evaluation of aquatic plants for phytoremediation

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

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© 2009 Qin Lu

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To my husband, Diangao, and my son, Xuanning

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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 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 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 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.

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http://soils.ifas.ufl.edu/personnel/li.html

http://soils.ifas.ufl.edu/personnel/daroub.html

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 joy and happiness into our life. They are the endless

source of strength I can always rely on.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS ...............................................................................................................4

LIST OF TABLES ...........................................................................................................................9

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

ABSTRACT ...................................................................................................................................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 ................................................................................................................25

2 NUTRIENT REMOVAL POTENTIAL OF WATER LETTUCE (PISTIA STRATIOTES L.) FROM STORMWATER IN DETENTION SYSTEMS ..................................................27

Introduction .............................................................................................................................27 Materials and Methods ...........................................................................................................28

Experimental Design .......................................................................................................28 Chemical Analysis ...........................................................................................................31 Data Treatment and Data Analysis ..................................................................................32

Results and Discussion ...........................................................................................................33 General Water Quality Improvement ..............................................................................33 Nitrogen and P Concentration Reduction ........................................................................39 Nitrogen and P Removal Potential by Plant Uptake .......................................................46 Physiological Limits ........................................................................................................48 System Management .......................................................................................................49

Conclusions .............................................................................................................................50

3 METAL REMOVAL POTENTIAL OF WATER LETTUCE (PISTIA STRATIOTES L.) FROM STORMWATER IN DETENTION SYSTEMS ........................................................51


Chemical Analysis ...........................................................................................................55 Data Treatment ................................................................................................................55

Results .....................................................................................................................................56 Metal Concentration Reduction in Water ........................................................................56 Metal Accumulation by Plant Root .................................................................................61

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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 .............................................................................................................................68

4 NITROGEN REQUIREMENT FOR WATER LETTUCE AND COMMON SALVINIA .............................................................................................................................69


Experimental Design .......................................................................................................70 Chemical Analysis ...........................................................................................................71 Statistical Analysis ..........................................................................................................71

Results and Discussion ...........................................................................................................71 Relationship between Plant Biomass Yield and N Concentration ..................................71 Relationship between Plant N and Solution N Concentration .........................................75 Plant Critical N Concentration ........................................................................................78

Conclusions .............................................................................................................................78

5 PHOSPHORUS REQUIREMENT FOR WATER LETTUCE AND common SALVINIA .............................................................................................................................80



Results and Discussion ...........................................................................................................82 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 .............................................................................................................................90

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



Results and Discussion ...........................................................................................................95 Plant Growth as Affected by a Salinity Gradient ............................................................95 Plant Biomass in Different Salinity .................................................................................95 Plant Nutrient Status under Different Salinity Conditions ..............................................98

Conclusions ...........................................................................................................................100

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

Introduction ...........................................................................................................................101 Materials and Methods .........................................................................................................102

Experimental Design .....................................................................................................102 Chemical Analysis .........................................................................................................103 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 ........................................................105

Conclusions ...........................................................................................................................109

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

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

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

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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. ...................................................70

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

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LIST OF FIGURES

Figure page 2-1 Experimental set up in the West Pond and the East Pond. ................................................30

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 Kjeldhal 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 levels. .................................................................................................................................73

4-2 Plant dry biomass yield at different N level treatments. ....................................................73

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

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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. .................................................89

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

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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

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control plots. Annual removal 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. 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 µS cm-1) of freshwater but its

biomass could be reduced by up to 30% by high salinity (1766 µS cm-1). This plant could not

survive in brackish water with salinity > 6937 µS cm-1. 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-1) to the present time

(223 Tg yr-1) 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 subject 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 µg P L-1 over the period 1992-1996 (Crouzet et al., 1999). Taking agricultural land

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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-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 Zealand’s shallow lakes were classified

as eutrophic (> 50 µg 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

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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 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, 114, 49 and

250 µg 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 major 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-

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 8.6×l05 kg of N, 9.1×105 kg of P, and 3.6×l08 kg of suspended solids

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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% (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

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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 (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

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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-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 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-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 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

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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 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 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 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-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 achieved 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 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 yr-1, with more eutrophic systems

achieving higher removal rate (Mitsch, 1992).

