal concentrations in the stormwater of detention

ponds. Water lettuce had great potential in concentrating metals from the surrounding water even

though the metal concentrations were under MDLs, with CF values ranging from 102 to 105. Of

the 14 metals investigated, only Ca had an R/S ratio in metal concentration less than 1, which

indicated a higher proportion of metal detected in the water lettuce plant remained in the root

instead of being transported up to the shoot. By periodic harvesting of plant biomass,

considerable amounts of metals, including macro- and micro-elements, were removed from the

stormwater. The DCB extraction method can be used to differentiate metals attached to the

external surface from those absorbed inside the root. More than 50% of Ca, Cd, Co, Fe, Mg, Mn,

and Zn recovered in the root were actually attached to the external surface, while more than 50%

of Al, Cr, Cu, Ni, and Pb was absorbed into the root. Water lettuce is a hyperaccumulator for Cr,

Cu, Fe, Mn, Ni, Pb, and Zn based on the bio-concentration factor (BCF) of 103 as a criterion.









CHAPTER 4
NITROGEN REQUIREMENT FOR WATER LETTUCE AND COMMON SALVINIA

Introduction

As nitrogen is a component of proteins and a part of chlorophyll, plants require a certain

level of external N for normal growth. This N level is called critical N concentration, below

which plant biomass yield, quality, or performance is unsatisfactory (Marschner, 1995). When

external N level is above the critical concentration, plant biomass production responds positively

to increased external N level to a point above which negative or no response in biomass yield

occurs (Petrucio and Esteves, 2000). This point in external N level is the optimum N

concentration for maximum biomass production.

For phytoremediation purpose, it is crucial that we apply water lettuce to water with

nutrient concentration above its nutrient critical level so that the plant can have net growth in

biomass after a certain growth period and by periodically harvesting nutrients or metals can be

removed from the water.

It is suggested that small-leaved floating plant, common salvinia, can be included in

polyculture systems with such large-leaved plant as water lettuce (Reddy and DeBusk, 1985)

because it is efficient in removing P with a narrow N/P ratio (Reddy and DeBusk, 1985). But if

common salvinia has a much higher nutrient critical level than water lettuce, the application of

the water lettuce-common salvinia polyculture system will have to be compromised.

The information on critical and optimum N level of both water lettuce and common

salvinia is important for management of phytoremediation systems using water lettuce or water

lettuce in combination with common salvinia, but it has not been well documented.

The obj ectives of this study were to:

*Find out the critical N concentrations of water lettuce and common salvinia;










*Investigate the possibility of a water lettuce-common salvinia polyculture system to
improve water treatment efficiency.

Materials and Methods

Experimental Design

Two free floating aquatic plants, water lettuce (Pistia stratiotes) and common salvinia

(Salvinia minima), were tested for their N requirements using a hydroponic study conducted in a

greenhouse. The experimental design was a completely randomized design with seven levels of

N and three replications for each N treatment of each plant species.

Healthy water lettuce and common salvinia seedlings of similar age and size were selected

and cultured in distilled water for three days before being transplanted in 8-L pots with modified

Hoagland nutrient solution (Reddy et al., 1983). Plants were so transplanted that all the pots with

the same plant species had very close initial plant biomass which was about 3.710.3 g in dry

weight for water lettuce and about 1.2910.17 g in dry weight for common salvinia. The nutrient

solution was prepared to provide sufficient amounts of essential nutrients except N for which a

series of concentrations were applied. Chemical concentrations in the nutrient solution were as

follows:

Table 4-1. Nutrient solution composition for N requirement study.
Nutrient Concentration (mg L^') Nutrient Concentration (mg L^1)
Ca 40.1 Mn 0.027
K 3.91 Mo 0.034
P 3.10 Fe 1.12
Mg 12.2 Zn 0.065
Cu 0.025 B 0.022
S 16.7 Cl 71.0

Nitrogen was added as NH4NO3. The seven levels of N were: 0.005, 0.025, 0.05, 0.25,

1.25, 2.5, and 5 mg N L Nutrient solutions were renewed every three days to maintain the

mentioned nutrient concentrations. When plants grew to occupy the whole water surface, some









mature plants were harvested to maintain an approximate %/ coverage so that new plants have

room to grow. Harvested plants from the same pot were pooled, weighed and oven-dried for

chemical analysis. For harvested water lettuce, root and shoot were separately weighed, dried,

and analyzed.

Chemical Analysis

After six weeks of growth (June 16-July 30, 2007), plants were removed from the pots,

rinsed with deionized water and blotted dry. Plant materials were oven-dried at 70 OC for three

days. Total plant weight from each pot was the sum of each harvest. Dried plant samples were

pulverized to <1 mm with a 4-Canister Ball Mill (Kleco Model 4200, Kinetic Laboratory

Equipment Company, Visalia, CA) prior to analysis for total N. Plant N concentration was

determined using a CN analyzer (vario Max CN, Elementar Analysensystem GmbH, Hanau,

Germany) .

Statistical Analysis

Data were subj ected to the analysis of variance (ANOVA) using the GLM procedure in

SAS software (SAS Institute, 2001). Differences between means were tested using the Tukey

method. All statistical analysis tests were performed using a significance level of 5%.

Results and Discussion

Relationship between Plant Biomass Yield and N Concentration

Typical N-deficiency symptoms such as senescence of older leaves and retarded growth

were found in the low N level treatments (0.005, 0.025, 0.05, and 0.25 mg N L^1, Figure 4-1).

Larger plant size resulted from more vegetative growth and more new individuals from vigorous

vegetative reproduction were observed in the high N level treatments (1.25, 2.5, and 5 mg N L^)~.

More plant dry biomass was obtained with higher N treatment (Figure 4-2). For common

salvinia, when the solution N concentration was 1.25 mg L^1 or lower, there was no statistical









difference in dry biomass between the five N levels. It produced significantly higher dry matter

when the solution N concentration was increased to 2.5 mg L^1 and above.

When the solution N concentration was 0.25 mg L^1 or lower, there was no statistical

difference in water lettuce dry biomass between the four N levels. Water lettuce produced

significantly higher dry matter yield when the solution N concentration was increased to 1.25 mg

L^ or above. The significant increase in water lettuce biomass in the treatments of 1.25, 2.5, and

5 mg N L^1 was mainly gained from the increase in above-water growth, which was

demonstrated by the changes in shoot/root (S/R) ratio of dry biomass with external N

concentration (Figure 4-3). When the solution N concentration was 0.25 mg L-1 or below, more

than half of the water lettuce' s biomass was accounted for by its root with S/R ratio below one.

Raising the solution N to 1.25 mg L^1 significantly increased S/R ratio (to higher than 1.5). Shoot

biomass was approximately three times greater than root biomass when external N concentration

was raised to 5 mg L^1

Regression analysis revealed that a quadratic model can well represent the relationship

between plant dry biomass yield and N concentration in the external solution (P < 0.05, Figure 4-

4). These results indicate that water lettuce or common salvinia, like many crop plants, has its

optimum N requirement for maximum biomass production. Solution N concentration higher than

the optimum concentration tended to cause a decrease in biomass production. The quadratic

regression curve predicts that the optimum N concentrations for water lettuce and common

salvinia to achieve a maximum biomass yield are approximately 4.3 and 5.3 mg L^1, respectively.

A close value of 5.5 mg L^1 was reported by Reddy et al. (Reddy et al., 1989) to be the optimum

N concentration for water hyacinth.











































SalviniaA








BB







5e-3 0.025 0.05 0.25 1.25 2.5 5

Solution N concentration (mg N L-1)


Water lettuce A
















5e-3 0.025 0.05 0.25 1.25 2.5 5

Solution N concentration (mg N L-1)


Figure 4-1. The growth performance of water lettuce and common salvinia under different N levels.


3.0


2.5

-
c 2.0




-c
1.0


Figure 4-2. Plant dry biomass yield at different N level treatments.













3.5

3.0


S2.0


S1.5

1. -
0.


0.0
5e-3 0.025 0.05 0.25 1.25 2.5 5
Solution N concentration (mg L 1)


Figure 4-3. The shoot/root ratio of water lettuce dry biomass at different N levels.


14
12 Water lettuce






0- 1 2





2.0



1. v=? -0.01x2 + 0.51Gx+ 0.649
.5 R = 0.89*




0 1 2 3 4 5 6

SolutionNV concentration (mg L-1)


Fiue4-.Rgrsincuv f ln dyboas ildv.souinN ocntain

Salvini74










Relationship between Plant N and Solution N Concentration

Higher plant N concentration was found in treatments with higher solution N

concentrations (Figure 4-5). Differences in N concentration between root and shoot of water

lettuce were not as big as those in some metal concentrations such as Cu and Fe (R/S>9, see

Chapter 3 and Figure 3-3). Root N concentration was higher than shoot N concentration when

the solution N concentration was 1.25 mg L^1 or lower. When the solution N concentration was

2.5 mg L^1 and higher, shoot had a higher N concentration than root. A significantly higher N

concentration was measured in plant with the treatments of solution N concentration of 2.5 mg L~

Sand above. Plant N concentrations were low when the solution N concentration was 1.25 mg L^1

or lower, and there were no statistical differences between these low N treatments. Compared to

plant dry biomass yield, a significant increase in plant N concentration occurred at a higher

external N concentration. This is likely due to the dilution effect on plant N concentration from

vigorous plant growth, and such effect diminishes under higher external N conditions.



30
mWater lettuce root
I Water lettuce shoot A







20
5e- 0.2 .5 025 12 .
Souto N ocntain m

Fiue -.Pln cnetrto a ifeet eeltetmns












35
Salvinia
30 -1 A





1 T BC
z cc





6e-3 0.025 0.05 0.25 1.25 2.5 5

Solution N concentration (mg L-1)

Figure 4-5. Continued.

Unlike water lettuce, no clear point of external N concentration was found for common

salvinia although higher plant N concentration was measured with treatments of higher N level

(Figure 4-5, lower graph). This might be caused by common salvinia's low ability in extracting

nutrients and competitiveness for growing space. Algae readily grew in pots with common

salvinia, especially in high N treatments, which might affect the growth performance of common

salvinia.

A linear model can be used to describe the relationship between plant N and solution N

concentration (P < 0.05, Figure 4-6). Unlike plant dry biomass yield, which responded positively

to increased external N concentration to a certain level and then negatively, plant N increased

continuously with increased external N concentration. Such luxury uptake of N, i.e. plant N

concentration increases without increasing plant biomass yield when external N concentration is

above plant optimum N concentration, was also reported by Petrucio and Esteves (2000) and

Gaudet (1973). As N is not needed for growth or other metabolic functions in luxury

consumption, it is converted to organic matter for later use in unfavorable times or under

76











environmental stress (Farahbakshazad and Morrison, 1997). It was also suggested by

Farahbakshazad and Morrison (1997) that in highly loaded water treatment systems luxury plant


uptake with rhizome storage dominates N removal.

25
's Heater lettuce root m


15 -1



z ~R-= 0.94l*





Solution N concentration (mg L-' )

30
r ~7Water lettuce shoot
25 -5

20



8 10,~ 1= .3.43x+ 6.8
z R-=0.98*'
E 5-


0 1 2 3 4 5 6
Solution N concentration (mg L- )

30
"a t Salvinial



1s 0 -1 *4,= 2.71x 112



5
O
0 1 2 3 4 5 6
Solution N concentration (mg L- )


Figure 4-6. Regression curve of plant N concentration vs. solution N concentration.

77









Plant Critical N Concentration

In the N treatments of 0.005, 0.025, 0.05, and 0.25 mg N L^1, water lettuce did not have net

growth after six weeks of culture with plant biomass being the same as that at the beginning of P

treatment (Figure 4-2), which indicated that water lettuce can survive, at least for six weeks, in

water with N concentration of 0.005-0.25 mg L-1 but can not support new growth. This is not

desirable for phytoremediation purpose. And more than half of its biomass was in root (Figure 4-

3), which indicated N stress (Reddy, 1984). Visually, plant in these N treatments showed clearly

typical symptoms of N deficiency with senescence of older leaves (Figure 4-1). Water lettuce in

1.25 mg L-1 treatment more than doubled its initial biomass (Figure 4-2). Plant showed healthy

bright green without yellowing of the old leaves (Figure 4-1). Shoot contributed more to the total

biomass than root as N was no longer a limiting factor (Figure 4-3). All these results indicate that

1.25 mg N L^1 is the critical external N concentration for normal growth of water lettuce.

For common salvinia, there was no net growth in the N treatments of 0.005, 0.025, 0.05,

0.25, and 1.25 mg N L^1 (Figure 4-2). Common salvinia in 2.5 mg L-1 treatment almost doubled

its initial biomass (Figure 4-2). These results indicate that 2.5 mg N L^1 was the critical external

N concentration for normal growth of common salvinia.

Conclusions

The critical N concentration for water lettuce to have net growth was 1.25 mg L^1. Water

lettuce may not be able to reduce N concentration in surrounding water to < 1.25 mg L1 below

which vegetative growth of water lettuce is minimal. At adequate N supply levels (> 1.25 mg L-

1), N uptake is mainly used for the above-water biomass production, and is the maj or contributor

for the significant increase in biomass. The critical N concentration for common salvinia to have

net biomass increase was 2.5 mg L^1










Based on regression model, the optimum N concentrations for maximum biomass

production are 4.3 and 5.3 mg L1 for water lettuce and common salvinia, respectively. Luxury

uptake of N by water lettuce and common salvinia may occur when N levels are higher than their

optimum levels.

Although it has been suggested to include such small-leaved aquatic plant as common

salvinia to a system based on large-leaved plants, i.e. water lettuce, to improve P removal

efficiency, such a system may not work to our purpose as common salvinia requires a higher N

concentration for net growth.









CHAPTER 5
PHOSPHORUS REQUIREMENT FOR WATER LETTUCE AND COMMON SALVINIA

Introduction

Phosphorus, as a maj or plant nutrient, is associated with its function in energy storage and

transfer as the maj or constituent of the "energy currency", adenosine di- and tri-phosphates

(ADP and ATP). Energy obtained from photosynthesis and metabolism of carbohydrates is

stored in these phosphorus compounds. It is also an important structural component of many

other biochemicals such as nucleic acids, coenzymes, nucleotides, phosphoproteins,

phospholipids, and sugar phosphates (Tisdale et al., 1993). Therefore, P deficiency retards

overall growth of plants.

Plants have their critical P level and optimum P level for normal growth and maximum

growth, respectively. For phytoremediation purpose, plant should be applied to water with P at

its optimum level so that best performance of the plant can be achieved. If that is not possible, it

is crucial that P concentration in the water is higher than its nutrient critical level so that the plant

can have net growth in biomass after a certain growth period and nutrients or metals can be

removed from the water by periodically harvesting the plant biomass.

The information on critical and optimum P level of both water lettuce and common

salvinia is important for management of phytoremediation systems using water lettuce or water

lettuce in combination with common salvinia, but it has not been well documented.

The obj ectives of this study were to:

* Find out the critical P concentrations of water lettuce and common salvinia;

* Investigate the possibility of a water lettuce-common salvinia polyculture system to
improve water treatment efficiency.












Experimental Design

Two free floating aquatic plants, water lettuce (Pistia stratiotes) and common salvinia

(Salvinia minima), were tested for their P requirements using hydroponic studies conducted in a

greenhouse. The experiment was a completely randomized design with six levels of P and three

replications of each treatment for each plant species.

Healthy water lettuce and common salvinia seedlings of similar age and size were selected

and cultured in distilled water for three days before being transplanted in 8-L pots with modified

Hoagland nutrient solution (Reddy et al., 1983). Plants were so transplanted that all the pots with

the same plant species had very close initial plant biomass which was about 3.310.3 g in dry

weight for water lettuce and about 1.1410.15 g in dry weight for common salvinia. The nutrient

solution was prepared to provide sufficient essential nutrients for plant growth except P for

which a series of concentrations were used. Phosphorus was added as KH2PO4, K was

compensated with K2SO4 in l0w P treatments to ensure equal K concentrations in all treatments.

Chemical concentrations in the solution are provided in Table 5-1:

Table 5-1. Nutrient solution composition for P requirement hydroponic study
Nutrient Concentration (mg L^') Nutrient Concentration (mg L^')
N 9.52 Cu 0.0254
K 6.31 Mn 0.0275
Ca 40.1 Mo 0.0336
Mg 12.2 Fe 1.12
S 16.7 Zn 0.0654
Cl 71.0 B 0.0216

The six levels of P were: 0.01, 0.05, 0.1, 0.5, 1, and 5 mg L 1. Nutrient solutions were

renewed every three days to maintain the aforementioned nutrient concentrations. When plants

grew to occupy the whole water surface, some mature plants were harvested to maintain an

approximate %/ coverage so that new plants have room to grow. Harvested plants were weighed


Materials and Methods









and oven-dried for chemical analysis. For harvested water lettuce, root and shoot were separately

weighed, dried, and analyzed.

Chemical Analysis

After seven weeks of growth (September 24-November 1 1, 2007), plants were removed

from the pots, rinsed with deionized water and blotted dry. Plant materials were oven-dried at 70

oC for three days. Total plant weight from each pot was the sum of each harvest. Dried plant

samples were pulverized to <1 mm with a 4-Canister Ball Mill (Kleco Model 4200, Kinetic

Laboratory Equipment Company, Visalia, CA) prior to analysis for total P. Pulverized plant

sample (0.400 g) was digested with 5 mL of concentrated HNO3 in digestion tube using a block

digestion system (AIM 500-C, A.I. Scientific Inc., Australia), and P concentration in the digested

solution was determined using ICP-OES (Ultima, JY Horiba Inc. Edison, NJ).

Statistical Analysis

Data were subj ected to the analysis of variance (ANOVA), using the GLM procedure in

SAS software (SAS Institute, 2001). Differences between means were tested using the Tukey

method. All statistical analysis tests were performed using a significance level of 5%.

Results and Discussion

Relationship between Plant Biomass Yield and Solution P Concentration

Retarded growth occurred in the low P treatments (0.01 and 0.05 mg P L^)~. In the high P

treatments (0.1, 0.5, 1, and 5 mg P L^)~, plants looked more healthy in bright green, and vigorous

vegetative reproduction resulted in lots of new individuals (Figure 5-1).

More plant dry biomass yield was obtained in the higher P level treatments (Figure 5-2).

When the solution P concentration was 0.05 mg L^1 or lower, there was no statistical difference

in water lettuce dry biomass between the two P levels. Water lettuce produced significantly

higher biomass yield than those in the 0.01 and 0.05 mg P L-1 treatments when the solution P









concentration was increased to 0.1 mg L^1 and above. Similar to N, the significant increase in

water lettuce biomass in the treatments of 0.5, 1, and 5 mg P L^1 was mainly from the increased

above-water growth, which was demonstrated by the changes in shoot/root (S/R) ratio of dry

biomass with external P concentration (Figure 5-3). Unlike N, more shoot dry biomass was

produced even at low P levels with S/R ratio in dry biomass being close to 2 and plant shoot

biomass (above water part) at high P treatments (0.5, 1, and 5 mg L^1) was more than 3-5 times

that of root biomass (under water part).

For common salvinia, biomass increased with increasing external P concentration, but

there was no such a clear point of P concentration as for water lettuce to differentiate significant

plant growth between treatments. As discussed in the previous chapter, this may be due to the

competition from algae growth. For a narrower and more precise critical range, further study

should be carried out with P levels set closed at 0.05-0.5 mg L-1 but with a shorter distance in

terms of P concentration, between treatments. And measures need to be taken to selectively

inhibit algae growth.

The relationship between plant dry biomass yield and P concentration in the nutrient

solution was well described by a quadratic model (Figure 5-4). This indicates that water lettuce

or common salvinia, like many other crop plants, has its optimum P requirement for maximum

biomass production. Solution P concentration higher than the optimum concentration may cause

a decrease in biomass production. Based on the quadratic regression, the optimum P

concentrations for both water lettuce and common salvinia to achieve maximum biomass yield

are around 2.9 mg L^1






















































0.05 0.1 0.5 1

Solution P concentration (mg P L-1)


G~A~a~
001mnPT,~1


~T
01 mn P I,


~C~~TIT~I


C~~TIT~I


Figure 5-1. Growth performance of water lettuce and common salvinia under different P levels.


12 -


10 -






d -



2-


0


A
Water lettuce








iAA


0.01 0.05 0.1 0.5 1 5 0.01

Solution P concentration (mg P L-1)


Figure 5-2. Plant dry biomass weights of different P level treatments.














6A



.E 4 AB
5 Bc

DD
2- D






0.01 0.05 0.1 0.5 1 5
Solution P concentration (mg L-1)



Figure 5-3. Water lettuce shoot/root in dry biomass under different P level.







































85












~i16

E 12




i 4

0


0123 4


Solution P concenltration (mg1 L- )


4






1

0


Solution P concenltration (mg1 L- )

Figure 5-4. Regression curves of plant dry biomass vs. solution P concentration.









Relationship between Plant P Concentration and Solution P Concentration

Higher plant P concentration was found in the treatments with higher solution P

concentrations (Figure 5-5). Differences in P concentration between root and shoot of water

lettuce were not as big as those found with some metal concentrations such as Cu and Fe (R/S>9,

see Chapter 3 and Figure 3-3). Root P was higher than shoot P when the solution P concentration

was 0.5 mg L^1 or lower. When the solution P concentration was 1 mg L^1 and higher, shoot had a

higher P concentration than root. Significantly higher P concentration was measured in plants

treated with P concentration of 0.5 mg L^1 and above (Figure 5-5). Compared to plant dry

biomass yield, a significant increase in plant P concentration required a higher external P

concentration, which is likely due to the dilution effect on plant P concentration from vigorous

plant growth, and such effect diminishes at higher external P concentrations.

When the solution P concentration was 0.1 mg L^1 and below, P concentrations in common

salvinia plant were low (< 1 g kg- ) and there were no statistical differences among these three P

treatments. When the solution P was increased to 0.5 mg L^1, P concentration in common salvinia

plant was significantly increased (close to 4 mg L^)~. Plant P concentration continued to increase

with increasing solution P concentration (P < 0.05, Figure 5-5).

The relationship between plant P and solution P concentration was well described by a

quadratic model (R2 = 0.99) (Figure 5-6). Like plant dry biomass yield, plant P concentration

increased positively with increasing external P concentration to a certain level and then

negatively. The optimum P concentration for both root and shoot of water lettuce was around

3.2, and about 4.2 for common salvinia. The decrease in plant P concentration at high external P

level might be caused by limit of other nutrients.
















555 Water lettuce rootA
I ""I Water lettuce shoot -













DE E D



0.01 0.05 0.1 0.5 1 5

Solution P concentration (mg L-1)



A
Salvinia


















0.01 0.05 0.1 0.5 1 5

Solution P concentration (g L )


Figure 5-5. Plant P concentration in treatments with different solution P level.































88


6-








2-

o-








16

14

S12


8o


8 6

4-

2

0
























0 1 2 3 4 5 6
Solution P concenltration (mgi L- )


1 2 3 4 5 6


Solution P concentration (m1g. L-1)


0 1


2 3 4 5 6


Solution P concenltration (mg1 L-')


Figure 5-6. Regression curve of plant P vs. solution P concentration.






89









Plant Critical P Concentration

In the P treatments of 0.01 and 0.05 mg L^1, water lettuce did not have net growth after

seven weeks with plant biomass being the same as that at the beginning of P treatment (Figure 5-

2), which indicated that water lettuce can survive, at least for seven weeks, in water with P

concentration of 0.01-0.05 mg L-1 but can not support new growth. This is not desirable for

phytoremediation purpose. Visually, plant in these two P treatments showed yellowing of older

leaves and growth was retarded (Figure 5-1). Water lettuce in the 0.1 mg P L-1 treatment more

than tripled its weight compared to the 0.01 mg P L^1 and almost doubled compared to the 0.05

mg P L-1 treatment (Figure 5-2), which indicated that plant in this P treatment not only survived

but also had surplus P to support new growth. All these indicate that 0.1 mg P L^1 is the critical

external P concentration for water lettuce in order to have net growth, which is important for its

application in phytoremediation.

