ACUTE TOXICITY OF THE AGRICULTURAL CHEMICALS

ENDOSULFAN AND COPPER SULFATE TO A

FRESHWATER SHRIMP, PALAEMONETES PALUDOSUS

by

Prajakta N. Kamthe

A Thesis Submitted to the Faculty of

The Charles E. Schmidt College of Science

in Partial Fulfillment of the Requirements for the Degree of

Master of Science

Florida Atlantic University

Boca Raton, Florida

August 2002 Copyright by Prajakta N. Kamthe 2002

11 ACUTE TOXICITY OF THE AGRICULTURAL CHEMICALS ENDOSULFAN AND COPPER SULFATE TO A FRESHWATER SHRIMP, PALAEMONETES PALUDOSUS

by Prajakta N. Kamthe

This thesis was prepared under the direction of the candidate's thesis advisor, Dr. John Baldwin, Department of Biology, and has been approved by the members of her supervisory committee. It was submitted to the faculty of The Charles E. Schmidt College of Science and was accepted in partial fulfillment of the requirements for the degree of Master of Science.

SUPERVISORY COMMITTEE: & ?;(?&?--:__ ~ Thesis Advisor w~ ~ 15rJ4 / [/?J · ector, Environmental Sciences Program

7 I 7 0 Z Vice Provost Date

lll ACKNOWLEDGEMENTS

I would like to thank my advisor Dr. John Baldwin and the members of my committee

Dr. Craig Byrdwell and Dr. Bill Louda for all their help. Without them this study would not be possible. I would also like to thank my family and my friends for all their support and understanding.

IV ABSTRACT

Author: Prajakta N. Kamthe

Title: Acute Toxicity of the Agricultural Chemicals Endosulfan and Copper

Sulfate to a Freshwater Shrimp, Palaemonetes paludosus

Institution: Florida Atlantic University

Thesis Advisor: Dr. John Baldwin

Degree: Master of Science

Year: 2002

The toxicity of endosulfan, a restricted use pesticide, and copper sulfate, an anti-algal agent, ranks among the highest in all insecticides. Both chemicals, used in agriculture fields of southern

Florida, are known to be highly toxic to aquatic invertebrates. Since Palaemonetes pugio, an extensively studied brackish water shrimp species, has proven to be excellent for toxicological analysis, a closely related freshwater shrimp species, Palaemonetes paludosus, was used as the test species in this study to determine the toxicity of endosulfan and copper sulfate in freshwater.

A series of static renewal 96 h bioassays (renewed every 24 h) performed on juvenile P. paludosus resulted in the 96 h lethal concentration (LC50) estimates of 0.24 11g/L (0.24 ppb) for endosulfan and 0.79 mg/L (0.79 ppm)for copper sulfate. The results of this study, when compared to other studies, indicate that P. paludosus is more sensitive to endosulfan and copper sulfate than other similar aquatic invertebrate species and is therefore an excellent toxicological indicator.

v TABLE OF CONTENTS

Page

LIST OFT ABLES ...... vii

LIST OF FIGURES ...... viii

INTRODUCTION ...... 1

MATERIALS AND METHODS ...... 6

RESULTS ...... 9

DISCUSSION ...... 11

REFERENCES ...... 18

Vl LIST OF TABLES

Table Page

1. Comparison of acute toxicity estimates (LC50) for ...... 21

Palaemonetes paludosus and other aquatic species for 48 and 96 h

exposure to endosulfan.

2. Comparisons of the LC50 values between different time endpoints ...... 22

for endosulfan

3. Comparisons of the LC50 values between different time endpoints ...... 22

for copper sulfate

4. Comparison of acute toxicity estimates (LC50) for ...... 22

Palaemonetes paludosus and other aquatic species for 48 and 96 h

exposure to copper sulfate using static renewal bioassays.

