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Effects of Sevin (Carbaryl Insecticide) on Early Life Stages of Zebrafish (Danio rerio)
Ecotoxicology and Environmental Safety 53, 267}272 (2002) Environmental Research, Section B doi:10.1006/eesa.2002.2231
Effects of Sevin (Carbaryl Insecticide) on Early Life Stages of Zebrafish (Danio rerio) Nancy E. Todd and Maryke Van Leeuwen Department of Biology, Manhattanville College, 2900 Purchase Street, Purchase, New York 10577 Received August 24, 2001
Sevin brand carbaryl insecticide is one of the most commonly used insecticides in the United States, with great potential for leaching into ground- and surface water reserves. Its possible teratogenic e4ects were tested on zebra5sh eggs in four dilutions of decreasing concentration. The average mortality rate was low, indicating that Sevin does not directly kill embryos at these concentrations. Eggs and embryos were consistently smaller than the control starting at 24 h after spawning until hatching. Embryos in the highest concentration took up to twice as long to hatch as the control. This delayed hatching time increases vulnerability to predation. In addition, as minnows are lower on the food chain, bioaccumulation of Sevin in tissues may increase in larger predators, a4ecting their metabolism and reproduction. 2002 Elsevier Science (USA) Key Words: zebra5sh; teratogen; insecticide; development.
INTRODUCTION
Aquatic ecosystems that run through agricultural or industrial areas have high probability of being contaminated by runo! and groundwater leaching by a variety of chemicals. In this study, Sevin brand carbaryl insecticide was diluted to serve as a proxy for trace amounts in runo! to test its teratogenic e!ects on the development of Danio rerio embryos. Sevin is very harmful and potent to nontarget species. It is one of the three most commonly used insecticides in the United States, with 10}15 million pounds being used annually (Cox, 1993). Sevin has been registered for use on 100 di!erent crops, animals, ornamental plants, and indoor areas since 1958. It contains 21.3% 1-naphthyl-N-methylcarbamate (approximately 2 lb per gallon of solution), and is slightly colored, mostly odorless, and a crystalline solid, but can be found in a concentrated liquid (which is used in this study). According to Carpenter et al. (1961) the melting point is 1423C, vapor pressure is less then 0.005 mm of Hg at To whom correspondence should be addressed. E-mail: toddn@ mville.edu. Fax: (914) 323-5480.
263C, and density is 1.232 at 203C. Sevin is only 0.01% soluble in water while it is 40% soluble in organic solvents (Carpenter et al., 1961). Sevin is slightly soluble in hydrocarbons, more soluble in chlorinated hydrocarbons and alcohols, moderately soluble in ketones, and very soluble in dimethylformamide, pyridine, diethanolamine, and dimethylsulfoxide (Carpenter et al., 1961). Sevin as a chemical is a cholinesterase inhibitor that is free of phosphorus (Carpenter et al., 1961). It also inhibits the action of the enzyme acetylcholinesterase (AChE), which controls the chemical reaction that transforms acetylcholine into choline involved in the transmission of nerve impulses across neuromuscular junctions (Cox, 1993). Inhibition of AChE causes the loss of normal muscle control. In low concentrations, Sevin has physiological and behavioral e!ects in "sh along with a decrease in amino acid levels in muscle, damage to gills and liver cells, kidney lesions, and slowing of "n regeneration (Cox, 1993). In higher concentrations, Sevin causes the inhibition of the synthesis of proteins in "sh livers, decreased respiration rate, alterations in feeding and social behavior, and decreased growth rate (Cox, 1993). Fish embryos have shown reduced pigmentation, slowed development, vision abnormalities, and reduction of the size of the embryo (Cox, 1993). The Environmental Protection Agency (EPA) has identi"ed carbaryl-based pesticides having great potential to leach into groundwater. Carbaryl has been found in groundwater in California, Missouri, New York, Rhode Island, Virginia, and Wisconsin, and in one-"fth of rivers in New Jersey (Cox, 1993). Previous research has examined the e!ects of a variety of pesticides and other chemicals on zebra"sh development. These studies can be divided into several areas: acute toxicity studies, physiological studies on adult "sh, reproductive behavior studies involving numbers and viability of eggs spawned by adult females, and developmental abnormalities in eggs and larvae. While chemicals used as pesticides have been tested in their pure forms on zebra"sh development, the goal of this research is to examine the e!ects of commonly used, commercially available products that
267 0147-6513/02 $35.00 2002 Elsevier Science (USA) All rights reserved.
