INTEGRATED INTERPRETATION OF SURVEY AND EXPERIMENTAL APPROACHES FOR DETERMINING NUTRIENT THRESHOLDS FOR MACROALGAE IN FLORIDA SPRINGS

LABORATORY EXPERIMENTS AND DISTURBANCE STUDY

DELIVERABLES FOR TASKS 2.1-2.8

DEP CONTRACT NUMBER S0291

SUBMITTED TO THE FLORIDA DE PARTMENT OF ENVIRONMENTAL PROTECTION October 31, 2007

AUTHORS: AGNIESZKA PINOWSKA R. JAN STEVENSON JAMES O. SICKMAN ANDREA ALBERTIN MARTIN ANDERSON2 Integrated interpretation of survey and experimental approaches for determining

nutrient thresholds for macr oalgae in Florida springs.

Description of deliverables for Task 2.1-2.8

Task 2.1-2.3

The deliverable for this task consists of 2 papers: one on Lyngbya wollei and one on Vaucheria sp. The papers describe the resu lts of laboratory experiments on the light, conductivity and microelement requirements of the two taxa. Effects of light, conductivity and microelements on the growth of Lyngbya wollei. Effects of light, conductivity and microelements on the growth of Vaucheria sp.

Task 2.4

The deliverable consists of a short description of the results of field experiments on the recovery of mats of Lyngbya wollei and Vauc heria sp. after complete biomass removal. Recovery of filamentous al gal mats of Lyngbya wollei and Vaucheria sp. after

disturbance.

Task 2.5-2.8

The deliverable for this task consists of 2 papers: one on Lyngbya wollei and one on Vaucheria sp. The papers desc ribe the results of laboratory experiments on the effects of3 nitrogen (in the form of n itrate and ammonia) and phosphorus on the growth of the two

taxa. Differential effect of PO 4 , NO 3 and NH 4 on the growth of Lyngbya wollei

Differential effect of PO 4 , NO 3 and NH 4 on the growth of Vaucheria sp.

In the Vaucheria sp. paper, not all of th e results from the laboratory experiments

conducted in small microcosms were include d. Vaucheria sp. did not respond well to the handling procedure we originally used. In so me of the experiments we observed a large variability within our data, and we did not observe any effects of the experimental treatments. When cutting off tips of Vaucheri a sp., it is necessary to observe whether the protoplast was damaged in the process. It often can be seen unde r the dissecting scope when the tip is cut off. Our final solution to this concern was to cut tips off 2 days before the experiment and only use tips that were sti ll healthy 48 hours later. The quality of the algal mat used for the experiments was cruc ial. If the Vaucheria sp. collected was healthy and vigorously growing in the field and sent to us within 24 hours from collection following the protocol that we developed, we we re very successful with the experiments. All mats that I personally collected resulted in healthy growing tips for the experiments and good experimental results. Lyngbya wolle i filaments have thick cell walls and can take more abuse than filaments of Vaucheria sp.4 A protocol for collection of Vaucheria sp. for use in experiments A kit to collect live Vaucheria sp. consists of a cooler, two ice packs and two 1 gallon Ziploc® bags. A mat that is intensively grow ing, has a bright green color, and is free of debris, sediments and invertebrates should be selected for collection. A fragment of a mat should be gently detached and rinsed in the site water by shaking while submersed. A 1 gallon bag should be filled with the site water and a well rinsed mat should be placed inside. The mat should have plenty of water an d should not fill more than half of the bag when it returns to its original volume, leaving half of the bag filled just with the site water. The first bag should be placed inside the sec ond bag, sealed and placed on an ice pack in a cooler and covered with an ice pack. If th ere is any empty space le ft, it should be filled with bubble wrap to make sure nothing is loose inside the cooler. Th e lid should be taped and sealed to the cooler using duct tape. The cooler should be shipped overnight and

received within 24 hours from collection to assure that the algae will be alive and growing. Upon receipt of the shipment, the bag with algae should be taken out of the

cooler and left on the count er for 2-3 hours until its te mperature adjusts to room temperature. Then the algae can be examined, any debris should be removed, and the algae can be transferred to the growth media. The growth medium used to maintain fresh

collections is a Z8Fl Fe200 medium modified to reflect the median chemistry of fresh water in Florida springs with reduced N and P concentration (Appendix 1).5 Effects of light, conductivity and microelements on the growth of Lyngbya wollei .

Introduction Most of the algae nutrition studies focus on the most important macronutrients: nitrogen and phosphorus. In the case of Lyngbya wollei , a cyanobacterium forming nuisance mats in lakes, ponds, rivers, streams and springs , studies have focused on the effects of nitrogen, phosphorus, salinity and light (Turner 1990, Speziale and Dyck 1992, Cowell and Botts 1994, Yin et al. 1997, Cowell and Dawe s 2004). None of the studies looked at growth at very low light levels and at growth in different conductivity water (at the levels found in fresh water springs). There also are no studies on the effects of iron and micronutrients on the growth of L. wollei. The growth media used in other studies were BG11, Z8 and LM6E. Most of these media we re developed to sustain algal growth in long-term batch cultures, however, not to optim ize the short term growth. In this study we developed a new medium based on the Z8 medium that reflects the median chemistry of freshwater Florida springs. We also test ed a new microcosm syst em for the growth of filamentous algae consisting of pol yethylene microcentrifuge tubes. The objectives of this study were: (i) to compare L. wollei growth rates in natural spring water to the ones in the growth medium and to test whether single or multiple filaments should be used while growing algae in micro centrifuge tubes, (ii) to investigate the growth of L. wollei at different levels of water conductiv ity, (iii) to investigate the growth

of L. wollei under different light levels, (iv) to study the growth of L. wollei in media with

different iron concentrations a nd (v) to compare the growth of L. wollei in the media with

and without the additi on of micronutrients.6 Materials and methods Five experiments were conducted using small algal microcosms (Table 1). The goal of any experiment investigating th e effects of nutrients on algal growth is to achieve high replication, to test as many nutrient levels as possible, and to grow algae in a continuous

culture with constant nutrient levels. To achieve high replication and a large number of treatments, we used microcentrifuge tubes as microcosms in which to grow algae. To achieve semi-continuous culture conditions, we kept the ratio between the algal biovolume and the volume of the growth medium very low, and we frequently changed growth media.

Experimental setup

Filaments of Lyngbya wollei were grown in 1.5 ml sterile microcentrifuge tubes filled with 1 ml of growth media. Growth media were changed every 48 hours. Racks made of clear polyethylene were placed on an orbital shaker (60 RPM). Microcentrifuge tubes

with L. wollei were incubated under grow lights providing 25 μmol/m 2 /s of light with the exception of the light gradient experiment. The unobstructed grow lights provided 250 μmol/m 2 /s, thus, the desired light intensity wa s obtained by coveri ng the racks with a neutral density film. The initial filament length was 1-2 mm. The filaments were photographed using a digital video came ra system installed on a Leica MZ12.5 Stereomicroscope under 8x magnification at the beginning and at the end of each experiment. Filament length was measured on the digital images using Image J software (Rasband 2003). Incubation time was 11 or 12 days. Each treatment was replicated 107 times. Since some of the filaments were lo st during media changes, the final number of replicates used for the data analyses wa s lower than set at the beginning of the experiment. All experiments had a complete factorial ANOV A design with 1 or 2 factors. Experiment 1 had a 2 factor design with 3 media treatme nts (spring water from Alexander, spring water from Silver Glen and Z8Fl medium) and 2 algal biomass treatments (1 and 5

filaments of L. wollei in each microcentrifuge tube). Due to an error in preparation of the Z8Fl medium for this experiment, no nitrate was added to the medium . Experiment 2 had a 1 factor design with 5 conductivity treatments (100, 300, 500, 1000 and 3000 μS/cm). The desired water conductivity was obtained by a combination of salts reflecting the chemistry of spring water with different conducti vity levels (Table 2). Experiment 3 had a 2 factor design with 15 tr eatments: a combination of 3 conductivity levels (100, 300, 1000 μS/cm) and 5 light intensities (1, 2.5, 5, 25, 250 μmol/m 2 /s). Experiment 4 had 2 treatments: a growth medium with and without the addi tion of microelements. Experiment 5 had a 1 factor design, with 4 ir on concentrations (0, 20, 200, 600 μg Fe/L). If not stated differently, all treatments had the same concentration of microelements, Fe, N, P, Ca, Mg, CO3 as in the st andard Z8Fl medium (Appendix 1).

Growth media A new growth medium was developed based on the Z8 medium (Kotai 1972). The Z8

medium was modified to simulate the medi an chemistry of macronutrients in Florida spring water (referred to as medium Z8Fl ) (Appendix 1). All growth media were

autoclaved or filter sterilized.8 Water analyses Ammonia nitrogen, Kjeldahl nitrogen, nitrite -nitrate nitrogen and total phosphorus concentrations were analyzed in the prepared growth media by FDEP (Table 3). A single sample of each growth medium was sent for the analyses. When the concentration of a compound was below the detection limit of the laboratory method, the method’s detection limit is reported (Table 4). None of the water analyses results were designated as Q or Y. Total nitrogen was calculated as a sum of nitrite-nitrate nitrogen (nitrates) and Kjeldahl nitrogen. Water conductivity was measured using YSI 556 MSP.

Experimental conditions All experiments were conducted in the walk-i n growth room. The temperature in the growth room was set for 22.5oC to reflect th e average water temper ature of the Florida springs. The minimum and maximum temperat ures were recorded daily on a max-min

thermometer.

Relative growth rates Algal relative growth rate (RGR) in the small microcosm experiments was calculated using the following formula (Hunt 1990):

Data analyses Data were analyzed using R statistical so ftware (R Development Core Team 2006). Analysis of variance was conducted using a ge neral linear model with car package. If days of # Length) ln(Initial Length) ln(Final

= RGR −9 necessary, data were square root or 1/square root transforme d to meet the assumptions of normality and variance homogeneity.

Results

Experiment 1 - growth of L. wollei in spring water and in growth media Both growth media and the number of filame nts in the microcosm affected the growth

rate of L. wollei (ANOVA for medium effect p<0.026 a nd for number of filaments effect p<0.022) (Fig. 1). When 5 filaments were pres ent in the microcosm, their growth rates

were lower than in the microcosms with 1 filament. Overall, the growth of L. wollei was affected by the nitrate concentration in the water (Table 5). Due to an error in the preparation of the Z8Fl medium for this expe riment, we were not ab le to truly test the medium in comparison to spring water, but in later experiments using the Z8Fl medium, we observed growth rates higher than the one s in spring water in this experiment.

Experiment 2 - growth of L. wollei in water with d ifferent conductivity Lyngbya wollei grew well in water with conducti vity from 100 to 1000 μS/cm. Its growth was reduced at 3000 μS/cm (ANOVA for conductivity effect p<0.022) (Fig. 2).

Actual conductivities meas ured in the treatments were: 105, 331, 516, 963 and 2942 μS/cm. All treatments had the same nitrog en and phosphorus concentrations, indicating that the observed growth differe nces were the result of differe nt conductivity levels in the growth media (Table 5).10 Experiment 3 -growth of L. wollei under different light l evels and water conductivities Both light and conductivity a ffected the growth rates of L. wollei (ANOVA for light effect p<0.001; for conductivity effect p< 0.040 and for interac tion light*conductivity p<0.004) . Lyngbya wollei grew even at very low light levels, but it had the highest growth rates at a light intensity of 25 μmol/m 2 /s or higher (Fig. 3). Only at the lowest light level (1 μmol/m 2 /s) water conductivity had an e ffect on the growth rates of L. wollei and at that light level filaments in 100 μS/ cm conductivity water grew better than filaments in 1000 μS/cm conductivity water (TukeyHSD p<0.010).

Experiment 4 - growth of L. wollei in the media with and wit hout micronutrients added Surprisingly, addition of microelements to th e growth media resulted in reduced growth rates (ANOVA for micronutrient effect p<0.001 ) (Fig. 4). The Gaffron’s micronutrient mix used in the Z8 medium has relatively low micronutrient concentrations compared to other freshwater growth media. There wa s no difference in the nitrogen and phosphorus

concentration between the differe nt growth media (Table 5).

Experiment 5 - growth of L. wollei in different iron concentrations Iron is an important micronutrient for the growth of L. wollei . (ANOVA for the iron effect p<0.001) (Fig. 5). When no iron was adde d to the growth media, very low growth

rates of L. wollei were observed. Surprisingly, an iron concentration of 600 μg/L, found in the standard Z8 medium, had a negative effect on the growth of L. wollei . High growth rates were observed at 20 and 200 μg Fe/L. There were no differences in the nitrogen and phosphorus concentrations between the different growth media (Table 5).11 Discussion Lyngbya wollei is clearly a freshwater species, since it did not grow well in high conductivity water (Fig. 2). Speziale and Dy ck (1992) and Cowell and Botts (1994) also observed reduced growth rates and loss of pigment when L. wollei was grow n in growth

media to which salt or sea water had been added . Lyngbya wollei grew well at low light levels (Fig.3), as also described by Turner (1990) and Yin et al . (1997). It did not respond well to the addition of microelements and to a high concentration of iron in the growth media (600 μg Fe/L) (Fi g. 5). This is opposite to Lyngbya majuscula, which has a very high iron requirement, 1400-6000 μg/L (G ross and Martin 1996). Microcentrifuge tubes used as microcosms for the growth of algal filaments worked very well, and using one 1-2 mm long filament worked better then using multiple filaments. Even though all chemicals used to prepare media were ACS certi fied, they have microelements in them as contaminants, and these are probably sufficient for L. wollei growth. Further study on the effects of particular microelements is n eeded to determine which micronutrient in Gaffron’s mix is causing the reduction of gr owth rates. When conducting experimental

work on L. wollei, growth media with reduced iron and micronutrient concentrations should be used as proposed in Appendix 1.