Vll LIST OF FIGURES

Figure Page l. Isomers of endosulfan and its degradation products ...... 23

2. The 'ANA' area ...... 24

3. LC50 values of endosulfan toxicity at 24, 48, 72, and 96 hrs ...... 25

4. Survival in relation to the concentration of insecticide- endosulfan ...... 26

5. LC50 values of copper sulfate toxicity at 24, 48, 72, and 96 hrs ...... 27

6. Survival in relation to the concentration of insecticide- copper sulfate ...... 28

Vlll INTRODUCTION

South Florida's sensitive wetland habitats receive a direct impact from one of the region's major economic activities, agriculture. Primary agriculture activities include extensive cultivation of sugarcane and rice in the Everglades Agricultural Area (EAA) south of Lake Okeechobee and the cultivation of citrus, watermelon, tomatoes, and green peppers in the Big Cypress region of south Florida. In all major agricultural operations, a large variety of chemicals are used to enhance production of crops, as well as to protect them from a variety of insects and diseases. These chemicals include fertilizers, herbicides, insecticides and fungicides. Irrigation and heavy seasonal rains common in south Florida promote the transfer of these xenobiotic chemicals into the surrounding aquatic habitats, resulting in exposure of the aquatic inhabitants to the toxicants. In general, insecticides are designed to target terrestrial arthropods; however, they may also affect non-target aquatic crustaceans, depending on their mode of action (Koeman et al.,

1978; Raizada et al., 1981 ). One of the compounds commonly used in green pepper fields to control insects is Thiodan, which has endosulfan as an active ingredient.

Endosulfan (6, 7, 8, 9, 10, 10-hexachloro-1, 5, Sa, 6, 9, 9a-hexahydro-6, 9-methano-2,

4, 3-benzodioxathiepin-3-oxide) (Fig. 1) was chosen for this study, as it is one of the most commonly used insecticides for agricultural crops in southern Florida (Baughman,

1986). It is used as an acaricide (kills insects and spiders) and miticide (kills ticks and mites) to control a variety of pests affecting fruits, vegetables, and ornamental plants (Berrill et al., 1998; Leight and Van Dolah, 1999). Since endosulfan is known to be

highly toxic to freshwater aquatic invertebrates (Table 1) such as the crayfish

Procambarus clarkii (Cebrian et al., 1992) and Daphnea magna, a cladoceran (Macek et

al., 1976), it is classified as an EPA Class I Restricted Use Pesticide (EPA, Restricted use

products report, 1995). Endosulfan is an organochlorine and a member of the cyclodiene

class of insecticides (Fig. 1). It is made up of a mixture of two isomers, a and ~.both of

which have similar insecticidal properties (Goebel et al., 1982). Technical endosulfan

contains 70% a-endosulfan and 30% ~-endosulfan (Goebel eta!., 1982).

Endosulfan and both its isomers work by inhibiting the Na+ - K+ ATPase (enzyme

responsible for regulating the Na+- K+ channels along nervous system pathways) and the

mitochondrial Mg2+ ATPase (enzyme responsible for regulating the ATP production in

the mitochondria: Goebel et al., 1982). Exposure to this chemical initially results in:

(i) increased excitation, restlessness, trembling, uncontrolled body and extremity

movements, (ii) followed by general exhaustion, incapacity of translocation, arching of the body, and convulsive tremors, (iii) and ultimately lead to immobility, lethargic reaction to strong stimuli, collapse of the abdomen, and death (Goebel et al., 1982). In studies conducted on the fish species Oreochromis mossambicus and Clarias batrachus, endosulfan caused a reduction in the blood's affinity for oxygen, which generated anoxia and necrosis of the gill epithelium (Rangaswamy and Naidu, 1999; Berrill et al., 1998).

Although no studies have been conducted on Palaemonetes paludosus (grass shrimp) concerning this topic, one can predict that a similar reaction may occur when shrimp are subjected to endosulfan, as crustaceans have similar mechanisms for oxygen intake as fish (Cochran and Burnett, 1996).

2 In an aquatic environment, endosulfan is degraded mostly by hydrolysis. Peterson and

Batley (1993) state that of the two isomeric products, ~-endosulfan hydrolyzes faster than

a-endosulfan. However, a-endosulfan is also removed efficiently by other mechanisms

such as volatilization, isomerization, or biological hydrolysis. It is relatively more water-

soluble than the ~-isomer, which is typically a more persistent compound. Thus, in an

aqueous environment where many factors in addition to hydrolysis play a role in the

degradation of endosulfan, a-endosulfan tends to degrade much faster than ~-endosulfan.

In freshwater, with a pH of 7 .0, the half-life of a-endosulfan is approximately 5 days,

while the half-life of ~-endosulfan is 15 days (Peterson and Batley, 1993).

The degradation of endosulfan (a and~) yields the products endosulfan sulfate and

endosulfan-diol (Peterson and Batley, 1993), each exhibiting different toxicity properties.