268
TODD AND VAN LEEUWEN
contain these chemicals as ingredients on zebra"sh embryo development to hatching. Previous studies have examined the acute toxicity of various pesticide chemicals to eggs and early-life-stage development (Elonen et al., 1998; Gallo et al., 1995; Groth et al., 1993; Henry et al., 1997; Samson and Shenker, 2000; Westerlund et al., 2000; Wiegand et al., 2000). The research reported here focuses speci"cally on egg development under exposure to Sevin. So, the aforementioned studies are not summarized. Sastry and Siddiqui (1982) looked at the e!ects of Sevin on metabolism in freshwater snakehead "sh blood, liver, muscles, kidneys, intestine, brain, and gills. No mortality was recorded, but at 30 and 60 days after exposure, the "sh were hyperglycemic and hyperlactemic. Sevin increased the blood sugar levels and decreased liver and muscle glycogen. The liver showed an increase in lactic acid. Fa grvus-Van Ree and Payne (1997) studied the e!ects of toxaphene, a mixture of polychlorinated terpenes, on the reproduction of zebra"sh. While their study focused on adult females, they also found signi"cant mortality and deformation in embryos from fertilized eggs laid by the females at concentrations of 2.2 g toxaphene/g daily. Ensenbach and Nagel (1995) mixed lindane and 3,4-dichloroaniline with Rhine River water and tap water to examine the e!ects of complex chemical mixtures in di!erent zebra"sh life stages. Egg hatching was not a!ected by lindane or 3,4-dichloroaniline, but survival of juvenile "sh in both chemicals in river and tap water was a!ected to varying degrees. In addition, growth appeared to be reduced in the mixtures, but was di$cult to quantify. Zebra"sh are used as a proxy for the species of Cyprinidae inhabiting the Hudson River. This family is by far the largest in the New York region with about 48 di!erent species (Decker et al., 1985). Minnows provide food for many larger "sh, and their consumption can lead to bioaccumulation of chemicals in tissues of larger species. Zebra"sh are related to the daces (Clinostomus spp.), and the most common in New York is the redside dace (Clinostomus elongatus), seen in many of the tributaries that lead into the Hudson River (Decker et al., 1985). The blacknose dace (Rhinichthys atratulus), longnose dace (Rhinichthys cataractae), and northern redbelly dace (Phoxinus eos) are also common (Decker et al., 1985). MATERIALS AND METHODS
Breeding and Egg Collection A 5-gal breeding tank was established with a 3:2 ratio of females to males for optimal breeding conditions. Fish were fed #aked food once a day and supplemented with bloodworms three times a day. Adults were kept in treated (aged) tap water and not exposed to Sevin. Eggs were removed by siphoning the marbles between 2 and 6 h after spawning.