References Cowell, B. C., and P. S. Botts. 1994. Factor s Influencing the Dist ribution, Abundance and Growth of Lyngbya-Wollei in Cent ral Florida. Aquatic Botany 49 :1-17. Cowell, B. C., and C. J. Dawes. 2004. Growth and nitrate-nitrogen uptake by the cyanobacterium Lyngbya wollei. Journa l of Aquatic Plant Management 42 :69-71. Gross, E. D., and D. F. Martin. 1996. Iron dependence of Lyngbya majuscula . Journal of Aquatic Plant Management 34 :17-20. Hunt, R. 1990. Basic growth analysis. Unvwin Hyman Ltd., London.12 Kotai, J. 1972. Instructions fo r preparation of modified nutrient solution Z8 for algae. Norwegian Institute for Water Researc h, publication B-11/69. Blindern, Oslo. R Development Core Team. 2006. R: a langu age and environment for statistical computing. Vienna, Austria. Rasband, W. 2003. ImageJ v 1.61. National Inst itutes of Health, Bethesda, Maryland. Speziale, B. J., and L. A. Dyck. 1992. Lyngbya infestations: comparative taxonomy of Lyngbya wollei comb. nov. (Cyanobacteria). Journal of Phycology 28 :693-706. Stevenson, R. J., A. Pinowska, and Y. K. Wang. 2004. Ecological c onditions of algae and nutrients in Florida springs . Final report. Florida Department of Environmental Protection contract number WM858, Michigan State University, East Lansing, MI. Turner, G. 1990. Studies on the biology of Lyngbya wollei . Master's Thesis. Clemson University, South Carolina. Yin, Q. Q., W. W. Carmichael , and W. R. Evans. 1997. Fact ors influencing growth and toxin production by culture s of the freshwater cyanobacterium Lyngbya wollei Farlow ex Gomont. Journal of Applied Phycology 9 :55-63.13 Fig. 1 Box plot showing relative growth rates (RGR) or L. wollei in a factorial mesocosm experiment with 3 growth media (spring wate r from Alexander, spring water from Silver Glen and Z8Fl growth medium but with very low nitrogen and phos phorus content) and 2 biomass levels: 1 or 5 filaments (n=57).14 Fig. 2 Box plots showing relative growth rates (RGR) of L. wollei in different conductivity water (n=45). Boxes sharing the same letter do not differ (Tukey’s test p<0.05).15 Fig. 3 Box plots showing relative grow th rates (RGR) in a factoria l mesocosm experiment with 5 light levels and 3 conduc tivity levels (n=121).16 Fig. 4 Box plots showing relati ve growth rates (RGR) L. wollei in growth media with and without micronutrients added (n=17).17 Fig. 5 Box plot showing relative growth rates (RGR) of L. wollei in 4 iron concentrations (n=37). Boxes sharing the same lett er do not differ (Tukey’s test p<0.05).18 Table 1 Lyngbya wollei experiments. Experiment numbers in pa renthesis refer to numbers used in the 2004 report (Stevenson et al. 2004).

Experiment

(project experiment

number) Treatments Algae collection location Season Growth medium Mean min and max temperature (oC) 1 (6) 2 (13) 3 (13) 4 (14) 5 (14) Spring water, media and number of filaments in the tube Conductivity Light and conductivity Microelements Iron Ichetucknee

Ichetucknee Ichetucknee Ichetucknee Ichetucknee Summer

Summer Summer Summer Summer Z8Fl

Z8Fl Z8Fl Z8Fl Z8Fl 20.6 & 24.9

20.4 & 24.8 20.4 & 24.8 20.6 & 24.8 20.6 & 24.8

Table 2 Chemical composition of a component of growth media in experiments 2 and 3 responsible for desired conduc tivity in the medium.

Conductivity

(μS/cm) CaCl 2 ·2H 2 O (mM) MgSO 4 ·7H 2 O (mM) Na 2 CO 3 (mM) KCl (mM) NaCl (mM) 100 300 500 1000 3000 0.116 0.496 0.747 1.618 2.739 0.101 0.203 0.284 0.284 0.284 0.198 0.792 1.293 1.293 1.293 0 0 0 0.123 0.875 0 0 0 0.123 0.875

Table 3 Methods used for analyses of water samples. Parameter Method Description Ammonia-N EPA 350.1 (1983) Ammonia in aquaeous matrices as mg N/L NO2NO3-N EPA 353.2 (1983) Nitrite/Nitrate in aqueous matrices as mg N/L N_KJEL_TOT EPA 351.2 (1983) Total Kjeldahl Nitrogen in aqueous matrices Total-P EPA 365.4 Total Phosphorus in aqueous matrices19 Table 4 Detection limits of water chemical analyses . When the concentration of a compound was below the detection limit of the laboratory method, the method detection limit is reported.

Compound Detection limit (mg/L) Ammonia nitrogen 0.010 Total Kjeldahl nitrogen 0.060 Nitrates 0.004 Total phosphorus 0.004

Table 5

Measured concentration of n itrate, ammonia, total nitroge n and total phosphorus in the growth media used in the experiments.

Treatments NH 4 -N (μg/L) NO 3 -N (μg/L) TN (μg/L) TP (μg/L) Experiment 1: Alexander spring water Silver Glen spring water Z8Fl Experiment 2 – conductivity: 100 μS/cm 300 μS/cm 500 μS/cm 1000 μS/cm 3000 μS/cm Experiment 4 – microelements: not added added Experiment 5 – Fe: 0 μg/L 20 μg/L 200 μg/L 600 μg/L 22 13 <10

<10 18 <10 <10 11

<10 13

18 16 17 16 53 19 6

1000 1000 1000 990 990

1100 1000

1000 1000 1000 1000 220 180 270

1360 1350 1360 1320 1370

1370 1200

1120 1190 1240 1220 41 24 42

94 74 73 77 69

110 100

95 94 80 11020 Effects of light, conductivity and microelements on the growth of Vaucheria sp.

Introduction

Vaucheria sp. (Xanthophyce) is a common filamentous mat-forming species in streams, rivers and lakes (Entwisle 1990, Mc Intire et al. 1994, Necchi et al. 1994). There are many studies on the ecological and environmental requirements of Vaucheria sp. (Lakatos 1978, Gómez et al. 1994, Aboal et al. 1996). Many species of Vaucheria show high tolerance to different water salinities (M oreno et al. 2001), although they do prefer sites with high light availa bility (Ensminger et al. 2005) (Leukart and Hanelt 1995).

Vaucheria sp. is also commonly used in studies on cell structure (Kataoka and Watanabe 1993, Tornbom and Oliveira 1993, Raven and Brownlee 2001, Takahashi et al. 2001). However, there are very few la boratory studies on the nutrition requirements of

Vaucheria sp., and these studies focus on macr onutrients such as nitrogen and phosphorus (Sudlow 1936, Ozimek et al. 1991). The objectives of this study were: (i) to compare Vaucheria sp. growth rates in natural spring water to the ones in the grow th medium and to test whether single or multiple filaments should be used while growin g algae in microcentrifuge tubes, (ii) to investigate the growth of Vaucheria sp. at different levels of wa ter conductivity, (iii) to investigate the growth of Vaucheria sp. under different light le vels, (iv) to study the growth of Vaucheria sp. in media with different iron c oncentrations and (v) to compare the growth of Vaucheria sp. in media with and without the addition of micronutrients.21 Materials and methods Five experiments were conducted using small algal microcosms (Table 1). The goal of any experiment investigating th e effects of nutrients on algal growth is to achieve high replication, to test as many nutrient levels as possible, and to grow algae in a continuous

culture with constant nutrient levels. To achieve high replication and a large number of treatments, we used microcentrifuge tubes as microcosms in which to grow algae. To achieve semi-continuous culture conditions, we kept the ratio between the algal biovolume and the volume of the growth medium very low, and we frequently changed growth media.

Experimental setup

Filaments of Vaucheria sp. were grown in 1.5 ml sterile microcentrifuge t ubes filled with 1 ml of growth media. Growth media were changed every 48 hours. Racks made of clear polyethylene were placed on an orbital shaker (60 RPM). Microcentrifuge tubes

with Vaucheria sp. were incubated under grow lights providing 25 μmol/m 2 /s of light, with the exception of the light gradient experiment. The unobstructed grow lights provided 250 μmol/m 2 /s, thus, the desired light inte nsity was obtained by covering the racks with a neutral density film. The initia l filament length was 1-2mm. The filaments were photographed using a digital video camera system installed on a Leica MZ12.5 Stereomicroscope under 8x magnification at the beginning and at the end of each experiment. Filament length was measured on the digital images using Image J software (Rasband 2003). Incubation time was 11 or 12 days. Each treatment was replicated 10

times. Since some of the filaments were lo st during media changes, the final number of22 replicates used for the data analyses wa s lower then set at the beginning of the experiment. All experiments had a complete factoria l ANOVA design with 1 or 2 factors. Experiment 1 had a 2 factor design with 3 media treatments (spring water from Alexander, spring water from Silver Glen and Z8Fl medium) and 2 algal biomass treatments (1 and 5 filaments of Vaucheria sp. in each microcentrifuge tube). Due to an error in preparation of the Z8Fl medium for this experiment, no nitrate was added to the medium. Experiment 2 had a 1 factor desi gn with 5 conductivity treatments (100, 300, 500, 1000 and 3000 μS/cm). The desired water conductivity was obtained by a combination of salts reflecting the chemistry of spring water with different conductivity levels (Table 2). Experiment 3 had a 2 fact or design with 15 treatments: a combination of 3 conductivity levels (100, 300, 1000 μS/cm) and 5 light intensities (1, 2.5, 5, 25, 250 μmol/m 2 /s). Experiment 4 had 2 treatments: a growth medium w ith and without the addition of microelements. Experiment 5 had a 1 factor design, with 4 iron concentrations (0, 20, 200, 600 μg Fe/L). If not stated differently, all treatments had the same concentration of microelements, Fe, N, P, Ca, Mg, CO 3 as in the standard Z8Fl medium (Appendix 1).

Growth media A new growth medium was developed based on the Z8 medium (Kotai 1972). The Z8

medium was modified to simulate the medi an chemistry of macronutrients in Florida spring water (referred to as medium Z8Fl ) (Appendix 1). All growth media were

autoclaved or filter sterilized.23 Water analyses Ammonia nitrogen, Kjeldahl nitrogen, nitrite -nitrate nitrogen and total phosphorus concentrations were analyzed in the prepared growth media by FDEP (Table 3). A single sample of each growth medium was sent for the analyses. When the concentration of a compound was below the detection limit of the laboratory method, the method’s detection limit is reported (Table 4). None of the water analyses results were designated as Q or Y. Total nitrogen was calculated as a sum of nitrite-nitrate nitrogen (nitrates) and Kjeldahl nitrogen. Water conductivity was measured using YSI 556 MSP.

Experimental conditions All experiments were conducted in the walk-in grow room. The temperature in the grow room was set for 22.5oC to reflect the average water temperature of the Florida springs. The minimum and maximum temperatures were recorded daily on a max-min

thermometer.

Relative growth rates Algal relative growth rate (RGR) in the small microcosm experiments was calculated using the following formula (Hunt 1990):

Identification of Vaucheria species Since the presence of reproductive structures is necessary to identify the species of Vaucheria, we incubated a small fragment of the mat (Ott and Oldham-Ott 2003). The days of # Length) ln(Initial Length) ln(Final

= RGR −24 mat fragment was placed onto a Petri dish fill ed with Z8Fl growth medium with 100 μg/L P and 1000 μg/L N and placed by the window. We were successful in observing reproductive structures in the samples collect ed from Manatee spring. The species was Vaucheria geminata (Prescott 1982).

Data analyses Data were analyzed using R statistical so ftware (R Development Core Team 2006). Analysis of variance was conducted using a ge neral linear model with car package. If necessary, data were square root or 1/square root transforme d to meet the assumptions of normality and variance homogeneity.

Results

Experiment 1 - growth of Vaucheria sp. in spring water and in growth media Neither the growth media nor the number of filaments in the microcosm had an effect on the growth rate of Vaucheria sp. (ANOVA for medium effect p<0.091 and for number of filaments effect p<0.622) (Fig. 1). Due to an error in the pr eparation of the Z8Fl medium for this experiment, we were not able to tr uly test the medium in comparison to spring water. In later experiments using the Z8Fl medium, however, we observed growth rates higher than the ones in spring water in Experiment 1.

Experiment 2 - growth of Vaucheria sp. in water with different conductivity

Vaucheria sp. grew well in water with conduc tivity at 100, 300 and 1000 μS/cm, but its growth was reduced in water with c onductivity at 500 and 3000 μS/cm (ANOVA for25 conductivity effect p<0.016) (Fig. 2). Howeve r, when the means were compared using the Tukey HSD test, none of the differences among the means was significant at p=0.05. At the levels of conductivity tested, the effect of conduc tivity on growth of Vaucheria sp. was indefinite. Actual conductivities m easured in the treatments were: 105, 331, 516, 963 and 2942 μS/cm. All treatments had the sa me nitrogen concentrations and the same phosphorus concentrations, indicating that the observed growth differences resulted from different conductivity levels in the growth media (Table 5).