Endosulfan sulfate, which is as toxic as its parent compound, has a half-life of 4 weeks in

freshwater, while endosulfan-diol, which is non-toxic, persists in an aqueous environment

with a half-life of 14 weeks in freshwater (Miles and Moy, 1979). Although technical endosulfan degrades rapidly in an aquatic environment, its toxic degradation products

(endosulfan sulfate and endosulfan-diol: Fig. 1) are found in large quantities near areas of application and in surface waters throughout the entire United States (Berrill et al.,

1998).

Sensitivity to endosulfan has been examined in several species of marine and estuarine crustaceans, such as the salt marsh inhabitants Palaemonetes pugio (grass shrimp)

Gammarus palustris (amphipod), and the marine inhabitant Callinectes sapidus (crab).

Only a few studies have been conducted on freshwater crustaceans, but include the cladoceran D. magna and P. clarkii, a crayfish (Table 1). The brackish-saltwater shrimp 3 species P. pugio has been extensively studied and has proven to be an excellent model for toxicology research (Baughman, 1986; Scott et al., 1987; Scott et al., 1990; Rayburn and

Fisher, 1999). Therefore, P. paludosus, a related yet little studied freshwater shrimp species, was chosen for this study in order to determine the risks endosulfan pose to freshwater aquatic crustaceans found in close proximity to agriculture. P. paludosus is a dominant freshwater macroinvertebrate and is widespread in the United States from New

Jersey to Texas and New Mexico (Fitzpatrick, 1983). It is an important member of the aquatic community, as it serves to be a primary food source for higher organisms. Since salinity is known to affect the degradation rate of endosulfan (Leight and Van Dolah,

1999), the fate and effects of endosulfan on freshwater organisms is hypothesized to differ from its effects on marine and estuarine organisms, thus the effects of endosulfan on P. paludosus may differ from the effects on P. pugio.

Copper sulfate, which is an EPA Class I General Use Pesticide, obviously contains copper which is ranked among the most toxic metals in the aquatic environment (Burton and Fisher, 1990). It is used on crops to control bacterial and fungal diseases such as mildew, leaf spots, and blights (Extoxnet, 2002). Copper sulfate was chosen for this study, as a reference toxicant, since it is a well-researched chemical with many studies on similar aquatic invertebrate species such asP. pugio (Burton and Fisher, 1990). It has widespread applications in the agriculture field and can persist as an element in the environment indefinitely (Extoxnet, 2002). The introduction of sulfate into freshwater habitats is also reported to suppmt the sulfate-reducing bacteria/methanogenic archae consortia responsible for the methylation of mercury in the Everglades (Bates et al.,

2002; Cleckner et al., 1999).

4 Experiments were designed to establish the percent mortality of P. paludosus to varying concentrations of endosulfan and copper sulfate. They were also designed to determine whether P. paludosus is as good an indicator for toxicological assays when compared to the saltwater species P. pugio.

5 MATERIALS AND METHODS

Juvenile P. paludosus ( 10- 15 mm in length) were collected from aquatic vegetation along the banks of south Florida freshwater canals using a dipnet. The collected specimens were then transported to the laboratory in a 5-gallon aerated container, where they were stored in 30-gallon tanks at a constant water temperature of 25°C ± 1o c. They were fed dry fish food pellets as required and an 18:6 light/dark cycle was maintained.

The tanks were aerated continuously. A holding time of two weeks was allotted for the juvenile P. paludosus to go through at least one molting cycle. Juvenile shrimp were separated from the holding tank into another tank two days prior to toxicity tests, where they were held without food. The purging tanks contained 'Artificial Agricultural Water'

(AA W), prepared so as to mimic the water parameters (as determined Cole, 2001) of areas in south Florida that are highly impacted by agricultural runoff, such as the 'AN A'

5 6 5 (Fig. 2: NH4Cl = 7.85 X 10- mol/L, KN03 = 1.9 X 10- mol/L, Na3P04 = 1.58 X 10-

4 5 mol/L, Na2Si03* 9H20 = 1.69 X 10- mol/L, KCL = 7.9 X 10- mol/L, Ca (CH3COO) 2 =

5 5 5 8.9 X 10- mol/L, MgS04 = 3.7 X 10- mol/L, NaCI = 5.1 X 10- mol/Land CaCb = 2.9

X 10-5 mol/L).