The eggs were split into "ve equal groups consisting of a control and four dilutions. Dilutions A stock solution of 16 ml of Sevin to 3000 ml of aged water was established and used to mix four dilutions: Dilution 4 (1/1000 stock to aged tapwater), Dilution 3 (1/750 stock to aged tapwater), Dilution 2 (1/500 stock to aged tapwater), and Dilution 1 (1/250 stock to aged tapwater). The control was aged tapwater. Egg Observation and Data Collection Eggs were suspended in dark, nylon pantyhose in 3 liters of solution to keep them separate from each other. Each solution was aerated and maintained at room temperature. Every 24 h the eggs were removed from the solutions for observation. The eggs were staged, measured, checked for viability, and placed back into solution. When hatching started to occur, eggs were removed from containers and placed in Petri dishes with appropriate dilutions. Each egg was observed every 24 h until all eggs were hatched. The maximum hatching time was 144 h, and this marked the end of the experiment. This method allowed us to track the development of each individual egg until hatching. Eggs were staged using the reference stages listed in Kimmel et al. (1995) and by information in Wester"eld (1995). Not all stages were represented in the eggs in this study, as observations were done every 24 h. Eggs were measured using a microscope with an ocular micrometer. Diameter measurements are of the embryo only, not the entire egg. Length measurements are of the entire length of the hatched embryo. All eggs from trials 1}4 (total of 26 eggs) were combined into a single data set for analysis. RESULTS
Viability The highest overall mortality rate (31%) was in Dilution 1. The lowest mortality rate (15%) occurred in Dilutions 3 and 4. The control had a mortality rate of 19% and Dilution 2 had a rate of 23%. Dilutions 3 and 4 initially declined at 24 and 48 h, respectively, but then remained constant for the duration of the experiment. Dilutions 1 and 2 declined steadily starting at 24 h until the end of the 144 h (Table 1, Fig. 1). Embryo Size Average diameter and length are plotted in line charts to illustrate growth from 0 to 144 h. Diameter measures and length measures for each dilution are plotted on the same
EFFECTS OF SEVIN ON ZEBRAFISH
TABLE 1 Average Egg Viability for Control and All Dilutions 0h
24 h
48 h
72 h
96 h
269
72 h and in Dilution 4 (0.0031) at 96 h (diameter). Overall, there was good consistency in size among all eggs.
120 h 144 h
Stages Control Dilution Dilution Dilution Dilution
Control Dilution Dilution Dilution Dilution
4 3 2 1
4 3 2 1
A. Raw numbers (of 26 total for 26 24 22 26 25 23 26 23 23 26 24 22 26 23 22 B. Percentage 100 92 100 96 100 88 100 92 100 88
each dilution) 21 21 21 22 22 22 22 22 22 22 21 20 21 20 20
viability 85 81 88 85 88 85 85 81 85 81
81 85 85 81 77
81 85 85 77 77
21 22 22 20 18
81 85 85 77 69
chart. The break indicates the hatching time for each dilution (Fig. 2). The descriptive statistics are summarized in Table 2. The highest variance (0.012) occurred in the control at 120 h (length) and in Dilution 3 (0.011) at 72 h (diameter). The lowest variance (0.000052) occurred in the control at
Compared with the control, eggs in Dilution 1 were delayed in stage development, with the initial delays beginning at 24 h. The control started hatching at 72 h, but eggs in Dilution 1 did not hatch until 144 h after spawning. At 24 h, the majority of the controls were at 18}21 somites, Dilution 4 eggs at 18 somites, Dilution 3 at 18}14 somites, Dilution 2 at 14 somites, and Dilution 1 at 10 somites. At 48 h the majority of control eggs were at long and high pec, Dilution 4 eggs at high pec, Dilution 3 at prim-22, Dilution 2 at prim-16, and Dilution 1 at prim-6. At 72 h the majority of the controls were at stages hatched pec "n and pec "n, while Dilution 4 eggs were at long pec and hatched pec "n, Dilution 3 at high pec and pec "n, Dilution 2 at high pec, and Dilution 1 at prim-22. At 96 h the controls had all hatched and had a protruding mouth, Dilution 4 eggs had hatched with a protruding mouth and hatched pec "n, Dilution 3 were at pec "n, Dilution 2 at long pec, and Dilution 1 at high and long pec. At 120 h all the controls still had a protruding mouth along with all of Dilution 4 "sh.
FIG. 1. Viability percentages from 0 to 144 h indicated the smallest decrease in egg viability of 15% for Dilutions 3 and 4, and the largest decrease of 31% for the highest dilution, No. 1.
270
TODD AND VAN LEEUWEN
FIG. 2. Mean diameter and length from 0 to 144 h. The values are based on the average from all eggs.
The majority of Dilution 3 eggs had hatched and had a protruding mouth; some had not hatched but had a protruding mouth. Dilution 2 and Dilution 3 eggs were similar, while Dilution 1 eggs had not hatched and were at pec "n and protruding mouth. At the "nal 144 h, all eggs had hatched and had reached the "nal stage of protruding mouth. DISCUSSION
Based on the appearance of stages in development, Sevin a!ected the developing embryos by retarding stage progression. Eggs in Dilution 1 were delayed the longest, about 48}72 h, followed by Dilutions 4, 3, and 2 which were delayed 24}48 h. The threshold concentration of Sevin at which developmental and growth retardation occurred was Dilution 2. The control eggs were the "rst to hatch at 72 h, which is the normal hatching time of zebra"sh. Hatching of eggs in Dilutions 4 and 3 was delayed until 96}120 h. Dilution 2 eggs took 120 h to hatch after the control and Dilution 1 eggs took the longest to hatch at 144 h after the control.