For common salvinia, there was no net growth in the P treatments of 0.01, 0.05, 0. 1, and

0.5 mg P L^1, with plant biomass being the same as that at the beginning of the P treatments

(Figure 5-2). Common salvinia in 1 mg P L-1 treatment almost doubled its initial biomass (Figure

5-2), indicating that 1 mg P L^1 was the critical external P concentration for common salvinia to

have net growth.

Conclusions

The critical P concentration for water lettuce to have net growth was 0.1 mg L^1. Water

lettuce may not be able to reduce P concentration in surrounding water to < 0.1 mg L1 below

which vegetative growth of water lettuce is minimal. Shoot contributes more to total biomass

when external P concentration is raised. The critical P concentration for common salvinia to have

net biomass increase was 1 mg L^1









Based on regression model, the optimum P concentrations for maximum biomass

production of water lettuce and common salvinia are the same, approximately 2.9 mg L^1

As common salvinia has a much higher P requirement for net growth than water lettuce,

and a concentration of 1 mg P L^1 or above is rarely found in stormwater, this plant may not be

useful for removing nutrients, especially P, from surface waters. In addition, it may not be

feasible to develop a polyculture system of remediation using water lettuce and common salvinia

because P concentration in stormwater is mostly lower than the critical level for common

salvinia.









CHAPTER 6
EFFECT OF SALINITY ON GROWTH OF WATER LETTUCE

Introduction

Stormwater vary in salinity which is affected by soil properties, irrigation water quality,

fertilization, and also by the sea environment in the coastal regions.

Terrestrial plants differ greatly in their tolerance of salinity. For example, barley and cotton

have considerable salt tolerance, while carrot and celery are salt sensitive (Tisdale et al., 1993).

Aquatic plants also vary in salinity tolerance. Large-leaved floating species are reported to be

most susceptible to salinity, submersed species can tolerant high salinity than large-leaved ones,

and small-leaved ones are the least susceptible of the three (Haller et al., 1974).

The tolerance of aquatic plants to salinity will directly influence their performance in water

treatment as decreases in transpiration and total dry weight will occur with increasing salinity

and death at toxic salinity level (Haller et al., 1974). Results from our field study in the two

stormwater detention ponds (see Chapters 2 and 3 of this dissertation) indicated that the less

satisfactory performance of the plant in the East Pond might be due to the high EC in the water

(Table 6-1), which negatively affected water lettuce's growth and consequently its performance.

Table 6-1. EC and ions contributing to water salinity in the waters of the East and West Ponds
(time period: 9/13/2005-3/28/2008, n=122).
Location EC Cl~ SO4-S Ca K Mg Na
CIS cml ------------------------------------ mg L^ --------------------
East
Pond 60613 72a) 81.8162.2 41 .7146.6 42.0124.8 7.4113.82 14.418.6 48.3133.3
West
Pond 229148 35.0113.7 1.5 713.35 22.213.7 4.0010.99 2.8011.24 16.515.8
a) Data shown are mean a standard deviation.

There are extensive data in the literature on the tolerance of terrestrial plants especially

crops to salinity. But few researches have been done on aquatic plants' salt tolerance especially

on those promising plants for water remediation. Utilization of such invasive aquatic plant as










water lettuce in stormwater detention ponds involves the possibility of its escape from the

detention systems into the lagoons or estuaries. From both points of utilization and disaster

prevention, we need to know water lettuce's salinity tolerance.

The obj ective of this study was to evaluate the effects of salinity on the growth

performance of water lettuce.

Materials and Methods

Experimental Design

Water lettuce (Pistia stratiotes) was tested for its salinity tolerance using hydroponic

studies conducted in a greenhouse. The experimental design was a completely randomized

design with six salinity treatments and three replications for each treatment.

Healthy water lettuce seedlings of similar age and size were selected and cultured in

distilled water for three days before being transplanted in 8-L pots with modified Hoagland

nutrient solution (Reddy et al., 1983). The solution was prepared to provide sufficient amounts of

essential nutrients for plant growth. Chemical compositions of the solution are provided in Table

6-2:

Table 6-2. Nutrient solution composition for the salinity tolerance study
Nutrient Concentration (mg L^') Nutrient Concentration (mg L^')
N 49.0 Mn 0.0275
P 9.29 Mo 0.0336
K 46.9 Cu 0.0254
Ca 40.1 Fe 1.12
Mg 12.2 Zn 0.0654
S 16.7 B 0.0216

Our preliminary study revealed that salinity level of 6000 mg NaCl L^ (equivalent to 9696

CIS cml in EC) was so toxic to water lettuce that the plant could not survive (Figure 6-1).

Therefore, the salinity level treatments chosen for this study were: 0, 800, 1600, 2400, 3200, and

4000 mg NaCl L^. Taking into account of salts from the nutrient solution itself, the six salt









treatments were: 293, 1093, 1893, 2693, 3493, and 4293 mg L^1 or 473, 1766, 3059, 4351, 5644,

and 6937 CIS cml in EC. Nutrient solutions were renewed every three days to maintain the

above-mentioned nutrient concentrations. When plants grew to occupy the whole water surface,

some mature plants were harvested to maintain an approximate %/ coverage so that new plants

have room to grow. Harvested plants from the same pot were pooled, weighed and oven-dried

for chemical analysis.


4000


Figure 6-1. Growth performance of water lettuce in water with gradient salinity.

Chemical Analysis

After about 3 weeks of growth (from September 21 to October 10, 2008), surviving plants

were removed from the pots, rinsed with deionized water and blotted dry. Total fresh weights

from each pot were recorded. Plants were oven-dried at 70 OC for three days and then plant dry

weights were measured. Dried plant samples were pulverized to <1 mm with a 4-Canister Ball









Mill (Kleco Model 4200, Kinetic Laboratory Equipment Company, Visalia, CA) prior to analysis

for nutrients (N, P, and metals). Plant N concentration was determined using a C/N analyzer

(vario Max CN, Elementar Analysensystem GmbH, Hanau, Germany). Pulverized plant sample

(0.400 g) was digested with 5 mL of concentrated HNO3 in digestion tube using a block

digestion system (AIM 500-C, A.I. Scientific Inc., Australia), and P and metal concentrations in

the digested solution was determined using the ICP-OES (Ultima, JY Horiba Inc. Edison, NJ).

Statistical Analysis

Data were subj ected to analysis of variance (ANOVA) using the GLM procedure in SAS

software (SAS Institute, 2001). Differences between means were tested using the Tukey method.

All statistical analysis tests were performed using a significance level of 5%.

Results and Discussion

Plant Growth as Affected by a Salinity Gradient

A prominent effect of salinity on water lettuce growth was that it inhibited vegetative

growth but promoted the reproduction of new small-sized individuals. This effect was more

pronounced with increasing salinity (Figure 6-2). Suppression of leaf expansion was recognized

as one of the several morphological and physiological effects of salinity (Nieman, 1964). It is

associated with the loss of cell turgor that exerts its effect on cell extension and /or division

(Greenway and Munns, 1980). Significant reduction in leaf area (from 1 192 cm2 in COntrol to

503 cm2) by high water salinity was also reported by Pascale et al. (1997). Chlorotic leaf

margins, which indicated salinity stress, were visually observed in the high salinity treatments.

Plant Biomass in Different Salinity

Salinity had a significant effect on plant dry biomass production (Figure 6-3). More new

individuals of small size plant could not compensate for the biomass reduction due to inhibited

vegetative growth at high salinity treatments. Plant dry matter yield was reduced by










approximately 30% in the 1766 CIS cm-l treatment as compared to the control (473 CIS cm- ), and

was further reduced (by about 50%) in the higher salinity treatments (Figure 6-3). Inhibited

biomass production (biomass reduced by more than 60%) by water salinity was also observed by

Pascale et al. (1997). And water availability has been considered to be one of the most important

factors that affect plant growth under saline conditions. Salinity inhibits plant water uptake by

decreasing the osmotic potential of the water.

9/22/2008 (Day 2)


6937 CLS cm-1


1__1 _
~1~+1 ~S
I~r r LC! ~~qi t ;I~ P~
rr ~... f
*I' 1F
r.
)' ~. ...
~; "$:

473 1766 3059 4351 5644

Figure 6-2. Growth performance of water lettuce in water with gradient salinity.


473 1766 3059 4351 5644 6937 ILS cm-1~

10/10/2008










Fresh water, by definition, contains less than 1000 mg L^1 of salts (1616 CIS cm- ) and

commonly less than 500 mg L^1 (808 CIS cm- ) (Sandia, 2003), but brackish water can have salt

concentration from 500-30000 mg L^1 (808-48480 CIS cm- ) (Greenlee et al., 2009). If the EC in

the 473 CIS cm-l treatment stands for a typical one for surface runoff, stormwater, or fresh water

and the EC in the 1766 CIS cm-l stands for a high one for stormwater or a low one for brackish

water, we can conclude that water lettuce can tolerant the salinity found in stormwater but its

biomass production may be reduced by up to 30% by high salinity. And water lettuce's escape

into lagoons and estuaries is a concern only when the brackish water has an EC less than 6937

CIS cm-l if we use a criterion of 50% reduction in biomass production compared to that in fresh

water and other conditions are favorable. According to Penfound and Earle (1948), large-leaved

plant, water hyacinth, when found near brackish water, is confined to the protected shorelines of

inflowing freshwater streams.

In the field study, the EC in the East Pond was high compared to the West Pond (Chapter

2). In seasons when EC in the East Pond rose and got close to or higher than 1766 CIS cm- plant

growth was negatively affected, which may have contributed to the less satisfactory performance

of the plants in the East Pond.


3.5

3.0-


S2.0- BC BC




0.5-

0.0
473 1766 3059 4351 5644 6937
EC (uS cm )

Figure 6-3. Plant dry biomass of water lettuce with different salinity treatments.










Plant Nutrient Status under Different Salinity Conditions

Although uptake of some nutrient was inhibited by high salinity, for instance, Ca, K, and

Mn uptake decreased significantly in the high salinity treatments, uptake of most other nutrients

was not significantly reduced. These elements included N, P, Mg, Fe, B, Cu, Mo, and Zn. For

nutrients whose uptake was inhibited, their concentrations in the plant still fell in the normal

range (Figure 6-4). For example, although plant Ca concentration was reduced by 75% in the

6937 CIS cm-l treatment, as compared to the control, its value, 6.13 g kg- still indicated adequate

Ca nutrient (plant Ca concentration ranges from 0.2 to 1.0% (Tisdale et al., 1993). It is unlikely

that salinity-induced nutrient deficiency might cause any severe inhibition of plant growth.

Therefore, besides water availability, the negative effect of salinity on plant growth might be

related to direct toxicity from Na+ and Cl- Excess Na+ might have caused metabolic

disturbances in those processes where low Na+ and high K+ or Ca2+ are required for optimum

function (Marschner, 1995). For example, when Na+ replaces Ca2+ in the cell membrane, cell

membrane function may be compromised, resulting in increased cell leakiness (Orcutt and

Nilsen, 2000). High Na+ also causes a decrease in nitrate reductase activity, inhibition of

photosystem II (Orcutt and Nilsen, 2000), and chlorophyll breakdown (Krishnamurthy et al.,

1987).


50 12




473N 176 35 31 54 9743 16 09 45 64 63
EC~~cm) E~u~m
Figre 6-4 Pln ntien cocnrain wit diffrenaiiyramns





















K



6(*




4(*







2[



473 1766 3059 4351 5644 6937

EC (uS cm )





to. Fe

1.6


















473 1766 3059 4351 5644 6937

EC (uS cm )


Ca










o I I I I








473 1766 3059 4351 5644 6937

EC (uS cml)





Mg l


100

B






0p 6[



0 4[








473 1766 3059 4351 5644 6937

EC (uS cm )


473 1766 3059 4351 5644 6937

EC (uS cm )


473 1766 3059 4351 5644 6937

EC (uS cm )


Figure 6-4. Continued.


Y"
s
c


c
8
S
o
r

m
a


-i
P
Cn
c
.P


a

s













70 60
-I Mn Mo

60 50 ~. II I ~
5( 4(*I
I I II
















47 1766 305 435 564 6937I I I


EC(uScm" )Cuc"


Fiue -. otiud

Coclsin

Saint ha infcn ffc ntegot o ae etc.Wae etc ims

















to the plan. Cniud









CHAPTER 7
EFFECT OF PH ON GROWTH OF WATER LETTUCE

Introduction

An important factor in plant growth is pH. In growth medium with low pH, plants often

suffer from hydrogen ion ( T) injury. Excess H' in the growth medium inhibits root elongation,

lateral branching, and water absorption. Hydrogen ions affect root ion fluxes via competition

with base cations for uptake, and causes damage to the ion-selective carrier in root membranes

(Pessarakli, 1999).

Other negative effects of low pH on plant growth are often associated with nutrient

availability. High availability of Al and Mn under low pH conditions causes toxicity, while low

pH-induced deficiency of Mg, Ca, P, and Mo also constrains plant growth.

Zinc and Mn deficiency are often the reason why the growth of plants in high pH medium

is inhibited. Sometimes, low availability of Fe and P is also a constraining factor.

Generally, a pH range of 5.5-7.0 provides the most satisfactory or balanced plant nutrient

levels for most plants. In the limited literature on interaction between pH and aquatic plants,

optimum pH ranges of 6.5-7.5 and 5.8-6.0 were reported for water hyacinth (El-Gendy et al.,

2004; Hao and Shen, 2006). Macroalga Chlorella sorokiniana grew best at pH 7-8 (Moronta et

al., 2006). According to Dyhr-Jensen and Brix (1996), although Typha latifolia L. had the

highest growth rates at pH 5.0 to 6.5, and growth was only slightly depressed at pH 8.0 but

completely stopped at pH 3.5. Documentation on effects of pH on water lettuce was minimal.

Although extreme pH values are seldom found in natural water bodies, they are not

uncommon in mine drainages and wastewaters which are often remediated using aquatic plants.

Whether the prospective aquatic plants can thrive in water with an extreme pH is the key to the

success of phytoremediation of contaminated waters.










The obj ective of this study was to determine the optimum pH range at which water lettuce

can grow normally and produce satisfactory amounts of biomass.

Materials and Methods

Experimental Design

Water lettuce (Pistia stratiotes) was tested for its optimum pH range using hydroponic

studies conducted in a greenhouse. The experimental design was a completely randomized

design with six pH treatments and three replications for each treatment.

Healthy water lettuce seedlings of similar age and size were selected and cultured in

distilled water for three days before being transplanted in 8-L pots with modified Hoagland

nutrient solution (Reddy et al., 1983). The nutrient solution was prepared to provide sufficient

amounts of essential nutrients for plant growth. Chemical composition in the nutrient solution is

provided in Table 7-1.

Table 7-1. Chemical composition of nutrient solution for pH effect study
Nutrient Concentration (mg L^') Nutrient Concentration (mg L^')
N 49.0 Mn 0.0275
P 9.29 Mo 0.0336
K 46.9 Cu 0.0254
Ca 40.1 Fe 1.12
Mg 12.2 Zn 0.0654
S 16.7 B 0.0216

The six pH treatments were: 3, 4.5, 6, 7.5, 9, and 10.5. Nutrient solutions were renewed

every three days to maintain the mentioned nutrient concentrations. Solution pH in each pot was

adjusted daily to the designed value by adding 0.1 mol L1 NaOH or 0.1 mol L^1 HC1. When

plants grew to occupy the whole water surface, some mature plants were harvested to maintain

an approximate %/ coverage so that new plants have room to grow. Harvested plants from the

same pot were pooled, weighed and oven-dried for chemical analysis.









Chemical Analysis

After about 4 weeks of growth (October 17-November 14, 2008), surviving plants were

removed from the pots, rinsed with deionized water and blotted dry. Total fresh weights from

each pot were recorded. Plants were oven-dried at 70 OC for three days and then plant dry

weights were measured. Dried plant samples were pulverized to < 1 mm with a 4-Canister Ball

Mill (Kleco Model 4200, Kinetic Laboratory Equipment Company, Visalia, CA) prior to analysis

for nutrients including non-metals (N, P, B and Mo) and metals (Ca, K, Mg, Cu, Zn, Fe, and

Mn). Plant N concentration was determined using a CN analyzer (vario Max CN, Elementar

Analysensystem GmbH, Hanau, Germany). Pulverized plant sample (0.400 g) was digested with

5 mL of concentrated HNO3 in digestion tube using a block digestion system (AIM 500-C, A.I.

Scientific Inc., Australia), and P and metal concentrations in the digested solution was

determined using the ICP-OES (Ultima, JY Horiba Inc. Edison, NJ).

Statistical Analysis

Data were subj ected to analysis of variance (ANOVA) using the GLM procedure in SAS

software (SAS Institute, 2001). Differences between means were tested using the Tukey method.

All statistical analysis tests were performed using a significance level of 5%.

Results and Discussion

Plant Growth in Water at Different pH

Although water lettuce leaves turned yellow in only two days after the pH 10.5 treatment

began, plant survived with a marginal increase in biomass. For the 3.0 pH treatment, plants did

not survive and died in about two weeks after transplanting (Figure 7-1). Growth of Typha

latifolia L. was also reported to completely stop at pH 3.5 (Dyhr-Jensen and Brix, 1996).








1 0/31~/2008


pH 3 pH 4.5 pH 6 pH 7.5 pH 9 pH 10.5


11~/07/2008


pH 4.5 pH 6 pH 7.5 pH 9 pH 10.5

Figure 7-1. Growth of water lettuce under different pH treatments.









Plant Biomass Yield at Different pH Treatments

Plant dry biomass yield increased with increasing solution pH from 4.5 to 9 and then

decreased at pH 10.5 (Figure 7-2). Regression analysis revealed that the relationship between

plant dry biomass and solution pH can be described by a quadratic model (Figure 7-3). Based on

regression analysis, the optimum pH for water lettuce growth was about 9, which indicates water

lettuce prefers a relatively alkaline environment. There are reports stating that aquatic plants

grow best at pH 8.0 (Dyhr-Jensen and Brix, 1996; Moronta et al., 2006), pH 9.0 is commonly

considered as the optimum pH for some algae (Ogbonda et al., 2007) and bacteria (Sanjib

Ghoshal et al., 2003). In this study although the highest plant biomass was measured in the pH

9.0 treatment, pH 9.0 might not truly represent the pH of these pots due to pH dynamic change

during the period of plant growth. It is well documented that plant roots excrete organic acids

which can acidify the growth medium and H' is released when plant roots take up NH4' Or other

cations. As a result, daily pH adjustment might not be able to maintain the designed solution pH

long enough as evidenced by the fact that each time the pH in the pots had dropped to 7-8 before

pH adjustment. A more sophisticated technique that can steadily maintain the selected pH in the

growth medium is needed for future study. However, we can still conclude that water lettuce

prefers and provides best growth in neutral to slightly alkaline waters.

Plant Nutrition Status at Different pH Treatments

Plant N, Mg, and Ca concentrations were similar for different pH treatments (Figure 7-4).

There were no differences in plant P and K concentrations among different pH treatments except

for pH 10.5 at which plant P and K were significantly lower. Water lettuce had the highest B and

Mn concentrations at pH 6 and the lowest at pH 10.5. Plant Fe, Zn, and Mo concentrations

decreased continuously with increasing pH in the solution (Figure 7-4).















S2.5




E 1.0
0.5


0.0 1 I I I I
3 4.5 6 7.5 9 10.5
pH


Figure 7-2. Dry biomass yield of water lettuce at different pH.


v,= -0.049u2 + 0.86xu 2.33
R- = 0.64*


1.5 3 4._5 6 7.5 9 10.5 12 13.5

pH


Figure 7-3. Regression curve of water lettuce dry biomass vs. solution pH.






































4.5 6 7.5 9 10.5

pH


300

Mn
250


20[













4.5 6 7.5 9 10.5

pH


70


4.5 6 7.5 9 10.5

pH




P
T -
















4 5 6 7 5 9 10 5

pH


4.5 6 7.5 9 10.5

pH


N


I~ I


Ca


50-
-

rn4[



20






10












12

-(


4.5 6 7.5 9 10.5


Figure 7-4. Plant nutrient concentration of water lettuce at different pH treatments.




















107


5
Y 30-
"
c
o

h
~2[
e
s
a
~1(
1


5(





.3(

2(

































































































Figure 7-4. Continued.



For the nutrients of Mn, P, K, B, Mo, Zn, and Fe, the lowest plant concentrations were



found in the treatments of highest pH, which might be due to the low concentrations of nutrients



in the solution (Table 7-2). Adjusting solution pH to 10.5 using 0.1 mol L1 NaOH might have



resulted in precipitation of some nutrients, which was visually observed in the pH 10.5 pots. At



108


140


120 -


100 -




60-


40


20



4 5 6 7 5 9 10 5

pH


50



40 -



30 -



20 -



10 -




4.5 6 7.5 9 10.5

pH


10

Fe


100

B

80 -






4(



2(


00

-O



00



00



00


us









5


4

3

2n

1i


4.5 6 7.5

pH


9 10.5


4.5 6 7.5 9 10.5

pH


4.5 6 7.5

DH


9 10.5









high pH, such micronutrients as Fe, Mn, Zn, and Cu react with OH- and precipitate as

hydroxides from the solution, leading to very low availability to the plant. Analysis of the

nutrient solution after pH adjustment confirmed the observation (Table 7-2). Iron was the most

affected element by high pH. Its availability at pH 10.5 was nearly 3 orders of magnitude lower

than that at pH 6.0-7.5, which is commonly found in most natural water bodies. Availability of

Mn, Zn, and P was also markedly lower in the pH 10.5 treatment.

The reason why water lettuce did not survive at pH 3.0 could be due to H+ injury

(Pessarakli, 1999). From the results of the salinity study (Chapter 6), it is clear that salinity

should not be critical for water lettuce's death, since EC in the pH 3.0 treatment was only 785 CIS

cml which is far below its toxic level.

Conclusions

Water pH has a significant effect on the growth of water lettuce. Water lettuce could not
survive at pH 3.0 or lower, which may be due to H+ inuy Wae letuc bims yil increased


with increasing pH up to 9, and then dropped at pH 10.5. For phytoremediation purpose, this

plant was recommended to be applied to neutral to slightly alkaline waters.










Table 7-2. Nutrient concentration and related properties of the nutrient solution at different pH levels.
Treatment EC Cl NO3-N PO4-P B Ca Cu Fe K Mg Mn Mo Zn

CIS cm --- --- ------ ---- --- -- --- ---m g
pH 3.0 785 29.4 66.96 12.46 0.011 37.8 0.12 1.14 46.4 14.5 0.027 0.015 0.12
pH 4.5 547 5.71 65.55 12.38 0.011 37.0 0.05 1.04 45.7 14.2 0.027 0.015 0.09
pH 6.0 547 5.20 59.41 10.14 0.011 37.6 0.03 0.89 46.7 15.1 0.026 0.016 0.09
pH 7.5 567 0.96 72.69 12.76 0.013 39.1 0.03 0.81 48.6 16.0 0.026 0.017 0.04
pH 9.0 551 0.26 69.45 6.58 0.012 30.6 0.03 0.75 48.2 15.1 0.007 0.018 0.01
pH 10.5 584 0.08 18.38 1.81 0.011 21.5 0.02 0.001 45.2 11.0 0.002 0.016 0.01









CHAPTER 8
SUMMARY AND CONCLUSIONS

Agricultural activities and urbanization have accelerated the input of nutrients and metals

in various water bodies, thus resulting in water eutrophication and the degradation of aquatic

ecosystems. Like many other places in the world, south Florida is facing challenges with surface

water eutrophication and drinking water depletion. Monitoring studies by He et al. (2003; 2006b)

indicated that surface runoff water from agricultural Hields in the Indian River area was enriched

with N and P, and that Cu and Zn were also transported from agricultural Hields in runoff waters

to receiving surface waters and the accumulation of Cu and Zn in the sediments of the St. Lucie

Estuary has been accelerated in the last two decades.