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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 µg 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 all 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 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 Nwajei, 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-1) were found to be 1621

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

21

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.

22

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-1 (Rogers and Davis, 1972) and as high as 7629 kg

N ha-1 yr-1 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

23

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 plant(s) can help

better utilize the plant(s) without bringing disaster. Salt concentrations of 1660 and 2500 mg kg-1

24

(equivalent to 2683 and 4040 µS cm-1) 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

sorokiniana 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

25

26

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 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 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 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 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 subject 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 major 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 8.6×l05 kg of N, 9.1×105 kg of

P, and 3.6×l08 kg of suspended solids to the estuary annually (Graves and Strom, 1992).

27

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 project (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 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 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

28

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 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 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.

29

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.

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 Kjeldahl 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 70°C 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 Scientific Inc., Fort Myers, FL) on the unfiltered water sample.

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

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

31

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 filter paper for TKN measurement, in which the filtrate 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 µm 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 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, IL) 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.

32

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.

33

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

34

35

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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).

Location Treatment Turbidity Total Solid PO4-P Total dissolved P Total P NO3-N NH4-N Total Kjeldhal

N

NTU g L-1 --------------------------------------------------------- mg L-1 ------------------------------------------------------East Pond Control 20.5±19.7ab) 0.413±0.295a 0.132±0.597a 0.123±0.215a 0.410±1.056a 0.015±0.025a 0.099±0.138a 1.78±0.72a Remediation 6.80±3.91b 0.330±0.238b 0.055±0.108b 0.087±0.144b 0.229±0.441b 0.007±0.009b 0.069±0.126b 1.40±0.74b Reduction 67% 20% 58% 29% 44% 52% 31% 21% West Pond Control 21.4±16.2a 0.260±0.211a 0.220±0.369a 0.277±0.485a 0.458±0.687a 0.022±0.046a 0.080±0.132a 1.05±0.63a Remediation 7.25±4.90b 0.212±0.175b 0.157±0.331b 0.228±0.478b 0.320±0.533b 0.006±0.005b 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.

36

Treatment Plot Control Plot

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 µS cm-1 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 from the water and raised water pH (Reddy

37

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).

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Figure 2-6. Water pH in the 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

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 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, 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).

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0.25

0.30


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

40

41

Sampling date

01/0

1/20

05

07/0

1/20

05

01/0

1/20

06

07/0

1/20

06

01/0

1/20

07

07/0

1/20

07

01/0

1/20

08

07/0

1/20

08

01/0

1/20

09

NH

4-N

(mg

L-1)

0.0

0.2

0.4

0.6

0.8

1.0

1.2


Sampling date

01/0

1/20

05

07/0

1/20

05

01/0

1/20

06

07/0

1/20

06

01/0

1/20

07

07/0

1/20

07

01/0

1/20

08

07/0

1/20

08

01/0

1/20

09

NH

4-N

(mg

L-1)

0.0

0.2

0.4

0.6

0.8

1.0

1.2


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

Sampling date

01/0

1/20

05

07/0

1/20

05

01/0

1/20

06

07/0

1/20

06

01/0

1/20

07

07/0

1/20

07

01/0

1/20

08

07/0

1/20

08

01/0

1/20

09

Tota

l Kje

ldha

l N (m

g L-1

)

0

1

2

3

4

5

6


Sampling date

01/0

1/20

05

07/0

1/20

05

01/0

1/20

06

07/0

1/20

06

01/0

1/20

07

07/0

1/20

07

01/0

1/20

08

07/0

1/20

08

01/0

1/20

09

Tota

l Kje

ldha

l N (m

g L-1

)

0

1

2

3

4

5

6


Figure 2-9. Total Kjeldhal N in the waters of the East and West Ponds.