Experiment 3 -growth of Vaucheria sp. under different light levels and water

conductivities Light affected the growth rates of Vaucheria sp. and conductivity did not (ANOVA for light effect p<0.001; for conductivity effect p<0.657 and for interaction light*conductivity p<0.046) . Vaucheria sp. grew even at very low light levels, but the growth rates were very low (F ig. 3). The highest growth ra tes were observed at a light intensity of 25 μmol/m 2 /s or higher.

Experiment 4 - growth of Vaucheria sp. in the media with and without micronutrients

added

The addition of microelements to the growth media had no e ffect on the growth rates of

Vaucheria sp. (ANOVA for micronutrient effect p<0.382) (Fig. 4). There was no difference in the nitrogen and phosphorus concen tration in the growth media (Table 5).

Experiment 5 - growth of Vaucheria sp. in different iron concentrations Iron is an important micronutrient for the growth of Vaucheria sp. (ANOVA for the iron effect p<0.001) (Fig. 5). When no iron was a dded to the growth media and when the iron26 concentration in the media was 20 μg/L, very low growth rates of Vaucheria sp. were observed. Surprisingly, the iron concentrati on of 600 μg/L found in the standard Z8 medium had a negative effect on the growth of Vaucheria sp. High growth rates were observed at 200 μg Fe/L. There were no di fferences in the nitrogen and phosphorus concentrations measured in the growth media (Table 5).

Discussion

Vaucheria sp. had comparable growth rates in spring water and in the artificial growth medium, and there were no differen ces in the growth rate between 1 and 5 filaments incubated in 1 microcentrifuge tube (F ig. 1). We also did not observe a trend in the response of Vaucheria sp. to different conductivities of water (Fig. 2). Many species

of Vaucheria are adapted to changes in water co nductivity and are found from fresh water to marine environments ( Mo reno et al. 2001, Johanson 2005). Vaucheria sp. did not grow well at very low light levels, 1 and 2.5 μmol/m 2 /s. The highest growth rates were found at a light intensity of 25 μmol/m 2 /s. In the streams, Vaucheria sp. prefers areas well exposed to light and does not adapt to lo w light levels (Ensminge r et al. 2005). We did not observe a difference in the growth rate of Vaucheria sp. incubated in the growth

media with or without the addition of micronutrients. Vaucheria sp. is tolerant to high levels of metals in the water, much hi gher than the concentrations used in our experiments (Pirszel et al. 1995, Skowronski et al. 1998, Alessa and Oliveira 2001). We were, however, surprised that it did not prefer the media with added micronutrients. The optimum iron concentration for the growth of Vaucheria sp. was 200 μg/L. It did not grow well in the iron concentration of 600 μg/L found in the Z8 growth medium.27 In our experiments, Vaucheria sp. showed a large variability of growth rates in all of the treatments. Also, in all of the e xperimental treatments, we found many filaments that did not grow at all (0 re lative growth rate). This was due to the process of cutting short 1-2 mm tips of Vaucheria sp. for the experiments. Even though Vaucheria sp. is known for its ability to heal wounded areas, no t all of the tips surv ived the process of being cut off. (Tornbom and Oliveira 1993) found that after excising a 3 mm long vegetative filament of Vaucheria longicaulis, the wound-healing process took several hours and new growth occurred 43 hours afte r wounding. Therefore, we changed our methodology in further experiments on Vaucheria sp. and incubated 1-2mm tips for 2 days in the Z8Fl growth medium (Appendix 1) before using them in the experiments.

References Aboal, M., M. Prefasi, and A. D. Asencio. 1996. The aquatic microphytes and macrophytes of the transvase Tajo-Segura irrigation system, southeastern Spain. Hydrobiologia 340 :101-107. Alessa, L., and L. Oliveira. 2001. Aluminum toxi city studies in Vauche ria longicaulis var. macounii (Xanthophyta, Tribophyceae). I. Effects on cytoplasmic organization. Environmental and Experimental Botany 45 :205-222. Ensminger, I., J. Foerster, C. Hagen, and W. Braune. 2005. Plasticity and acclimation to light reflected in temporal and spatia l changes of small-scale macroalgal distribution in a stream. Jour nal of Experimental Botany 56 :2047-2058. Entwisle, T. J. 1990. Macroalgae in the U pper Yarra and Watts River Catchments - Distribution and Phenolo gy. Australian Journal of Marine and Freshwater Research 41 :505-522. Gómez, R., M. R. Vidal-Abarca, and M. L. Suárez. 1994. Bioavailability of phosphorus in Ajauque Stream wetland (SE Spain) . Verh. Internat. Verein. Limnol. 25 :1357- 1360. Hunt, R. 1990. Basic growth analysis. Unvwin Hyman Ltd., London. Johanson, L. R. 2005. Phylum Xanthophyt a (yellow-green algae). Pages 245-270 in D. M. John, B. A. Whitton, and A. J. Brook, editors. The freshwater algal flora of the British Isles. Cambridge Univ ersity Press, Cambridge. Kataoka, H., and M. Watanabe. 1993. Negative Phototropism in Vaucheria-Terrestris Regulated by Calcium .3. The Role of Calcium Characterized by Use of a High-28 Power Argon-Ion Laser as the Source of Unilateral Blue-Light. Plant and Cell Physiology 34 :737-744. Kotai, J. 1972. Instructions fo r preparation of modified nutrient solution Z8 for algae. Norwegian Institute for Water Researc h, publication B-11/69, Blindern, Oslo. Lakatos, G. 1978. The phenomenon and significa nce of benthonic eutrophication in Lake Velencei, Hungary. Acta Biological Debrecina 15 :147-168. Leukart, P., and D. Hanelt. 1995. Light requir ements for photosynthesis and growth in several macroalgae from a small soft-wat er stream in the Spessart Mountains, Germany. Phycologia 34 :528-532.McIntire, C. D., H. K. Phinney, G. L. Larson, and M. Buktenica. 1994. VerticalDistribution of a Deep-Wat er Moss and Associated Epiphytes in Crater Lake, Oregon. Northwest Science 68 :11-21. Moreno, J. L., M. Aboal, M. R. Vidal-Ab arca, and M. L. Suarez. 2001. Macroalgae and submerged macrophytes from fresh and sali ne waterbodies of ephemeral streams ('ramblas') in semiarid south-eastern Spain. Marine and Freshwater Research 52 :891-905. Necchi, O., D. Pascoaloto, and L. H. Z. Branco. 1994. Distribution of Macroalgae in a Tropical River Basin from Southeastern Brazil. Archiv Fur Hydrobiologie 129 :459-471. Ott, D. W., and C. K. Oldham-Ott. 2003. Eustigmatophyte, raphidophyte, and tribophyte algae. Pages 423-469 in J. D. Wehr and R. G. Sheat h, editors. Freshwater algae of North America. Academic Press, San Diego. Ozimek, T., E. Pieczynska, and A. Hankiewi cz. 1991. Effect of filamentous algae on submerged macrophyte growth: a laborat ory experiment. Aquatic Botany 41 :309- 315. Pirszel, J., B. Pawlik, and T. Skowronski . 1995. Cation-Exchange Capacity of Algae and Cyanobacteria - a Parameter of Their Metal Sorption Abilities. Journal of Industrial Microbiology 14 :319-322. Prescott, G. W. 1982. Algae of the Western Great Lakes Area. Otto Koeltz Science Publishers, Koenigstein. R Development Core Team. 2006. R: a langu age and environment for statistical computing. in , Vienna, Austria. Rasband, W. 2003. ImageJ v 1.61. in. National Institutes of Health, Bethesda, Maryland. Raven, J. A., and C. Brownlee. 2001. Unders tanding membrane function. Journal of Phycology 37 :960-967. Skowronski, T., J. A. de Knecht, J. Simons, and J. A. C. Verkleij. 1998. Phytochelatin synthesis in response to cadmium uptake in Vaucheria (Xanthophyceae). European Journal of Phycology 33 :87-91. Stevenson, R. J., A. Pinowska, and Y. K. Wang. 2004. Ecological c onditions of algae and nutrients in Florida springs . Final report. Florida Department of Environmental Protection contract number WM858, Michigan State University, East Lansing, MI. Sudlow, P. L. 1936. Growth of Vaucheria in culture. Master's Thesis. Ohio State University. Takahashi, F., T. Hishinuma, and H. Ka taoka. 2001. Blue light-i nduced branching in Vaucheria. Requirement of nuclear accumu lation in the irradiated region. Plant and Cell Physiology 42 :274-285.29 Tornbom, L., and L. Oliveira. 1993. Wound-H ealing in Vaucheria-Longicaulis Hoppaugh Var Macounii Blum .2. Cytoplasmic Str eaming and Organelle Movement. New Phytologist 124 :135-148.30 Fig. 1 Box plot showing relative growth rates (RGR) or Vaucheria sp. in a factorial mesocosm experiment with 3 growth media (spring wate r from Alexander, spring water from Silver Glen and Z8Fl growth medium but with very low nitrogen and phos phorus content) and 2 biomass levels: 1 or 5 filaments (n=50).31 Fig. 2 Box plots showing relative growth rates (RGR) of Vaucheria sp. in different conductivity water (n=48).32 Fig. 3 Box plots showing relative growth rates (RGR) of Vaucheria sp. in a factorial mesocosm experiment with 5 light levels and 3 conductivity levels (n=121).33 Fig. 4 Box plots showing relative growth rates (RGR) of Vaucheria sp. in growth media with and without micronutrients added (n=18).34 Fig. 5 Box plot showing relative growth rates (RGR) of Vaucheria sp. in 4 iron concentrations (n=39). Boxes sharing the same lett er do not differ (Tukey’s test p<0.05).35 Table 1

Vaucheria sp. experiments. Experiment numbers in pa renthesis refer to numbers used in the 2004 report (Stevenson et al. 2004).

Experiment

(project experiment

number) Treatments Algae collection location Season Growth medium Mean min and max temperature (oC) 1 (6) 2 (13) 3 (13) 4 (14) 5 (14) Spring water, media and number of filaments in the tube Conductivity Light and conductivity Microelements Iron Manatee

Manatee Manatee Manatee Manatee Summer

Summer Summer Summer Summer Z8Fl

Z8Fl Z8Fl Z8Fl Z8Fl 20.7 &25.0

20.4 & 24.7 20.4 & 24.7 20.6 & 24.8 20.6 & 24.8

Table 2 Chemical composition of a component of growth media in experiments 2 and 3 responsible for desired conduc tivity in the medium.

Conductivity

(μS/cm) CaCl 2 ·2H 2 O (mM) MgSO 4 ·7H 2 O (mM) Na 2 CO 3 (mM) KCl (mM) NaCl (mM) 100 300 500 1000 3000 0.116 0.496 0.747 1.618 2.739 0.101 0.203 0.284 0.284 0.284 0.198 0.792 1.293 1.293 1.293 0 0 0 0.123 0.875 0 0 0 0.123 0.875

Table 3 Methods used for analyses of water samples. Parameter Method Description Ammonia-N EPA 350.1 (1983) Ammonia in aquaeous matrices as mg N/L NO2NO3-N EPA 353.2 (1983) Nitrite/Nitrate in aqueous matrices as mg N/L N_KJEL_TOT EPA 351.2 (1983) Total Kjeldahl Nitrogen in aqueous matrices Total-P EPA 365.4 Total Phosphorus in aqueous matrices36 Table 4 Detection limits of water chemical analyses . When the concentration of a compound was below the detection limit of the laboratory method, the method detection limit is reported.

Compound Detection limit (mg/L) Ammonia nitrogen 0.010 Total Kjeldahl nitrogen 0.060 Nitrates 0.004 Total phosphorus 0.004

Table 5

Measured concentration of nitr ates, ammonia, tota l nitrogen and tota l phosphorus in the growth media used in the experiments.

Treatments NH 4 -N (μg/L) NO 3 -N (μg/L) TN (μg/L) TP (μg/L) Experiment 1: Alexander spring water Silver Glen spring water Z8Fl Experiment 2 – conductivity: 100 μS/cm 300 μS/cm 500 μS/cm 1000 μS/cm 3000 μS/cm Experiment 4 – microelements: not added added Experiment 5 – Fe: 0 μg/L 20 μg/L 200 μg/L 600 μg/L 22 13 <10

<10 18 <10 <10 11

<10 13

18 16 17 16 53 19 6

1000 1000 1000 990 990

1100 1000

1000 1000 1000 1000 220 180 270

1360 1350 1360 1320 1370

1370 1200

1120 1190 1240 1220 41 24 42

94 74 73 77 69

110 100

95 94 80 11037 Recovery of filament ous algal mats of Lyngbya wollei and Vaucheria sp. after

disturbance.

Introduction Algae in Florida springs are subject to physical disturba nce, most commonly flooding and physical disturbance by large animals (large fish, manatees and alligators) or human activity (swimming, snorkeling, SC UBA diving). This results in sediment deposition which reduces light availability or direct bi omass removal. The goal of this investigation was to simulate the effects of large animal herbivory and disturbance and the effects of sever flooding with turbid wate r that result in mass algal die off. In this study we monitored the regrowth of filamentous algal mats of Lyngbya wollei and Vaucheria sp. after direct biomass removal.