Of the technologies available for remediating contaminated soil and water,

phytoremediation using aquatic plants is promising because of its low cost compared to

conventional physical or chemical methods, fewer negative effects, and suitability for removal of

low concentration pollutants at a large scale.

Phytoremediation of eutrophic stormwater in detention systems using water lettuce (Pistia

stratiotes L.) was evaluted for its effectiveness. Water lettuce plants were grown in the treatment

plots (with water lettuce) of two detention ponds (the East and West Pond). Water samples were

weekly collected from both the treatment plots and the control plots (without any plants) and

analyzed for water quality parameters including total solids, turbidity, pH, EC, nutrient and metal

concentrations. Plants were monthly sampled for nutrient concentration analysis. Three-fouth

coverage of the water surface in the treatment plots was maintained by periodically harvesting.

Nutrient and metal removal by harvesting was quantified.

Data from this three-year study showed that growing water lettuce improved water quality

by decreasing total solids and water turbidity. Total solids in the water column were decreased









by an average of approximately 20% in the treatment plots compared with that in the control

plots. On average, turbidity was reduced by 65% in the treatment plots as compared to the

control plots. Ammonium-N and NO3-N concentrations in water of the treatments plots were 3 1-

45% and 52-72% lower than those in the control plots, respectively. Reductions in PO4-P, total

dissolved P, and total P concentrations in water were 18-58%, as compared to the control plots.

By periodic harvesting, water lettuce removed 190-329 kg N ha-l and 25-34 kg P ha-l annually

from the waters. Water lettuce had great potential in concentrating metals from the surrounding

water even when the metal concentration was extremely low (under method detection limits)

with concentration factor (CF) from 102 to 105. By periodic harvesting, considerable amounts of

metals, including macro- and micro-elements, were removed from the stormwater. The

dithionite-citrate-bicarbonate (DCB) extraction method was applied to differentiate metals

attached to the external surface from those absorbed into the root and the results revealed that

besides plant uptake, precipitation and adsorption of metals onto the root surface were the other

two important mechanisms by which water lettuce removed metals from water column. More

than 50% of Ca, Cd, Co, Fe, Mg, Mn, and Zn recovered in the root were actually attached to the

external surface, while more than 50% of Al, Cr, Cu, Ni, and Pb was absorbed into the root.

To investigate the possibility of including another free floating aquatic plant, common

salvinia (Salvinia minima), in a polyculture system with water lettuce to further improve P

removal efficiency, hydroponic studies on these two species' N and P requirements were

conducted in a greenhouse. Seven N levels, 0.005, 0.025, 0.05, 0.25, 1.25, 2.5, and 5 mg N L^1,

and six P levels, 0.01, 0.05, 0.1, 0.5, 1, and 5 mg L^1 were applied, respectively.

Critical N concentrations required for net plant growth in biomass after a certain period

were 1.25 and 2.5 mg L^1 for water lettuce and salvinia, respectively. Critical P concentrations









required for water lettuce and common salvinia to have net growth in biomass were 0.1 and 1 mg

L^, respectively. These results revealed higher N and P requirements for common salvinia to

have net growth, which is not desirable when considering including common salvinia in a

polyculture system with water lettuce.

Water lettuce has optimum N and P concentrations of 4.3 and 2.9 mg L^1, respectively, as

predicted from regression analysis, indicating that this plant would work best in waters with N

and P concentrations close to these levels.

Waters differ in their properties such as pH and salinity, which may have marked effects

on plant performance as indicated in the stormwater detention pond study. To better utilize water

lettuce to remediate polluted water, it is critical that the plant can tolerate the pH and salinity of

the water and still give satisfactory performance.

To investigate how water salinity affect water lettuce's performance, a greenhouse

hydroponic study was conducted with six salinity treatments, 473, 1766, 3059, 4351, 5644, and

6937 CIS cm- Water lettuce biomass yield decreased significantly with increasing water salinity,

by approximately 30% in the 1766 CIS cm-l treatment as compared to the control (473 CIS cm )~,

and was further reduced (by about 50%) in the higher salinity treatments (>1766 CIS cm )~.

A hydroponic study with six pH treatments, 3, 4.5, 6, 7.5, 9, and 10.5, was conducted to

determine how water lettuce performs in waters with different pH. Water lettuce could not

survive in pH 3.0 or lower. Although water lettuce could survive in pH 4.5-10.5, it produced

most biomass in neutral and slightly alkaline.

All these studies proved that phytoremediation using water lettuce can efficiently remove

nutrients and metals from eutrophic stormwater in detention systems and improve water quality.









This is encouraging in that detention systems have been widely used for decades

throughout Florida and remain strongly recommended by the Florida Department of

Environmental Protection (FDEP) on sites where conditions favor their use (i.e. shallow

groundwater) (Breitrick, 2008). To address growing concerns about over-enrichment of Florida' s

waters (including surface waters, ground waters, and springs) by nutrients, the FDEP has

initiated the proposed Statewide Stormwater Treatment Rule to increase the level of nutrient

removal required of stormwater treatment systems serving new development, including urban

redevelopment (FDEP, 2009). Larger and deeper ponds are considered as one of the promising

BMPs.

In the N and P requirement studies, the range in N or P concentration between two

consecutive levels was so big that the conclusions on critical N and P concentrations for the

plants could be higher than the true values. Take water lettuce critical N concentration for

example, we concluded it was the level of 1.25 mg L- but it could be a value between 0.25-1.25

mg L^1, say, 0.5 mg L^1, where water lettuce may still have net growth. As previously discussed,

algal growth in the pots containing common salvinia interfered with the growth of common

salvinia and consequently the results. For more accurate and convincing results, further research

should be conducted with N and P concentrations between the ranges of 0.25-1.25 and 0.05-0. 1

mg L^1, respectively. In addition, effective measures need to be developed to inhibit algal growth

in pots with plants of less competitiveness.










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BIOGRAPHICAL SKETCH

Qin Lu was born in 1976 in the hilly city of Xinyi, Guangdong, south China. She received

her bachelor' s degree in agriculture, with specialization in soil science from China Agricultural

University, Beijing, China, in 1999. After she received her Master of Science degree in soil

quality from the Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China, in

2002, she worked for three years as a scientific editor at the Editorial Office of PEDOSPHERE,

Nanjing, China. In 2005, she j oined the University of Florida, Department of Soil and Water

Science, for doctoral study in water quality and received her Ph.D. in summer 2009.





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EVALUATION OF AQUATIC PLANTS FOR PHYTOREMEDIATION OF EUTROPHIC STORMWATERS By QIN LU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009 1

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2009 Qin Lu 2

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To my husband, Diangao, and my son, Xuanning 3

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ACKNOWLEDGMENTS First of all, I would like to express my deepest thanks to my advisor, Dr. Zhenli L. He, for his encouragement, trust, and patience as my mentor. He has not only provided me professional opportunities and offered me numerous insightful suggestions for my research, but also, as a role model, he has shown me that hardworking and pe rsistence as well as creativity and independent thinking are ingredients of success. I am very grateful to my co-advisor, Dr. Donald A. Graetz. He arranged for my airport pick up, social security number applic ation, and first term registration, all of which helped me adjust sm oothly to a whole new environment and feel at home. He has been giving invaluable suggestions and comments for my research. I would also like to give my sincere thanks to Drs. Peter J. Stoffella, Yuncong Li and Samira Daroub for serving on my advisory committee and maki ng major contributions to my research. Special thanks go to the late Dr. Dolen Morris, who had always been prompt in helping me improve my writing. I profoundly appreciate So uth Florida Water Management District for funding the research. I thank Dr. Min Liu and Ms. Yu Wang, Lace y, Katrina, Leighton, and Brandon in Dr. Graetzs lab and Sampson and many other friends in Gainesville for their help and friendship which made my stay in Gainesville a pleasant one. I wish to thank the faculty, staff, and students of the Soil and Water Scie nce Department for thei r assistance and support. Dr. Charles A. Powell of Indian River Research and Education Center at the University of Florida is acknowledged for making hi s laboratory facilitie s available for my use. I wish to thank all the faculty, staff, and students, especially Mr s. Youjian Lin, Hai Lu, Mrs. Cuifeng Hu, Mrs. Maria Solis, Drs. Peter J. Van Blokland and Sandr a B. Wilson, at Indian River Research and Education Center of University of Florida. Th eir kindness and help in many ways made my stay in Fort Pierce a memorable one. 4

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I thank Dr. Xiaoe Yang for providing insight, expertise, and s upport. Special thanks go to Drs. Guochao Chen, Jinyan Yang, Yuangen Yang, Frederico Vieira, Wenrong Chen, Yangbo Wang, Mr. Douglas J. Banks, Mrs. Shaoqin Lu, and PhD students Jinghua Fan and Bruno Pereira for providing assistance in laboratory analysis, expertise and laughter over the past three years. Without their help, successful completion of my PhD study is impossible. I have always felt fortunate to be part of Dr. He s group where I have learned, enjoyed and benefited from team work. I wish to express my appreciation to Dr. Xiao chang Wang for his contin ued interest in my progress, encouragement and support. I am very grateful to my parents, parents-in-law, and siblings for their love, support, encouragement, and confidence in me, which have been the driving force for me to pursue my dreams. I am greatly indebted to my loving husband, Diangao, who has sacrificed so much to be with me here in the United States and helped me in the field and in the lab. I thank my adorable son, Xuanning, who has brought so much joy and ha ppiness into our life. They are the endless source of strength I can always rely on. 5

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF TABLES ...........................................................................................................................9LIST OF FIGURES .......................................................................................................................10ABSTRACT ...................................................................................................................... .............12 CHAPTER 1 LITERATURE REVIEW .......................................................................................................14Water Quality: A Worldwide Concern ...................................................................................14Phytoremediation of Contaminated Water Using Aquatic Plants ..........................................17Stormwater Treatment with Floating Aquatic Plants .............................................................22Growth Factors of Aquatic Plants ...........................................................................................23Research Objectives ........................................................................................................... .....252 NUTRIENT REMOVAL POTENTIAL OF WATER LETTUCE ( PISTIA STRATIOTES L.) FROM STORMWATER IN DETENTION SYSTEMS ..................................................27Introduction .................................................................................................................. ...........27Materials and Methods ...........................................................................................................28Experimental Design .......................................................................................................28Chemical Analysis ...........................................................................................................31Data Treatment and Data Analysis ..................................................................................32Results and Discussion ........................................................................................................ ...33General Water Quality Improvement ..............................................................................33Nitrogen and P Concentration Reduction ........................................................................39Nitrogen and P Removal Potential by Plant Uptake .......................................................46Physiological Limits ........................................................................................................48System Management .......................................................................................................49Conclusions .............................................................................................................................503 METAL REMOVAL POTENTIAL OF WATER LETTUCE ( PISTIA STRATIOTES L.) FROM STORMWATER IN DETENTION SYSTEMS ........................................................51Introduction .................................................................................................................. ...........51Materials and Methods ...........................................................................................................54Chemical Analysis ...........................................................................................................55Data Treatment ................................................................................................................55Results .....................................................................................................................................56Metal Concentration Reduction in Water ........................................................................56Metal Accumulation by Plant Root .................................................................................61 6

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Metal Distribution in Plant ..............................................................................................61Estimation of Annual Metal Removal .............................................................................62Metal Uptake and Surface Adsorption ............................................................................63Metal Bio-concentrated by Plant .....................................................................................64Discussion .................................................................................................................... ...........66Conclusions .............................................................................................................................684 NITROGEN REQUIREMENT FO R WATER LETTUCE AND COMMON SALVINIA ...................................................................................................................... .......69Introduction .................................................................................................................. ...........69Materials and Methods ...........................................................................................................70Experimental Design .......................................................................................................70Chemical Analysis ...........................................................................................................71Statistical Analysis .......................................................................................................... 71Results and Discussion ........................................................................................................ ...71Relationship between Plant Bioma ss Yield and N Concentration ..................................71Relationship between Plant N and Solution N Concentration .........................................75Plant Critical N Concentration ........................................................................................78Conclusions .............................................................................................................................785 PHOSPHORUS REQUIREMENT FOR WATER LETTUCE AND common SALVINIA ...................................................................................................................... .......80Introduction .................................................................................................................. ...........80Materials and Methods ...........................................................................................................81Experimental Design .......................................................................................................81Chemical Analysis ...........................................................................................................82Statistical Analysis .......................................................................................................... 82Results and Discussion ........................................................................................................ ...82Relationship between Plant Biomass Yield and Solution P Concentration ....................82Relationship between Plant P Concentration and Solution P Concentration ..................87Plant Critical P Concentration .........................................................................................90Conclusions .............................................................................................................................906 EFFECT OF SALINITY ON GROWTH OF WATER LETTUCE .......................................92Introduction .................................................................................................................. ...........92Materials and Methods ...........................................................................................................93Experimental Design .......................................................................................................93Chemical Analysis ...........................................................................................................94Statistical Analysis .......................................................................................................... 95Results and Discussion ........................................................................................................ ...95Plant Growth as Affected by a Salinity Gradient ............................................................95Plant Biomass in Different Salinity .................................................................................95Plant Nutrient Status under Different Salinity Conditions ..............................................98Conclusions ...........................................................................................................................100 7

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7 EFFECT OF PH ON GR OWTH OF WATER LETTUCE ..................................................101Introduction .................................................................................................................. .........101Materials and Methods .........................................................................................................102Experimental Design .....................................................................................................102Chemical Analysis .........................................................................................................103Statistical Analysis ........................................................................................................10 3Results and Discussion ........................................................................................................ .103Plant Growth in Water at Different pH .........................................................................103Plant Biomass Yield at Di fferent pH Treatments ..........................................................105Plant Nutrition Status at Different pH Treatments ........................................................105Conclusions ...........................................................................................................................1098 SUMMARY AND CONCLUSIONS ...................................................................................111LIST OF REFERENCES .............................................................................................................115BIOGRAPHICAL SKETCH .......................................................................................................127 8

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LIST OF TABLES Table page 2-1 Water quality improvement in the treatm ent plots of the East and West Ponds. ...............362-2 Annual removal amounts of plant dry biom ass, N, and P from the East and West Ponds. .................................................................................................................................473-1 Annual metal removal rates by periodic harvesting of water lettuce. ................................654-1 Nutrient solution composition for N requirement study. ...................................................705-1 Nutrient solution composition for P requirement hydroponic study. ................................816-1 EC and ions contributing to water salinity in the waters of the East and West Ponds. .....926-2 Nutrient solution composition fo r the salinity tolerance study. .........................................937-1 Chemical composition of nutrien t solution for pH effect study. .....................................1027-2 Nutrient concentration and related properties of the nutri ent solution at different pH levels. ....................................................................................................................... ........110 9

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LIST OF FIGURES Figure page 2-1 Experimental set up in the West Pond and the East Pond. ................................................302-2 Total solid concentrations in the waters of the East and West Ponds. ...............................342-3 Turbidity in the East and West Ponds. ...............................................................................352-4 Water samples from treatment plot and control plot. ........................................................372-5 Water EC in the East and West Ponds. ..............................................................................382-6 Water pH in the East and West Ponds. ..............................................................................392-7 Nitrate-N in the waters of the East and West Ponds. .........................................................402-8 Ammonium-N in the waters of the East and West Ponds. .................................................412-9 Total Kjeldhal N in the waters of the East and West Ponds. .............................................422-10 Water PO4-P in the East and West Ponds. .........................................................................432-11 Total dissolved P in the waters of the East and West Ponds. ............................................442-12 Total P in the waters of the East and West Ponds. .............................................................452-13 Nitrogen concentrations in plant roots and shoots from the East and West Ponds. ...........472-14 Phosphorus concentrations in plant roots and shoots from the East and West Ponds. ......483-1 Total dissolved metal concentrations in the treatment and control plots of the East and West Ponds duri ng 2005-2007 (n=122).. ....................................................................563-2 Plant metal concentration factors (CFs) in the East and West Ponds. ...............................613-3 Metal root/shoot ratio in concentration of the East and West Ponds. ................................623-4 Distribution of metals outside and inside of water lettuce root. ........................................643-5 Plant metal bio-concentration factors (BCFs) in the East and West Ponds. ......................664-1 The growth performance of water lettuce and common salvinia under different N levels. ....................................................................................................................... ..........734-2 Plant dry biomass yield at different N level treatments. ....................................................734-3 The shoot/root ratio of water lettu ce dry biomass at di fferent N levels. ............................74 10

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4-4 Regression curve of plant dry bioma ss yield vs. solution N concentration. ......................744-5 Plant N concentration at di fferent N level treatments. .......................................................754-6 Regression curve of plant N concentr ation vs. solution N concentration. .........................775-1 Growth performance of water lettuce and common salvinia under different P levels. ......845-2 Plant dry biomass weights of different P level treatments. ................................................845-3 Water lettuce shoot/root in dry biomass under different P level. ......................................855-4 Regression curves of plant dry bi omass vs. solution P concentration. ..............................865-5 Plant P concentration in treatment s with different solution P level. ..................................885-6 Regression curve of plant P vs. solution P concentration. .................................................896-1 Growth performance of water lettuce in water with gradient salinity. ..............................946-2 Growth performance of water lettuce in water with gradient salinity. ..............................966-3 Plant dry biomass of water lettuce with different salinity treatments. ...............................976-4 Plant nutrient concentrations w ith different salinity treatments. .......................................987-1 Growth of water lettuce unde r different pH treatments. ..................................................1047-2 Dry biomass yield of wate r lettuce at different pH. .........................................................1067-3 Regression curve of water lettu ce dry biomass vs. solution pH. .....................................1067-4 Plant nutrient concentration of water lettuce at different pH treatments. ........................107 11

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EVALUATION OF AQUATIC PLANTS FOR PHYTOREMEDIATION OF EUTROPHIC STORMWATERS By Qin Lu August 2009 Chair: Zhenli L. He Cochair: Donald A. Graetz Major: Soil and Water Science Water quality impairment by nutrient and metal enrichment from agricultural activities has been a concern worldwide. Phytoremediation te chnology using aquatic plants was evaluated for its efficacy in removing N, P, and metals from stormwater in detention ponds. Water lettuce ( Pistia stratiotes ) plants were grown in treatment plots in two stormwater detention ponds and water quality in both ponds was monitored. To be tter utilize water lettuce and investigate the possibility of a water lettuce-common salvinia (Salvinia minima ) polyculture system, water lettuce and common salvinia were tested for their N and P requirements for normal growth with hydroponic studies conducted in a greenhouse. Water lettuce was also evaluated for its growth performance in water with differe nt pH and salinity levels. Water quality in both ponds was improved by phytoremediation with water lettuce, as evidenced by decreased turbidity, total solids and nutrient concentr ations. Turbidity was decreased by more than 65%. Total solids decreased by about 20%. Ammonium-N and NO3-N concentrations in the treatments plots were 31-72% lower than those in th e control plots (without plants), and total Kjeldhal N was decreas ed by more than 20%. Reductions in PO4-P, total dissolved P, and total P concentrations in wa ter were approximately 18-58% compared to the 12

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13 control plots. Annual rem oval of N and P from the water was 190 and 25 kg ha-1, respectively in the East Pond, and 329 and 34 kg ha-1, respectively in the West Pond by harvesting plant biomass. Compared to the control plots, Al, Fe, and Mn concentrations were reduced by an average of 20%, and K by 10% in the treatment plots. Ca lcium, Mg, and Na concentrations were also reduced by 5-10%. Metals were substantially accumu lated in the roots of water lettuce. A larger proportion of Ca, Cd, Co, Fe, K, Mg, Mn, and Zn was attached to external root surfaces by adsorption or surface deposition while more Al, Cr, Cu, Ni, and Pb were absorbed and accumulated into the root. The critical N concentrations required for wa ter lettuce and common salvinia to have net growth in biomass were 1.25 and 2.5 mg L-1, respectively, and the critic al P concentrations were 0.1 and 1 mg L-1, respectively. Higher N and P requi rements make common salvinia less desirable for a polyculture system with water lettuce. Water lettuce could tolerate th e salinity level (< 1766 S cm-1) of freshwater but its biomass could be reduced by up to 30% by high salinity (1766 S cm-1). This plant could not survive in brackish water with salinity > 6937 S cm-1. We can also expect optimum performance from this plant in neut ral and slightly alkaline water.