42

Sampling date

01/0

1/20

05

07/0

1/20

05

01/0

1/20

06

07/0

1/20

06

01/0

1/20

07

07/0

1/20

07

01/0

1/20

08

07/0

1/20

08

01/0

1/20

09

PO4-

P (m

g L-1

)

0.0

0.5

1.0

1.5

2.0

2.5


Sampling date

01/0

1/20

05

07/0

1/20

05

01/0

1/20

06

07/0

1/20

06

01/0

1/20

07

07/0

1/20

07

01/0

1/20

08

07/0

1/20

08

01/0

1/20

09

PO4-

P (m

g L-1

)

0.0

0.5

1.0

1.5

2.0

2.5


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

43

Sampling date

01/0

1/20

05

07/0

1/20

05

01/0

1/20

06

07/0

1/20

06

01/0

1/20

07

07/0

1/20

07

01/0

1/20

08

07/0

1/20

08

01/0

1/20

09

Tota

l dis

solv

ed P

(mg

L-1)

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5


Sampling date

01/0

1/20

05

07/0

1/20

05

01/0

1/20

06

07/0

1/20

06

01/0

1/20

07

07/0

1/20

07

01/0

1/20

08

07/0

1/20

08

01/0

1/20

09

Tota

l dis

solv

ed P

(mg

L-1)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5


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

44

45

Sampling date

01/0

1/20

05

07/0

1/20

05

01/0

1/20

06

07/0

1/20

06

01/0

1/20

07

07/0

1/20

07

01/0

1/20

08

07/0

1/20

08

01/0

1/20

09

Tota

l P (m

g L-1

)

0

1

2

3

4

5


Sampling date

01/0

1/20

05

07/0

1/20

05

01/0

1/20

06

07/0

1/20

06

01/0

1/20

07

07/0

1/20

07

01/0

1/20

08

07/0

1/20

08

01/0

1/20

09

Tota

l P (m

g L-1

)

0

1

2

3

4

5


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

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-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 (Figure 2-14). Nitrogen and P concentration typically

averaging 15-40 g N and 4-10 g P kg-1 for such large-leaved floating 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 reported, for instance, 1980 kg N and 322 kg P ha-1

46

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 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 Dry biomass N P Mg ha-1 ------------------- kg ha-1 ------------------- East Pond 9 190 25 West Pond 15 329 34

Sampling date

01/0

1/20

05

07/0

1/20

05

01/0

1/20

06

07/0

1/20

06

01/0

1/20

07

07/0

1/20

07

01/0

1/20

08

Plan

t N c

once

ntra

tion

(g k

g-1)

0

10

20

30

40

50

East - RootEast - Shoot

Sampling date

01/0

1/20

05

07/0

1/20

05

01/0

1/20

06

07/0

1/20

06

01/0

1/20

07

07/0

1/20

07

01/0

1/20

08

Plan

t N c

once

ntra

tion

(g k

g-1)

0

10

20

30

40

50

West - RootWest - Shoot

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

47

Sampling date

01/0

1/20

05

07/0

1/20

05

01/0

1/20

06

07/0

1/20

06

01/0

1/20

07

07/0

1/20

07

01/0

1/20

08

Plan

t P c

once

ntra

tion

(g k

g-1)

0

1

2

3

4

5

6

7

8

9

East - RootEast - Shoot

Sampling date

01/0

1/20

05

07/0

1/20

05

01/0

1/20

06

07/0

1/20

06

01/0

1/20

07

07/0

1/20

07

01/0

1/20

08

Plan

t P c

once

ntra

tion

(g k

g-1)

1

2

3

4

5

6

7

8

9

West - RootWest - Shoot

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

48

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 µS cm-1 in the East and

West Ponds, respectively) between the two ponds. It was reported that an EC of 2683 µS cm-1

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 majority 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).

49

50

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-1 yr-1 and 25-34 kg P ha-1 yr-1 from the waters.

CHAPTER 3 METAL REMOVAL POTENTIAL OF WATER LETTUCE (PISTIA STRATIOTES L.) FROM

STORMWATER IN 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 major 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 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) (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 (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

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

52

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 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 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 (Na2S2O4) as a strong reducing

agent, sodium citrate (Na3C6H5O7·2H2O) as a chelating agent to maintain the extracted metals in

53

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 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 lettuce in stormwater detention ponds and to

understand the mechanisms of metal removal by this plant.


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.

54

Chemical Analysis

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

through a 0.45 µm 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, Na3C6H5O7·2H2O, 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 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).

55

Results

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-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 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).

Plot

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

Con

cent

ratio

n (m

g 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 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.