Materials and methods This study was conducted at two locations: Manatee spring and Blue Hole in the Ichetucknee spring system. The dominant mat-forming filamentous alga at Manatee spring is Vaucheria sp (Stevenson et al. 2004). The Mana tee spring waters are frequently turbid due to flooding from the Suwannee River, and as its name suggests, the spring is also frequently visited by mana tees that graze on algae. The Blue Hole spring bottom is covered by macrophytes and mats of Lyngbya wollei . The Blue Hole spring stays clear even during severe flooding events. This study was conducted from January to May 2006. Three sites were selected within each spring. Each site consisted of 238 experimental plots (50cm x 50cm each) de lineated by PVC poles driven into the

sediments. All of the sites were within rest ricted areas that are not affected by direct human activity. At each site, the initial thic kness of the mat was m easured. There were no differences in the pretreatment mat thic kness for the disturbed and control plots, indicating that there were no si te-specific differences between the plots. After the initial measurement of mat thickness, one plot within each site was disturbed. Using a rake, all visible filamentous algae were removed down to the sediments. The recolonization of disturbed areas and algal growth on the c ontrol (undisturbed) plots were initially monitored every 2 weeks, and later less freque ntly depending on the recolonization rates. Mat thickness was measured with a 1⁄4” diameter dowel at 13 evenly distributed points on each plot. On each observation date, th e water depth, dissolved oxygen (DO), pH, conductivity and temperature were measured. Data were analyzed using SAS statistical software (SAS 2000) (repeated measures ANOVA with LS means test) and R statistica l software (R Development Core Team 2006). Data were square root transformed to reduce deviations from normality and homoscedasticity.

Results The two populations studied had very different recovery rates after disturbance. The

control mats of Lyngbya wollei did not change in thickness during the course of this study. Disturbed plots of Lyngbya wollei slowly grew back but did not recover from thedisturbance even after 108 days (rep eated measures ANOVA: time effect p<0.0013, disturbance effect p< 0.0021) (Fig. 1).39 Fig. 1 Average thickness of the mats of Lyngbya wollei at Blue Hole in Ic hetucknee springs. Disturbed sites were complete ly scraped on the 31 of January and control sites were

undisturbed.

Vaucheria sp. in the control plots had the thickest mats on February 17. Later, mat thickness decreased and in Ap ril control plots were se nescing and decomposing.

Disturbed Vaucheria sp. recovered very quickly, a nd after 24 days there was no40 difference in the thickness of the mat at th e disturbed and control sites (LS means for 24 Feb p=0.2067, for 3 Mar p=0.3108, for 10 Mar p=0.2600, 7 Apr p=0.8269). By the end of our study, the control mats had begun to senesce and disintegrate (Fig. 2).

Fig. 2 Average thickness of the mats of Vaucheria sp. at Manatee spring. Disturbed sites were completely scraped on January 31 and control sites were undisturbed.41 The two sites studied had comparable wate r parameters, however, Manatee springs is characterized by a higher am plitude of changes in conductivity and dissolved oxygen (Table 1).

Table 1 Water parameters at the two studied sites.

Spring Conductivity (μS/cm) Dissolved oxygen (mg/L) pH Temperature (oC)

Blue Hole 279-312 2.3-2.6 6.8-7.7 21.35-21.43

Manatee 285-502 1.0-2.4 6.7-7.8 21.35-22.1

Discussion Even though in our lab experiment s the relative growth rates of Vaucheria sp. and Lyngbya wollei were similar, Vaucheria sp. recovered very quickly after disturbance and Lyngbya wollei did not recover even af ter 108 days. Both sites studied are characterized by high nutrient levels (Stevenson et al. 2004). The difference between the two taxa in the speed of recovery after disturbance can be explained by their different means of reproduction and the differences in their growth forms. Vaucheria sp. can reproduce asexually by fragmentation of the mat and through production of zoospores, akinets and aplanospores (Starmach 1972, Johanson 2005). It can also reproduce sexually by formation of antheridia and oogonia. The ma t is formed from multinucleate filaments42 and attached by colorless rhizoids to the substrate. Lyngbya wollei reproduces only by fragmentation of trychomes (Komárek a nd Anagnostidis 2005). The fact that Vaucheria sp. can reproduce much faster than L. wollei probably explains why Vaucheria sp. is the dominant taxon in springs along the Suwannee River. Manatee, Fanning and Guaranto springs are dominated by mats of Vaucheria sp. These springs also frequently experience the intrusion of turbid waters from the Suwannee River during floods.

References

Johanson, L. R. 2005. Phylum Xanthophyt a (yellow-green algae). Pages 245-270 in D. M. John, B. A. Whitton, and A. J. Brook, editors. The freshwater algal flora of the British Isles. Cambridge Univ ersity Press, Cambridge. Komárek, J., and K. Anagnostidis. 2005. Cy anoprokaryota. 2. Teil: Oscillatoriales. Gustav Fischer, Jena. R Development Core Team. 2006. R: a langu age and environment for statistical computing. in Vienna, Austria. SAS. 2000. SAS/STAT. in SAS Institute, Cary, N.C. Starmach, K. 1972. Chlorophyta III. Zieleni ce nitkowate: Ulotrhichales, Ulvales, Prasiolales, Shaeropleales, Cladophor ales, Chaetophorales, Trentepohliales, Siphonales, Dichotomosiphonales. Pans twowe Wydawnictwo Naukowe, Kraków. Stevenson, R. J., A. Pinowska, and Y. K. Wang. 2004. Ecological c onditions of algae and nutrients in Florida springs . Final report. Florida Department of Environmental Protection. Contract number WM858, Michigan State Un iversity, East Lansing, MI.43 Differential effect of PO 4 , NO 3 and NH 4 on the growth of Lyngbya wollei

Introduction Lyngbya wollei (Farlow ex Gomont), family Oscillato riace, a filamentous blue green alga with unusually thick, often over 60 μm wide, trichomes, forms nuisance growths in many Florida springs. In the algae surveys of 68 Florida spring sites conducted in 2003 and 2006 (Stevenson et al. 2004), and unpublished data) it was found that large mats were most commonly formed by L. wollei. This filamentous alga is found in water bodies throughout the southeastern United States (Speziale and Dyck 1992, Cowell and Botts 1994, Cowell and Dawes 2004), Texas (Owens et al. 2005), Canada (Macbeth 2004) and

other locations. Lyngbya wollei can fix atmospheric nitrogen (Phlips et al. 1992) but large mats are often found at sites with hi gh nitrate concentrati ons (Cowell and Botts 1994, Stevenson et al. 2004). In the natural environment the growth of f ilamentous algal mats is highly affected by many biotic and abiotic factors. Even though L. wollei mats are not readily consumed by herbivores (Camacho and Thacker 2006) in Florida springs the mats are disturbed by large vertebrates and people visiting the spri ngs. To successfully study nutrient effects on the growth of algae, the mats have to be moved to controlled laboratory conditions. Several studies investigated the effects of nitrogen and phosphorus on the growth of L.

wollei (Turner 1990, Cowell and Botts 1994, Yin et al. 1997, Cowell and Dawes 2004). These studies used only a few concentrations of nutrients in their experiments and none of them investigated the threshold le vels of nutrients for the growth of L. wollei.

.44 This study focused only on L. wollei from Florida springs. The objectives of this study were: (i) to investigate th e effects of a wide range of concentrations of nitrogen (in

the form of nitrate and ammonia) and phosphorus on the growth of L. wollei , (ii) to

determine whether nitrate or ammonia is a preferred source of nitrogen for L. wollei , (iii) to study the importance of the size of algal mats when L. wollei was grown under different nutrient levels, and (iv) to investigate whether L. wollei populations show seasonal differences in their growth rates a nd whether populations from different Florida springs differ in their growth rates as well.

Materials and methods Two experimental systems were used to st udy the growth of filamentous algae: small microcosms and large microcosms. The sma ll microcosm system allowed a large number of replicates but restricted the experimental unit to a short algal filament. The large microcosm system could accommodate small fila mentous algal mats of varying sizes but restricted the number of repl icates. We conducted 9 small microcosm experiments and 3 large microcosm experiments (Table 1). The nutrient levels used in the experiments reflected phosphate and nitrate concentrati ons observed in Florida springs water. Ammonia concentrations used in the experi ments were higher than those observed in Florida springs water but in the ra nge of those observed inside the L. wollei mats (Task 3 of the report – the study conducted by Albertin and Sickman).45 Small microcosm experiments

Filaments of L. wollei were grown in 1.5 ml microcentr ifuge tubes filled with 1 ml of growth media. Growth media were cha nged every 48 hours. Racks made of clear polyethylene were placed on an orbital shaker (60 RPM) and incubated under grow lights providing 25 μmol/m 2 /s of light. To achieve this light intensity, the racks were covered with neutral density film. Light intensity of 25 μmol/m 2 /s was found to be the optimal intensity for the growth of i ndividual filaments in microcentrifuge tubes. The initial filament length was 1-2mm. The filament wa s photographed using a digital video camera system installed on a Leica MZ12.5 Stereo microscope under 8x magnification at the beginning and at the end of each experiment . Filament length was measured on the digital images using Image J software (Rasband 2003). Incubation time was 11 or 12 days. Each treatment was replicated 10 times, or 5 times if L. wollei from two locations (2 different springs) was used. Since some of the filaments were lost during the media changes, the final number of replicates used for the data analyses was lower than set at the beginning of the experiment. All experiments had a complete factorial ANOVA design with 1 or 2 factors. Experiments 1, 2 and 3 had a 2 factor design with 10 nutrient treatments each (PO 4 -P concentrations: 1, 5, 10, 20, 30, 40, 60, 80, 100 and 250 μg P/L) and 2 population location treatments. The NO 3 -N concentration in experiment 1, 2 and 3 was 1000 μg/L. Experiment 5 had a one fact or design with 11 nutrient treatments (NO 3 - N concentrations: 10, 30, 60, 125, 250, 500, 750, 1000, 1500, 2500 and 5000 μg N/L) and experiments 6, 7 and 8 had a 2 factor design with 10 nutrien t treatments (NO 3 -N concentrations: 10, 30, 60, 125, 250, 500, 750, 1000, 2000 and 5000 μg N/L) and 2 population location treatments. Experiment 10 had a 1 factor design with 11 nutrient46 treatments (NH 4 -N concentrations: 10, 30, 60, 125, 250, 500, 750, 1000, 1500, 2500 and 5000 μg N/L). Experiment 12 had a 2 factor design with 16 nutrient treatments: a

combination of 4 ammonia concentrations (20, 100, 500 and 1000 μg N/L) and 4 nitrate concentrations (20, 100, 500 and 1000 μg N/L). The PO 4 -P concentration in experiments 5, 6, 7, 8, 10 and 12 was 100 μg/L.

Large microcosm experiments The large microcosm system consisted of a flat platform with Plexiglas cylindrical raceways (Manoylov 2005). The platform was pl aced on an orbital shaker table (60RMP) in a walk-in growth room. The platform was illuminated with fluorescent grow lights providing 150 μmol/m 2 /s of light. Each microcosm wa s filled with 100 ml of growth medium. The media were changed daily. At the beginning and at the end of the experiment, small fragments of L. wollei mats were held between a Nitex screen and gently patted dry with a paper towel to remove excess water, then weighed (fresh mass). At the end of the experiment, all samples were processed for dry mass (DM). At the beginning of each experiment, 10 algal samples from each initial mass treatment were patted dry in between a Nitex screen, weighed to measure the initial fresh mass (FM), dried in the oven, and weighed again (DM). This allowed the calculation of the initial dry mass of the experimental samples based on their initial fresh mass. All samples for

DM were dried in the oven at 105oC for 24 hours (Eaton et al. 1995). All experiments had a complete factorial ANOVA design w ith 2 experimental factors: nutrient

concentration and initial bioma ss. Experiment 4 had a total of 15 treatments that were a combination of 5 phosphate concentrations (PO 4 -P concentrations: 2, 10, 30, 60, 150 μg47 P/L) and 3 initial fresh ma ss levels (0.01g, 0.05g, 0.2g). The NO 3 -N concentration in experiment 4 was 1000 μg/L. Experiment 9 had a total of 15 treatments that were a

combination of 5 nitrat e concentrations (NO 3 -N concentrations: 20, 125, 500, 1000, 2500 μg N/L) and 3 initial fresh mass levels ( 0.01g, 0.05g, 0.2g). Experiment 11 had a total of

15 treatments that were a combinati on of 5 ammonia concentrations (NH 4 -N concentrations: 20, 125, 500, 1000, 2500 μg N/L) a nd 3 initial fresh mass levels (0.01g, 0.05g, 0.2g). The PO 4 -P concentration in experiment 9 and 11 was 100 μg/L.

Relative growth rates Algal relative growth rate (RGR) in the large microcosm experiments was calculated using the following formula (Hunt 1990):

In the small microcosm experiments, algal initial and final filament length was used instead of algal mass.