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CHAPTER 1 LITERATURE REVIEW Water Quality: A Worldwide Concern To meet the requirement of a burgeoning human population, fertilizers and chemicals have been extensively used to boost crop producti on. Of the nitrogen (N ) taken up by plants, approximately 70% is provided by inorganic fe rtilizers (Singh and Verma, 2007). Nitrogen loading to the land has doubled from the pre-industrial period (111 Tg yr-1) to the present time (223 Tg yr-1) due to anthropogeni c activities (Green et al., 20 04). Manures and biosolids are usually applied based on crop N requirements, which provides phosphorus (P) in excess of crop needs. Many fungicides contain he avy metals such as copper (Cu) and zinc (Zn). Repeated use of the fungicides in citrus and vegetable crop pr oduction systems has resulted in accumulation of Cu and Zn in the soils (Zhu and Alva, 1993). Off-site migration of these nutrients and me tals by runoff to surface water is a worldwide concern because of the resulting degradation of the aquatic ecosystems and decreased water availability. Urbanization also contributes to the deterioration of the aquatic ecosystems by boosting sediment loads because of decreased surface area available for abso rption and infiltration of rainwater and snow melt and by incr easing heavy metal inputs from automobile usage. Fertilizers and chemicals applied on urban/suburban lawns, gardens, and golf course s are also subject to loss by surface runoff Runoff from agricultural fields or urban area carries inorganic nutrien ts (Caccia and Boyer, 2005). In Europe, 65% of the Atlantic coast shows vary ing degrees of eutrophication (Diaz and Rosenberg, 2008), and 55% of river stations had annual av erage dissolved P concentrations in excess of 50 g P L-1 over the period 1992-1996 (Crouzet et al., 1999). Taking agricultural land 14

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out of production brought both loads and concentr ations of soluble r eactive P and dissolved inorganic N down by about 90% in a first-order agricultural stream in a small rural watershed, Germany (Chambers et al., 2006). This strongly shows how much agriculture contributes to increased nutrient inputs into the waterways. Measurements in a lake (0.08-2.29 mg P L-1 total P (TP) and 3-15 mg N L-1 total Kjeldhal N (TKN)) and upstream to the lake (0.6-3.8 mg P L-1 and 10-22 mg N L-1 respectively) indicated eutrophication of lakes by receiving nutrient-rich surface runoff from urbanized areas of Central Africa (Kemka et al., 2006). Approximately 10% of New Zealands shallow lakes were classified as eutrophic (> 50 g TP L-1) (Cameron et al., 2002). An agricultural non-point source pollution su rvey in 18 townships in Fujian Province, China revealed that N and P were the primary cont aminants in the drainage area and that farm nutrient loss, aquaculture, lives tock and bird feces and urine were the largest three pollution sources (Huang et al., 2008). In another provin ce of south China, Guangdong, where fertilizers are heavily applied in the orch ards of its hilly and mountainou s area, 90.5% of the runoff water samples from the orchards in Dongyuan County ha d a total N (TN) concentration higher than 0.35 mg L-1 and 54.2% had a TP concentr ation higher than 0.1 mg L-1 (Zeng et al., 2008). According to Diaz and Rosenberg (2008), 78% of the continental US coastal area show varying degrees of eutrophication. An estimate of 45% of US waterways has impaired water quality due to nutrient enrichment according to the US Environmental Protection Agency (CEEP, 2001). An average NO3-N concentration of 6.6 mg L-1 in surface runoff resulted from corn production was measured in Lake Bloomington watershed, Illi nois in a nine-year (1993-2002) and 36-site monitoring study (Smiciklas et al., 2008). Both N and P concentrations above the 15

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eutrophic level in the receiving water bodies were observed by Yu et al. (2008) in a watershed associated with sugarcane production in L ouisiana. Lake Apopka in Florida was made hypereutrophic by P loading from fl oodplain farms (Coveney et al., 2002). An important source of heavy metals is highway runoff, especially in large cities such as Guangzhou in south China (Gan et al., 2008). Highway runoff on the island of Crete, Greece showed two-year (2005-2007 ) mean concentrations of Cu, Ni, Pb and Zn to be 56, 114, 49 and 250 g L-1, respectively (Terzakis et al., 2008). Coppe r was found to be the dominant metal in the surface runoff from a suburban parking lot near Portland, Oregon (Mesuere and Fish, 1989). Five times background levels of Cr, Cu, Ni, Pb and Zn concentrations were found in the sediment of River Murray, Australia (Thoms, 200 7). Higher metal concentrations in the river, lake, or coastal sediments were often associ ated with increased agricultural and urban development, accompanying with more anthropogenic activities (Amin et al., 2009). Water quality throughout south Florida has been a major concern for many years. Nutrient enrichment has been considered to impact ecologi cal functions of the Everglades National Park, Lake Okeechobee, and Indian River Lagoon (Capece et al., 2007; Ritter et al., 2007). Results from recent monitoring study in Indian River Lag oon (IRL) by He et al. (2006b) indicate that more than 50% of the surface runoff water samples contained TN of 1 to 5 mg L-1 and TP above 1.0 mg L-1. Mean concentrations of TN and TP in the runoff were 4.1 and 1.6 mg L-1, respectively, which are much greater than the US EPA critical levels for surface water (1.5 mg L1 for total N and 0.1 mg L-1 for total P) (U. S. Environm ental Protection Agency., 1976). The intricate network of Canals C-23, C-24, and C-44, that drain the surrounding urban and agricultural lands in the St. Lucie Basin and are connected to the IR L, are estimated to collectively deliver at least 8.6l05 kg of N, 9.1105 kg of P, and 3.6l08 kg of suspended solids 16

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to the estuary annually (Graves and Strom, 1992). Overall IRL total N load is projected (year 2010) to increase by 32% (WoodwardClyde Consultants, 1994). Repeated use of the fungicides in citrus and vegetable crop production systems has resulted in accumulation of Cu and Zn in the sediments of the St. Lucie Estuary (Haunert, 1988; He et al., 2003). High concentrations of Cu and Zn were measured in storm runoff water from these production systems (He et al., 2006a; Zhang et al., 2003). Phytoremediation of Contaminated Water Using Aquatic Plants Excessive nutrients (N and P) in surface runoff cause eutrophication in the receiving water, such as lakes and estuaries, and lead to alga l blooms and changes in species composition. The increased metals in the receiving water are t oxic to the living communities in the aquatic ecosystem, and also cause health problems in human. The aquatic ecosystems are degraded by the increased nutrients and metals, water quality is impaired, and wa ter availability is decreased. Actions are needed to remediate such polluted systems or to treat the surface runoff before it gets into the receiving water. Unlike point so urce water pollution, which is localized and easier to monitor and control (Smith et al., 1999), non-poi nt source pollution is of a diffuse nature. Conventional remediation methods suitable for po int source pollution may not be desirable or cost-effective when applied to non-point source po llution because of the relatively low pollutant concentrations and large source area. In addition to development of best manage ment practices (BMPs) to reduce losses of nutrients (N, P) and tran sport of contaminants (heavy metals and pesticides) from land to water, constructed wetlands such as st ormwater treatment areas (STAs) water detention systems, and retention ponds have been incr easingly built in South Florida to clean eutrophic water from agriculture or urban areas before they are discha rged to surface water systems such as Indian River Lagoon. The functions of these systems ar e to settle down susp ended solids and reduce 17

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concentrations of dissolved nutrien ts and contaminants in water where aquatic plants can play an important role. Phytoremediation has been increasingly used to clean up contaminated soil and water systems because of its lower costs and fewer negative effects than physical or chemical engineering approaches (Ignjat ovic and Marjanovic, 1985; Prasad and Freitas, 2003; Reddy and DeBusk, 1986). The principles of phytoremediation system to clean up stormwater include: 1) identification and implementation of efficient aq uatic plant systems; 2) uptake of dissolved nutrients including N and P and meta ls by the growing plants, and th e plants creating a favorable environment for a variety of complex chemical, bi ological and physical pro cesses that contribute to the removal and degradation of nutrients (B illore et al., 1998; Gu mbricht, 1993); and 3) harvest and beneficial use of the plant biomass produced from the remediation system. Because of their fast growth rates, simple growth requirements, and ability to accumulate biogenic elements and toxic substa nces, aquatic plants are utilized for nutrient and metal removal from water. Since the first rec ognition of their valu e in water quality improvement in the 1960s and the 1970s (Sheffield, 1967; Steward, 1970; Wooten and Dodd, 1976), aquatic plants have been widely used to treat wast ewaters or increasingly used to remediate eutrophic waters in forms of constructed wetlands or retention ponds This is a low-cost treatment with low land requirements, which is attractive to urban areas with high land prices. Aquatic plants are grouped into submerged, em ergent, and floating/fl oating-leaved aquatic plants according to their leafs relation with water. Among the submerged aquatic plants, coontail ( Ceratophyllum demersum L.), hydrilla ( Hydrilla verticillata ), southern naiad ( Najas guadalupensis ) are the most investigated (Badr and Fawzy, 2008; Bunluesin et al., 2004). Cattail ( Typha latifolia ), bullrush ( Scirpus lacustris), and common reed ( Phragmites australis ) are the 18

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most planted emergent plants in constructed we tlands to remove nutri ents such as N and P (Manab Das and Maiti, 2008). Among the floating/floating-leaved aquatic plants, water hyacinth ( Eichhornia crassipes ), water lettuce ( Pistia stratiotes), duckweed ( Lemna spp. and Spirodela polyrrhiza W. Koch), pennywort ( Hydrocotyle umbellata ), and common salvinia ( Salvinia minima baker) are the best candidates (John et al., 2008; Maine et al., 2004; Mishra et al., 2008; Sanchez-Galvan et al., 2008). With regard to the uptake capacity of aquatic plants, and subsequently the amount of nutrients or contamin ants that can be removed when the biomass is harvested, floating plants (especially large-leaved species) are in the lead, followed by emergent species and then submerged species Approximately 350 kg P and 2000 kg N ha-1 yr-1 were removed by large-leaved floating plants such as water hyacinths, whereas the capacity of submerged macrophytes was lower (<100 kg P and 700 kg N ha-1 yr-1) (Brix, 1997). Growing in waters with similar P concentrations, water hya cinth had an average P concentration almost twice that of hydrilla, hornwort, pondweed, eelgrass, or naiad, showing a much greater ability for P scavenging (Easley and Shirley, 1974). Emergent macrophytes are mostly in the range of 30 to 150 kg P ha-1 yr-1 and 200 to 2500 kg N ha-1 yr-1 (Brix, 1994; Gumbricht, 1993). Impressive removal rates of inorganic N (NO3-N, NH4-N, and total N) and P (PO4-P and total P) have been reported from all kinds of phytoremediation systems using aquatic plants especially when invasive floating aquatic plants such as water hyacinth we re utilized in nutrientor metal-rich wastewaters. A wide range of nut rient reduction in wastewaters containing water hyacinth has been reported. For inorganic N, Reddy et al. (1982) reporte d a reduction of about 80%, while Sheffield (1967) obser ved a 94% reduction. For orthoP, a 40-55% reduction was reported by Sheffield (1967). For total P, Reddy et al. (1982) measured about 32% reduction, while Ornes and Sutton (1975) achieved a much hi gher removal rate of 80% in their treatment 19

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pond. In a pilot scale study using a series of six tanks with water hyacinth for wastewater treatment, the mean decrease in total N and total P in the effluent as it flowed the six tank series was 27.6% and 4.48%, respectively (Bramwell a nd Devi Prasad, 1995). A pond containing water hyacinth, with an air stripping un it and a flocculation and settling unit, was reported to remove >99% ortho-P, 99% nitrate-N, and >99% amm onia-N (Sheffield, 1967). Plant uptake contributes a large proportion to the N and P removal for ve ry high uptake rates have been reported, for instance, 1980 kg N and 322 kg P ha-1 y-1 by Boyd (1970), 2500 kg N and 700 kg P ha-1 y-1 by Rogers and Davis (1972), and up to 5350 kg N ha-1 y-1 and 1260 kg P ha-1 y-1 by Reddy and Tucker (1983). Although at a lower rate compared to such la rge-leaved floating species as water hyacinth, small-leaved floating species such as duckweed can also remove a considerable amount of nutrients and have been utilized in remediation of wastewater s. Small tank polycultures of duckweed species (Lemna minor and Spirodela polyrhiza ) were found to remove 404 mg N m-2 day-1 (1460 kg N ha-1 yr-1) and 84 mg P m-2 day-1 (307 kg P ha-1 yr-1) from dairy barn wastewater (Whitehead et al., 1987). Phosphorus removal rates of 60-92.2% were achiev ed in a wastewater system utilizing Lemna gibba (Hammouda et al., 1995). Two species of Azolla ( Azolla filiculoides and Azolla pinnata ) removed N from mixed waste wate r resulting in more than 50% decrease in concentration (Elsharawy et al., 2004). According to Ruan et al. (2006), polluted rive r water was efficiently treated by pilot-scale constructed wetland systems planted with emergent aquatic plants, Typha latifolia and Scirpus lacustris with mean NH4-N removal rates of over 85%. Wetlands with emergent macrophytes were reported to remove P at rates from 0.4 to 4.0 g m-2 yr-1, with more eutrophic systems achieving higher removal rate (Mitsch, 1992). 20

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Tatrai et al. (2005) observed an increase in transparen cy and a decrease in the concentrations of P simultaneou sly with increased presence of submerged macrophytes in the lake. Aquatic plants also demonstrate tremendous potential in metal accumulation and removal from the surrounding waters. Free water surface a nd subsurface flow pilot-size wetlands were constructed to treat highway runoff with metal removal rates of 47%, 23%, 33%, and 61% for Cu, Ni, Pb and Zn, respectively, with their resp ective two-year mean concentrations of 56, 114, 49 and 250 g L-1 (Terzakis et al., 2008). Azolla filiculoides removed 91.0, 41.5, 82.5, 37.7, 12.1, 46.7 and 67.2% of the initial Fe, Zn, Cu, Mn, Co, Cd and Ni, respectively from mixture of waste waters, while Azolla pinnata removed 92.7, 83.0, 59.1, 65.1, 95.0, 90.0 and 73.1%, respectively (Elsharawy et al., 2004). Although al l three plants, water lettuce ( Pistia stratiotes L.), duckweed ( Spirodela polyrrhiza W. Koch), and water hyacinth ( Eichhornia crassipes) demonstrated high removal rates of Fe, Zn, Cu, Cr, and Cd (>90 %) without reduction in growth, water hyacinth were the most efficient followed by water le ttuce and duckweed (Mishra and Tripathi, 2008). Many researchers have reported th at high heavy metal concentrati ons (Cu, Cd, Mn, Pb, Hg, etc.) were measured in the tissues of aquatic pl ant growing in waters with elevated metal concentrations and no toxic eff ects or reduction in plant growth were observed (Badr and Fawzy, 2008; Mishra et al., 2008; Okafor and Nwajei, 2007). Common duckweed and water hyaci nth have been reported to be the top species as Cd accumulators (Wang et al., 2002; Zayed et al., 1998; Zhu et al., 1999). Both Salvinia herzogii and Pistia stratiotes efficiently removed Cr from water at the concentrations of 1, 2, 4, and 6 mg Cr L-1 (Maine et al., 2004). Lead con centrations in plant tissue (mg kg-1) were found to be 1621 and 1327 times those in the external solution (mg L-1) for C. demersum and C. caroliniana, 21

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respectively (Fonkou et al., 2005). Salvinia minima has been reported as a hyperaccumulator of Cd (Olguin et al., 2002) and Pb (Olguin et al ., 2005) with bioconcentration factors (metal concentration in plant tissue ove r that in external solution) of approximately 3000 for both heavy metals. Stormwater Treatment with Floating Aquatic Plants To enhance the performance of stormwater detention ponds, aquatic plants are often planted. Biomass production, growth rate, and ea siness of management and harvest are the considerations that should be take n into in selecting aquatic plants. Floating aquatic plants can grow in a verti cal as well as horizont al direction, thereby increasing the photosynthetic surface area. In addition, unlike submerged species, they photosynthesize in an aerial environment where CO2 is not a constraini ng factor and water supply is abundant. All these factors together make floating aqua tic plants, especially largeleaved species, one of the earths most produc tive communities. Their an nual primary production was estimated to be up to 85 Mt in dry matter pe r hectare in subtropical and tropical regions (Westlake, 1963). Floating plants are more favorable in terms of energy and machinery use in management. In addition, harvesting floating plan ts causes minimal disturbance to the system, thus reducing sediment re-suspension. Water hyacinth is a free-floati ng vascular aquatic plant found throughout the tropical and subtropical regions of the worl d (Holm et al., 1969). It extract s nutrients from the water through a system of fine, feathery roots. Water hyacint h is one of the earliest and most widely used floating aquatic plants with extensive publications on its biomass production, growth rate, nutrient uptake dynamic and ability. According to Knipling et al. (1970), the harvesting of one acre of water hyacinths would remove 170 kg of N and 60 kg of P from Lake Alice in Gainesville, Florida. 22

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Compared to water hyacinth, the other largeleaved free-floating aquatic plant, water lettuce, has a lower nutrient upt ake capacity and lower nutrient concentration. For example, the N, P and ash contents of biomass were about 1.5 times higher in water hyacinths than in water lettuce (Aoi and Hayashi, 1996). However, when considering management, the smaller biomass of water lettuce renders an easier re moval of biomass from water bodies. Biomass yields of small-leaved floating plants such as Salvinia Lemna and Azolla are significantly lower than those of large-leaved sp ecies, which makes these plants unsuitable for monoculture systems. But they were reported to have high P removal capacity (Sutton and Ornes, 1975) and low light requirements (We dge and Burris, 1982). Reddy and DeBusk (1985) suggested they be integrated into treatment sy stems based on large-leav ed species to improve overall nutrient removal efficiency. Growth Factors of Aquatic Plants For a phytoremediation system to work effici ently, optimal plant growth is the key. Many environmental factors can influence plant growth and its performance, such as temperature, nutrient concentration, pH, solar radiation, and salinity of the water. The weight and size of aquatic plants are a function of these factors. For example, growth of water hyacinth plants cultured in nutrient solution were significantly influenced by the seasonal changes in temperature and solar radiation, shorter time was required to reach maximum biomass yield in summer with high growth rate (Reddy et al., 1983). If maximum growth is obtained, one hectare of water hyacinths could remove about 2500 kg N yr-1 (Rogers and Davis, 1972) and as high as 7629 kg N ha-1 yr-1 was reported by Reddy and Tucker (1983) fo r water hyacinth cultured in a nutrient solution. Although large-leaved floating plants such as water hyacinth and water lettuce can produce high biomass and remove large amounts of nutrien ts and metals, they may not be suitable for 23

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temperate or frigid areas due to their sensitivity to cool temperature which significantly affects their performance (Clough et al ., 1987). Instead, duckweed or azo lla could be a better choice because of their tolerance to colder weather (Reddy et al., 1 983). This also explains why pennywort removed 20% more N and 30% more P fr om primary domestic effluent than water hyacinth during the winter in cent ral Florida (Clough et al., 1987). Nutrient availability affects the growth and performance of aquatic plants. Within the studied nutrient concentr ation ranges, mean number of ramets mean height and total biomass of water hyacinth significantly incr eased with increasing nutrient level (Zhao et al., 2006). A 200fold difference in dry weight of water lettu ce was reported by Aoi and Hayashi (1996) between cultivated in rain water and treated sewage water. Similar to terrestrial species, aquatic plants respond positively to nutrient con centration increases up to a certain point followed by no further response or a negative response. Five a nd a half mg N per liter and 1.06 mg P L-1 were such points reported for water hyacinth growth (Reddy et al., 1989; Reddy et al., 1990), while 20 mg N L-1 and 2 mg P L-1 were found for Salvinia molesta (Cary and Weerts, 1984). Not only nutrient concentration itself, but also ra tios between different nutrients pl ay an important role in plant growth. It was reported that th e highest production of water hyacinth occurs when the N:P ratio in the water was close to 3.6 (Reddy and Tucker, 1983). Stormwater varies in salinity which may have significant effects on aquatic plants growth and performance. Utilization of such invasive aquatic plants as water hyacinth and water lettuce has its advantages as discussed above and its co ncern of plant escape from the detention systems into the lagoons or estuaries. Knowledge on salinity tolerance of candidate plant(s) can help better utilize the pl ant(s) without bringing disaster. Salt concentra tions of 1660 and 2500 mg kg-1 24

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(equivalent to 2683 and 4040 S cm-1) were reported to have toxi c effects on water lettuce and water hyacinth, respectively (Haller et al., 1974). pH plays a role in plant growth directly by hydrogen ion (H+) injury at low pH and indirectly by affecting availabi lity and toxicity of mineral el ements(Pessarakli, 1999). Generally, plant grows best in the pH range of 5.5-7.0. Optimum pH ranges 6.5-7.5 and 5.8-6.0 were reported for water hyacinth (El-Gendy et al., 2004; Hao and Shen, 2006). Macroalga Chlorella sorokiniana grew best at pH 78 (Moronta et al., 2006). Research Objectives Taking their high biomass production and easiness in management into consideration, free floating aquatic plants were chosen for my di ssertation studies. Compared to water hyacinth, water lettuce has been overlooked with little i nvestigation. Compared to large-leaved floating plants, small-leaved floating plants such as azolla ( Azolla filiculoides and Azolla pinnata ), duckweed ( Spirodela polyrrhiza W. Koch), and common salvinia ( Salvinia minima ) produce much less biomass, which is a disadvantage for application to phytor emediation (Reddy and Bagnall, 1981; Reddy, 1984). But it was also shown th at these small-leaved floating plants have a narrower N/P ratio indicating they are effici ent in removing P (Reddy and DeBusk, 1985). It was suggested that small-leaved floating plants can be included in polyculture systems with large-leaved plants (Reddy and DeBusk, 1985). Among the small-leaved aquatic plants, common salvinia has been shown to produce dry bioma ss twice that of duckweed when cultured in nutrient solution (Olguin et al., 2002) and outcomp ete duckweed for growth surface in a mixed culture (Olguin et al., 2007). Comm on salvinia was also reported to be capable of removing over 70% of NH4-N and PO4-P from coffee processing effluent (Olguin et al., 2003). It was of our interest to compare water lettuce and common salvinia in terms of their nutrient uptake ability 25

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26 and determine the possibility of include common salvinia in a polyculture system with water lettuce. It is critical to select a ppropriate plants for water tr eatment taking into account the characteristics of the water to be remediated. Th e overall objective of this study was to evaluate water lettuces nutrient and me tal removal potential in stormwater detention ponds and its growth response to environmental factors. Spec ific objectives addressed in this dissertation include: Evaluation of water lettuce for its potential in N and P removal from stormwater; Investigation of water lettuce regarding its metal accumulation ability and mechanism, and metal distribution in the plant; Determining N requirements of water lettu ce and common salvinia for both net and maximum growth; Determining P requirement of water lettu ce and common salvinia for both net and maximum growth; Assessing the effects of salinity on the growth of water lettuce; Assessing the effects of pH on the growth of water lettuce.

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CHAPTER 2 NUTRIENT REMOVAL POTENTIAL OF WATER LETTUCE (PISTIA STRATIOTES L.) FROM STORMWATER IN DETENTION SYSTEMS Introduction Chemical fertilizers have been playing a very important role in agricultural production in the modern society. Because of crops quick resp onse to chemical fertilizers, to many farmers, fertilizer application seems to be the only guarantee of high crop yield. But the ever increasing use of fertilizer results in si gnificant build-up of nut rients, such as nitrogen (N) and phosphorus (P), in the soils (Smith et al., 2007). These nut rients are subject to loss to surface and ground water. Water quality is impaired and water av ailability is reduced because of accelerated eutrophication (Carpenter et al., 1998). Estuaries are among the most biologically productive ecosy stems in the world. The St. Lucie Estuary (SLE), rich in habitats and species, is one of the largest and most ecologically diverse estuaries located on the cen tral east coast of Florida and a major tributary to the Indian River Lagoon (IRL). Surrounded by a rapidly growing human populat ion, its health has been a concern for years due to growing pressures from anthropogenic sources of nutrients and pollutants (Chamberlain and Hayward, 1996; Phlips et al., 2002). Results from recent monitoring study in IRL by He et al. (2006b) indicate that more than 50% of th e surface runoff water samples contained TN of 1 to 5 mg L-1 and TP above 1.0 mg L-1. Mean concentrations of TN and TP in the runoff were 4.1 and 1.6 mg L-1, respectively, which are much greater than the USEPA critical levels for surface water (1.5 mg L-1 for total N and 0.1 mg L-1 for total P) (U. S. Environmental Protection Agency., 1976). The in tricate network of Canals C-23, C-24, and C44, that drain the surrounding urban and agricult ural lands in the St. Lucie Basin and are connected to the IRL, are estimated to collectively deliv er at least 8.6l05 kg of N, 9.1105 kg of P, and 3.6l08 kg of suspended solids to the estu ary annually (Graves and Strom, 1992). 27

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Best management practices (BMPs) have been implemented to reduce N and P export from urban area and agricultural fiel d and approximately 10-15% reduc tion may be realized based on our previous BMPs project (He et al., 2005). This reduction is still far below the goals (30-70% reduction in N and P) established in the Su rface Water Improvement and Management Plan (SWIM Plan) (SFWMD and SJRWMD, 1994) fo r the St. Lucie Estuary watershed. The stormwater needs to be further treated before it is dischargeable to the St. Lucie Estuary. Large constructed wetlands or stormwater treatment areas have been operating since early 1990s to filter nutrients in eu trophic stormwater from Evergl ades Agricultural Area (EAA) before they are drained into wa ter conservation area in the Evergl ades National Park. Stormwater detention systems are to be constructed in th e Indian River area for cleaning up nutrients and pollutants in stormwater from agriculture and urban area. Key to the performance of the constructed wetlands including STAs, water dete ntion systems, and retention ponds is the establishment and sustainability of desired vegetation communities. The primary objectives of this study were to evaluate the effectiveness of water lettuce ( Pistia stratiotes L.) in removing nutrients including N and P from stormwater in the constructed water detention systems and to quantify the poten tial of this plant in improving stormwater quality in detention pond system. Materials and Methods Experimental Design Two stormwater detention ponds (called the West Pond and the East Pond), located to the west and east side, respectively, of the Universi ty of Florida, Indian River Research and Education Center (IRREC) Facility in Fort Pierce, were selected for th is study (Figure 2-1). The East Pond has an area of approximately 2500 m2 and the West pond, approximately 5000 m2. The West Pond receives stormwater from I RREC teaching gardens. The land surrounding the 28

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East Pond was used for citrus production but has b een left fallow since the occurrence of canker five years ago. Besides receiving stormwater from the fallow land, the East Pond also receives stormwater from the ditch along Kings Highway. For each pond there were two plots, i.e. the c ontrol (without plants) and the treatment plot (with plants), which were separated from each other and from the rest part of the pond by a soft wall made of weather-resistant plastic material, which allows only water and dissolved ions to pass through. The bottom of the soft wall was in serted into the sediment by an impregnated stainless iron chain and its top was floated on water surface by means of a wrapped foam bump. The height of the soft wall is equal to the ma ximum water depth (2 m and 3 m in the East and West Pond, respectively) of the plot site when the pond is full of wa ter. Therefore, the height of soft wall can change according to water le vel. Each plot had an area of 72 m2 (12 m x 6 m). Water lettuce ( Pistia Stratiotes) was selected for this st udy because of its high yield potential and high uptake capacity for nutrients. Due to low levels of N and P in the two ponds at the time of project implementation, known amounts of N and P were spiked in both plots before water lettuce was planted into the treatment plots. Water lettuce was transplanted in the treatment plot of each pond on August 22, 2005, and was maintain ed to cover three-fourth of water surface of the plot. Known amounts of N and P were also spiked on January 27 and September 5, 2006 because of low N and P concentrations in both p onds without receiving stormwater for a certain period. For each spiking, the same amounts of the same nutrient (N or P) were added into both the control and treatment plots for each pond. Sampling of water and plant from both plots be gan after the full establishment of the plants in the treatment plots, which was approximate ly two months after th e experimental set up. 29

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30 West Pond Control Plot Treatment Plot East Pond Control Plot Treatment Plot Figure 2-1. Experimental set up in the West Pond and the East Pond.