This delayed development in stages is supported by statistical analysis. To compare the means of each dilution with that of the control, F tests for variance and two-tailed Student t tests were performed. The overall results of the F tests indicate that the control and the dilutions were not signi"cantly di!erent. The only signi"cant P values at 95% con"dence (P(0.05) were between the control and Dilution 4 at 48 h (diameter), the control and Dilutions 3 and 2 at 120 h (length), and the control and Dilutions 2 at 144 h (length) (Table 2). This indicates that the variances in the di!erent dilutions are comparable. To test whether the embryos in di!erent dilutions were signi"cantly di!erent in size from the controls, two-tailed t tests with equal or unequal variance (according to the F test) were performed. The results of the analysis show a signi"cant di!erence starting at 24 h (diameter) between the controls and Dilutions 2 and 1. From this period on, all the t tests were signi"cant for all dilutions and the controls except at 72 h (diameter) between the control and Dilutions 4 and 3 (Table 2). While all eggs were maintained at constant room temperature between 21 and 243C, this lower temperature may
271
EFFECTS OF SEVIN ON ZEBRAFISH
TABLE 2 t Test and F Test Results for Embryo Size in Control and All Dilutions until Hatching Dilution 4
Dilution 3
Dilution 2
Dilution 1
A. Diameter measurements 0h Control F test t test
0.8641 0.8853
0.6950 0.8772
0.9099 0.4297
0.9375 0.8558
24 h Control F test t test
0.3992 0.9216
0.6817 0.1234
0.8812 0.0028
0.0892 0.0000
48 h Control F test t test
0.0212 0.0004
0.0817 0.0000
0.2174 0.0000
0.1581 0.0000
72 h Control F test t test
1 0.2418
1 0.1995
0.9999 0.0000
1 0.0000
B. Length measurements 72 h Control F test t test
0.5211 0.0005
96 h Control F test t test
0.5523 0.0000
0.3797 0.0000
120 h Control F test t test
0.0549 0.0000
0.0010 0.0000
0.0021 0.0000
144 h Control F test t test
0.1548 0.0000
0.4314 0.0000
0.0218 0.0000
0.1388 0.0000
have a!ected development. Work is currently in progress to test the e!ect of temperature on embryo development and hatching time. Other conditions in Hudson River water, such as pH and turbidity, would need to be examined so that additional controls can be established. CONCLUSION
Sevin has a signi"cant e!ect on embryo size from the time the eggs are laid until they hatch. Embryos in the highest concentration, Dilution 1, developed more slowly and hatched later than the controls and eggs in other dilutions. Embryos are smaller than the controls even at the lowest
concentration of Sevin. Since the mortality percentage for this experiment was relatively low (mean of approximately 20%), these dilutions do not directly kill the embryos. However, Sevin does apparently retard development, a!ect embryo size, and delay hatching. Under normal conditions of 28.53C with 5}10 embryos/ml, zebra"sh take 48}72 h to develop from time of spawning to hatching (Kimmel et al., 1995). This satis"es a rapid developmental rate and generation time. Canalization is the uniformity and variation between batches of embryos (Bolker, 1994). This characteristic is very important in model systems and studies, because if the batches of eggs vary greatly the results will not be accurate and conclusive. Zebra"sh again satisfy this condition by spawning eggs that are similar in genotype when they develop consistently under the same environmental conditions. Bolker (1994) states that life history is important because with short generation times the evolutionary history of a species changes from genetics to other traits. This usually means a small adult size, making it easier to work with the species in the laboratory, but this also causes a problem. If the adults are not usually small and become small, there is usually a loss of structure, morphological novelties, and an increase in variation between individuals, making the uniformity unstable (Bolker, 1994). Although this study was not carried out in hatched larvae, we can hypothesize that larvae exposed to Sevin will be smaller than controls, and may su!er consequences suggested by Bolker (1994). When embryos are exposed to Sevin or other materials that delay hatching, the embryos are in the eggs longer, increasing their vulnerability to predators. Eggs exposed to Dilution 1 of Sevin (strongest dilution) took twice as long to hatch, thus increasing their chance of being eaten by predators. Another factor is bioaccumulation. It is assumed that Sevin penetrates the tissue of the a!ected "sh and becomes incorporated into tissues of predators that eat these "sh. Although Sevin cannot be directly linked to killing the "sh, the results indicate that the higher the concentration of Sevin, the lower the percentage viability. What will happen to the food chain and to the other species of "sh that depend on minnows as their food source when the overall population decreases because of pesticide use? If Sevin is indeed part of the runo! entering the Hudson River, these e!ects on "sh development would have a great impact on the overall population, breeding cycles, size of developing and adult minnows, and the entire food web. ACKNOWLEDGMENTS This was an undergraduate senior thesis project in the Department of Biology at Manhattanville College, and we acknowledge Dr. Annemarie Bettica and the resources of the department for funding this project. We also thank Alan Troast and Jenneen DeFiore for aid with "sh care and support, and Gary P. Aronsen for reading this manuscript.