Growth media A new growth medium was developed based on the Z8 medium (Kotai 1972). The Z8

medium was modified to simulate the medi an chemistry of macronutrients in Florida spring water (referred to as medium Z8Fl) (Appendix 1). We originally used a Z8Fl days of # Mass) ln(Initial Mass) ln(Final

= RGR −48 Fe600 medium that had the original concentr ation of iron (600 μg/L) found in the Z8

medium. Later we used Z8Fl Fe200 with th e iron concentration reduced to 200μg/L,

because we found an Fe concentration of 600μg/L to have a suppressing effect on the growth of L. wollei sp. We used the Gaffron micronutrien ts mix as used by Kotai (1972). To 1L of the final growth medi um we also added 10ml of CaCl 2 ·2H 2 O solution (stock solution 18.3g CaCl 2 ·2H 2 O in 1L of deionized water), 10ml of MgSO 4 ·7H 2 O solution (stock solution 7g MgSO 4 ·7H 2 O in 1L of deionized water), and 10ml of Na 2 CO 3 solution (stock solution 13.7g Na 2 CO 3 in 1L deionized water). We used NaNO 3 and NH 4 Cl as sources of nitrogen and K 2 HPO 4 as a source of phosphorus. When NaNO 3 gradient was investigated, NaCl was also added to keep the Na concentration constant in all the treatments. In the experiments with K 2 HPO 4, gradient KCl was added to keep the K

concentration constant in all of the trea tments, following the experimental design of (Ahlgren 1977).

Experimental conditions All experiments were conducted in the walk-i n growth room. The temperature in the growth room was set for 22.5oC to reflect the average water temp erature in Florida springs. The minimum and maximum temper ature was recorded daily on a max-min

thermometer.49 Water analyses Water samples of prepared growth media and from experimental macrocosms were sent to FDEP for analyses. On last day of experi ment a sample of water in which algae were growing for 24 hours was collected for analyses.

Data analyses Data were analyzed using SAS statistical software (SAS 2000) (repeated measures ANOVA) and R statistical so ftware (R Development Co re Team 2006). Logistic regression analysis was conducted using drc p ackage for R (Ritz and Streibig 2007). All final and initial filament length and small mat biomass data were first tested using repeated measures ANOVA to test the signifi cance of nutrient effect and time effect (indicating algal growth). Next, factorial analys is of variance was used to test the effect of nutrient concentration, season and L. wollei population location on relative growth rates (RGR). A lowess smooth line was fitted through the data for general assessment of the trends in the data. If a significant nutrient effect was found in the ANOVA analysis, a logistic curve was fitted through the RGR data. If the logistic curve did not provide a good fit and data were distribute d along a straight line, a linear regression was calculated. Various logistic models were fitted through the data (Ritz and Streibig 2005, Ritz and Streibig 2006) and the best fitting models are presented in the results. The best fitting models for our data were: four parameter logi stic model (l4( )), four parameter logistic

model with variance calculat ed as a power function of the mean to account for heterogeneity of the data (l4() varPower), Monod model (calculated using code for50 Michaelis-Menten model from the drc pack age) and five parameter Brain-Cousens hormesis model (Borchardt 1996, Schabenberger et al. 1999, Ritz and Streibig 2007). When seasonal effects were compared using factorial ANOVA, only data from experiments conducted using exactly the same growth media and for populations collected from the same locations during di fferent seasons were used. When population effects were compared, only experiments c onducted in the same media and season were

used. All data sets were ch ecked for normality and heterogeneity of variance, and, where necessary, data were transformed for the ANOV A analyses. All analyses using the drc package were conducted on untransformed RGR data.

Results Lyngbya wollei grew in all experiments under all nutrient concentra tions. Relative growth rates (RGR) varied from 0 to 0.40. Lo w nutrient levels resu lted in low growth rates and high nutrient levels resulted in high growth ra tes, with the exception of ammonia treatments. In these, the highest NH 4 concentrations in the small microcosm experiments resulted in reduced growth rates.

Effects of PO 4 In the small microcosm experiments, an increase in phosphate phosphorus had a significant effect on the growth of L. wollei (Fig 1). The relative growth rate data were well described by a logistic dose-response curve (Fig. 1, Table 2). The threshold concentration of phosphate phos phorus for the growth of L. wollei was 17.5 μg/L - the Effective Dose of 50% growth saturation (ED 50 ). The mean growth rate in the51 experiments conducted on the L. wollei population from the Ichetu cknee in the spring of the year (0.29±0.08 mm/mm/day) was highe r than in the fall (0.20±0.10 mm/mm/day) (ANOVA for the season effect, P< 0.001). The three populations of L. wollei studied did not differ in their RGRs (ANOVA for the location effect, P<0.543). In the large microcosm experiments, L. wollei mats with smaller initial biomass grew faster than larger mats, es pecially in high phosphate conc entrations (ANOVA for biomass effect, P<0.001) (Fig.2). A dose response curv e was successfully fitted only through the

lowest initial biomass data . The threshold phosphate phos phorus concentration was 49 μg/L (Fig.2, Table 3).

Effect of NO 3 In the small microcosm experiments, an incr ease in nitrate nitrogen had a positive effect on the growth of L. wollei (Fig. 3). The RGR data were we ll described by a logistic doseresponse curve (Fig. 3, Table 2). The thresh old nitrate nitrogen concentration for the growth of L. wollei was 88.1 μg/L (ED 50 ). The mean growth rate in the experiments

conducted on the L. wollei population from the Ichetuckne e in the spring of the year (0.27±0.08 mm/mm/day) was higher then in the fall (0.19±0.08 mm/mm/day) (ANOVA for the season effect, P<0.001). Among the three studied populations of L. wollei, the Ichetucknee population (0.22±0.10 mm/mm/day) had lower relative growth rates than the Silver Glen and Alexander popula tions (0.24±0.09 mm/mm/day and 0.24±0.08 mm/mm/day) (ANOVA for the location effect, P<0.001). In the large microcosm experiments, L. wollei mats with smaller initial biomass grew faster than larger mats (ANOVA for biom ass effect, P<0.001) (Fig.4). Dose response curves were fitted through the data. The th reshold nitrate nitrogen concentration was 51952 μg/L for the 0.01g initial biomass and 546 μg/L for the 0.05g initial biomass (Fig.4, Table 3).

Effect of NH 4 In the small microcosm experiments, an increase in ammonia nitrogen resulted in increased growth rates up to a 796.3 μg/L NH 4 -N concentration, where it reached the highest growth rate of 0.27 mm/mm/day (Fi g. 5). The RGR data were well described by the Brain-Cousens hormesis curve (Fig. 5, Table 2). In the large microcosm experiments, L. wollei mats with smaller initial biomass grew faster than larger mats in high ammoni a concentrations (ANOVA for biomass effect, P<0.001) (Fig.6). Dose response curves were fitted through the 0.01g and 0.05g initial

biomass data. The threshold ammonia nitr ogen concentration was 248 μg/L for the 0.01g initial biomass and 227 μg/L for the 0.05g initial biomass (Fig.6, Table 3).

Combined effect of NO 3 and NH 4 In the small microcosm experiments, nitrogen was added in the form of ammonia, nitrate

or a combination of those two forms. Both nitrate and ammonia nitrogen had a positive effect on the growth of L. wollei (ANOVA for NO 3 effect, P<0.001, for NH 4 effect P<0.001) (Fig.7). We also found that the form of nitrogen used did not matter and only

an absolute concentration of nitrogen had an effect on the growth rates of L. wollei . The

RGR data were well described by the Monod model (Fig. 8, Table 2)

. The half saturation

constant K μ was reached at a 104μg/L combined nitrogen concentration.53 Discussion In our experiments Lyngbya wollei grew in all experimental conditions (Table 4). A maximum doubling rate of 0.4 was lower or simila r to the growth rates of other species of filamentous algae (Borchardt 1996, Hall a nd Payne 1997). On the other hand. the doubling rates of L. wollei in our study were higher or si milar to the growth rates of L.

wollei observed by others (Cowell and Botts 1994, Yin et al. 1997, Cowell and Dawes 2004). Cowell and Dawes (2004) in vestigated the effect of n itrate on the growth of small

mats of L. wollei in a continuous culture and found that the growth rate increased when the nitrate concentration was equal to or above 600 μg N/L. When we examined the growth of L. wollei mats in different nitrate conc entrations, we found a threshold concentration of 518-546 μg N/L, which is in agreement with the results of Cowell and Dawes (2004). Turner (1990) a nd Yin et al. (1997) experimented with very high nutrient

concentrations. These concentrations were a bove the highest concentration used in this study. Some of the low growth rates observe d by Turner (1990) and Yin et al. (1997) were possibly the result of nutrient t oxicity. In our study we did not find L. wollei to show a preference for ammonia or nitrate as a source of nitrogen, which agrees with the results of Turner (1990). In this study growth rates of singl e filaments were saturated at lower nutrient levels than growth rates of algal mats. Also, different size mats of L.

wollei were used, and withlarger mat sizes, the saturating nutrient concentration was increased as well. Overall, for individual fila ments, the nitrate concentration in almost all Florida springs (Stevenson et al. 2004) were above threshold concentrations for the growth of L. wollei, and in many of the springs the nitr ate concentrations were above the threshold concentration for the growth and establishment of mats. Once mats develop,54 they can recycle nutrients within the mat, thus, their growth is less dependent on the

nutrients available from the water column. The water phos phate concentration in many Florida springs is above th e threshold concentration fo r the growth of individual filaments. However, it was below 50 μg/L, th e threshold concentration for the growth of mats, in 76% of the springs surveyed (Stevenson et al. 2004).

References Ahlgren, G. 1977. Growth of Oscillatoria-Agar dhii in Chemostat Culture .1. Nitrogen and Phosphorus Requirements. Oikos 29 :209-224. Borchardt, M. A. 1996. Nutrients. Pages 183-227 in R. J. Stevenson, M. L. Bothwell, and R. L. Lowe, editors. Algal ecology. Academic Press, Sand Diego. Camacho, F. A., and R. W. Thacker. 2006. Amphipod herbivory on the freshwater cyanobacterium Lyngbya wollei: Chemical stimulants and morphological defenses. Limnology and Oceanography 51 :1870-1875. Cowell, B. C., and P. S. Botts. 1994. Factor s Influencing the Dist ribution, Abundance and Growth of Lyngbya-Wollei in Cent ral Florida. Aquatic Botany 49 :1-17. Cowell, B. C., and C. J. Dawes. 2004. Growth and nitrate-nitrogen uptake by the cyanobacterium Lyngbya wollei. Journa l of Aquatic Plant Management 42 :69-71. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg, Eds. 1995. Standard methods for the examination of water and wastewater, 19th edition. American Public Health Association, Washington, D.C. Hall, J., and G. Payne. 1997. Factors contro lling the growth of field populations of Hydrodictyon reticulatum in New Zeal and. Journal of Applied Phycology 9 :229- 236. Hunt, R. 1990. Basic growth analysis. Unvwin Hyman Ltd., London. Kotai, J. 1972. Instructions fo r preparation of modified nutrient solution Z8 for algae. Norwegian Institute for Water Researc h, publication B-11/69., Blindern, Oslo. Macbeth, A. J. 2004. Investigation of an introduced subtropical alga ( Lyngbya wollei ) in Whiteshell Provincial Park, Manitoba. Mast ers Thesis. University of Manitoba, Winnipeg. Manoylov, K. M. 2005. Ecological strate gies of benthic diatoms for nutrient competition. Ph.D. Dissertation. Mi chigan State University, East Lansing. Owens, C. S., M. J. Grodwitz, and F. Nibling. 2005. A survey of the invasive aquatic and riparian plants of the lower Rio Gran de, 2004. U.S. Army Corps of Engineers, Washington, DC 20314-1000. Phlips, E. J., J. Ihnat, and M. Conroy. 1992. Nitrogen fixation by the benthic freshwater cyanobacterium Lyngbya wollei . Hydrobiologia 234 :59-64.55 R Development Core Team. 2006. R: a langu age and environment for statistical computing. in , Vienna, Austria. Rasband, W. 2003. ImageJ v 1.61. in . National Institute of Health, Bethesda, Maryland. Ritz, c., and J. C. Streibig. 2005. Bioassay an alysis using R. Jour nal of Statistical Software 12 :1-22. Ritz, C., and J. C. Streibig. 2006. Bioassay for allelochemicals: examples with R. Pages 24 in

. Ritz, C., and J. C. Streibig. 2007. The drc p ackage. Analysis of dose-response curves. in