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Water samples were collected weekly from the control and the treatment plots for two and a half years (September 2005-March 2008) and anal yzed for water quality parameters, including total P (TP) and total Kjeldahl N (TKN), NO3-N, NH4-N, ortho-P, pH, EC, turbidity, and total solids (TS). Water lettuce was sampled monthly from th e treatment plots. After being rinsed thoroughly with deionized water to remove adhe ring materials and blotted dry, root and shoot were separated and their fresh we ights were recorded. Plant samp les were oven-dried at 70C for three days and then pulverized to <1 mm with a 4-Canister Ball Mill (K leco Model 4200, Kinetic Laboratory Equipment Company, Visalia, CA) prior to analysis fo r N and P concentrations in both root and shoot. Besides monthly sampling, plants were also peri odically harvested to maintain three-fourth coverage of the water surface of the treatment plot. For each harvest, the total fresh weight of plant was recorded, plant moisture and nutrient (N and P) concentr ations of plant root and shoot were determined, and total amount of dry bi omass yield was calculated. Total amount of nutrients removed from the wate r by the harvested plant biomass was quantified as the sum of the amounts in both root and shoot. The amount of nutrients (N, P) in root or shoot was the product of root or shoot dry bi omass yield and the nutrient con centration in that plant part. Chemical Analysis Prior to filtration, pH and EC of the water samples were determined using a pH/ion/conductivity meter (pH/Conductivity Meter, Model 2 20, Denver Instrument, Denver, CO) following EPA method 150.1 and 120.1, respectively. Turbidity was measured using a turbidity meter (DRT-100B, HF Scientific Inc., Fort Myers, FL) on the unfiltered water sample. Total solids in unfiltered water samples were measured using a gravimetric method at 105C (EPA 160.3). Total P in the unfiltered water sa mple was determined by the molybdenum-blue 31

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method after digestion with acid ified ammonium persulfate (EPA method 365.1). As dissolved N and P were of primary interest in the phytorem ediation study, sub-samples of the water were filtered through a Whatman 42 filter paper for T KN measurement, in which the filtrate was digested with acidified cupric su lfate and potassium sulfate and NH4-N concentration in the digested solution was determined following EP A method 351.3 using an N/ P Discrete Analyser (Easychem Plus, Systea Scientific, LLC, Illinois, US A). Portions of the sub-samples were further filtered through a 0.45 m membrane filter for the measurement of NH4-N, NO3-N, total dissolved P (TDP), and PO4-P. Concentrations of NO3-N and PO4-P were measured within 48 h after sample collection using an ion chroma tography (DX 500; Dionex Corporation, Sunnyvale, CA) following EPA method 300.0. Concentration of NH4-N in water samples was determined using an N/P Discrete Analyser (Easychem Pl us, Systea Scientific, LLC, IL) following EPA method 351.3. Total dissolved P in water was de termined using inductively coupled plasma optical emission spectrometry (ICP-OES, Ultima, JY Horiba Inc. Edison, NJ) following EPA method 200.7. Plant N concentration was determined using a CN analyzer (vario Max CN, Elementar Analysensystem GmbH, Hanau, Germany). Subsam ples (each 0.400 g) of plant material were digested with 5 mL of concentrated HNO3 in digestion tube using a block digestion system (AIM 500-C, A.I. Scientific Inc., Australia), and P concentration in the digested solution was determined using the ICP-OES. Data Treatment and Data Analysis As very high concentrations of N and P were measured in the water after fertilizer spikes, data from the month following each nutrient spik e were discarded and not included in graph or data analysis. 32

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At times when N or P concentration in water was so low that it was below detection limit, half of its method detection limit (MDL) was used in graph or in calculation (USEPA, 2006). Differences between control and treatment we re tested using the MEANS procedure in SAS software (SAS Institute, 2001). All statis tical analysis tests we re performed using a significance level of 5%. Results and Discussion General Water Quality Improvement Total solids and turbidity in the waters of both plots varied seasona lly: increasing during the rainy season from late May to mid-Novemb er and decreasing during the dry season from mid-November to late May with values in the rainy season being several times higher than those in the dry season (Figures 2-2 and 2-3). The incr ease in these parameters during the rainy season was likely due to the input of stormwater, whic h carried soil particles and solutes, including nutrients. The growth of water lettuce improved water quality by significantly decreasing TS and turbidity in water of the treatment plots (Table 2-1). Total solids in the water column was decreased by an average of approximately 20% in the treatment plots, as compared with that in the control plots (Figure 2-2) due to better particle sedimentation in the plant-growing plots (Brix, 1997). In addition, the presence of plants decr eased water disturbance by wind, thus reducing sediment resuspension. On average, water tu rbidity was reduced by approximately 65% in treatment plot as compared to the control in both ponds (Figur e 2-3). Water lettuce growth blocks available sunlight for algae and phytoplankton growth, which, together with sedimentation, contributes to clear er water (Figure 2-4). The much larger decrease in turbidity than TS indicated that algae and phytoplankton contributed a high proporti on to water turbidity while minimal to TS due to thei r negligible biomass weight. 33

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Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 Total solid concentration (g L -1 ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 East Control East Treatment Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 Total solid concentration (g L -1 ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 West Control West Treatment Figure 2-2. Total solid concentrations in the waters of the East and West Ponds. 34

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35 Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 Turbidity (NTU) 0 20 40 60 80 100 120 140 East Control East Treatment Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 Turbidity (NTU) 0 20 40 60 80 100 120 140 West Control West Treatment Figure 2-3. Turbidity in th e East and West Ponds.

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Table 2-1. Water quality improvement in the treatment plots of the East and We st Ponds (time period: 9/13/2005-3/28/2008a), n=122). Location Treatment Turbidity Total Solid PO4-P Total dissolved P Total P N O3-N N H4-N Total Kjeldhal N N TU g L-1 --------------------------------------------------------mg L-1 -----------------------------------------------------East Pond Control 20.519.7ab) 0.4130.295a 0.1320.597a 0.1230.215a 0.410 1.056a 0.0150.025a 0.0990.138a 1.78.72a Remediation 6.80.91b 0.3300.238b 0.0550.108b 0.0 870.144b 0.2290.441b 0.0070.009b 0.0690.126b 1.40.74b Reduction 67% 20% 58% 29% 44% 52% 31% 21% West Pond Control 21.46.2a 0.2600.211a 0.2200.369a 0. 2770.485a 0.4580.687a 0.0220.046a 0.0800.132a 1.05.63a Remediation 7.25.90b 0.2120.175b 0.1570.331b 0.2 280.478b 0.3200.533b 0.0060.005b 0.0440.044b 0.75.44b Reduction 66% 18% 29% 18% 30% 72% 45% 29% a ) Not include data from Jan. 25 to Feb. 27 and from Sept. 8 to Oct. 8 of 2006, each of which was about one month after fertilizer spike. b) Data shown are mean standard deviation; different letters following the numbers denot e significant difference between the means of control and treatment. 36

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Treatment Plot Control Plot Figure 2-4. Water samples from tr eatment plot and control plot. Water lettuce growth decreased water EC in the treatment plot of both ponds (Figure 2-5), due to salt removal from the waters by plant upta ke or root adsorption. Compared to the West Pond, the EC of water from the East Pond was higher (close to 2000 S cm-1 in some seasons) with large fluctuations. The reas on could be due to the fact that besides receiving stormwater from the fallow land, the East Pond also rece ived stormwater from the ditch along Kings Highway and runoff from roads with heavy traffi c was reported to be enriched with salts including heavy metals (Gan et al ., 2008; Terzakis et al., 2008). High pH (8.5-9.7) was measured from April to June in the contro l plots (Figure 2-6). Water sampling was performed at approximately 2 pm when solar radiation was strong and temperature was high. Therefore, the increase in pH in the control plots compared to the treatment plots might result from the photosynt hetic activity of peri phyton and phytoplankton communities or algae which depleted dissolved CO2 from the water and raised water pH (Reddy 37

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38 and DeLaune, 2008). A pH value as high as 9.5 in the afternoon was documented in an aquatic system containing algae by Reddy and Patrick (1984). Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 EC (uS cm -1 ) 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 East Control East Treatment Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 EC (uS cm -1 ) 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 West Control West Treatment Figure 2-5. Water EC in th e East and West Ponds.

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Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 pH 5 6 7 8 9 10 East Control East Treatment Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 pH 5 6 7 8 9 10 West Control West Treatment Figure 2-6. Water pH in th e East and West Ponds. Nitrogen and P Concentration Reduction Changes of NH4-N, NO3-N, TKN, PO4-P, TDP, and TP in water for the period of September 2005 to March 2008 are shown in Figure 2-7 to Figure 2-12. Their average 39

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concentrations were calculat ed and shown in Table 2-1. Li ke TS and turbidity, nutrient concentrations in the waters showed seasonal changes during the year, which were affected by external inputs from stormwater. Higher NH4-N concentration than NO3-N in both ponds may indicate atmospheric input of NH4-N (Pauziah Hanum et al., 2009). Although there are many reports showi ng that aquatic plants, such as Salvinia molesta and Elodea densa preferred NH4-N to NO3-N (Reddy et al., 1987; Shimada et al., 1988) and theoretically NH4 + uptake is energetically more efficient than that of NO3 -, reduction rate of NH4-N (31 and 45% in the East and West Pond, respectively) was smaller than that of NO3-N (52 and 72% in the East and West Pond, resp ectively) in both ponds. Besides plant uptake, denitrification may also c ontribute to the decreased NO3-N concentration in the treatment plots as a more anaerobic condition (dissolved oxygen < 1.5 and 0.7 mg L-1 in the East and West Pond, respectively) at water surface was created by the growing plants. Other anaerobic microsites may also contribute to NO3-N removal through denitrif ication (Gumbricht, 1993; Reddy, 1983). Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 NO 3 -N (mg L -1 ) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 East Control East Treatment Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 NO 3 -N (mg L -1 ) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 West Control West Treatment Figure 2-7. Nitrate-N in the waters of the East and West Ponds. 40

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41 Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 NH 4 -N (mg L -1 ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 East Control East Treatment Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 NH 4 -N (mg L -1 ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 West Control West Treatment Figure 2-8. Ammonium-N in the wate rs of the East and West Ponds.

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Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 Total Kjeldhal N (mg L -1 ) 0 1 2 3 4 5 6 East Control East Treatment Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 Total Kjeldhal N (mg L -1 ) 0 1 2 3 4 5 6 West Control West Treatment Figure 2-9. Total Kjeldhal N in the wa ters of the East and West Ponds. 42

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Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 PO4-P (mg L -1 ) 0.0 0.5 1.0 1.5 2.0 2.5 East Control East Treatment Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 PO 4 -P (mg L -1 ) 0.0 0.5 1.0 1.5 2.0 2.5 West Control West Treatment Figure 2-10. Water PO4-P in the East and West Ponds. 43

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Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 Total dissolved P (mg L -1 ) -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 East Control East Treatment Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 Total dissolved P (mg L -1 ) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 West Control West Treatment Figure 2-11. Total dissolved P in the waters of the East and West Ponds. 44

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45 Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 Total P (mg L -1 ) 0 1 2 3 4 5 East Control East Treatment Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 07/01/2008 01/01/2009 Total P (mg L -1 ) 0 1 2 3 4 5 West Control West Treatment Figure 2-12. Total P in the waters of the East and West Ponds.

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Inorganic P (PO4-P) removal (58 and 29% in the East and West Pond, respectively) was as efficient as inorganic N (NH4-N and NO3-N) in the remediation plots of both ponds (Table 2-1). Sheffield (Sheffield, 1967) measured a much higher reduction rate (94%) in inorganic N than ortho-P (40-55%) in a water hyacinth system. Tota l P had a higher reduction than total dissolved P (Table 2-1), which indicates that the role aquatic plants play in such a remediation system is far more than uptake. More importantly, the aqua tic plants play a cruc ial role by providing additional surface and favorable environment in the root zone for microorganisms to grow and involve in a variety of complex ch emical, biological and physical pr ocesses, such as nitrification, that contribute to the removal and degradati on of nutrients, which was considered the most important functions of aquatic plants (Brix, 1997 ). A higher removal rate in total P than in dissolved total P may result from the additiona l sedimentation effect of plant growth on particulate P. Nitrogen and P Removal Potential by Plant Uptake Nitrogen and P concentrations in th e plant were averaged 17 and 3 g kg-1, respectively, with N concentration being higher in root than shoot (Figure 2-13) but only a minimal difference in P concentration between root and shoot (Fig ure 2-14). Nitrogen and P concentration typically averaging 15-40 g N and 4-10 g P kg-1 for such large-leaved floati ng plants as water lettuce and water hyacinth ( Eichhornia crassipes) (Aoi and Hayashi, 1996). Annual removal of N and P by water lettuce were 190 and 25 kg ha-1, respectively in the East Pond, and 329 and 34 kg ha-1, respectively in the West Ponds, with dry matter being approximately 9 (the East Pond) and 15 Mg ha-1 (the West Pond). Research has also been conducted on another invasive, large-leaf floating aquatic plant, water hyacinth ( Eichhornia crassipes). Very high uptake rates have been re ported, for instance, 1980 kg N and 322 kg P ha-1 46

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yr-1 by Boyd (1970), 2500 kg N and 700 kg P ha-1 yr-1 by Rogers and Davis (1972), and up to 5350 kg N ha-1 yr-1 and 1260 kg P ha-1 yr-1 by Reddy and Tucker (1983). Reasons behind this big difference in nutrient uptake rate between this study and those in th e literature include: 1) water hyacinth has a higher nutrient uptak e and biomass yield potential th an water lettuce, 2) previous studies were conducted using nutrient solution wi th nutrient concentratio ns much higher than those in the stormwater detention ponds, thus re sulting in higher removal rates, and 3) the high reported values were based on short-term experiments and extrapolated to one year, which often overestimates the nutrient uptake rate of the plant. As a 1.5-fo ld difference was reported by Aoi and Hayashi (Aoi and Hayashi, 1996), the much lower nutrient uptake values from this study also indicated that the water le ttuce in the stormwater detenti on ponds was far from reaching its maximum nutrient uptake potential. Table 2-2. Annual removal amounts of plant dry biomass, N, and P from the East and West Ponds. Location Dry biomass N P Mg ha-1 -----------------kg ha-1 -----------------East Pond 9 190 25 West Pond 15 329 34 Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 Plant N concentration (g kg -1 ) 0 10 20 30 40 50 East Root East Shoot Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 Plant N concentration (g kg -1 ) 0 10 20 30 40 50 West Root West Shoot Figure 2-13. Nitrogen concentrations in plant roots and shoots from the East and West Ponds. 47

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Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 Plant P concentration (g kg -1 ) 0 1 2 3 4 5 6 7 8 9 East Root East Shoot Sampling date 01/01/2005 07/01/2005 01/01/2006 07/01/2006 01/01/2007 07/01/2007 01/01/2008 Plant P concentration (g kg -1 ) 1 2 3 4 5 6 7 8 9 West Root West Shoot Figure 2-14. Phosphorus concentrations in plant roots and shoots from th e East and West Ponds. Physiological Limits Plant growth is influenced by many environm ental factors such as solar radiation and temperature, and thus nutrient removal efficiency as reflected in both nutrient concentrations in 48

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the plant and biomass yield of water lettuce, showed strong s easonal dependence (Figures 2-13 and 2-14). This seasonal variat ion in plant growth and thus nut rient removal capacity was also discussed by Reddy and Sutton (1984). They stated that in Florida, 50% of the annual biomass yield was produced from May to August and 34% from March to April and from September to October. The West Pond worked better than the East Pond in removing N and P from the waters (Table 2-2) by producing a much higher biomass, which could be related to the differences in total dissolved organic carbon (averages of 30 and 12 mg L-1 in the East and West Ponds, respectively) and EC of the wate rs (Figure 2-5, 180-2000 and 100-400 S cm-1 in the East and West Ponds, respectively) between the two ponds. It was repo rted that an EC of 2683 S cm-1 was toxic to water lettuce (Haller et al., 1974). High EC in the East Pond negatively affected water lettuces growth, leading to less nutrient removal from the water. System Management For efficient water treatment, some aquati c macrophyte biomass must be removed from water bodies to keep an optimum pl ant density (0.2-0.7 kg dry biomass m-2 was suggested by Reddy and DeBusk, 1984). If not harvested, the vast majority of the nutrients that have been incorporated into the plant tissu e would be returned to the wa ter by the decomposition processes (Brix, 1997). It was shown that more intensiv e management with more frequent and timely harvest of plant biomass usually leads to a higher nutrient removal rate (DeBusk and Reddy, 1991). In Florida during the wet season when temperature is also favorable for water lettuce growth, plants should be harveste d every other week to maintain about three-forth coverage of the water surface (DeBusk and Reddy, 1991). Harvested plant biomass, rich in nutrients and organic matter, can be used as a soil amendment, processed into livestock feed, or converted to methane (Reddy and Sutton, 1984). 49

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50 Conclusions Water lettuce worked well in such low nutrient systems as stormw ater detention ponds. Water quality in both ponds was improved, as ev idenced by significant decreases in turbidity, total solids and nutrient con centrations. Inorganic N (NH4-N and NO3-N) concentrations in treatments plots were more than 30% lower than those in the control pl ots (without plant). TKN was reduced by more than 20%. Reductions in PO4-P, TDP, and total P were approximately 1858%, as compared to the control plots. By pe riodic harvesting, water lettuce removed 190-329 kg N ha-1 yr-1 and 25-34 kg P ha-1 yr-1 from the waters.

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CHAPTER 3 METAL REMOVAL POTENTIAL OF WATER LETTUCE ( PISTIA STRATIOTES L.) FROM STORMWATER IN DETENTION SYSTEMS Introduction Intensive use of commercial fertilizers, limi ng materials and agro-chemicals in agriculture has resulted in heavy metal accumulation in the soils. Significantly higher concentrations of extractable Cu, Zn, Mn, Fe, Co, and Cr than thos e in forest soils (nonagricultural soils) were measured in soils from eleven field sites (seven at commercial citrus groves and four at vegetable production farms) in St. Lucie and Martin Counties, Florida (He et al., 2004). High concentrations of Cu and Zn were measured in storm runoff water from these production systems (He et al., 2006a; Zhang et al., 2003). Although medi an concentrations of Cd, Cu, Pb, and Zn in Ten Mile Creek, a major tributary of the In dian River Lagoon (IRL), were below U.S. EPA drinking water critical levels and the threshold levels recommended for aquatic organisms, their individual pulse concentrations were above U. S. EPA recommended lim its (Yang et al., 2008). Accumulation of Zn and Cu in the sediments of the St. Lucie Estuary has also been reported (Haunert, 1988; He et al., 2003). There are extensive studies on metal accumulation by aquatic plants. The aquatic plants include floating plants, such as Salvinia herzogii (Maine et al., 2004), water hyacinth ( Eichhornia crassipes ) (Mishra et al., 2008; Muramoto and Oki, 1983), duckweed (including Lemna polyrrhiza L., Lemna minor and Spirodela polyrrhiza W. Koch) (John et al., 2008; Mishra and Tripathi, 2008), mosquito fern ( Azolla pinnata R. Brown) (Mishr a et al., 2008), and water lettuce ( Pistia stratiotes) (Maine et al., 2004; Mi shra et al., 2008), emergent plants such as common cattail (Typha latifolia ) (Manab Das and Maiti, 2008), and submerged plants, such as pondweed ( Potamogeton pectinatus or Potamogeton crispus ) (Badr and Fawzy, 2008; Mishra et al., 2008), hydrilla ( hydrilla verticillata ) (Bunluesin et al., 2004; Mishra et al., 2008), and 51

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coontail ( Ceratophyllum demersum L.) (Badr and Fawzy, 2008; Bunluesin et al., 2004). Interested metals accumulated by these aquatic plants were mainly micronutrients or heavy metals, namely, Fe (Almeida et al., 2006; Mana b Das and Maiti, 2008), Mn (Mishra et al., 2008; Vardanyan and Ingole, 2006), Cu (Almeida et al., 2006; Badr and Fawz y, 2008), Ni (Manab Das and Maiti, 2008; Vardanyan and Ingole, 2006), Co (Vardanyan and Ingole, 2006), Zn (Manab Das and Maiti, 2008; Vardanyan and Ingole, 2006), Cd (Badr and Fawzy, 2008; Bunluesin et al., 2004; Mishra et al., 2008), Hg (Mis hra et al., 2008; Molisani et al., 2006), Cr (Almeida et al., 2006; Mishra and Tripathi, 2008), Ti (Vardanyan and Ingole, 2006), Ba (V ardanyan and Ingole, 2006), and Pb (Almeida et al., 2006; Ba dr and Fawzy, 2008; John et al., 2008). Most studies were conducted in laboratory or greenhouse settings using metal-enriched nutrient solutions (B unluesin et al., 2004; John et al., 2008; Maine et al., 2004; Mishra and Tripathi, 2008). Results from thes e studies were usually very impr essive with high metal uptake or accumulation (>90%, Mishra and Tripathi, 2008). However, it may be entirely different when these aquatic plants are applied to field condition such as lakes, reservoirs, and estuaries where both metals and nutrients are of much lower con centrations and other environmental factors are far less favorable. On the other ha nd, the performance of aquatic pl ants in natural water bodies is more meaningful as degradation of natural a quatic ecosystem is a wo rldwide concern and yet conventional physical or chemical treatments are not cost-effective due to the nature of non-point source pollution. Investigations have been conducted in natural water bodies such as lakes (Badr and Fawzy, 2008; Vardanyan and Ingole, 2006), reservoirs (Mishra et al., 2008; Molisani et al., 2006), and estuaries (Almeida et al., 2006). But related in formation on man-made wa ter bodies, stormwater detention ponds, is minimal. Stor mwater carries with it nutrients, heavy metals, and chemicals 52