Toxicity Tests

To determine the effects of both endosulfan and copper sulfate on the survival of juvenile P. paludosus, a series of static-renewal 96 h bioassays were performed (renewed

6 every 24 h). A test series contained different concentrations of the test chemical with 10

organisms per concentration, with each test organism placed separately in a 40-ml beaker

containing 20 ml solution of the test chemical and AA W. A control was assigned for

each test series consisting of 100% AA W. In all test series endosulfan was dissolved

using 0.01 % acetone, therefore an additional control series of 0.01 % acetone (no

pesticide) was set up to ascertain minimal toxicity of acetone in the assays. During the

tests, the water temperature was maintained at 25°C ± 1° C, and the 18:6 light/dark cycle

was also maintained.

The toxicity for the 96 h static-renewal tests was determined by mortality. At 24, 48,

and 72 hr, 90% of the test water was replaced with fresh test solutions and the numbers of

dead were noted. The organisms were considered dead if no physical response to stimuli,

such as prodding with a glass rod, was observed. Three replicate 96 h tests were

performed for each chemical.

Literature values indicated that for P. pugio, the 96 h Lethal Concentration (LC50)

using endosulfan were between 1.54 J.lg/L (1.54 ppb) to 0.25 J.lg/L (0.25 ppb: Table 1).

Based on these values, three replicate 96 h static renewal range-finding assays were

performed on P. paludosus, with the concentrations for endosulfan set at 0.00, 0.0001,

0.001 , 0.01, 0.10, 1.00, 10.00 J.lg/L (ppb). A 100% survival rate was observed at 0.00

J.lg/L (0.00 ppb) and a 0% survival rate was observed 1.00 J.lgiL (1.00 ppb). Thus, concentrations for endosulfan in this study were set at 0.00, 0.0001, 0.0005, 0.001, 0.01 ,

0.05, 0.10, 0.50, 1.00 J.lg!L (ppb ). A toxicant stock solution of 1.00 mg/L ( 1.0 ppm) endosulfan was made in the laboratory with a 0.01 % acetone concentration to use in dilutions. 7 Burton and Fisher (1990) indicated that for juvenile P. pugio the 48 h (LC~w), using copper sulfate was 2.1 mg/L (2.1 ppm: Table 4 ). Based on this value, three replicate 96 h static renewal range-finding assays were performed on P. paludosus, with the concentrations for copper sulfate set at 0.00, 0.1 0, l.OO, 1.50, 2.00, 2.50 mg/L (ppm). A

100% survival rate was observed at 0.00 mg/L (0.00 ppm) and a 0% survival rate was observed 1.50 mg!L ( 1.50 ppm). Thus, concentrations for copper sulfate in this study were set at 0.00, 0.1 0, 0.50, 1.00, 1.50 mg/L (ppm). A toxicant stock solution of 100 mg/L copper sulfate was prepared in the laboratory, to which specific amounts of AA W was added to make different concentrations.

Statistical Analysis

As an estimate of relative lethal toxicity, LC50 values and their respective 95 % confidence limits for both toxicants were calculated for the 24, 48, 72, and 96 h time endpoints for each of the test series using a linear regression analysis technique. Only those assays which gave a 100% control survival rate (for AA Wand acetone controls) were used in computing the estimates. Values for each individual test series were evaluated for every time endpoint and then averaged to calculate the mean LC50 (with a

95 % confidence interval around each mean). In order to determine the statistical difference (p$.0.05) between the survival rate of any consecutive set of toxicant concentrations, within the three replicates, and among the estimated LC50 values at the

24, 48, 72, and 96 h time endpoints, t-tests using the natural log of the estimated values were conducted.

8 RESULTS

Endosulfan

The LC50 estimates for the 96 h static renewal bioassays, averaged from 0.47 f.lg/L

(0.4 7 ppb) in the first 24 hours to 0.24 f.lg/L (0.24 ppb) at 96 h (Fig. 3 ). No significant difference (p$.0.05) was observed in LC50 values for all the time endpoints within the three replicates. A steady decline in the LC50 values of endosulfan was observed at each time endpoint, showing the expected dose effect (concentration x time: Fig. 3).

Significant differences (p<0.05) were observed among the estimated LC50 values at the

24, 48, 72, and 96 h time endpoints (Table 2).

The highest concentration of endosulfan tested ( 1.00 f.!g!L: 1.00 ppb) resulted in a

13.3% survival rate at 24 hand a 0% survival by 48 h (Fig. 4). No significant difference

(p$.0.05) in the rate of survival for all time endpoints was observed within the three test replicates. The endosulfan concentration was inversely related to the survival rate (Fig.