56

Plot


Con

cent

ratio

n (m

g L-1

)

0

5

10

15

20

25

K

Plot


Con

cent

ratio

n (m

g L-1

)

0

10

20

30

40

50

60

Mg

Plot


Con

cent

ratio

n (m

g L-1

)

0

50

100

150

200

250

Na

Figure 3-1.Continued.

57

Plot


Con

cent

ratio

n (m

g L-1

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Al

Plot


Con

cent

ratio

n (m

g 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


58

Plot


Con

cent

ratio

n (m

g L-1

)

0.00

0.01

0.02

0.03

0.04

0.05

0.06Cu

Plot


Con

cent

ratio

n (m

g L-1

)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14Mn


59

Plot


Con

cent

ratio

n (m

g L-1

)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14Ni

Plot


Con

cent

ratio

n (m

g 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 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.

60

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-1)

over that in the surrounding water (mg L-1). 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.

Element

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

(con

cent

ratio

n in

root

/con

cent

ratio

n in

wat

er)

102

103

104

105

106

East PondWest Pond

Figure 3-2. Plant metal concentration factors (CFs, metal concentration, mg kg-1, 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

61

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.

Metal

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

Roo

t/Sho

ot ra

tio in

met

al c

oncn

etra

tion

0

5

10

15

20

25

East PondWest Pond

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,

62

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 (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 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

63

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 main component of the iron plaque (St-Cyr and Campbell, 1996).

Metal

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

Perc

enta

ge (%

)

0

20

40

60

80

100

120

140

Outside the rootInside the root

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-1) over that in the surrounding water (mg L-1),

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 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

Element

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

BC

F (c

once

ntra

tion

with

in ro

ot/c

once

ntra

tion

in w

ater

)

102

103

104

105

106

East PondWest 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 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).

66

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, complexed 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.

67

68

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 objectives of this study were to:

• Find out the critical N concentrations of water lettuce and common salvinia;

69

• 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 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 3.7±0.3 g in dry

weight for water lettuce and about 1.29±0.17 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

mature plants were harvested to maintain an approximate ¾ 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 Max 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%.


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-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

72

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-1 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.

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 lettuce and common salvinia under different N levels.

Solution N concentration (mg N L-1)

5e-3 0.025 0.05 0.25 1.25 2.5 5

Plan

t dry

wei

ght (

g)

0

2

4

6

8

10

12

14Water lettuce

CC

CC

BB

A

Solution N concentration (mg N L-1)

5e-3 0.025 0.05 0.25 1.25 2.5 5

Plan

t dry

wei

ght (

g)

0.0

0.5

1.0

1.5

2.0

2.5

3.0Salvinia

BB

B B

B

AA

73

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

Solution N concentration (mg L-1)

5e-3 0.025 0.05 0.25 1.25 2.5 5

Wat

er le

ttuce

sho

ot/ro

ot in

dry

bio

mas

s

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 lettuce dry biomass at different N levels.

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

74

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-

1 and 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.


5e-3 0.025 0.05 0.25 1.25 2.5 5

Plan

t N c

once

ntra

tion

(g k

g-1)

0

5

10

15

20

25

30Water lettuce rootWater lettuce shoot

C

DC

DC

DC CD

C C

B B

A

A

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

75


5e-3 0.025 0.05 0.25 1.25 2.5 5

Plan

t N c

once

ntra

tion

(g k

g-1)

0

5

10

15

20

25

30

35

BC

C

BCBC

ABCAB

A

Salvinia


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.

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 L-1 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 major 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.

78

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 salvinia requires a higher N

concentration for net growth.

CHAPTER 5 PHOSPHORUS REQUIREMENT FOR WATER LETTUCE AND COMMON SALVINIA

Introduction

Phosphorus, as a major plant nutrient, is associated with its function in energy storage and

transfer as the major 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 objectives 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.

80


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 3.3±0.3 g in dry

weight for water lettuce and about 1.14±0.15 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 low 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-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 aforementioned nutrient concentrations. When plants

grew to occupy the whole water surface, some mature plants were harvested to maintain an

approximate ¾ coverage so that new plants have room to grow. Harvested plants were weighed

81

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 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).


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%.


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

83

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.

0.01 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 salvinia under different P levels.