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from urban area and agricultural fields and ma y contribute to the degr adations of aquatic ecosystems (Casey et al., 2005; He et al., 2006b). Stormwater detention ponds are constructed to collect and remediate eutrophic stormwater before it is discharged into water bodies such as estuaries. Aquatic plants are useful in enhanc ing the water treatment pe rformance of man-made and natural wetland systems. Knowledge on metal removal potential is necessary for better use of these plants for water quality improvement. Because of the greater availability of solubl e ferrous iron species in the anoxic conditions (Ponnamperuma, 1972) and leakage of O2 from the roots of aquatic plants (Armstrong, 1979), Fe tends to precipitate in the oxidized zone of root surface, forming Fe oxyhydroxides as coatings on roots, which is often termed iron plaque and has been widely observed in aquatic plants and terrestrial plants when subjected to flooding (Crowder and St-Cyr, 1991; Hansel et al., 2001; Otte et al., 1989; Ye et al., 1997). Once formed, the large surface area of the iron plaque (which is often in excess of 200 m2 g-1) provides a reactive s ubstrate to sequester metals such as Zn, Cu, and Ni (Otte et al., 1989; Taylor and Crowder, 1983b). As the partitioning of metals on the root surface, within the ro ot and shoot has an important implication for predicting th eir potential bioavail ability and/or movement upon changing physicochemical conditions, it is of our interest to differentiate meta l outside and with in the plant. In addition, such knowledge is necessary when making plant di sposal decisions. Among the methods used to extract the metals located on the external surfaces of the root, the dithionite-citrate-bicarbonate (DCB) extraction has been shown to be the best for removing all the external precipitate on root surface (M cLaughlin et al., 1985; Taylor and Crowder, 1983a). This technique involves the use of sodium dithionite (Na2S2O4) as a strong reducing agent, sodium citrate (Na3C6H5O7H2O) as a chelating agent to maintain the extracted metals in 53

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solution and sodium bicarbonate (NaHCO3) as a buffer. The DCB method is very efficient in removing the iron oxyhydroxide coat ing without damaging root tissu es (Bienfait et al., 1984; Otte et al., 1989) or leaving considerable Fe on the surface of the washed roots as other methods do. This method has been applied to rooted aqua tic plants such as submerged and emergent aquatic plants. No attempt has been made to apply this method to such free floating aquatic plants as water lettuce. Also in terests have been mainly on a few metals, namely Fe, Mn, Zn, and Pb, on characterization of the iron plaque, and on the interactions be tween iron plaque and metals. As DCB solution can remove not only Fe oxi de and its associated metals but also metals adsorbed on the surface, it can be used to quantif y the amount of metals on the external surface of root. In this study, we utilized the DCB method to differentiate metals outside from inside the root, so that we have better understanding of mechanisms involve d in the removal of metals by aquatic plants. Compared to heavy metals such as Cd, Cu, Zn, and Pb, non-heavy metals such as K, Ca, Na, Mg, and Al are usually overlooked. Although they are not as deteriorating as heavy metals, they also affect water quality and are factors in algal bloom. Also for recycling purpose, we need to monitor these metals concentrations in the plan t. Therefore, the objective of this study was to investigate the removal potential of both heavy metals (Fe, Mn, Zn, Cu, Cr, Ni, Pb, Cd, Co) and non-heavy metals (K, Ca, Na, Mg, Al) by water le ttuce in stormwater detention ponds and to understand the mechanisms of me tal removal by this plant. Materials and Methods Experimental design, weekly water sampli ng, monthly plant sampling, water and plant sample preparation and processing are the same as described in the Materials and Methods section of Chapter 2. 54

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Chemical Analysis For the measurement of total dissolved metal concentration, water samples were filtered through a 0.45 m membrane filter and pres erved at pH < 2.0 by adding concentrated HNO3 before analysis using inductively coupled plasma optical emission spectrometry (ICP-OES, Ultima, JY Horiba Inc. Edison, NJ) followi ng EPA method 200.7. Similar to N and P, total amount of each metal removed from the water by the harvested plant biomass was the sum of the amount in plant shoots and roots, which was calculated as the pr oduct of root/shoot dry biomass yield and root/shoot metal concentration. The plant samples (root or shoot) were oven-dried, pulverized and digested with concentrated nitric acid, metal concentrations in the digested solution were determined using the ICP-OES. To differentiate metals that were absorbed into the interior of root from those attached to the external surface of root, the DCB extraction technique was applied (McLaughlin et al., 1985; Taylor and Crowder, 1983a). Briefly, twenty-fiv e g of fresh roots were soaked in 450 mL of DCB solution (containi ng 400 mL 0.3 mol L-1 sodium citrate, Na3C6H5O7H2O, 50 mL 1.0 mol L-1 sodium bicarbonate, NaHCO3, and 3 g sodium dithionite, Na2S2O4) at 60 oC for 20 min. Then, the roots were removed, rinsed several times with deionized water and blotted dry before they were oven dried, pulverized, and analyzed for metal absorbed by the root using the ICPOES. The DCB extract was filtered and analyzed for metal concentrations using the ICP-OES. Metals dissolved in the DCB soluti on is considered as those attach ed to the external surfaces of the roots by adsorption or surface deposition. Data Treatment When metal concentration was below detecti on limit, half of its method detection limit (MDL) was used in graph or in calculation (USEPA, 2006). 55

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Results Metal Concentration Reduction in Water Figure 3-1 shows the total dissolved metal con centrations of water samples from both the treatment and control plots of the East and West Ponds. Aluminum, Ca, Fe, K, Mg, and Na were the main metals detected in the waters (about 0.2-50 mg L-1). Copper, Mn, Ni, and Zn were of very low concentrations. As Cd, Co, Cr, and Pb concentrations were mostly below MDLs, they were not shown in Figure 3-1. The two ponds had si milar concentrations in Al, Cu, Ni, and Zn, while the concentrations of Ca, Fe, K, Mg, Mn, and Na in the East Pond were about two times higher than those in the West Pond, which agreed with the EC measurement (see Chapter 2). Plot East-controlEast-treatment West-controlWest-treatment Concentration (mg L-1) 0 20 40 60 80 100 120 140 160 Ca Figure 3-1. Total dissolved metal concentrations in the treatment and control plots of the East and West Ponds during 2005-2007 (n=122). The middle line is the median value of the data range. The error bars represent th e 5 and 95 percentile of the data. The upper value of the box is the 75 percentile and the lower value of the box is the 25 percentile. The dots are outliners. 56

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Plot East-controlEast-treatmentWest-controlWest-treatment Concentration (mg L-1) 0 5 10 15 20 25 K Plot East-controlEast-treatmentWest-controlWest-treatment Concentration (mg L-1) 0 10 20 30 40 50 60 Mg Plot East-controlEast-treatmentWest-controlWest-treatment Concentration (mg L-1) 0 50 100 150 200 250 Na Figure 3-1.Continued. 57

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Plot East-controlEast-treatmentWest-controlWest-treatment Concentration (mg L-1) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Al Plot East-controlEast-treatmentWest-controlWest-treatment Concentration (mg L-1) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Fe Figure 3-1.Continued. 58

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Plot East-controlEast-treatmentWest-controlWest-treatment Concentration (mg L1 ) 0.00 0.01 0.02 0.03 0.04 0.05 0.06 Cu Plot East-controlEast-treatmentWest-controlWest-treatment Concentration (mg L-1) 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 Mn Figure 3-1.Continued. 59

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Plot East-controlEast-treatmentWest-controlWest-treatment Concentration (mg L1 ) 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 Ni Plot East-controlEast-treatmentWest-controlWest-treatment Concentration (mg L-1) 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 Zn Figure 3-1. Continued. Compared to the control plots, Fe, Mn, and Al concentrations in water were reduced by an average of more than 20% by planting water lett uce. Potassium was reduced by more than 10% in the treatment plots. Calcium, Mg, and Na concentration reduction in the water was close to 10% in the East Pond and about 5% in the We st Pond as compared to the control plots. 60

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Metal Accumulation by Plant Root Figure 3-2 shows the metal concentration f actor (CF) of water lettuce, which was calculated as the ratio of metal concentration in plant root regardless of mechanisms (mg kg-1) over that in the surrounding water (mg L-1). All the investigated meta ls (Al, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, and Zn) had a CF higher than 102, with Al, Cd, Co, Cr, Fe, Mn, and Pb having a CF higher than 104. The CF values of the 14 metals changed in the following order for the East Pond: Cr > Mn > Co > Pb > Fe > Zn > Cd > Al > Ni > Cu > K > Ca > Mg > Na. For the West Pond the order was: Cr > Fe > Mn > Co > Al > Pb > Cd > Ni > K > Zn > Cu > Mg > Ca >Na. Element Al Ca Cd Co Cr Cu Fe KMg Mn NaNi Pb Zn CF (concentration in root/concentration in water) 102103104105106 East Pond West Pond Figure 3-2. Plant metal concentration f actors (CFs, metal concentration, mg kg-1, in root divided by metal concentration in th e surrounding water, mg L-1) in the East and West Ponds. Metal Distribution in Plant Most of the metals investigated were not eff ectively transported to shoot from root, with a root/shoot (R/S) ratio in metal concentration high er than 1 (Figure 3-3) Of the 14 metals, only Ca had an R/S ratio less than 1, which means higher Ca concentrations were in the shoot than in 61

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the root. Potassium, Mg, and Na had an R/S ratio close to 1. For Cr, Cu, Fe, and Ni, more than 80% of their accumulation occurred in the root, with an R/S ratio close to or higher than 6. This was most prominent in the case of Fe with an R/S ratio higher than 17. Much higher concentrations of the above four elements in th e root than in the shoot were also observed by Jayaweera et al. (2008) and many other researchers (Maine et al., 2004; Manab Das and Maiti, 2008; Qian et al., 1999). Some physio logical barriers were believed to play a role in preventing their transport to the aerial ti ssues (Zhu et al., 1999), which is one of the mechanisms protecting the aerial part (where photosynt hesis takes place) from being damaged by excessive metals (Fe, Cu, Ni, and Cr). Although Fe, Cu, and Ni are essential for plant growth, when at high concentrations, they are toxic to plant. For h eavy metals which are not essential and toxic to plant such as Cd, Co, and Pb, they were onl y detected in the root of water lettuce. Metal CaKMgNaAlCdCoCrCuFeMnNiPbZn Root/Shoot ratio in metal concnetration 0 5 10 15 20 25 East Pond West Pond Figure 3-3. Metal root/shoot ra tio in concentration of the East and West Ponds. Estimation of Annual Metal Removal Periodic harvesting of water lettuce plant is n ecessary not only for ma intaining an optimum growth density, but also for effec tive removal of nutrients (N and P) and metals from the waters, 62

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otherwise the nutrients and metals would be released back into the water system after the plant died and decomposed. Harvesting was mainly conducted in the summer when both temperature and rainfall were high and the plant growth rate was the high est during a year. Water lettuce removed a considerable amount of macroelements such as Ca, K, and Mg, and a sizable amount of microelements such as Fe and Mn from the st ormwater (Table 3-1). Hi gh metal concentrations in the roots of water lettuce have be en reported elsewh ere (1038 mg Cu kg-1 (Qian et al., 1999), 9.43 mg Co kg-1, 27.07 mg Pb kg-1, 107.32 mg Cr kg-1 (Vardanyan and Ingole, 2006)). In comparison, water lettuce in the two ponds were far from reaching its poten tial in removing trace metals, especially for Cd, Co, Ni, and Pb because of their low concentrations in the waters. For both dry matter and in most cases, individual elem ents, the West Ponds annual removal rate was twice that of the East Pond. The higher rate in the West Pond wa s related to higher biomass yield due to more favorable conditions, as high total organic carbon and EC in the East Pond might have negatively affected the growth of water lettuce (see Chapter 2 for discussion). Metal Uptake and Surface Adsorption According to their distribution between outside and inside the root (Figure 3-4), the 12 metals (as Na is a component of the DCB solution and the highly mobile nature of K in plant, these two elements are excluded) can be groupe d into 2 categories: 1) higher proportion was located on the external surfaces of the root: Ca, Cd, Co, Fe, Mg, Mn, and Zn, and 2) higher proportion was located inside the root: Al, Cr Cu, Ni, and Pb. Many studies have been conducted on elements such as Fe, Mn, Cd, Pb, Cu, and Zn (Hansel et al., 2001; Vesk et al., 1999). The distribution patterns of Fe, Mn, and Zn agree with thos e from St-Cyr and Campbells research (St-Cyr and Campbell, 1996). As a plant essential nutrient, Ni was found mainly inside the root (> 90%). Although Cr is a non-esse ntial element, more than 90% of the plant accumulated Cr had made its way into the root. This part of Cr could have been strongly bound 63

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64 by the cell wall to prevent possible damage to the plant (Maine et al ., 2004). Magnesium was equally distributed outside and inside the root. About 80% of the Fe was located on the external surface of the root as the ma in component of the iron pla que (St-Cyr and Campbell, 1996). Metal AlCaCdCoCrCuFeMgMnNiPbZn Percentage (%) 0 20 40 60 80 100 120 140 Outside the root Inside the root Figure 3-4. Distribution of metals outside and inside of wa ter lettuce root. Metal Bio-concentrated by Plant As a portion of the metals taken up by plant from water was actually located on the external surfaces of the roots by adsorption or deposition instead of being absorbed into the plant, the CFs previously calculated based on the total amount of meta l removed by plant may not accurately indicate the bio-accu mulation capacity of a plant for certain metals. Therefore, it is necessary to make some corrections. Another index, bio-concentration factor (BCF), the ratio of metal concentration within plant root (mg kg-1) over that in the surrounding water (mg L-1), which can more accurately reflect the plants uptake potential, was calculated (Figure 3-5). For metals such as Cd, Fe, and Mn with a large proportion being adsorbed on the external surfaces of the roots, their BCF value was much smaller than the respective CF value. For metals like Cr and Ni with a large proportion being absorbed into the roots, the difference between their BCF and CF value was small.

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Table 3-1. Annual metal removal rates by periodic harvesting of water lettuce. Location Dry matter Al Ca Fe K Mg Mn Na Zn Cd Co Cr Cu Ni Pb --------------------------------kg ha-1 ------------------------------------------------------g ha-1 -------------------East Pond 10455 16 357 29 344 70 5.3 138 1.3 4.0 4.9 92 107 31 51 West Pond 26005 55 546 57 853 134 5.3 370 1.2 11 10 189 336 52 110 65 65

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Element Al Ca Cd Co Cr Cu Fe Mg Mn Ni Pb Zn BCF (concentration within root/concentration in water) 102103104105106 East Pond West Pond Figure 3-5. Plant metal bio-concentration factors (BCFs), the ratio of metal concentration within plant root (mg kg-1) over that in the surrounding water (mg L-1), in the East and West Ponds. Discussion Planting water lettuce in the stormwater de tention ponds not only improved water quality by decreasing turbidity, total solids and nutrients (N and P) in the water as shown in Chapter 2, but also by removing metals (Fi gure 3-1). Better metal removal performance by aquatic plants was reported by many researchers w ith removal rates close to or higher than 90% (Mishra and Tripathi, 2008; Mungur et al., 1997 ). But the high removal rates were usually associated with laboratory or greenhouse experiments which provid ed more favorable environmental conditions for plant growth in terms of light, temperatur e, and nutrient concentr ations. In addition, high spiked metal concentrations in the water were used in those studies (Ingole and Bhole, 2003; Maine et al., 2001). High metal removal rates are al so common when aquatic plants were applied to the remediation of wastewater which usually contains high con centrations of metals (Kao et al., 2001). 66

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The plant is one of the sinks for metals in wa ter column. As the metals except Ca were not effectively transported to shoot from root (Figure 3-3), the root is the im portant final destination for the metals. High concentrations of such metals as Cd, Co, Cr, and Pb in the plant can pose a hazard to the plant. Fortunately, only a portion of the total metal located in root can made its way into the root while the remainder stayed on th e external surface of the root, complexed or adsorbed. This was confirmed qualitatively by Ha nsel et al. (2001) applying X-ray microprobe and X-ray fluorescence microtomography to free ze-dried root cross-sectional slice and quantitatively by the DCB extractio n in this study (Figure 3-4). A plant is commonly defined as a hyperaccumulato r of a metal if the CF of that metal is over 103 (Bunluesin et al., 2004). According to this definition, water lettuce can be considered a hyperaccumulator of such trace metals as Cr, Cu, Fe, Mn, Ni, Pb, and Zn. But when we talk about hyperaccumulation, we tend to emphasize the amount of metals accumulated within the plant by absorption. Therefore, the BCF, which excludes the portion of metals on the external surfaces of the roots, is a more appropriat e index than CF for the differentiation of hyperaccumulation, accumulation or non-accumulati on plants for metals. Based on the BCF index of 103 as the criterion, we found that water lettuce is a hyperaccumulator for Cr, Cu, Fe, Mn, Ni, Pb, and Zn. Many reported BCFs are actua lly CFs without excluding that portion of metals on the external surfaces of the roots (B unluesin et al., 2004; Zayed et al., 1998), although this may not change the conclusion regarding a hyperaccumulator for certain metals, as is the case in this study. However, it is importan t that a BCF is used for differentiating a hyperaccumulator from a regular plant based on plant physiology principle. In addition, this differentiation help understand the mechanisms of metal accumulation and detoxification by plants. 67

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68 Conclusions Growth of water lettuce reduced metal concen trations in the stormwater of detention ponds. Water lettuce had great potential in concen trating metals from the surrounding water even though the metal concentrations were under MDLs, with CF values ranging from 102 to 105. Of the 14 metals investigated, only Ca had an R/S ra tio in metal concentration less than 1, which indicated a higher proportion of metal detected in the water lettuce plant remained in the root instead of being transported up to the shoot By periodic harvesting of plant biomass, considerable amounts of metals, including macroand micro-elements, were removed from the stormwater. The DCB extraction method can be used to differentiate metals attached to the external surface from those absorbed inside the root. More than 50% of Ca, Cd, Co, Fe, Mg, Mn, and Zn recovered in the root were actually attached to the extern al surface, while more than 50% of Al, Cr, Cu, Ni, and Pb was absorbed into the root. Water lettuce is a hyperaccumulator for Cr, Cu, Fe, Mn, Ni, Pb, and Zn based on the bio-concentration factor (BCF) of 103 as a criterion.

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CHAPTER 4 NITROGEN REQUIREMENT FOR WATE R LETTUCE AND COMMON SALVINIA Introduction As nitrogen is a component of protei ns and a part of chlorophyll, plants require a certain level of external N for normal growth. This N level is called critical N concentration, below which plant biomass yield, quality, or performance is unsatisfactory (Marschner, 1995). When external N level is above the critical concen tration, plant biomass production responds positively to increased external N level to a point above which negative or no response in biomass yield occurs (Petrucio and Esteves, 2000). This poi nt in external N level is the optimum N concentration for maximum biomass production. For phytoremediation purpose, it is crucial th at we apply water lettuce to water with nutrient concentration above its nutrient critical level so that the plant can have net growth in biomass after a certain growth period and by period ically harvesting nutrie nts or metals can be removed from the water. It is suggested that smallleaved floating plant, common salvinia, can be included in polyculture systems with such large-leaved plant as water lettuce (Reddy and DeBusk, 1985) because it is efficient in removing P with a na rrow N/P ratio (Reddy and DeBusk, 1985). But if common salvinia has a much higher nutrient critical level than water lettuce, the application of the water lettuce-common salvinia polyculture system will have to be compromised. The information on critical and optimum N level of both water lettuce and common salvinia is important for management of phytor emediation systems using water lettuce or water lettuce in combination with common salvinia, but it has not been well documented. The objectives of this study were to: Find out the critical N concentrations of water lettuce and common salvinia; 69

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Investigate the possibility of a water lett uce-common salvinia polyculture system to improve water treatment efficiency. Materials and Methods Experimental Design Two free floating aquatic plants, water lettuce ( Pistia stratiotes ) and common salvinia ( Salvinia minima ), were tested for their N requiremen ts using a hydroponic study conducted in a greenhouse. The experimental design was a comple tely randomized design with seven levels of N and three replications for each N treatment of each plant species. Healthy water lettuce and common salvinia seed lings of similar age a nd size were selected and cultured in distilled water for three days before bei ng transplanted in 8L pots with modified Hoagland nutrient solution (Reddy et al., 1983). Plants were so transp lanted that all the pots with the same plant species had very close initial plant biomass which was about 3.7.3 g in dry weight for water lettuce and about 1.29.17 g in dry weight for common salvinia. The nutrient solution was prepared to provide sufficient amounts of essential nutrients except N for which a series of concentrations were applied. Chemical concentrations in the nutrient solution were as follows: Table 4-1. Nutrient solution composition for N requirement study. Nutrient Concentration (mg L-1) Nutrient Concentration (mg L-1) Ca 40.1 Mn 0.027 K 3.91 Mo 0.034 P 3.10 Fe 1.12 Mg 12.2 Zn 0.065 Cu 0.025 B 0.022 S 16.7 Cl 71.0 Nitrogen was added as NH4NO3. The seven levels of N were: 0.005, 0.025, 0.05, 0.25, 1.25, 2.5, and 5 mg N L-1. Nutrient solutions were renewed every three days to maintain the mentioned nutrient concentrations When plants grew to occupy the whole water surface, some 70

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mature plants were harvested to maintain an a pproximate coverage so that new plants have room to grow. Harvested plants from the same pot were pooled, weighed and oven-dried for chemical analysis. For harvested water lettuce, root and shoot were separately weighed, dried, and analyzed. Chemical Analysis After six weeks of growth (June 16-July 30, 2007), plants were removed from the pots, rinsed with deionized water and blotted dry. Plant materials were oven-dried at 70 C for three days. Total plant weight from each pot was the sum of each harvest. Dried plant samples were pulverized to <1 mm with a 4-Canister Ball Mill (Kleco Model 4200, Kinetic Laboratory Equipment Company, Visalia, CA) prior to analysis for total N. Plant N concentration was determined using a CN analyzer (vario Ma x CN, Elementar Analysensystem GmbH, Hanau, Germany). Statistical Analysis Data were subjected to the analysis of variance (ANOVA) using the GLM procedure in SAS software (SAS Institute, 2001). Differences between means were tested using the Tukey method. All statistical analysis tests were performed using a significance level of 5%. Results and Discussion Relationship between Plant Biom ass Yield and N Concentration Typical N-deficiency symptoms such as sene scence of older leaves and retarded growth were found in the low N level treatments (0.005, 0.025, 0.05, and 0.25 mg N L-1, Figure 4-1). Larger plant size resulted from more vegetative growth and more new individuals from vigorous vegetative reproduction were observed in the high N level treatments (1.25, 2.5, and 5 mg N L-1). More plant dry biomass was obtained with higher N treatment (Figure 4-2). For common salvinia, when the solution N concentration was 1.25 mg L-1 or lower, there was no statistical 71

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72 difference in dry biomass between the five N levels. It produced signifi cantly higher dry matter when the solution N concentration was increased to 2.5 mg L-1 and above. When the solution N concentration was 0.25 mg L-1 or lower, there was no statistical difference in water lettuce dry biomass between the four N levels. Water lettuce produced significantly higher dry matter yiel d when the solution N concentr ation was increased to 1.25 mg L-1 or above. The significant increa se in water lettuce biomass in the treatments of 1.25, 2.5, and 5 mg N L-1 was mainly gained from the increas e in above-water growth, which was demonstrated by the changes in shoot/root (S /R) ratio of dry biomass with external N concentration (Figure 4-3). When the solution N concentration was 0.25 mg L-1 or below, more than half of the water lettuces biomass was accounted for by its root with S/R ratio below one. Raising the solution N to 1.25 mg L-1 significantly increased S/R ra tio (to higher than 1.5). Shoot biomass was approximately three times greater than root biomass when external N concentration was raised to 5 mg L-1. Regression analysis revealed that a quadratic model can well represent the relationship between plant dry biomass yield and N c oncentration in the external solution ( P < 0.05, Figure 44). These results indicate that water lettuce or common salvinia like many crop plants, has its optimum N requirement for maximum biomass pr oduction. Solution N concentration higher than the optimum concentration tended to cause a decrease in biomass production. The quadratic regression curve predicts that the optimum N concentrations for water lettuce and common salvinia to achieve a maximum biomass yield are approximately 4.3 and 5.3 mg L-1, respectively. A close value of 5.5 mg L-1 was reported by Reddy et al. (Reddy et al., 1989) to be the optimum N concentration for water hyacinth.