. SAS. 2000. SAS/STAT. in . SAS Institute, Cary, N.C. Schabenberger, O., B. E. Tharp, J. J. Kells , and D. Penner. 1999. Statistical tests for hormesis and effective dosages in herb icide dose response. Agronomy Journal 91 :713-721. Speziale, B. J., and L. A. Dyck. 1992. Lyngbya infestations: comparative taxonomy of Lyngbya wollei comb. nov. (Cyanobacteria). Journal of Phycology 28 :693-706. Stevenson, R. J., A. Pinowska, and Y. K. Wang. 2004. Ecological c onditions of algae and nutrients in Florida springs . Final report. Florida Department of Environmental Protection contract number WM858, Michigan State University, East Lansing, MI. Turner, G. 1990. Studies on the biology of Lyngbya wollei . Master Thesis. Clemson University, South Carolina. Yin, Q. Q., W. W. Carmichael , and W. R. Evans. 1997. Fact ors influencing growth and toxin production by culture s of the freshwater cyanobacterium Lyngbya wollei Farlow ex Gomont. Journal of Applied Phycology 9 :55-63.56 Fig 1 Relative growth rates (RGR) of Lyngbya wollei in different phosphate phosphorus concentrations (n=271). A four parameter logistic curve, with variance as a power function of the mean, was fitted through the combined data from small microcosm experiments (experiments 1, 2 and 3). Curve parameters are in Table 2.57 Fig. 2 Relative growth rates (RGR) of Lyngbya wollei in a factorial experi ment with 3 initial biomass levels (0.01g, 0.05g, 0.2g) and 5 phospha te phosphorus concentrations (n=45). A four parameter logistic curve and 2 regression lines (in itial biomass 0.05g R 2 =0.05, P<0.41; initial biomass 0.2g R 2 =0.21, P<0.086) were fitted through the data from a large microcosm experiment (experiment 4). Curve parameters are in Table 3.5859 Fig. 3 Relative growth rates (RGR) of Lyngbya wollei in different n itrate nitrogen concentrations (n=380). A four parameter logistic curve, with variance as a power function of the mean, was fitted through the combined data from small microcosm experiments (experiments 5, 6, 7 and 8). Curve parameters are in Table 2.60 Fig. 4 Relative growth rates (RGR) of Lyngbya wollei in a factorial experi ment with 3 initial biomass levels (0.01g, 0.05g, 0.2g) and 5 nitrate nitrogen concentrations (n=45). Four parameter logistic curves were fitted through the data from a large microcosm experiment (experiment 9). Curve parameters are in Table 3.61 Fig. 5 Relative growth rates (RGR) of Lyngbya wollei in different ammonia nitrogen concentrations (n=101). A five parameter Brain-Cousens hormesis logistic model was fitted through the data from a small microcosm experiment (experiment 10). Curve parameters are in Table 2.62 Fig. 6 Relative growth rates (RGR) of Lyngbya wollei in a factorial experi ment with 3 initial biomass levels (0.01g, 0.05g, 0.2g) and 5 ammoni a nitrogen concentrations (n=45). Two four parameter logistic curves and a regression line (in itial biomass 0.2g R 2 =0.45, P<0.006) were fitted through the data from a large microcosm experiment (experiment 11). Curve parameters are in Table 3.63 Fig. 7 Boxplot of relative growth rates (RGR) of Lyngbya wollei in a combination of different ammonia and nitrate nitrogen c oncentrations (experiment 12).64 Fig. 8 Relative growth rates (RGR) of Lyngbya wollei in different nitrogen (sum of NO 3 -N and

NH 4 -N) concentrations (n=153). A M onod logistic curve starting at y 0 =0 was fitted through data from small microcosm experime nt (experiment 12). The assumption that

y 0 =0 was used since there were no data in the low range of concentrations. Curve parameters are in Table 2.65 Table 1 Lyngbya wollei experiments. Experiment numbers in parenthesis refer to the numbers used in the 2006 report.

Experiment (project experiment number) Micro cosm Nutrient gradient Algae collection location Season Growth medium Mean min and max temperature (oC) 1 (18) 2 (19) 3 (21) 4 (8) 5 (3) 6 (16) 7 (17) 8 (20) 9 (9) 10 (4) 11 (12) 12 (2) Small Small Small Large Small Small Small Small Large Small Large Small PO 4 -P PO 4 -P PO 4 -P PO 4 -P NO 3 -N NO 3 -N NO 3 -N NO 3 -N NO 3 -N NH 4 -N NH 4 -N N (NH 4 and NO 3 ) Ichetucknee and Silver Glen Ichetucknee and Silver Glen Ichetucknee and Alexander Silver Glen Silver Glen Ichetucknee and Silver Glen Ichetucknee and Silver Glen Ichetucknee and Alexander Ichetucknee Silver Glen Silver Glen Silver Glen Fall Fall Spring Spring Spring Fall Fall Spring Spring Spring Fall Spring Z8Fl Fe200 Z8Fl Fe200 Z8Fl Fe200 Z8Fl Fe600 Z8Fl Fe600 Z8Fl Fe200 Z8Fl Fe200 Z8Fl Fe200 Z8Fl Fe600 Z8Fl Fe600 Z8Fl Fe600 Z8Fl Fe600 20.8 &24.3 21.1 &25.3 21.5 &24.9 21.1 & 25.4 21.9 & 24.2 20.9 & 24.5 20.8 & 24.3 21.5 &24.9 20.6 & 22.3 21.5 & 24.5 22.0 & 26.7 21.0 & 24.366 Table 2 Parameters of the logistic dose- response model applied to the Lyngbya wollei RGR data from small microcosm experiments, where d and c denote the upper and lower limit of algae growth rates at infinitely low a nd high concentrations of nutrient. ED 50 (also called

e ) denotes the concentration re quired to increase algal grow th rate by half between the upper and lower limits and b is proportional to the slope (the rate of change) of the curve

around ED 50 . Significant parameters are in boldface. ED 10 and ED 90 denote the concentration required for 10% and 90% gr owth saturation. For the Brain-Cousens hormesis model, c and d are the upper and lower asym ptote of the response. The b and e columns are related to the rate of change and point of inflection of the curve, respectively;

and f indicates the initial rate of increase at low concentrations. For the Monod model,

μ max is the maximum specific growth rate and K μ is the half saturation constant. Significant parameters are in boldface.

Nutrient Model d or μ max (mm/mm/ day) c (mm/mm/ day) b ED 50 (μg/L) e or K μ ED 90 (μg/L) or f ED 10 (μg/L)

PO 4 NO 3 NH 4 N (NO 3 and NH 4 ) l4() varPower l4() varPower Brain-Cousens Monod 0.28±0.01 0.26±0.00 0.09±0.01 0.19±0.01 0.14±0.01 0.10±0.01 0.11±0.01

- -4.65±1.39 -2.29±0.31 5.18±0.71

- 17.50±1.39 88.10±6.14 796.3±43.5 104.2±21.5 28.10±3.80 230.05±32.06 0.00037±0.00004

- 10.90±2.00 33.74 ± 5.28

-

-67 Table 3 Parameters of the logistic dose- response model applied to the Lyngbya wollei growth response data from large microcosm experiments, where d and c denote the upper and lower limit of algae growth rates at infinite ly low and high concentr ations of nutrient.

ED 50 denotes the concentration re quired to increase the algal gr owth rate by half between the upper and lower limits and b is proportional to the slope of the curve around ED 50 . Significant parameters are in boldface. ED 10 and ED 90 denote the concentration required for 10% and 90% growth saturation.

Nutrient and biomass Model d (g/g/day) c (g/g/day) b ED 50 (μg/L) ED 90 (μg/L) ED 10 (μg/L)

PO 4 x 0.01g NO 3 x0.01g NO 3 x0.05g NO 3 x0.2g NH 4 x 0.01g NH 4 x 0.05g l4() l4() l4() l4() l4() l4() 0.23±0.01 0.27±0.01 0.24±0.03 0.15±0.03 0.23±0.01 0.18±0.01 0.15±0.01 0.15±0.01 0.11±0.02 0.11±0.02 0.11±0.01 0.11±0.01 -3.05±1.41 -4.78±4.00 -1.19±0.87 -0.83±1.19 -1.90±0.47 -3.11±1.63 49.15±9.74 518.6±47.9 546.0±250.0 808.2±2015 247.51±39.26 226.95±41.86 100.9±42.8 820.6±353.7 3440.0±5490.0 11283±61207 788.5±246.4 460.2±205.8 23.9±8.3 327.7±116.1 86.66±106.1 57.88±396.8 77.70±26.74 111.9±42.7868 Table 4 Summary of L. wollei growth parameters.

Single filaments Mats Parameter PO 4 -P NO 3 -N NH 4 -N PO 4 -P NO 3 -N NH 4 -N Lowest nutrient concentration tested Average growth rate at lowest nutrient concentration Highest nutrient concentration tested Average growth rate at highest nutrient concentration Average maximum growth rate Lowest concentration at maximum growth rate Minimum concentration at which growth was observed Difference between maximum and minimum growth rate Average threshold concentration 1 μg/L 0.15 250 μg/L

0.27

0.30 60 μg/L 1 μg/L

0.15 17.5 μg/L 10 μg/L 0.10 5 mg/L

0.28

0.28 5 mg/L 10 μg/L

0.18 88μg/L 10 μg/L 0.08 5 mg/L

0.12

0.26 0.5 mg/L 10 μg/L

0.18 796 μg/L 2 μg/L 0.15 150 μg/L

0.19

0.19 150 μg/L

2 μg/L

0.04 49 μg/L 20 μg/L 0.11 2.5 mg/L

0.22

0.22 2.5 mg/L 20 μg/L

0.11 532μg/L 20 μg/L 0.11 2.5 mg/L

0.16

0.19 1.5 mg/L 20 μg/L

0.09 238 μg/L69 Differential effect of PO 4 , NO 3 and NH 4 on the growth of Vaucheria sp.

Introduction

Vaucheria sp. (Xanthophyce) is a co mmon filamentous mat-forming species in streams, rivers and lakes (Entwisle 1990, McIntire et al. 1994, Necchi et al. 1994, Aboal et al. 1996). It is often found at hi gh nutrient sites, and its pr esence can be a symptom of system eutrophication (Lakatos 1978, Simons et al. 1994, Biggs 1996). Vaucheria sp. can also be found in the springs and areas of ground water input into streams (Davis and Gworek 1973, Moreno et al. 2001). In a survey of 60 sites within 28 spring systems in Northern and Central Florida, we found Vaucheria sp. mats at 33 sites (Stevenson et al. 2004). At Manatee, Fanning and Guaranto spri ngs, more than 70% of the spring bottoms were covered by Vaucheria sp. Even though Vaucheria sp. is commonly found in the fresh, brackish and marine water systems around the world, there are few laboratory studies on its nutrient requireme nts (Sudlow 1936, Ozimek 1990). This study was conducted on Vaucheria sp. in Florida springs. The objectives of the study were: (i) to investigate th e effects of a wide range of concentrations of nitrogen (in

the form of nitrate and ammonia) and phosphorus on the growth of Vaucheria sp., (ii) to

determine whether nitrate or ammonia is a preferred source of nitrogen for Vaucheria sp., (iii) to study the importance of the size of algal mats when Vaucheria sp. was grown under different nutrient levels, a nd (iv) to investigate whether Vaucheria sp. populations from different Florida springs differ in their growth rates.70 Materials and methods Two experimental systems were used to st udy the growth of filamentous algae: small microcosms and large microcosms. The sma ll microcosm system allowed a large number of replicates but restricted the experimental unit to short algal filaments. The large microcosm system could accommodate small fila mentous algal mats of varying sizes but restricted the number of repl icates. We conducted 4 small microcosm experiments and 3 large microcosm experiments (Table 1).

Small microcosm experiments

Filaments of Vaucheria sp. were grown in 1.5 ml microcen trifuge tubes filled with 1ml of growth media. Growth media were change d every 48 hours. Racks made of clear polyethylene were placed on an orbital shaker (60 RPM) and incubated under grow lights providing 25 μmol/m 2 /s of light. To achieve this light intensity, racks were covered with neutral density film. A li ght intensity of 25 μmol/m 2 /s was found to be the optimal intensity for the growth of individual filaments in microcentrifuge tubes. Healthy, growing tips of Vaucheria sp. 1-2mm in length were cut fr om the filaments. Originally these tips were used directly for the experiments. Howeve r, we found that many of the cut off tips of Vaucheria sp. were damaged in the proce ss and did not grow. Therefore, we changed our methodology and incubated 1- 2mm tips for 2 days in Z8Fl growth medium (Appendix 1) with 100μg/L P and 1000μg/L N before we used them in the experiments. For experiment 1 and 3, tip s were pre-incubated for 48 hours, and for experiments 4 and 6, tips were used right after cutting. A tip of Vaucheira sp. was photographed using a digital video came ra system installed on a Leica MZ12.5 Stereomicroscope under 8x magnification at the beginning and at the end of experiment.71 Filament length was measured on the digital images using ImageJ software (Rasband 1997-2006). Incubation time was 11 or 12 days. Each treatment was replicated 5 times if

Vaucheria from two locations (2 different springs) was used. If Vaucheria from one location was used, each treatment was replicated 10 times. . Since some of the filaments were lost during the media changes, the fina l number of replicates used for the data analyses was lower then the number set at the beginning of the experiments. All experiments had a complete factorial ANOVA design with one or two factors. Experiment 1 had a two-factor desi gn with 10 nutrient treatments (PO 4 -P concentrations: 1, 5, 10, 20, 30, 40, 60, 80, 100 and 250 μg P/L) and 2 population location treatments.

The NO 3 -N concentration in experiment 1 was 1000 μg/L. Experiment 3 had a twofactor design with 10 nutrient treatments (NO 3 -N concentrations: 10, 30, 60, 125, 250, 500, 750, 1000, 2000 and 5000 μg N/L) and 2 popul ation location treatments. The PO 4 -P concentration in experiment 3 was 100 μg/L. Experiment 5 had a one-factor design with 11 nutrient treatments (NH 4 -N concentrations: 10, 30, 60, 125, 250, 500, 750, 1000, 1500, 2500 and 5000 μg N/L). The PO 4 -P concentration in experiment 5 was 100 μg/L. Experiment 7 had a two-factor design with 16 nutrient treatments : a combination of 4 ammonia concentrations (20, 100, 500 and 1000 μg N/L) and 4 nitrate concentrations (20, 100, 500 and 1000 μg N/L).