4). There was no significant difference observed between the survival rate of any consecutive set of toxicant concentrations except between concentrations, 0.0005 f.lg/L, =

0.50 ppt vs. 0.001 f.lg/L, = 1.00 ppt (p = 0.03) and 0.10 f.lg/L, = 0.1 ppb vs. 0.50 f.lg/L, =

0.50 ppb (p = 0.05).

9 Copper sulfate

The LC50 estimates for the 96 h static renewal bioassay averaged from 1.34 mg/L

(1.64 ppm) in the first 24 hours to 0.79 mg/L (0.79 ppm) at 96 h (Fig. 5). No significant difference (p~0.05) was observed in the LC50 values for all the time endpoints within the three replicates. A steady decline in the LC50 values of copper sulfate was observed at each time endpoint, showing the expected dose effect (concentration x time: Fig. 5). The estimated LC50 values within any of the consecutive time endpoints also displayed no significant difference (Table 3).

The highest concentration of copper sulfate tested ( 1.50 mg/L: 1.50 ppm) resulted in a

30% survival rate at 24 h, a 10% survival rate at 48 hand a 3.3% survival rate at 72 and

96 h (Fig. 6). No significant difference (p~0.05) was observed in the survival rate for all time endpoints within the three replicates. The copper sulfate concentration was inversely related to the survival rate (Fig. 6). There was no significant difference observed between the survival rates for any consecutive set of toxicant concentrations.

10 DISCUSSION

There have been no previous toxicity studies conducted on the freshwater shrimp species P. paludosus and few studies have been conducted on comparable species using similar assays. However, we can gain some insight by comparing this study with studies conducted on P. pugio, a closely related brackish water shrimp species that has proven to be an excellent candidate for toxicological analysis (Rayburn and Fisher, 1999).

Endosulfan toxicity infreshwater

The 96 h LC50 value for the freshwater shrimp P. paludosus, obtained in this study using a static renewal bioassay, was 0.24 j..tg!L (0.24 ppb) endosulfan (Table 1). This value is much smaller than the 120 j..tg/L (120 ppb) 96 h LC50 value obtained by Cebrian et al. (1992) for P. clarkii (a freshwater crayfish species), using the 96 h static non­ renewal exposure (Table 1). The difference may be species or age/stage specific or related to the protocol used. Cebrian et al. ( 1992) used a static non-renewal exposure, whereas in a static renewal exposure the treatment water is replaced at a certain time interval (after every 24 h in this study), which reduces the amount of highly toxic ammonia in the assay, caused due to metabolic activity. However this type of exposure also replenishes the toxicant load every 24 hours and may reduce the amount of degraded endosulfan products present in the assay, resulting in a higher level of toxicity to endosulfan.

1 l Another endosulfan toxicity study performed by Macek et al. (1976) on the cladoceran

D. magna produced a 48 h LC50 value of 16.60 ~g/L ( 16.60 ppb ), using a flow-through exposure (Table 1). This value is significantly greater compared to the estimated 48 h

LC50 value of 0.37 ~giL (0.37 ppb) for P. paludosus, using a static renewal exposure

(Table 1). The higher amount of endosulfan required to produce a LC50 in D. magna may be species specific or related to the flow through technique used by Macek et al. (1976).

The flow through technique, designed to provide a continuous supply of treatment water to the assay, reduces the accumulation of endosulfan degradation products, such as endosulfan sulfate and endosulfan-diol, from the assay. This type of an exposure should increase the sensitivity to endosulfan, as the degraded products are constantly replaced by fresh loads of the toxicant. However, the constantly replenished treatment water also provides an ample oxygen supply, necessary for the organism's survival, since one of the effects of endosulfan is reduction of the blood's affinity for oxygen (Rangaswamy and

Naidu, 1999; Berrill et al., 1998). This type of exposure also nullifies toxic ammonia buildup, which is due to metabolic activity, and normally causes increased stress levels in organisms (McCulloch, 1990; Frias-Espericueta et al., 1999). In conjunction, ample oxygen supply and reduced ammonia levels may account for the increased tolerance to endosulfan seen in the Macek et al. (1976) study. However, in this experiment, a 96 h static renewal technique was used, and this also replenishes the toxicant load every 24 h and reduces ammonia buildup, although not as efficiently as the flow-through technique.