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0.005 mg N L-1 0.025 mg N L-1 0.05 mg N L-1 0.25 mg N L-1 1.25 mg N L-1 2.5 mg N L-1 5 mg N L-1 Figure 4-1. The growth performance of water lettu ce and common salvinia under different N levels. Solution N concentration (mg N L -1 ) 5e-30.0250.050.251.252.55 Plant dry weight (g) 0 2 4 6 8 10 12 14 Water lettuce C C C C B B A Solution N concentration (mg N L -1 ) 5e-30.0250.050.251.252.55 Plant dry weight (g) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Salvinia B B B B B A A 73 Figure 4-2. Plant dry biomass yield at different N level treatments.

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Solution N concentration (mg L-1) 5e-30.0250.050.251.252.55 Water lettuce shoot/root in dry biomass 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 D D D D C B A Figure 4-3. The shoot/root ratio of water le ttuce dry biomass at different N levels. Figure 4-4. Regression curve of plant dry bi omass yield vs. solution N concentration. 74

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Relationship between Plant N and Solution N Concentration Higher plant N concentration was found in treatments with higher solution N concentrations (Figure 4-5). Differences in N concentration between root and shoot of water lettuce were not as big as thos e in some metal concentrations such as Cu and Fe (R/S>9, see Chapter 3 and Figure 3-3). Root N concentrati on was higher than shoot N concentration when the solution N concentration was 1.25 mg L-1 or lower. When the solution N concentration was 2.5 mg L-1 and higher, shoot had a higher N concentr ation than root. A significantly higher N concentration was measured in pl ant with the treatmen ts of solution N concentration of 2.5 mg L1 and above. Plant N concentrations were low when the solution N concentration was 1.25 mg L-1 or lower, and there were no stat istical differences between these low N treatments. Compared to plant dry biomass yield, a signifi cant increase in plant N concentration occurred at a higher external N concentration. This is likely due to the dilution effect on pl ant N concentration from vigorous plant growth, and such effect dimi nishes under higher external N conditions. Solution N concentration (mg L-1) 5e-30.0250.050.251.252.55 Plant N concentration (g kg-1) 0 5 10 15 20 25 30 Water lettuce root Water lettuce shoot C D C D C D C CD C C B B A A Figure 4-5. Plant N concentration at different N level treatments. 75

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Solution N concentration (mg L -1 ) 5e-30.0250.050.251.252.55 Plant N concentration (g kg -1 ) 0 5 10 15 20 25 30 35 BC C BC BC ABC AB A Salvinia Figure 4-5. Continued. Unlike water lettuce, no clear poi nt of external N concentr ation was found for common salvinia although higher plant N c oncentration was measured with treatments of higher N level (Figure 4-5, lower graph). This might be caused by common salvinias low ability in extracting nutrients and competitiveness for growing space. Algae readily grew in pots with common salvinia, especially in high N treatments, which might affect the growth performance of common salvinia. A linear model can be used to describe the relationship between plant N and solution N concentration ( P < 0.05, Figure 4-6). Unlike plant dry bi omass yield, which responded positively to increased external N concentr ation to a certain level and then negatively, plant N increased continuously with increased external N concentr ation. Such luxury uptake of N, i.e. plant N concentration increases without in creasing plant biomass yield when external N concentration is above plant optimum N concentr ation, was also reported by Petr ucio and Esteves (2000) and Gaudet (1973). As N is not needed for grow th or other metabolic functions in luxury consumption, it is converted to organic matter for later use in unfavorable times or under 76

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environmental stress (Farahbakshazad and Morrison, 1997). It was also suggested by Farahbakshazad and Morrison (1997) that in highl y loaded water treatment systems luxury plant uptake with rhizome storage dominates N removal. Figure 4-6. Regression curve of plant N con centration vs. soluti on N concentration. 77

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Plant Critical N Concentration In the N treatments of 0.005, 0.025, 0.05, and 0.25 mg N L-1, water lettuce did not have net growth after six weeks of culture with plant biom ass being the same as th at at the beginning of P treatment (Figure 4-2), which indicated that water lettuce can survive, at least for six weeks, in water with N concentration of 0.005-0.25 mg L-1 but can not support new growth. This is not desirable for phytoremediation purpose. And more than half of its biomass was in root (Figure 43), which indicated N stress (Re ddy, 1984). Visually, plant in these N treatments showed clearly typical symptoms of N deficiency with senescence of older leaves (Figure 4-1). Water lettuce in 1.25 mg L-1 treatment more than doubled its initial biomass (Figure 4-2). Plant showed healthy bright green without yellowing of the old leaves (Figure 4-1). Shoot contributed more to the total biomass than root as N was no longer a limiting fact or (Figure 4-3). All these results indicate that 1.25 mg N L-1 is the critical external N concentra tion for normal growth of water lettuce. For common salvinia, there was no net grow th in the N treatments of 0.005, 0.025, 0.05, 0.25, and 1.25 mg N L-1 (Figure 4-2). Common sa lvinia in 2.5 mg L-1 treatment almost doubled its initial biomass (Figure 4-2). Th ese results indicate that 2.5 mg N L-1 was the critical external N concentration for normal growth of common salvinia. Conclusions The critical N concentration for water lettuce to have net growth was 1.25 mg L-1. Water lettuce may not be able to reduce N concen tration in surrounding water to < 1.25 mg L-1 below which vegetative growth of water lettuce is minima l. At adequate N supply levels (> 1.25 mg L1), N uptake is mainly used for the above-water biomass production, and is the major contributor for the significant increase in biomass. The cri tical N concentration for common salvinia to have net biomass increase was 2.5 mg L-1. 78

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79 Based on regression model, the optimum N concentrations for maximum biomass production are 4.3 and 5.3 mg L-1 for water lettuce and common salvinia, respectively. Luxury uptake of N by water lettuce and common salvinia may occur when N levels are higher than their optimum levels. Although it has been suggested to include such small-leaved aquatic plant as common salvinia to a system based on large-leaved plants, i.e. water lettuce, to improve P removal efficiency, such a system may not work to our purpose as common salvin ia requires a higher N concentration for net growth.

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CHAPTER 5 PHOSPHORUS REQUIREMENT FOR WA TER LETTUCE AND COMMON SALVINIA Introduction Phosphorus, as a major plant nutrient, is associ ated with its function in energy storage and transfer as the major constituent of the ener gy currency, adenosine diand tri-phosphates (ADP and ATP). Energy obtained from photos ynthesis and metabolism of carbohydrates is stored in these phosphorus compounds. It is also an important structur al component of many other biochemicals such as nucleic acids, coenzymes, nucleotides, phosphoproteins, phospholipids, and sugar phosphates (Tisdale et al., 1993). Therefore, P deficiency retards overall growth of plants. Plants have their critical P level and optimum P level for normal growth and maximum growth, respectively. For phytoremediation purpose, plant should be applied to water with P at its optimum level so that best pe rformance of the plant can be achie ved. If that is not possible, it is crucial that P concentration in the water is higher than its nutrient critical level so that the plant can have net growth in biomass after a certain growth period and nutrients or metals can be removed from the water by periodically harvesting the plant biomass. The information on critical and optimum P level of both water lettuce and common salvinia is important for management of phytor emediation systems using water lettuce or water lettuce in combination with common salvinia, but it has not been well documented. The objectives of this study were to: Find out the critical P concentrations of water lettuce and common salvinia; Investigate the possibility of a water lett uce-common salvinia polyculture system to improve water treatment efficiency. 80

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Materials and Methods Experimental Design Two free floating aquatic plants, water lettuce ( Pistia stratiotes ) and common salvinia ( Salvinia minima ), were tested for their P requiremen ts using hydroponic studies conducted in a greenhouse. The experiment was a completely randomized design w ith six levels of P and three replications of each treatm ent for each plant species. Healthy water lettuce and common salvinia seedlings of similar age and size were selected and cultured in distilled water for three days before bei ng transplanted in 8L pots with modified Hoagland nutrient solution (Reddy et al., 1983). Plants were so transp lanted that all the pots with the same plant species had very close initial plant biomass which was about 3.3.3 g in dry weight for water lettuce and about 1.14.15 g in dry weight for common salvinia. The nutrient solution was prepared to provide sufficient esse ntial nutrients for plant growth except P for which a series of concentrations we re used. Phosphorus was added as KH2PO4, K was compensated with K2SO4 in low P treatments to ensure equal K concentrations in all treatments. Chemical concentrations in the so lution are provided in Table 5-1: Table 5-1. Nutrient solution composition for P requirement hydroponic study. Nutrient Concentration (mg L-1) Nutrient Concentration (mg L-1) N 9.52 Cu 0.0254 K 6.31 Mn 0.0275 Ca 40.1 Mo 0.0336 Mg 12.2 Fe 1.12 S 16.7 Zn 0.0654 Cl 71.0 B 0.0216 The six levels of P were: 0.01, 0.05, 0.1, 0.5, 1, and 5 mg L-1. Nutrient solutions were renewed every three days to maintain the aforem entioned nutrient concentrations. When plants grew to occupy the whole water surface, some ma ture plants were harvested to maintain an approximate coverage so that new plants have room to grow. Harvested plants were weighed 81

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and oven-dried for chemical analysis. For harvested water lettuce, root and shoot were separately weighed, dried, and analyzed. Chemical Analysis After seven weeks of growth (September 24-November 11, 2007), plants were removed from the pots, rinsed with deionized water and blotted dry. Plant materials were oven-dried at 70 C for three days. Total plant weight from each pot was the sum of each harvest. Dried plant samples were pulverized to <1 mm with a 4Canister Ball Mill (Kleco Model 4200, Kinetic Laboratory Equipment Company, Visalia, CA) prior to analysis fo r total P. Pulverized plant sample (0.400 g) was digested with 5 mL of concentrated HNO3 in digestion tube using a block digestion system (AIM 500-C, A.I. Scientific Inc., Australia), and P concentration in the digested solution was determined using ICP-OES (Ultima, JY Horiba Inc. Edison, NJ). Statistical Analysis Data were subjected to the analysis of variance (ANOVA), using the GLM procedure in SAS software (SAS Institute, 2001). Differences between means were tested using the Tukey method. All statistical analysis tests were performed using a significance level of 5%. Results and Discussion Relationship between Plant Biomass Yield and Solution P Concentration Retarded growth occurred in the low P treatments (0.01 and 0.05 mg P L-1). In the high P treatments (0.1, 0.5, 1, and 5 mg P L-1), plants looked more healthy in bright green, and vigorous vegetative reproduction resulted in lots of new individuals (Figure 5-1). More plant dry biomass yield was obtained in the higher P level treatments (Figure 5-2). When the solution P concentration was 0.05 mg L-1 or lower, there was no statistical difference in water lettuce dry biomass between the two P levels. Water lettuce produced significantly higher biomass yield than those in the 0.01 and 0.05 mg P L-1 treatments when the solution P 82

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83 concentration was increased to 0.1 mg L-1 and above. Similar to N, the significant increase in water lettuce biomass in the tr eatments of 0.5, 1, and 5 mg P L-1 was mainly from the increased above-water growth, which was demonstrated by the changes in shoot/root (S/R) ratio of dry biomass with external P concentration (Figure 5-3). Unlike N, more shoot dry biomass was produced even at low P levels with S/R ratio in dry biomass being close to 2 and plant shoot biomass (above water part) at high P treatments (0.5, 1, and 5 mg L-1) was more than 3-5 times that of root bioma ss (under water part). For common salvinia, biomass increased with increasing external P concentration, but there was no such a clear point of P concentration as for water lettuce to differentiate significant plant growth between treatments. As discussed in the previous chapter, this may be due to the competition from algae growth. For a narrower an d more precise criti cal range, further study should be carried out with P levels set closed at 0.05-0.5 mg L-1 but with a shorter distance in terms of P concentration, between treatments. A nd measures need to be taken to selectively inhibit algae growth. The relationship between plant dry biomass yield and P concentra tion in the nutrient solution was well described by a quadratic model (F igure 5-4). This indicat es that water lettuce or common salvinia, like many other crop plants, has its optim um P requirement for maximum biomass production. Solution P concentration higher than the optimum concentration may cause a decrease in biomass production. Based on the quadratic regression, the optimum P concentrations for both water lettuce and comm on salvinia to achieve maximum biomass yield are around 2.9 mg L-1.

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0.01 mg P L-1 0.05 mg P L-1 0.1 mg P L-1 0.5 mg P L-1 1 mg P L-1 5 mg P L-1 Figure 5-1. Growth performance of water lettuce and common salvin ia under different P levels. Solution P concentration (mg P L -1 ) 0.010.050.10.515 Plant dry weight (g) 0 2 4 6 8 10 12 Water lettuce C C B A A A Solution P concentration (mg P L -1 ) 0.010.050.10.515 Plant dry biomass (g) 0 1 2 3 4 Salvinia B AB AB AB A A 84 Figure 5-2. Plant dry biomass weights of different P level treatments.

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Solution P concentration (mg L -1 ) 0.010.050.10.515 Water lettuce shoot/root in dry weight 0 1 2 3 4 5 6 CD D D BC AB A Figure 5-3. Water lettuce shoot/root in dry biomass under different P level. 85

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Figure 5-4. Regression curves of plant dr y biomass vs. solution P concentration. 86

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Relationship between Plant P Concentr ation and Solution P Concentration Higher plant P concentration was found in the treatments with higher solution P concentrations (Figure 5-5). Differences in P concentration between root and shoot of water lettuce were not as big as those found with some metal concentrations such as Cu and Fe (R/S>9, see Chapter 3 and Figure 3-3). R oot P was higher than shoot P wh en the solution P concentration was 0.5 mg L-1 or lower. When the soluti on P concentration was 1 mg L-1 and higher, shoot had a higher P concentration than root Significantly higher P concentration was measured in plants treated with P concentration of 0.5 mg L-1 and above (Figure 5-5). Compared to plant dry biomass yield, a significant increase in plant P concentration required a higher external P concentration, which is likely due to the diluti on effect on plant P concentration from vigorous plant growth, and such effect diminishes at higher external P concentrations. When the solution P concentration was 0.1 mg L-1 and below, P concentrations in common salvinia plant were low (< 1 g kg-1) and there were no statistical differences among these three P treatments. When the solution P was increased to 0.5 mg L-1, P concentration in common salvinia plant was significantly increased (close to 4 mg L-1). Plant P concentration continued to increase with increasing solution P concentration (P < 0.05, Figure 5-5). The relationship between plant P and solutio n P concentration was well described by a quadratic model ( R2 = 0.99) (Figure 5-6). Like plant dr y biomass yield, plant P concentration increased positively with increasing external P concentration to a certain level and then negatively. The optimum P concentration for bot h root and shoot of water lettuce was around 3.2, and about 4.2 for common salvinia. The decrease in plant P concentration at high external P level might be caused by limit of other nutrients. 87

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Solution P concentration (mg L -1 ) 0.010.050.10.515 Plant P concentration (g kg -1 ) 0 2 4 6 8 Water lettuce root Water lettuce shoot DE D E D D D C C B B A A Solution P concentration (g L-1) 0.010.050.10.515 Plant P concentration (g kg -1 ) 0 2 4 6 8 10 12 14 16 Salvinia D D D C B A Figure 5-5. Plant P concentration in treat ments with different solution P level. 88

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Figure 5-6. Regression curve of plan t P vs. solution P concentration. 89

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Plant Critical P Concentration In the P treatments of 0.01 and 0.05 mg L-1, water lettuce did not ha ve net growth after seven weeks with plant biomass being the same as that at the beginning of P treatment (Figure 52), which indicated that water lettuce can surviv e, at least for seven weeks, in water with P concentration of 0.01-0.05 mg L-1 but can not support new growth. This is not desirable for phytoremediation purpose. Visuall y, plant in these two P treatmen ts showed yellowing of older leaves and growth was retarded (Figur e 5-1). Water lettuce in the 0.1 mg P L-1 treatment more than tripled its weight co mpared to the 0.01 mg P L-1 and almost doubled compared to the 0.05 mg P L-1 treatment (Figure 5-2), which indicated that plant in this P treatment not only survived but also had surplus P to support new grow th. All these indicate that 0.1 mg P L-1 is the critical external P concentration for water lettuce in order to have net growth, which is important for its application in phytoremediation. For common salvinia, there was no net grow th in the P treatments of 0.01, 0.05, 0.1, and 0.5 mg P L-1, with plant biomass being the same as th at at the beginning of the P treatments (Figure 5-2). Common salvinia in 1 mg P L-1 treatment almost doubled its initial biomass (Figure 5-2), indicating that 1 mg P L-1 was the critical external P concentration for common salvinia to have net growth. Conclusions The critical P concentration for water lettuce to have net growth was 0.1 mg L-1. Water lettuce may not be able to reduce P concen tration in surrounding water to < 0.1 mg L-1 below which vegetative growth of water lettuce is mini mal. Shoot contributes more to total biomass when external P concentration is raised. The critical P concentration for common salvinia to have net biomass increase was 1 mg L-1. 90

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91 Based on regression model, the optimum P concentrations for maximum biomass production of water lettuce and common salvin ia are the same, approximately 2.9 mg L-1. As common salvinia has a much higher P requirement for net growth than water lettuce, and a concentration of 1 mg P L-1 or above is rarely found in stormwater, this plant may not be useful for removing nutrients, especially P, fr om surface waters. In addition, it may not be feasible to develop a polyculture system of remediation using water lettuce and common salvinia because P concentration in stormwater is mos tly lower than the cri tical level for common salvinia.

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CHAPTER 6 EFFECT OF SALINITY ON GROWTH OF WATER LETTUCE Introduction Stormwater vary in salinity which is affected by soil properties, irrigation water quality, fertilization, and also by the sea environment in the coastal regions. Terrestrial plants differ greatly in their tolera nce of salinity. For example, barley and cotton have considerable salt toleran ce, while carrot and celery are salt sensitive (Tisdale et al., 1993). Aquatic plants also vary in salinity tolerance. Large-leaved floating sp ecies are reported to be most susceptible to salinity, submersed species can tolerant high salinity than large-leaved ones, and small-leaved ones are the least suscep tible of the three (Haller et al., 1974). The tolerance of aquatic plants to salinity will directly influence their performance in water treatment as decreases in transpiration and tota l dry weight will occur with increasing salinity and death at toxic salinity leve l (Haller et al., 1974). Results from our field study in the two stormwater detention ponds (see Chapters 2 and 3 of this dissertation) indicated that the less satisfactory performance of the plant in the East Pond might be due to the high EC in the water (Table 6-1), which negatively affected water le ttuces growth and conseque ntly its performance. Table 6-1. EC and ions contributing to water salin ity in the waters of the East and West Ponds (time period: 9/13/2005-3/28/2008, n=122). Location EC ClSO4-S Ca K Mg Na S cm-1 ---------------------------------mg L-1 -------------------------------------East Pond 606a) 81.8.2 41.7.6 42.0.8 7.41.82 14.4.6 48.3.3 West Pond 229 35.0.7 1.57.35 22.2.7 4.00.99 2.80.24 16.5.8 a ) Data shown are mean standard deviation. There are extensive data in th e literature on the tolerance of terrestrial plants especially crops to salinity. But few researches have been done on aquatic plants salt tolerance especially on those promising plants for water remediation. Utilization of such invasive aquatic plant as 92

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water lettuce in stormwater detention ponds invo lves the possibility of its escape from the detention systems into the lagoons or estuaries. From both points of utilization and disaster prevention, we need to know water lettuces salinity tolerance. The objective of this study was to evaluate the effect s of salinity on the growth performance of water lettuce. Materials and Methods Experimental Design Water lettuce ( Pistia stratiotes) was tested for its salin ity tolerance using hydroponic studies conducted in a greenhouse. The experi mental design was a completely randomized design with six salinity treatments and three replications for each treatment. Healthy water lettuce seedlings of similar age and size were selected and cultured in distilled water for three days before being transplanted in 8-L pots with modified Hoagland nutrient solution (Reddy et al., 1983). The solution was prepar ed to provide sufficient amounts of essential nutrients for plant growth. Chemical co mpositions of the solution are provided in Table 6-2: Table 6-2. Nutrient solution compositi on for the salinity tolerance study. Nutrient Concentration (mg L-1) Nutrient Concentration (mg L-1) N 49.0 Mn 0.0275 P 9.29 Mo 0.0336 K 46.9 Cu 0.0254 Ca 40.1 Fe 1.12 Mg 12.2 Zn 0.0654 S 16.7 B 0.0216 Our preliminary study revealed that salinity level of 6000 mg NaCl L-1 (equivalent to 9696 S cm-1 in EC) was so toxic to water lettuce that the plant could not survive (Figure 6-1). Therefore, the salinity level treatments chosen for this study were: 0, 800, 1600, 2400, 3200, and 4000 mg NaCl L-1. Taking into account of salts from th e nutrient solution itself, the six salt 93

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treatments were: 293, 1093, 1893, 2693, 3493, and 4293 mg L-1 or 473, 1766, 3059, 4351, 5644, and 6937 S cm-1 in EC. Nutrient solutions were rene wed every three days to maintain the above-mentioned nutrient concentrations. When pl ants grew to occupy the whole water surface, some mature plants were harvested to maintain an approximate coverage so that new plants have room to grow. Harvested plants from th e same pot were pooled, weighed and oven-dried for chemical analysis. NaCl: 0 2000 4000 6000 mg L-1 Figure 6-1. Growth performance of water le ttuce in water with gradient salinity. Chemical Analysis After about 3 weeks of growth (from Septem ber 21 to October 10, 2008), surviving plants were removed from the pots, rinsed with deio nized water and blotted dry. Total fresh weights from each pot were recorded. Plants were oven-dr ied at 70 C for three days and then plant dry weights were measured. Dried plant samples were pulverized to <1 mm with a 4-Canister Ball 94

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Mill (Kleco Model 4200, Kinetic Laboratory Equi pment Company, Visalia, CA) prior to analysis for nutrients (N, P, and metals). Plant N con centration was determined using a C/N analyzer (vario Max CN, Elementar Analysensystem GmbH Hanau, Germany). Pulverized plant sample (0.400 g) was digested with 5 mL of concentrated HNO3 in digestion tube using a block digestion system (AIM 500-C, A.I. Scientific Inc., Australia), a nd P and metal concentrations in the digested solution was determined using the ICP-OES (Ultima, JY Horiba Inc. Edison, NJ). Statistical Analysis Data were subjected to analysis of vari ance (ANOVA) using the GLM procedure in SAS software (SAS Institute, 2001). Differences between means were tested using the Tukey method. All statistical analysis tests were perf ormed using a significance level of 5%. Results and Discussion Plant Growth as Affected by a Salinity Gradient A prominent effect of salinity on water lettuce growth was that it inhibited vegetative growth but promoted the reproduction of new sm all-sized individuals. Th is effect was more pronounced with increasing salinity (Figure 6-2). Suppression of leaf expansion was recognized as one of the several morphologi cal and physiological effects of salinity (Nieman, 1964). It is associated with the loss of cell turgor that exerts its effect on cell extension and /or division (Greenway and Munns, 1980). Significant re duction in leaf area (from 1192 cm2 in control to 503 cm2) by high water salinity was also reported by Pascale et al. (1997). Chlorotic leaf margins, which indicated salinity stress, were visu ally observed in the high salinity treatments. Plant Biomass in Different Salinity Salinity had a significant effect on plant dr y biomass production (Figure 6-3). More new individuals of small size plant could not compensate for the bi omass reduction due to inhibited vegetative growth at high salinity treatm ents. Plant dry matter yield was reduced by 95