Large microcosm experiments The large microcosm system consisted of a flat platform with Plexiglas cylindrical raceways (Manoylov 2005). The platform was pl aced on an orbital shaker table (60RMP) in a walk-in growth room. The platform wa s illuminated with fluorescent growth lights providing 150 μmol/m 2 /s of light. Each microcosm wa s filled with 100 ml of growth72 medium. The media were changed daily. At the beginning and at the end of the experiment small fragments of Vaucheria sp. mats were gently patted dry with a paper towel, while held in between a Nitex screen, to remove excess water, and weighed (fresh mass). At the end of the experiment all sa mples were processed for dry mass (DM). At the beginning of each experiment 10 algal samples from each initial mass treatment were patted dry in between a Nitex screen, weighed to measure in itial fresh mass (FM), dried in the oven, and weighed again (DM). This allo wed for the calculation of the initial dry mass of the experimental samples based on thei r initial fresh mass. All samples for DM were dried in the oven at 105oC for 24 hours (E aton et al. 1995). All experiments had a complete factorial ANOVA design with 2 experi mental factors: nut rient concentration and initial biomass. Experiment 2 had a total of 15 treatments that were a combination of 5 phosphate concentrations (PO 4 -P concentrations: 2, 10, 30, 60, 150 μg P/L) and 3 initial fresh mass levels (0.01g, 0.05g, 0.2g). The NO 3 -N concentration in experiment 2 was 1000 μg/L. Experiment 4 had a total of 15 treatme nts that were a combination of 5 nitrate concentrations (NO 3 -N concentrations: 20, 125, 500, 1000, 2500 μg N/L) and 3 initial fresh mass levels (0.01g, 0.05g, 0.2g). The PO 4 -P concentration in experiment 4 was 100 μg/L. Experiment 6 had a total of 15 treatm ents that were a combination of 5 ammonia concentrations (NH 4 -N concentrations: 20, 125, 500, 1000, 2500 μg N/L) and 3 initial fresh mass levels (0.01g, 0.05g, 0.2g). The PO4- P concentration in experiment 6 was 100 μg/L.

Relative growth rates Algal relative growth rate (RGR) in the large microcosm experiments was calculated using the following formula (Hunt 1990):73 In the small microcosm experiments, the alga l initial and final fila ment length was used instead of algal mass.

Growth media A new growth medium was developed based on the Z8 medium (Kotai 1972). The Z8

medium was modified to simulate the medi an chemistry of macronutrients in Florida spring water (referred to as medium Z8Fl) (A ppendix 1). We originally used Z8Fl Fe600 medium that had the original concentration of iron (600 μg /L) found in the Z8 medium.

Later we used Z8Fl Fe200 with the iron con centration reduced to 200μg/L, because we found an Fe concentration of 600μg/L to ha ve a suppressing effect on the growth of

Vaucheria sp. We used the Gaffron micronutrients mix as used by Kotai (1972). To 1L of the final growth medium we also added 10ml of CaCl 2 ·2H 2 O solution (stock solution 18.3g CaCl 2 ·2H 2 O in 1L of deionized water), 10ml of MgSO 4 ·7H 2 O solution (stock solution 7g MgSO 4 ·7H 2 O in 1L of deionized water), and 10ml of Na 2 CO 3 solution (stock solution 13.7g Na 2 CO 3 in 1L of deionized water). We used NaNO 3 and NH 4 Cl as sources of nitrogen and K 2 HPO 4 as a source of phosphorus. When the NaNO 3 gradient was investigated, NaCl was also added to keep the Na concentr ation constant in all of the treatments. In the experiments with K 2 HPO 4, gradient KCl was added to keep the K

concentration constant in all of the trea tments, following the experimental design of (Ahlgren 1977). days of # Mass) ln(Initial Mass) ln(Final

= RGR −74 Water analyses Water samples of prepared growth media and from experimental macrocosms were sent to FDEP for analyses. On last day of experi ment a sample of water in which algae were growing for 24 hours was collected for analyses.

Experimental conditions All experiments were conducted in the walk-i n growth room. The temperature in the growth room was set for 22.5oC to reflect th e average water temper ature of the Florida springs. The minimum and maximum temperat ures were recorded daily on a max-min

thermometer.

Identification of Vaucheria species Since the presence of reproductive structures is necessary to identify the species of

Vaucheria from each batch of algae used in the experiments, we incubated a small fragment of the mat (Ott and Oldham-Ott 2003) . The mat fragment was placed onto a Petri dish filled with Z8Fl growth me dium with 100μg/L P and 1000μg/L N and placed by the window. We were successful in obs erving reproductive stru ctures only in the samples collected from Manatee spring. The species was Vaucheria geminata (Prescott 1982)

Data analyses Data were analyzed using SAS statistical software (SAS 2000) (repeated measures ANOVA) and R statistical so ftware (R Development Co re Team 2006). Logistic regression analysis was conducted using drc p ackage for R (Ritz and Streibig 2007). All final and initial filament length and small mat biomass data were first tested using75 repeated measures ANOVA to test the signifi cance of nutrient effect and time effect (indicating algal growth). Next, factorial analys is of variance was used to test the effect

of nutrient concentration and Vaucheria sp. population location on relative growth rates (RGR). A lowess smooth line was fitted thr ough the data for general assessment of the trends in the data. If a significant nutrie nt effect was found in the ANOVA analysis, a logistic curve was fitted through the RGR data. If the logistic curve did not provide a good fit and data were distribute d along a straight line, a linear regression was calculated. Various logistic models were fitted through the data (Ritz and Streibig 2005, Ritz and Streibig 2006) and the best fitting models are presented in the results. For our data, the best fitting models were: the four paramete r logistic model (l4()), the four parameter logistic model with variance calculated as a power function of the mean to account for heterogeneity of the data (l4() varPower), the Monod model (calculated using code forthe Michaelis-Menten model from the drc package) and the five parameter BrainCousens hormesis model (Borchardt 1996, Scha benberger et al. 1999, Ritz and Streibig 2007). In allof the treatments in the small microcosm experiments we had filaments of

Vaucheria sp. that did not grow at al l. This is probably because Vaucheria sp. did not like to be handled. Precutting tips 2 days before the experime nt partially resolved that problem, but we still had tips that did not gr ow. All initial data analysis was conducted on a complete dataset. With the 0 growth sample s in our data set, we were not able to fit any of the logistic models. The results fr om the small microcosm experiments presented here are based on the dataset with the 0 grow th samples removed (less then 10% of the76 whole dataset). The 0 growth samples were randomly found in our datasets. There was no relationship between the expe rimental treatments and number of 0 growth samples. All data sets were checked for normality and heterogeneity of variance, and where necessary, data were transformed for the ANOVA analyses. All analyses using drs package were conducted on the untransformed RGR data.

Results

Vaucheria sp. grew in all experiments under all nu trient concentrations. Relative growth rates (RGR) varied from 0 to 0.35. In th e small microcosm experiments, probably partially due to the cutt ing of filaments, we observed larg e variability in the growth rates for each treatment. In the large microcosm e xperiments, high nutrient levels resulted in high RGRs of Vaucheria sp.

Effects of PO 4 In the small microcosm experiments, an increase in phosphate phosphorus had a significant effect on the growth of Vaucheria sp. (Fig 1). The relative growth rate data were well described by a logist ic dose-response curve (Fig. 1, Table 2). The threshold concentration of phosphate phos phorus for the growth of Vaucheria sp. was 11.6 μg/L - the Effective Dose of 50% growth saturation (ED 50 ). The two populations of Vaucheria sp. that were studied did not differ in their RG Rs (ANOVA for the location effect, P<0.418). In the large microcosm experiments, Vaucheria sp. mats with smaller initial biomass grew faster than larger mats in high p hosphate concentrations (ANOVA for biomass77 effect, P<0.001) (Fig.2). Dose response curves were fitted through the 0.01g and 0.05g

initial biomass data. The threshold nitr ogen concentration was 36.7 μg/L for the 0.01g initial biomass and 33.7 μg/L for the 0.05g initial biomass (Fig.2, Table 3).

Effect of NO 3 Inthe small microcosm experiments, an increase in nitrate nitrogen did not have an effect on the growth of Vaucheria sp. (Fig. 3). The two populations of Vaucheria sp. that were

studied did not differ in their RGRs (ANOVA for the location effect, P<0.742). In the large microcosm experiments, Vaucheria sp. mats with smaller initial biomass grew faster than larger mats (ANOVA for biomass effect, P<0.001) (Fig.4). Dose response curves were fitted through the data. The threshold nitrogen concentrations were 210 μg/L for the 0.01g initial biomass, 208 μg /L for the 0.05g initial biomass, and 499 μg/L for the 0.2g initial biomass (Fig.4, Table 3).

Effect of NH 4 In the small microcosm experiments, an increase in ammonia nitrogen resulted in increased growth rates up to approximately 500 μg/L NH 4 -N concentration, where it reached the highest growth rate of 0.25 mm /mm/day (Fig. 5). The RGR data were well described by the Brain-Cousens hor mesis curve (Fig. 5, Table 2). In the large microcosm experiments, Vaucheria sp. mats with smaller initial biomass grew faster than larger mats in 200, 500 and 1500 μg/L ammonia concentrations (ANOVA for biomass effect, P<0.001) (Fig.6). Brain-Cousens hormesis curves were fitted through the data, and a good fit was achieved for the initial biomass of 0.01g and 0.05g (Fig.6, Table 3). A high ammonia concen tration of 5000 μg/L resulted in reduced78 growth rates and, in 4 cases, death of th e mats and/or due to take over by coccoid cyanobacteria.

Combined effect of NO 3 and NH 4 In the small microcosm experiment, nitrogen was added as a combination of the two

forms: ammonia and nitrate. Neither ammoni a nor nitrate nitrogen had an effect on the growth of Vaucheria sp. (ANOVA for NO 3 effect, P<0.797, for NH 4 effect P<0.584) (Fig.7). Further, no effect was observed when both forms of nitrogen were summed and the growth of Vaucheria sp. was analyzed as a function of an absolute concentration of nitrogen (Fig. 8).

Discussion

Vaucheria sp. is usually found at locations with high nutrient concentrations. Leukart and Hanelt (1995) found Vaucheria bursata in a stream in Germany at phosphate concentrations above 200 μg/L and nitrate co ncentrations above 1700 μg/L Sabater et al. (2003), in his study of algal mats in the Llobregat River in Spain, found Vaucheria

bursata at even higher levels of phosphate (400 μg/L) and nitrate (3900 μg/L). In this laboratory study, we found a threshold concen tration of phosphorus to be in the range from 11.6 to 36.7 μg P/L, and we also found th at larger mats required higher phosphorus

levels. The nitrate threshol d concentrations were from 208 to 499 μg N/L. When ammonia was used as a source of nitrogen, we observed an increase in growth rates above 50 μgN/L. Above 1000 μgN/L, however, ammonia became toxic and growth rates were reduced (Fig. 5). Vaucheria sp. used ammonia and nitrate as a source of nitrogen79 and we did not observe any preference for either. Ozimek (1990), in laboratory experiments, grew mat fragments of Vaucheria dichotoma and found higher growth rates in higher nutrient concentra tions. The relative growth rates calculated based on Ozimek’s (1990) data were in the lower ra nge of those observed in this study ((0.01-0.05 mg/mg/day dry weight).80 References Aboal, M., M. Prefasi, and A. D. Asencio. 1996. The aquatic microphytes and macrophytes of the transvase Tajo-Segura irrigation system, southeastern Spain. Hydrobiologia 340 :101-107. Ahlgren, G. 1977. Growth of Oscillatoria-Agar dhii in Chemostat Culture .1. Nitrogen and Phosphorus Requirements. Oikos 29 :209-224. Biggs, B. J. F. 1996. Patterns in benthic algae of streams. Pages 31-56 in R. J. Stevenson, M. L. Bothwell, and R. L. Lowe, editors. Algal ecology. Academic Press, San Diego. Borchardt, M. A. 1996. Nutrients. Pages 183-227 in R. J. Stevenson, M. L. Bothwell, and R. L. Lowe, editors. Algal ecology. Academic Press, Sand Diego. Davis, J. S., and W. F. Gworek. 1973. Rotifer Parasitizing Vaucheria in a Florida Spring. Transactions of the American Microscopical Society 92 :135-140. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg, Eds. 1995. Standard methods for the examination of water and wastewater, 19th edition. American Public Health Association, Washington, D.C. Entwisle, T. J. 1990. Macroalgae in the U pper Yarra and Watts River Catchments - Distribution and Phenolo gy. Australian Journal of Marine and Freshwater Research 41 :505-522. Hunt, R. 1990. Basic growth analysis. Unvwin Hyman Ltd., London. Kotai, J. 1972. Instructions fo r preparation of modified nutrient solution Z8 for algae. Norwegian Institute for Water Researc h, publication B-11/69., Blindern, Oslo. Lakatos, G. 1978. The phenomenon and significa nce of benthonic eutrophication in Lake Velencei, Hungary. Acta Biological Debrecina 15 :147-168. Leukart, P., and D. Hanelt. 1995. Light requir ements for photosynthesis and growth in several macroalgae from a small soft-wat er stream in the Spessart Mountains, Germany. Phycologia 34 :528-532. Manoylov, K. M. 2005. Ecological strategies of benthic diatoms for nutrient competition. Ph.D. Dissertation. Michigan St ate University, East Lansing.McIntire, C. D., H. K. Phinney, G. L. Larson, and M. Buktenica. 1994. VerticalDistribution of a Deep-Wat er Moss and Associated Epiphytes in Crater Lake, Oregon. Northwest Science 68 :11-21. Moreno, J. L., M. Aboal, M. R. Vidal-Ab arca, and M. L. Suarez. 2001. Macroalgae and submerged macrophytes from fresh and sali ne waterbodies of ephemeral streams ('ramblas') in semiarid south-eastern Spain. Marine and Freshwater Research 52 :891-905. Necchi, O., D. Pascoaloto, and L. H. Z. Branco. 1994. Distribution of Macroalgae in a Tropical River Basin from Southeastern Brazil. Archiv Fur Hydrobiologie 129 :459-471. Ott, D. W., and C. K. Oldham-Ott. 2003. Eustigmatophyte, raphidophyte, and tribophyte algae. Pages 423-469 in J. D. Wehr and R. G. Sheat h, editors. Freshwater algae of North America. Academic Press, San Diego. Ozimek, T. 1990. Aspects of the ecology o fila mentous alga in a eutrophicated lake. Hydrobiologia 191 :23-27.81 Prescott, G. W. 1982. Algae of the Western Great lakes Area. Otto Koeltz Science Publishers, Koenigstein. R Development Core Team. 2006. R: a langu age and environment for statistical computing. in , Vienna, Austria. Rasband, W. S. 1997-2006. ImageJ. in . U.S. National Institutes of Health, Bethesda, Maryland, USA. Ritz, c., and J. C. Streibig. 2005. Bioassay an alysis using R. Jour nal of Statistical Software 12 :1-22. Ritz, C., and J. C. Streibig. 2006. Bioassay for allelochemicals: examples with R. Pages 24 in