Solution P concentration (mg P L-1)

0.01 0.05 0.1 0.5 1 5

Plan

t dry

wei

ght (

g)

0

2

4

6

8

10

12

Water lettuce

C

C

B

A

A A

Solution P concentration (mg P L-1)

0.01 0.05 0.1 0.5 1 5

Plan

t dry

bio

mas

s (g)

0

1

2

3

4Salvinia

B

AB

ABAB

A

A

84

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

Solution P concentration (mg L-1)

0.01 0.05 0.1 0.5 1 5

Wat

er le

ttuce

sho

ot/ro

ot in

dry

wei

ght

0

1

2

3

4

5

6

CD

DD

BCAB

A

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

85

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

86

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-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 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.

87

Solution P concentration (mg L-1)

0.01 0.05 0.1 0.5 1 5

Plan

t P c

once

ntra

tion

(g k

g-1)

0

2

4

6

8Water lettuce rootWater lettuce shoot

DED

ED

DD

C C

B

B A

A

Solution P concentration (g L-1)

0.01 0.05 0.1 0.5 1 5

Plan

t P c

once

ntra

tion

(g k

g-1)

0

2

4

6

8

10

12

14

16Salvinia

D D D

C

B

A

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

88

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 L-1 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.

90

91

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 µS cm-1 ------------------------------------ mg L-1 ---------------------------------------- East Pond 606±372a) 81.8±62.2 41.7±46.6 42.0±24.8 7.41±3.82 14.4±8.6 48.3±33.3West Pond 229±48 35.0±13.7 1.57±3.35 22.2±3.7 4.00±0.99 2.80±1.24 16.5±5.8 a) Data shown are mean ± 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

92

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 objective of this study was to evaluate the effects of salinity on the growth

performance of water lettuce.


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-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

µS 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 the nutrient solution itself, the six salt

93

treatments were: 293, 1093, 1893, 2693, 3493, and 4293 mg L-1 or 473, 1766, 3059, 4351, 5644,

and 6937 µS cm-1 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 ¾ coverage so that new plants

have room to grow. Harvested plants from the 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 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 °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

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).


Data were subjected 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%.


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 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 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

95

approximately 30% in the 1766 µS cm-1 treatment as compared to the control (473 µS cm-1), 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)

473 1766 3059 4351 5644 6937 µS cm-1

10/10/2008

473 1766 3059 4351 5644 6937 µS cm-1

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

96

Fresh water, by definition, contains less than 1000 mg L-1 of salts (1616 µS cm-1) and

commonly less than 500 mg L-1 (808 µS cm-1) (Sandia, 2003), but brackish water can have salt

concentration from 500-30000 mg L-1 (808-48480 µS cm-1) (Greenlee et al., 2009). If the EC in

the 473 µS cm-1 treatment stands for a typical one for surface runoff, stormwater, or fresh water

and the EC in the 1766 µS cm-1 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

µS cm-1 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 µS 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)473 1766 3059 4351 5644 6937

Wat

er le

ttuce

dry

bio

mas

s (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 lettuce with different salinity treatments.

97

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 µS cm-1 treatment, as compared to the control, its value, 6.13 g kg-1, 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-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 nitrate reductase activity, inhibition of

photosystem II (Orcutt and Nilsen, 2000), and chlorophyll breakdown (Krishnamurthy et al.,

1987).

EC (uS cm-1)

473 1766 3059 4351 5644 6937

Plan

t N c

once

ntra

tion

(g k

g-1)

0

10

20

30

40

50

60

N

EC (uS cm-1)

473 1766 3059 4351 5644 6937

Plan

t P c

once

ntra

tion

(g k

g-1)

0

2

4

6

8

10

12P

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

98

EC (uS cm-1)

473 1766 3059 4351 5644 6937

Plan

t Ca

conc

entr

atio

n (g

kg-1

)

0

5

10

15

20

25

Ca

EC (uS cm-1)

473 1766 3059 4351 5644 6937

Plan

t K c

once

ntra

tion

(g k

g-1)

0

20

40

60

80

K

EC (uS cm-1)

473 1766 3059 4351 5644 6937

Plan

t Mg

conc

entr

atio

n (g

kg-1

)

0

1

2

3

4

5

6

Mg

EC (uS cm-1)

473 1766 3059 4351 5644 6937

Plan

t Fe

conc

entr

atio

n (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)