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approximately 30% in the 1766 S cm-1 treatment as compared to the control (473 S cm-1), and was further reduced (by about 50%) in the highe r salinity treatments (F igure 6-3). Inhibited biomass production (biomass reduced by more than 60%) by water salinity was also observed by Pascale et al. (1997). And water av ailability has been considered to be one of the most important factors that affect plant growth under saline conditions. Salinity inhibits plant water uptake by decreasing the osmotic potential of the water. 9/22/2008 (Day 2) 473 1766 3059 4351 5644 6937 S cm-1 10/10/2008 473 1766 3059 4351 5644 6937 S cm-1 Figure 6-2. Growth performance of water le ttuce in water with gradient salinity. 96

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Fresh water, by definition, contains less than 1000 mg L-1 of salts (1616 S cm-1) and commonly less than 500 mg L-1 (808 S cm-1) (Sandia, 2003), but brackish water can have salt concentration from 500-30000 mg L-1 (808-48480 S cm-1) (Greenlee et al., 2009). If the EC in the 473 S cm-1 treatment stands for a typical one for surface runoff, stormwater, or fresh water and the EC in the 1766 S cm-1 stands for a high one for stormw ater or a low one for brackish water, we can conclude that wa ter lettuce can tolerant the salinity found in stormwater but its biomass production may be reduced by up to 30% by high salinity. And water lettuces escape into lagoons and estuaries is a concern only when the brackish water has an EC less than 6937 S cm-1 if we use a criterion of 50% reduction in biomass production compared to that in fresh water and other conditions are favorable. Accord ing to Penfound and Earle (1948), large-leaved plant, water hyacinth, when found near brackish water, is confined to the pr otected shorelines of inflowing freshwater streams. In the field study, the EC in the East Pond wa s high compared to the West Pond (Chapter 2). In seasons when EC in the East Pond ro se and got close to or higher than 1766 S cm-1, plant growth was negatively affected, which may have contributed to the less satisfactory performance of the plants in the East Pond. EC (uS cm-1) 47317663059435156446937 Water lettuce dry biomass (g) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 A B BC BC BC C Figure 6-3. Plant dry biomass of water lett uce with different salinity treatments. 97

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Plant Nutrient Status under Different Salinity Conditions Although uptake of some nutrient was inhibited by high salinity, for instance, Ca, K, and Mn uptake decreased significantly in the high sali nity treatments, uptake of most other nutrients was not significantly reduced. These elements in cluded N, P, Mg, Fe, B, Cu, Mo, and Zn. For nutrients whose uptake was inhibi ted, their concentrations in th e plant still fell in the normal range (Figure 6-4). For example, although plant Ca concentration was reduced by 75% in the 6937 S cm-1 treatment, as compared to the control, its value, 6.13 g kg-1, still indicated adequate Ca nutrient (plant Ca concentra tion ranges from 0.2 to 1.0% (Tisda le et al., 1993). It is unlikely that salinity-induced nutrient deficiency might cause any severe inhibition of plant growth. Therefore, besides water availability, the negativ e effect of salinity on plant growth might be related to direct toxicity from Na+ and Cl-1. Excess Na+ might have caused metabolic disturbances in those processes where low Na+ and high K+ or Ca2+ are required for optimum function (Marschner, 1995). For example, when Na+ replaces Ca2+ in the cell membrane, cell membrane function may be compromised, resulting in increased cell leakiness (Orcutt and Nilsen, 2000). High Na+ also causes a decrease in nitrat e reductase activity, inhibition of photosystem II (Orcutt and Nils en, 2000), and chlorophyll brea kdown (Krishnamurthy et al., 1987). EC (uS cm-1) 47317663059435156446937 Plant N concentration (g kg-1) 0 10 20 30 40 50 60 N EC (uS cm-1) 47317663059435156446937 Plant P concentration (g kg-1) 0 2 4 6 8 10 12 P Figure 6-4. Plant nutrient concentrations with different salinity treatments. 98

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EC (uS cm-1) 47317663059435156446937 Plant Ca concentration (g kg-1) 0 5 10 15 20 25 Ca EC (uS cm-1) 47317663059435156446937 Plant K concentration (g kg-1) 0 20 40 60 80 K EC (uS cm-1) 47317663059435156446937 Plant Mg concentration (g kg-1) 0 1 2 3 4 5 6 Mg EC (uS cm-1) 47317663059435156446937 Plant Fe concentration (g kg-1) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Fe EC (uS cm-1) 47317663059435156446937 Plant B concentration (mg kg-1) 0 20 40 60 80 100 B EC (uS cm-1) 47317663059435156446937 Plant Cu concentration (mg kg-1) 0 20 40 60 80 Cu Figure 6-4. Continued. 99

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100 EC (uS cm-1) 47317663059435156446937 Plant Mn concentration (mg kg-1) 0 10 20 30 40 50 60 70 Mn EC (uS cm-1) 47317663059435156446937 Plant Mo concentration (mg kg-1) 0 10 20 30 40 50 60 Mo EC (uS cm-1) 47317663059435156446937 Plant Zn concentration (mg kg-1) 0 50 100 150 200 250 Zn Figure 6-4. Continued. Conclusions Salinity has a significant effect on the grow th of water lettuce. Water lettuce biomass production decreases significantly with increasin g salinity. Although water lettuce survived the salinity span (< 1616 S cm-1) of fresh water, its biomass production was reduced by up to 30% by high salinity. At a salinity of 6937 S cm-1, as with brackish water, its biomass production was reduced by more than 50%. Effects of salinity are mainly related to plant water availability (or relative water potential between salty wate r and the plant) and di rect toxicity of Na+ and Clto the plant.

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CHAPTER 7 EFFECT OF PH ON GR OWTH OF WATER LETTUCE Introduction An important factor in plant growth is pH. In growth medium with low pH, plants often suffer from hydrogen ion (H+) injury. Excess H+ in the growth medium inhibits root elongation, lateral branching, and water abso rption. Hydrogen ions affect root ion fluxes via competition with base cations for uptake, and causes damage to the ion-selective carrier in root membranes (Pessarakli, 1999). Other negative effects of low pH on plant growth are often associated with nutrient availability. High availability of Al and Mn unde r low pH conditions causes toxicity, while low pH-induced deficiency of Mg, Ca, P, a nd Mo also constrains plant growth. Zinc and Mn deficiency are often the reason why the growth of plants in high pH medium is inhibited. Sometimes, low availability of Fe and P is also a constraining factor. Generally, a pH range of 5.5-7.0 provides the most satisfactory or balanced plant nutrient levels for most plants. In the limited literatur e on interaction between pH and aquatic plants, optimum pH ranges of 6.5-7.5 and 5.8-6.0 were reported for water hyacinth (El-Gendy et al., 2004; Hao and Shen, 2006). Macroalga Chlorella sorokiniana grew best at pH 7-8 (Moronta et al., 2006). According to Dyhr-Jen sen and Brix (1996), although Typha latifolia L. had the highest growth rates at pH 5.0 to 6.5, and growth was only slight ly depressed at pH 8.0 but completely stopped at pH 3.5. Documentation on e ffects of pH on water lettuce was minimal. Although extreme pH values ar e seldom found in natural water bodies, they are not uncommon in mine drainages and wastewaters whic h are often remediated using aquatic plants. Whether the prospective aquatic plants can thrive in water with an extreme pH is the key to the success of phytoremediation of contaminated waters. 101

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The objective of this study was to determine the optimum pH range at which water lettuce can grow normally and produce satisfactory amounts of biomass. Materials and Methods Experimental Design Water lettuce ( Pistia stratiotes) was tested for its optim um pH range using hydroponic studies conducted in a greenhouse. The experi mental design was a completely randomized design with six pH treatments and th ree replications for each treatment. Healthy water lettuce seedlings of similar age and size were selected and cultured in distilled water for three days before being transplanted in 8-L pots with modified Hoagland nutrient solution (Reddy et al., 1983). The nutrient solution wa s prepared to provide sufficient amounts of essential nutrients for plant growth. Chemical composition in the nutrient solution is provided in Table 7-1. Table 7-1. Chemical composition of nut rient solution for pH effect study. Nutrient Concentration (mg L-1) Nutrient Concentration (mg L-1) N 49.0 Mn 0.0275 P 9.29 Mo 0.0336 K 46.9 Cu 0.0254 Ca 40.1 Fe 1.12 Mg 12.2 Zn 0.0654 S 16.7 B 0.0216 The six pH treatments were: 3, 4.5, 6, 7.5, 9, and 10.5. Nutrient solutions were renewed every three days to maintain the mentioned nutrient concentrations. Solu tion pH in each pot was adjusted daily to the designe d value by adding 0.1 mol L-1 NaOH or 0.1 mol L-1 HCl. When plants grew to occupy the whole water surface, so me mature plants were harvested to maintain an approximate coverage so that new plants have room to grow. Harvested plants from the same pot were pooled, weighed and ov en-dried for chemical analysis. 102

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Chemical Analysis After about 4 weeks of growth (October 17-November 14, 2008), surviving plants were removed from the pots, rinsed with deionized water and blotted dry. Total fresh weights from each pot were recorded. Plants were oven-dried at 70 C for three days and then plant dry weights were measured. Dried plant samples were pulverized to < 1 mm with a 4-Canister Ball Mill (Kleco Model 4200, Kinetic Laboratory Equi pment Company, Visalia, CA) prior to analysis for nutrients including non-metals (N, P, B and Mo) and metals (Ca, K, Mg, Cu, Zn, Fe, and Mn). Plant N concentration was determined us ing a CN analyzer (vario Max CN, Elementar Analysensystem GmbH, Hanau, Germany). Pulveriz ed plant sample (0.400 g) was digested with 5 mL of concentrated HNO3 in digestion tube using a block digestion system (AIM 500-C, A.I. Scientific Inc., Australia), and P and metal concentrations in the digested solution was determined using the ICP-OES (Ultima JY Horiba Inc. Edison, NJ). Statistical Analysis Data were subjected to analysis of vari ance (ANOVA) using the GLM procedure in SAS software (SAS Institute, 2001). Differences between means were tested using the Tukey method. All statistical analysis tests were perf ormed using a significance level of 5%. Results and Discussion Plant Growth in Water at Different pH Although water lettuce leaves tu rned yellow in only two days after the pH 10.5 treatment began, plant survived with a marginal increase in biomass. For the 3.0 pH treatment, plants did not survive and died in about two weeks after transplanting (Fi gure 7-1). Growth of Typha latifolia L. was also reported to completely stop at pH 3.5 (Dyhr-Jensen and Brix, 1996). 103

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10/31/2008 pH 3 pH 4.5 pH 6 pH 7.5 pH 9 pH 10.5 11/07/2008 pH 4.5 pH 6 pH 7.5 pH 9 pH 10.5Figure 7-1. Growth of water lettuce under different pH treatments. 104

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Plant Biomass Yield at Different pH Treatments Plant dry biomass yield increased with incr easing solution pH from 4.5 to 9 and then decreased at pH 10.5 (Figure 7-2). Regression anal ysis revealed that the relationship between plant dry biomass and solution pH can be descri bed by a quadratic model (Figure 7-3). Based on regression analysis, the optimum pH for water le ttuce growth was about 9, which indicates water lettuce prefers a relatively alkali ne environment. There are reports stating that aquatic plants grow best at pH 8.0 (Dyhr-Jensen and Brix, 1996; Moronta et al., 2006), pH 9.0 is commonly considered as the optimum pH for some al gae (Ogbonda et al., 2007) and bacteria (Sanjib Ghoshal et al., 2003). In this study although the hi ghest plant biomass was measured in the pH 9.0 treatment, pH 9.0 might not tr uly represent the pH of these pots due to pH dynamic change during the period of plant growth. It is well doc umented that plant roots excrete organic acids which can acidify the growth medium and H+ is released when plant roots take up NH4 + or other cations. As a result, daily pH ad justment might not be able to maintain the designed solution pH long enough as evidenced by the fact that each time the pH in the pots had dropped to 7-8 before pH adjustment. A more sophisticat ed technique that can steadily ma intain the selected pH in the growth medium is needed for future study. Howeve r, we can still conclude that water lettuce prefers and provides best gr owth in neutral to slightly alkaline waters. Plant Nutrition Status at Different pH Treatments Plant N, Mg, and Ca concentrations were sim ilar for different pH treatments (Figure 7-4). There were no differences in plant P and K con centrations among different pH treatments except for pH 10.5 at which plant P and K were significan tly lower. Water lettuce had the highest B and Mn concentrations at pH 6 and the lowest at pH 10.5. Plant Fe, Zn, and Mo concentrations decreased continuously w ith increasing pH in the solution (Figure 7-4). 105

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pH 34.567.5910.5 Water lettuce dry biomass (g) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 C BC ABC AB A ABC Figure 7-2. Dry biomass yield of water lettuce at different pH. Figure 7-3. Regression curve of water lettuce dry biomass vs. solution pH. 106

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pH 4.5 6 7.5 910.5 Plant N concentration (g kg 1 ) 0 10 20 30 40 50 60 N pH 4 567 591 0 5 Plant Ca concentration (g kg-1) 0 10 20 30 40 Ca pH 4 567 591 0 5 Plant Mg concentration (g kg-1) 0 2 4 6 8 10 Mg pH 4.5 6 7.5 910.5 Plant Mn concentration (mg kg-1) 0 50 100 150 200 250 300 Mn pH 4.5 6 7.5 910.5 Plant P concentration (g kg-1) 0 2 4 6 8 10 12 P pH 4.5 6 7.5 910.5 Plant K concentration (g kg-1) 0 10 20 30 40 50 60 70 K Figure 7-4. Plant nutrient concen tration of water lettuce at different pH treatments. 107

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pH 4.567.5910.5 Plant Cu concentration (mg kg -1 ) 0 20 40 60 80 100 120 140 Cu pH 4.5 6 7.5 910.5 Plant B concentration (mg kg-1) 0 20 40 60 80 100 B pH 4.5 6 7.5 910.5 Plant Mo concentration (mg kg-1) 0 10 20 30 40 50 Mo pH 4.5 6 7.5 910.5 Plant Zn concentration (mg kg-1) 0 100 200 300 400 500 Zn p H 4.5 6 7.5 910.5 Plant Fe concentration (g kg-1) 0 2 4 6 8 10 Fe Figure 7-4. Continued. For the nutrients of Mn, P, K, B, Mo, Zn, a nd Fe, the lowest plant concentrations were found in the treatments of highest pH, which might be due to the low concentrations of nutrients in the solution (Table 7-2). Adjus ting solution pH to 10.5 using 0.1 mol L-1 NaOH might have resulted in precipitation of some nutrients, which was visually observed in the pH 10.5 pots. At 108

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109 high pH, such micronutrients as Fe, Mn, Zn, and Cu react with OHand precipitate as hydroxides from the solution, leading to very low availability to the plant. Analysis of the nutrient solution after pH adjust ment confirmed the observation (T able 7-2). Iron was the most affected element by high pH. Its availability at pH 10.5 was nearly 3 orders of magnitude lower than that at pH 6.0-7.5, which is commonly found in most natural water bodies. Availability of Mn, Zn, and P was also markedly lower in the pH 10.5 treatment. The reason why water lettuce did not su rvive at pH 3.0 could be due to H+ injury (Pessarakli, 1999). From the resu lts of the salinity study (Chapter 6), it is clear that salinity should not be critical for water lettuces death, since EC in the pH 3.0 treatment was only 785 S cm-1 which is far below its toxic level. Conclusions Water pH has a significant effect on the grow th of water lettuce. Water lettuce could not survive at pH 3.0 or lower, which may be due to H+ injury. Water lettuce biomass yield increased with increasing pH up to 9, and then dropped at pH 10.5. For phytoremediation purpose, this plant was recommended to be applied to neutral to slightly alkaline waters.

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Table 7-2. Nutrient concentration and related properti es of the nutrient solution at different pH levels. Treatment EC Cl NO3-N PO4-P B Ca Cu Fe K Mg Mn Mo Zn S cm-1 --------------------------------------------------mg kg-1 ---------------------------------------------pH 3.0 785 29.4 66.96 12.46 0.011 37.8 0.12 1.14 46.4 14.5 0.027 0.015 0.12 pH 4.5 547 5.71 65.55 12.38 0.011 37.0 0.05 1.04 45.7 14.2 0.027 0.015 0.09 pH 6.0 547 5.20 59.41 10.14 0.011 37.6 0.03 0.89 46.7 15.1 0.026 0.016 0.09 pH 7.5 567 0.96 72.69 12.76 0.013 39.1 0.03 0.81 48.6 16.0 0.026 0.017 0.04 pH 9.0 551 0.26 69.45 6.58 0.012 30.6 0.03 0.75 48.2 15.1 0.007 0.018 0.01 pH 10.5 584 0.08 18.38 1.81 0.011 21.5 0.02 0.001 45.2 11.0 0.002 0.016 0.01 110

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CHAPTER 8 SUMMARY AND CONCLUSIONS Agricultural activities and urbanization have acc elerated the input of nutrients and metals in various water bodies, thus resulting in water eutrophication and the degr adation of aquatic ecosystems. Like many other places in the world, south Florida is facing challenges with surface water eutrophication and drinking water depletion. Monitoring st udies by He et al. (2003; 2006b) indicated that surface runoff water from agricultural fields in the Indian River area was enriched with N and P, and that Cu and Zn were also transported from agri cultural fields in runoff waters to receiving surface waters and th e accumulation of Cu and Zn in the sediments of the St. Lucie Estuary has been accelerated in the last two decades. Of the technologies available for remediating contaminated soil and water, phytoremediation using aquatic plants is promis ing because of its low cost compared to conventional physical or chemical methods, fewer negative effects, and suitability for removal of low concentration pollutants at a large scale. Phytoremediation of eutrophic stormwater in detention systems using water lettuce ( Pistia stratiotes L.) was evaluted for its effectiveness. Water lettuce plants were grown in the treatment plots (with water lettuce) of tw o detention ponds (the East and West Pond). Water samples were weekly collected from both the treatment plots and the control plots (w ithout any plants) and analyzed for water quality parameters including total solids, turbidity, pH EC, nutrient and metal concentrations. Plants were monthly sampled fo r nutrient concentration analysis. Three-fouth coverage of the water surface in the treatment plots was maintained by periodically harvesting. Nutrient and metal removal by harvesting was quantified. Data from this three-year study showed that growing water lettuce improved water quality by decreasing total solids and water turbidity. To tal solids in the water column were decreased 111

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by an average of approximately 20% in the treatment plots compar ed with that in the control plots. On average, turbidity was reduced by 65% in the treatm ent plots as compared to the control plots. Ammonium-N and NO3-N concentrations in water of the treatments plots were 3145% and 52-72% lower than those in the cont rol plots, respectively. Reductions in PO4-P, total dissolved P, and total P concentrations in water were 18-58%, as compared to the control plots. By periodic harvesting, water lettuce removed 190-329 kg N ha-1 and 25-34 kg P ha-1 annually from the waters. Water lettuce had great potential in concentrating metals from the surrounding water even when the metal concentration wa s extremely low (under method detection limits) with concentration f actor (CF) from 102 to 105. By periodic harvesting, considerable amounts of metals, including macroand micro-elements, were removed from the stormwater. The dithionite-citrate-b icarbonate (DCB) extraction method was applied to differentiate metals attached to the external surface from those absorbed into the root and the results revealed that besides plant uptake, precipitation and adsorption of metals onto the root surface were the other two important mechanisms by which water lettuce removed metals from water column. More than 50% of Ca, Cd, Co, Fe, Mg, Mn, and Zn recove red in the root were actually attached to the external surface, while more than 50% of Al, Cr, Cu, Ni, and Pb was absorbed into the root. To investigate the possibility of includi ng another free floating aquatic plant, common salvinia ( Salvinia minima ), in a polyculture system with water lettuce to further improve P removal efficiency, hydroponic studies on thes e two species N and P requirements were conducted in a greenhouse. Seven N le vels, 0.005, 0.025, 0.05, 0.25, 1.25, 2.5, and 5 mg N L-1, and six P levels, 0.01, 0.05, 0.1, 0.5, 1, and 5 mg L-1 were applied, respectively. Critical N concentrations required for net plan t growth in biomass after a certain period were 1.25 and 2.5 mg L-1 for water lettuce and salvinia, respectively. Critical P concentrations 112

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required for water lettuce and common salvinia to have net growth in biomass were 0.1 and 1 mg L-1, respectively. These results revealed higher N and P requirements for common salvinia to have net growth, which is not desirable when considering including common salvinia in a polyculture system with water lettuce. Water lettuce has optimum N and P concentrations of 4.3 and 2.9 mg L-1, respectively, as predicted from regression analysis, indicating that this plant would work best in waters with N and P concentrations close to these levels. Waters differ in their properties such as pH and salinity, which may have marked effects on plant performance as indicated in the stormw ater detention pond study. To better utilize water lettuce to remediate polluted water, it is critical that the plant can tolerate the pH and salinity of the water and still give satisfactory performance. To investigate how water salinity affect water lettuces performance, a greenhouse hydroponic study was conducted with six sa linity treatments, 473, 1766, 3059, 4351, 5644, and 6937 S cm-1. Water lettuce biomass yield decreased significantly with increasing water salinity, by approximately 30% in the 1766 S cm-1 treatment as compared to the control (473 S cm-1), and was further reduced (by about 50%) in the higher salinity treatments (>1766 S cm-1). A hydroponic study with six pH treatments 3, 4.5, 6, 7.5, 9, and 10.5, was conducted to determine how water lettuce performs in waters with different pH. Water lettuce could not survive in pH 3.0 or lower. Although water le ttuce could survive in pH 4.5-10.5, it produced most biomass in neutral and slightly alkaline. All these studies proved that phytoremediation using water le ttuce can efficiently remove nutrients and metals from eutrophi c stormwater in detention syst ems and improve water quality. 113

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114 This is encouraging in that detention systems have been widely used for decades throughout Florida and remain strongly r ecommended by the Florida Department of Environmental Protection (FDEP) on sites wher e conditions favor thei r use (i.e. shallow groundwater) (Breitrick, 2008). To address growing concerns about over-enrichment of Floridas waters (including surface waters, ground waters, and springs) by nutrients, the FDEP has initiated the proposed Statewide Stormwater Trea tment Rule to increase the level of nutrient removal required of stormwater treatment syst ems serving new development, including urban redevelopment (FDEP, 2009). Larger and deeper ponds are considered as one of the promising BMPs. In the N and P requirement studies, the ra nge in N or P concen tration between two consecutive levels was so big th at the conclusions on critical N and P concentrations for the plants could be higher than the true values. Take water lettuce critical N concentration for example, we concluded it was the level of 1.25 mg L-1, but it could be a value between 0.25-1.25 mg L-1, say, 0.5 mg L-1, where water lettuce may still have net growth. As previously discussed, algal growth in the pots cont aining common salvinia interfered with the growth of common salvinia and consequently the results. For more accurate and convincing results, further research should be conducted with N and P concentra tions between the ranges of 0.25-1.25 and 0.05-0.1 mg L-1, respectively. In addition, effective measures n eed to be developed to inhibit algal growth in pots with plants of less competitiveness.

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BIOGRAPHICAL SKETCH Qin Lu was born in 1976 in the hilly city of Xinyi, Guangdong, south China. She received her bachelors degree in agricu lture, with specialization in soil science from China Agricultural University, Beijing, China, in 1999. After she recei ved her Master of Science degree in soil quality from the Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China, in 2002, she worked for three years as a scientific editor at the Editorial Office of PEDOSPHERE, Nanjing, China. In 2005, she joined the University of Florida, Department of Soil and Water Science, for doctoral study in water qua lity and received her Ph.D. in summer 2009. 127