Endosulfan toxicity in brackish water

Many studies on endosulfan toxicity have been conducted on brackish water

12 organisms such as the shrimp species, P. pugio (Baughman, 1986; Scott et al., 1987;

Scott et al., 1990; Mayer, 1987). Using the 96 h static renewal exposures, various life stages of P. pugio produced LC50 values ranging from 0.25 1-1g/L to 1.54 1-1g/L (0.25 ppb to 1.54 ppb: Table 1). The average 96 h LC50 value of 0.24 1-1g/L (0.24 ppb) for juvenile

P. paludosus (a freshwater species), derived from this study using the same type of exposure, fell just below the 96 h LC50 value of 0.39 /-!giL (0.39 ppb) for juvenile P. pugio (a brackish water species: Table 1), indicating that P. paludosus is more sensitive to endosulfan than P. pugio. The difference between the two values may be species or age/stage specific or related to the salinity of the treatment water. One study showed that in freshwater with a pH of 7 .0, the half-life due to hydrolysis alone was 5 days for a­ endosulfan and 15 days for ~-endosulfan (Peterson and Batley, 1993). Another study showed that in sterile seawater with 32 ppm salinity, the half-life of a-endosulfan was 4.2 days (Cotham and Bidelman, 1989), which indicates a somewhat faster degradation rate than the half-life of a-endosulfan in freshwater. As the degradation rate of endosulfan in saltwater is a bit faster than the degradation rate in freshwater (Leight and Van Dolah,

1999), higher salinity may have been the cause of the lower level of toxicity to endosulfan found in P. pugio.

A study conducted by Mayer ( 1987) on P. pugio, using the flow though exposure, reported a 96 h LC50 value of 1.30 !-!giL (1.30 ppb: Table 1). This value is greater than the estimated 0.24 /-!giL (0.24 ppb) 96 h LC50 value on P. paludosus, produced using the static renewal exposure. The difference may be related to the different protocols used.

Flow-through exposures, due to their constant replenishment of treatment water, should

13 result in higher total exposure to endosulfan, but since a continuous supply of oxygen exists in the system and little to no ammonia and other waste products are allowed to accumulate. The organisms therefore experience less stress and an increased tolerance for the toxicant.

Leight and Van Dolah (1999), using static renewal exposures, reported a 96 h LC50 value of 0.54 !lg/L (0.54 ppb) for G. palustris, a brackish water amp hi pod (Table 1) .

Sanders (1969, 1972) studied the brackish water amp hi pods Gammarus lacustris and fasciatus using static non-renewal exposures, and reported 96 h LC50 values of 5.80 !lg/L

(5.80 ppb) for G. lacustris and 6.00 11g1L (6.00 ppb) for G.fasciatus (Table 1). These values are much higher than the 96 h LC50 value of 0.24 !lg/L (0.24 ppb) found for P. paludosus in this study using static renewal exposures (Table 1). This indicates that P. paludosus is generally more sensitive to endosulfan than the Gammarus species regardless of the protocols used.

Sensitivity to endosulfan

Comparing the results of the freshwater P. paludosus and brackish water P. pugio, a higher level of sensitivity to endosulfan was observed in P. paludosus than in P. pugio. It is fairly common for different species of the same genus to show different sensitivities to a particular toxin (Berrill et al., 1998). Thus, one of the conclusions that may be drawn from this comparison is that P. pugio and P. paludosus simply differ in tolerance since they are adapted to different environments. A similar trend is observed in the varying

LC50 values between different species of amphipods of the genera Gammarus (Leight and

Van Dolah, 1999; Sanders, 1969; Sanders, 1972). A second conclusion that may be 14 derived from the comparison between the two congeners is that water chemistry, such as salinity, via its component parts plays a role in the toxicity to endosulfan (Goebel et al.,

1982). The relative loss of toxicity, caused by a faster degradation of endosulfan in the marine environment (degradation time of 4.2 days in saltwater compared to 5 days in freshwater for a-endosulfan: Cotham and Bidleman, 1989; Eichelberger and

Lichtenberg, 1971; Goebel et al., 1982), may be the cause of the lower levels of sensitivity to endosulfan found in P. pugio.

Sensitivity to endosulfan also varies according to the type of exposures used.