473 1766 3059 4351 5644 6937

Plan

t B c

once

ntra

tion

(mg

kg-1

)

0

20

40

60

80

100

B

EC (uS cm-1)

473 1766 3059 4351 5644 6937

Plan

t Cu

conc

entr

atio

n (m

g kg

-1)

0

20

40

60

80

Cu


99

100

EC (uS cm-1)

473 1766 3059 4351 5644 6937

Plan

t Mn

conc

entr

atio

n (m

g kg

-1)

0

10

20

30

40

50

60

70

Mn

EC (uS cm-1)

473 1766 3059 4351 5644 6937

Plan

t Mo

conc

entr

atio

n (m

g kg

-1)

0

10

20

30

40

50

60

Mo

EC (uS cm-1)

473 1766 3059 4351 5644 6937

Plan

t Zn

conc

entr

atio

n (m

g kg

-1)

0

50

100

150

200

250

Zn


Conclusions

Salinity has a significant effect on the growth of water lettuce. Water lettuce biomass

production decreases significantly with increasing salinity. Although water lettuce survived the

salinity span (< 1616 µS cm-1) of fresh water, its biomass production was reduced by up to 30%

by high salinity. At a salinity of 6937 µS 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 water and the plant) and direct toxicity of Na+ and Cl-

to the plant.

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 (H+) 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.

101

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.


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-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. Solution pH in each pot was

adjusted daily to the designed 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, some mature plants were harvested to maintain

an approximate ¾ coverage so that new plants have room to grow. Harvested plants from the

same pot were pooled, weighed and oven-dried for chemical analysis.

102

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 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).


Data were subjected 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%.


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).

103

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 10.5

Figure 7-1. Growth of water lettuce under different pH treatments.

104

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).

105

pH

3 4.5 6 7.5 9 10.5

Wat

er le

ttuc

e dr

y bi

omas

s (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

pH

4.5 6 7.5 9 10.5

Plan

t N c

once

ntra

tion

(g k

g-1)

0

10

20

30

40

50

60

N

pH

4.5 6 7.5 9 10.5

Plan

t Ca

conc

entr

atio

n (g

kg-1

)

0

10

20

30

40

Ca

pH

4.5 6 7.5 9 10.5

Plan

t Mg

conc

entr

atio

n (g

kg-1

)

0

2

4

6

8

10

Mg

pH

4.5 6 7.5 9 10.5

Plan

t Mn

conc

entr

atio

n (m

g kg

-1)

0

50

100

150

200

250

300

Mn

pH

4.5 6 7.5 9 10.5

Plan

t P c

once

ntra

tion

(g k

g-1)

0

2

4

6

8

10

12

P

pH

4.5 6 7.5 9 10.5

Plan

t K c

once

ntra

tion

(g k

g-1)

0

10

20

30

40

50

60

70

K

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

107

pH

4.5 6 7.5 9 10.5

Plan

t Cu

conc

entra

tion

(mg

kg-1

)

0

20

40

60

80

100

120

140Cu

pH

4.5 6 7.5 9 10.5

Plan

t B c

once

ntra

tion

(mg

kg-1

)

0

20

40

60

80

100

B

pH

4.5 6 7.5 9 10.5

Plan

t Mo

conc

entr

atio

n (m

g kg

-1)

0

10

20

30

40

50

Mo

pH

4.5 6 7.5 9 10.5

Plan

t Zn

conc

entr

atio

n (m

g kg

-1)

0

100

200

300

400

500Zn

pH

4.5 6 7.5 9 10.5

Plan

t Fe

conc

entr

atio

n (g

kg-1

)

0

2

4

6

8

10

Fe


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 L-1 NaOH might have

resulted in precipitation of some nutrients, which was visually observed in the pH 10.5 pots. At

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109

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 µS

cm-1 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+ 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.

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

µS 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

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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 fields in the Indian River area was enriched

with N and P, and that Cu and Zn were also transported from agricultural fields 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

111

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 31-

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-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 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

112

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 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 µS cm-1. Water lettuce biomass yield decreased significantly with increasing water salinity,

by approximately 30% in the 1766 µS cm-1 treatment as compared to the control (473 µS cm-1),

and was further reduced (by about 50%) in the higher salinity treatments (>1766 µS 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 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.

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114

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-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 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 joined 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

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