. Ritz, C., and J. C. Streibig. 2007. The drc p ackage. Analysis of dose-response curves. Sabater, S., E. Vilalta, A. Gaudes, H. Guasch, I. Munoz , and A. Romani. 2003. Ecological implications of mass growth of benthic cyanobacteria in rivers. Aquatic Microbial Ecology 32 :175-184. SAS. 2000. SAS/STAT. in . SAS Institute, Cary, N.C. Schabenberger, O., B. E. Tharp, J. J. Kells , and D. Penner. 1999. Statistical tests for hormesis and effective dosages in herb icide dose response. Agronomy Journal 91 :713-721. Simons, J., M. Ohm, R. Daalde r, P. Boers, and W. Rip. 1994. Restoration of Botshol (the Netherlands) by Reduction of External Nutr ient Load - Recovery of a Characean Community, Dominated by Char a-Connivens. Hydrobiologia 276 :243-253. Stevenson, R. J., A. Pinowska, and Y. K. Wang. 2004. Ecological c onditions of algae and nutrients in Florida springs . Final report. Florida Department of Environmental Protection contract number WM858, Michigan State University, East Lansing, MI. Sudlow, P. L. 1936. Growth of Vaucheria in culture. Master Thesis. Ohio State University.82 Fig 1 Relative growth rate (RGR) of Vaucheria sp. in different phosphate phosphorus concentrations (n=65). A four parameter logi stic curve was fitted through the data from the small microcosm experiments (experiment 1). Curve parameters are in Table 2.83 Fig. 2 Relative growth rate (RGR) of Vaucheria sp. in a factorial experiment with 3 initial biomass levels (0.01g, 0.05g, 0.2g) and 5 phosphor us concentrations (n=45). A four parameter logistic curve and a re gression line (initial biomass 0.2g R 2 =0.048, P<0.433) were fitted through the data from a large microcosm experiment (experiment 2). Curve parameters are in Table 3.8485 Fig. 3 Relative growth rate (RGR) of Vaucheria sp. in different nitrate nitrogen concentrations (n=68). Linear regression was fitted through the data from a small microcosm experiment (experiment 3) (R 2 =0.01, P<0.414).86 Fig. 4 Relative growth rate (RGR) of Vaucheria sp. in a factorial experiment with 3 initial biomass levels (0.01g, 0.05g, 0.2g) and 5 nitrogen concentrations (n=45). Four parameter logistic curves were fitted through the data from a large microcosm experiment (experiment 4). Curve parameters are in Table 3.87 Fig. 5 Relative growth rate (RGR) of Vaucheria sp. in different ammonia nitrogen concentrations (n=58). Five parameter Brai n-Cousens hormesis logistic model was fitted through the data from small microcosm experi ment (experiment 5). Since missing data

did not allow the calculation of th e full model, point of inflection e was set at 500 μg/L after analysis of lowess smooth line. Curve parameters in Table 2.8889 Fig. 6 Relative growth rate (RGR) of Vaucheria sp. in a factorial experiment with 3 initial biomass levels (0.01g, 0.05g, 0.2g) and 5 ammo nia concentrations (n=41). Five parameter Brain-Cousens hormesis logistic model was fitted thr ough the data from a large microcosm experiment (experiment 6). Since missing data from the 5000 μg/L

treatment did not allow the calculation of the full model, point of inflection e was set at 800 μg/L after analysis of lowess smooth lin es. Curve parameters are in Table 3.9091 Fig. 7 Boxplot of relative growth rates (RGR) of Vaucheria sp. in a combination of different ammonia and nitrate nitrogen c oncentrations (experiment 7).92 Fig. 8 Relative growth rate (RGR) of Vaucheria sp. in different nitrogen (sum of NO 3 -N and

NH 4 -N) concentrations (n=99). Linear regr ession was fitted through the data from a small microcosm experiment (experiment 7) (R 2 =0.03, P<0.097).93 Table 1

Vaucheria sp. experiments. Experiment numbers in pa renthesis refer to numbers used in the 2006 report.

Experiment (project experiment number) Micro cosm Nutrient gradient Algae collection location Season Growth medium Mean min and max temperature (oC) 1 (21) 2 (7) 3 (20) 4 (9) 5 (4) 6 (11) 7 (2) Small Large Small Large Small Large Small PO 4 -P PO 4 -P NO 3 -N NO 3 -N NH 4 -N NH 4 -N N (NH 4 and NO 3 ) Ichetucknee and Alexander Ponce de Leon Ichetucknee and Alexander Alexander Manatee Ponce de Leon Manatee Spring Winter Spring Spring Summer Winter Fall Z8Fl Fe200 Z8Fl Fe200 Z8Fl Fe200 Z8Fl Fe200 Z8Fl Fe600 Z8Fl Fe200 Z8Fl Fe600 21.4 &24.9 20.2 & 24.0 21.3 &24.9 20.8 & 22.3 20.7 & 24.8 20.2 & 23.8 20.8 & 24.494 Table 2 Parameters of the logistic dose- response model applied to the Vaucheria sp. RGR data from small microcosm experiments, where d and c denote the upper and lower limit of algae growth rates at zero and infi nite concentrations of nutrient. ED 50 (also called e ) denotes the concentration require d to increase the algal grow th rate by half between the upper and lower limits and b is proportional to the slope (the rate of change) of the curve

around ED 50 . Significant parameters are in boldface. ED 10 and ED 90 denote the concentration required for 10% and 90% gr owth saturation. For the Brain-Cousens hormesis model, c and d are the upper and lower asym ptote of the response. The columns designated b and e are related to the rate of cha nge and the point of inflection of the curve respectively; and f indicates the initial rate of increase at low concentrations.

Nutrien t Model d (mm/mm/ day) c (mm/mm/ day) b ED 50 (μg/L) e ED 90 (μg/L) or f ED 10 (μg/L)

PO 4 NH 4 l4() Brain-Cousens 0.27±0.01 0.03±0.01 0.16±0.01 0.08±0.01 -3.54±1.68 5.40±1.54 11.59±2.59 set at 500 21.53±8.19 0.0004±0.0001 6.24±2.22

-95 Table 3 Parameters of the logistic dose- response model applied to the Vaucheria sp. growth response data from large microcosm experiments, where d and c denote the upper and lower limit of algae growth rates at zero a nd infinite concentrations of nutrient. ED 50 denotes the concentration require d to increase the algal grow th rate by half between the upper and lower limits and b is proportional to the slope of the curve around ED 50 . Significant parameters are in boldface. ED 10 and ED 90 denote the concentration required for 10% and 90% growth saturation. Fo r the Brain-Cousens hormesis model, c and d are the upper and lower asymptote of the response. The columns designated b and e are related to the rate of change and point of inflection of the curve respectively; and f indicates the initial rate of increase at low concentrations.

Appendix 1 Z8Fl medium was derived from Z8 medium (Kotai 1972) for the cu lture of filamentous algae from Florida springs. It was adopted to reflect the Florida springs’ water chemistry. The concentrations of nitrogen and phosphorus were reduced and the concentrations of calcium, magnesium and bicarbonat e were increased. The original Z8 medium had an Fe concentration of 600μg/L. We f ound that this concentration reduced the growth rates of the algae, and we later used a concentration of 200μg/L. We used the original Nutrient and biomass Model d (g/g/day) c (g/g/day) b ED 50 (μg/L) e ED 90 (μg/L) of

f ED 10 (μg/L)

PO 4 x 0.01g PO 4 x 0.05g NO 3 x0.01g NO 3 x0.05g NO 3 x0.2g NH 4 x 0.01g NH 4 x 0.05g l4() l4() l4() l4() l4() Brain-Cousens Brain-Cousens 0.15±0.02 0.11±0.02 0.25±0.01 0.19±0.01 0.11±0.01 0.11±0.01 0.07±0.01 0.02±0.01 0.06±0.01 0.10±0.01 0.07±0.01 0.05±0.01 0.06±0.00 -0.02±0.0 -2.89±1.34 -2.71±3.60 -1.96±0.42 -1.25±0.43 -4.13±2.87 1.78±0.31 1.67±0.59 36.68±8.95 33.67±20.61 210.3±30.4 207.8±56.6 499.2±68.9 set at 800 set at 800 78.37±40.24 75.78±107.9 644.0±202.2 1200±797.0 849.9.5±315.1 0.00028±0.00007 0.00024±0.00001 17.17±5.55 14.96±15.31 68.67±16.62 35.97±23.92 293.2±122.4

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-96 concentration of microelements, but for futu re work this concentration probably should be reduced to one-half of the original con centration, because we found that the original

concentration of microelement s was limiting the growth of Lyngbya wollei . Solutions 1

and 2 were filter sterilize d. Solution 5 was autoclaved.

To prepare 1 L of Z8Fl medium, fill a fl ask with 957ml of deionized water (DIH 2 O), then add solutions 3, 4, 6 and 7 and autoclave. After autoclaving add solutions 1, 2, and 5. The prepared medium should be left for 24 hours for the gases to equalize. The target pH concentration should be around 8.5. The pH might have to be adjusted to that value.

1. Fe-EDTA 10ml (can be used at reduced level, then use 3ml) 10ml ● FeCl 3 solution (2.8 g FeCl 3 ·6H 2 O dissolved in 100ml 0.1N HCl) and 9.5ml from an ● EDTA-solution (3.9g EDTA-Na 2 dissolved in 100 ml 0.1N NaOH) mixed and filled up to 1000ml with distilled water. 2. Gaffron’s trace element solution 1ml (can be used at reduced level, then use 0.5ml) Stock solutions (kept separate) Each in g in 100ml DIH 2 O

● ZnSO 4 ·7H 2 O 0.287 g

● (NH 4 ) 6 Mo 7 O 24 ·4H 2 O 0.088 g

● Co(NO 3 ) 2 ·6H 2 O 0.146 g

● VOSO 4 ·6H 2 O 0.035 g

● Al 2 (SO 4 ) 3 K 2 SO 4 ·2H 2 O 0.474 g

● NiSO 4 (NH 4 ) 2 SO 4 ·6H 2 O 0.198 g

● Cd(NO 3 ) 2 ·4H 2 O 0.155 g

● Cr(NO 3 ) 3 ·7H 2 O 0.037 g

● Na 2 WO 4 ·2H 2 O 0.033 g

● KBr 0.119 g

● KI 0.083 g

● Cu(SO 4 )·5H 2 O 0.125 g Take 10ml of each solution.

● H 3 BO 3 3.10 g and MnSO 4 ·H 2 O 1.69 g in 1L DIH 2 O. Take 100ml.97 Final trace element stock solution: 12x10ml + 100ml + DIH 2 O (780 ml) to 1L.

3. NaNO 3 solution 1ml ● 6.1g NaNO 3 in 1L DIH 2 O

4. K 2 HPO 4 solution 1ml ● 0.56g K 2 HPO 4 in 1L DIH 2 O

5. CaCl 2 ·2H 2 O solution 10ml ● 18.3g CaCl 2 ·2H 2 O in 1L DIH 2 O 6. MgSO 4 ·7H 2 O solution 10ml ● 7g MgSO 4 ·7H 2 O in 1L DIH 2 O 7. Na2CO3 solution 10ml ● 13.7 g Na 2 CO 3 in 1L DIH 2 O

References Kotai, J. 1972. Instructions fo r preparation of modified nutrient solution Z8 for algae. Norwegian Institute for Water Researc h, publication B-11/69., Blindern, Oslo.