Comparing the studies conducted on shrimp species, static renewal exposures produced the highest LC50 values ranging from 0.24 ~-tg/L (0.24 ppb) to 1.54 ~-tg!L ( 1.54 ppb ), static non-renewal exposures produced a medium LC50 value of 0.20 ~-tg!L (0.20 ppb ), and flow through exposures produced the lowest LC50 value of 0.04 ~-tg/L (0.04 ppb: Table 1).

Comparing these methodologies to the real world scenarios, one would gather that the flow through technique represents the real world more so than any other technique, as water has a constant flow . The real world volume to surface area is also best represented in this technique than any other technique.

Copper sulfate toxicity in freshwater versus brackish water

The 48 h LC50 value for the static renewal bioassay, performed on P. paludosus was estimated to be 0.90 mg!L (0.90 ppm: Table 4), which is a smaller value in comparison to the 2.10 mg/L (2.1 0 ppm) value for P. pugio, derived by Burton and Fisher (1990) using the 48 h static renewal exposure. Since the type of exposures used to determine copper sulfate toxicity in P. paludosus and P. pugio are the same, one may infer that 15 copper sulfate is more toxic toP. paludosus than toP. pugio. Using the model of Tartare et al ( 1997), which related higher free ion concentrations in the water to increasing relative metal toxicity, one can conclude that toxicity of copper sulfate may vary directly with the salinity of the water. However, when subjected to lower concentrations of free ions in freshwater (Tartare et al., 1997), P. paludosus (LC50 = 0.90 mg/L) was affected to a greater extent by copper sulfate's toxicity than P. pugio (LC50 = 2.1 mg/L). Thus, assuming similar toxicity in cogeners, the results of this study contradict the model put forth by Tartare et al (1997).

Summary

In this study, P paludosus was determined to be highly sensitive to endosulfan and copper sulfate, more so than its congner P. pugio. The difference in the values obtained from this study and previous studies on P. pugio may be species or age/stage specific, and/or may also be related to salinity, as salinity affects the breakdown rate of endosulfan and the toxicity of copper sulfate. This study has indicated that P. paludosus may be an excellent model for toxicology studies in freshwater, as P. pugio is in saltwater.

The sensitivity of P. paludosus to endosulfan and copper sulfate can be related to other freshwater crustaceans as they have similar biological structure and processes. In the wetlands of south Florida and any other place on the east coast where agriculture pesticides are applied near freshwater aquatic habitats, P. paludosus can be used as an adaptive monitoring tool for biological and environmental toxicology, in order to assess the problems caused by runoff of these pesticides from agricultural areas into the surrounding areas. Further studies on P. paludosus using other agriculture pesticides will

16 help establish a guideline for the use of these pesticides and may suggest replacement of some of these pesticides with less toxic alternatives wherever possible.

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20 Table 1: Comparison of acute toxicity estimates (LC50) for Palaemonetes paludosus and other aquatic species for 48 and 96 h exposure to endosulfan. LCSO Test Species Organism Type Habitat Test Type References Jlg/LI ppb 96 h Exoosures Palaemonetes paludosus Shrimp (juvenile) !Freshwater STRE 0.24 lfhis Study, 2002 Palaemonetes pugio Shrimp (juvenile) Brackish STRE 0.39 Baughman, 1986 Palaemonetes pugio Shrimp (newly hatched) Brackish STRE 0.25 Baughman, 1986 Palaemonetes pugio Shrimp Brackish STRE 0.25 Scott et al., 1987 Palaemonetes pugio Shrimp Brackish STRE 1.01 Scott et al., 1990 Palaemonetes pugio Shrimp (adult) Brackish STRE 1.54 Baughman, 1986 Palaemonetes pugio Shrimp Brackish Fr 1.30 Mayer, 1987 Gammarus palustris Amphipod Brackish STRE 0.54 Leight and Van Dolan, 1999 Gammarus palustris Amphipod Brackish ST 0.43 Leight and Van Dolan, 1999 Gammarus fascia tus Amphipod Brackish ST 6.00 Sanders, 1972 N ...... Gammarus lacustris Am phi pod Brackish ST 5.80 Sanders, 1969 Procambarus clarkii Crayfish Freshwater ST 120 ~ebrian et al., 1992 Penaeus duorarum Shrimp Marine FT 0.04 Mayer, 1987 Crangnon septemspinosa Shrimp Marine ST 0.20 McLeese and Metcalfe, 1980 48 h Exoosures Palaemonetes paludosus Shrimp (juvenile) Freshwater STRE 0.37 lfhis Study, 2002 Gammarus palustris Amphipod Brackish STRE 2.29 Leight and Van Dolah, 1999 Gammarus palustris Amphipod Brackish ST 5.63 Leight and Van Dolah, 1999 Gammarus lacustris Amphipod Brackish ST 6.40 Sanders, 1969 Daphnea magna Cladoceran Freshwater FT 16.60 Macek et al., 1976 Callinectes sapidus Crab Marine FT 19 Mayer, 1987 STRE: Static Renewal Bioassay ST: Static Non-Renewal Bioassay FT: Flow Through Bioassay Table 2: Comparisons of the LCso values between different time endpoints for endosulfan. 24 vs. 48 h 48 vs. 72 h 72 vs. 96 h value 0.009 0.03 0.01

Table 3: Comparisons of the LC50 values between different time endpoints for er sulfate. 24 vs. 48 h 48 vs. 72 h 72 vs. 96 h 0.14 0.22 0.34

Table 4: Comparison of acute toxicity estimates (LCSO) for Palaemonetes paludosus and aquatic species for 48 and 96 h exposure to copper sulfate using static renewal bioassays. Organism LCSO Test Species Habitat References Type mg/L/ppm 96 h Exposures Palaemonetes paludosus Shrimp (juvenile) !Freshwater 0.79 lfhis Study, 2002. 48 h Exposures Palaemonetes paludosus Shrimp (juvenile) !Freshwater 0.90 !This Study, 2002 'Palaemonetes pugio Shrimp (juvenile) Brackish 2.10 !Burton and Fisher, 1990

22 Figure 1: Isomers of endosulfan and its degradation products (Peterson and Batley, 1993).

o.-Endo .. ulfan I P- 1-:ndc-...uJfan

23 Figure 2: The 'ANA' area.

Pepper/ Citrus Fields

Sampling site

·~ ;_ : ~f¥t_A:.;I""'· f '.i:~ • ..H.; '{. w.,...._ _ .... ·~ ~ It The ANA Area

24 Figure 3: LC50 values of endosulfan toxicity at 24, 48, 72, and 96 hrs

0.6 -,------, Replicates ~ ,.Q 0. 0. 0.5 '-' -+-- 1 ~ :::1. 0.4 ---- 2 37 .~....= 3 ....f 0.3 Cl.l _ ---7(-- Average [ (j= 0 24 § 0.2 (j Q uon ....:l 0.1

0 24 Hrs 48 Hrs 72 Hrs 96 Hrs Time

LC50 values of endosulfan toxicity at 24 hrs = 0.47 J.lg!L

LC50 values of endosulfan toxicity at 48 hrs = 0.37 J.lg/L

LC50 values of endosulfan toxicity at 72 hrs = 0.31 J.lg/L

LC50 values of endosulfan toxicity at 96 hrs = 0.24 J.lg!L

25 Figure 4: Survival in relation to the concentration of the insecticide - endosulfan

100 Concentration 90 in J.lg/L

80 --+-- 0

70 . ---- 0.0001 -; 0.0005 .... 60 ·;:;: ~ 0.001 "" 50 00= ~ 0.01 ~ 40 ----- o.o5 -t- 0.1 30 - 0.5 20 - 1 10

0 0 Hrs 24 Hrs 48 Hrs 72 Hrs 96 Hrs

Time

26 Figure 5: LC50 values of copper sulfate toxicity at 24, 48, 72, and 96 hrs

2.5 ,-.., sc. c. '-' 2 Replicates ~s = 1.5 :.:0 1-+- 1 ~ ---- 2 ....r.. =QJ 3 <:.I =0 ~ Average <:.I 0.79 l c II) u 0.5 ~

0 - 24 Hrs 48 Hrs 72 Hrs 96 Hrs Time

LC50 values of copper sulfate toxicity at 24 hrs = 1.34 mg/L

LC50 values of copper sulfate toxicity at 24 hrs = 0.90 mg/L

LC50 values of copper sulfate toxicity at 24 hrs = 0.82 mg/L

LC50 values of copper sulfate toxicity at 24 hrs = 0.79 mg/L

27 Figure 6: Survival in relation to the concentration of the insecticide - copper sulfate

100

90 Concentration in mg!L 80

70 -+---- 0 '; 60 --o.1 ~ ·s: 0.5 s 50 rJ'J ~ 40 -

30

20 -

I 0 -

0 OHrs 24 Hrs 48 Hrs 72Hrs 96 Hrs

Time

28