272
TODD AND VAN LEEUWEN
REFERENCES Bolker, J. A. (1994). Model systems in developmental biology. BioEssays 17, 451}455. Carpenter, C. P., Weil, C. S., Palm, P. E., Woodside, M. W., Nair, J. H., III, and Smyth, H. F., Jr. (1961). Mammalian toxicity of 1-naphthyl-Nmethylcarbamate (Sevin insecticide). Insecticide ¹oxicol. Agric. Food Chem. 9, 30}39. Cox, C. (1993). The problems with Sevin (Carbaryl). J. Pestic. Reform. 13, 31}36. Decker, D. J., Howard, R. A., Everhart, W. H., and Kelley, J. W. (1985). Guide to Freshwater Fishes of New >ork. Cornell Cooperative Extension, New York. Elonen, G. E., Spehar, R. L., Holcombe, G. W., Johnson, R. D., Fernandez, J. D., Erickson, R. J., Tietge, J. E., and Cooke, P. M. (1998). Comparative toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin to seven freshwater "sh species during early life-stage development. Environ. ¹oxicol. Chem. 17, 472}483. Ensenbach, U., and Nagel, R. (1995). Toxicity of complex chemical mixtures: Acute and long-term e!ects on di!erent life stages of zebra"sh (Brachydanio rerio). Ecotoxicol. Environ. Saf. 30, 151}157. Fa grvus-Van Ree, G. E., and Payne, J. F. (1997). E!ect of toxaphene on reproduction of "sh. Chemosphere 34, 855}867. Gallo, D., Merendino, A., Keizer, J., and Vittozzi, L. (1995). Acute toxicity of two carbamates to the guppy (Poecilia reticulata) and the zebra"sh (Brachydanio rerio). Sci. ¹otal Environ. 171, 131}136.
Groth, G., Schreeb, K., Herdt, V., and Freundt, K. J. (1993). Toxicity studies in fertilized zebra"sh eggs treated with N-methylamine, N, N-dimethylamine, 2-aminoethanol, isopropylamine, chloroacetaldehyde, or cyclohexanol. Bull. Environ. Contam. ¹oxicol. 50, 878}882. Henry, T. R., Spitsbergen, J. M., Hornung, M. W., Abnet, C. C., and Peterson, R. E. (1997). Early life-stage toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebra"sh (Danio rerio). ¹oxicol. Appl. Pharmacol. 142, 56}68. Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., and Schilling, T. F. (1995). Stages of embryonic development of the zebra"sh. Dev. Dyn. 203, 263}310. Samson, J. C., and Shenker, J. (2000). The teratogenic e!ects of methylmercury on early development of the zebra"sh, Danio rerio. Aquat. ¹oxicol. 48, 343}354. Sastry, K. V., and Siddiqui, A. A. (1982). Chronic toxic e!ect of the carbamate pesticide Sevin on carbohydrate metabolism in a freshwater snakehead "sh, Channa punctatus. ¹oxicol. ¸ett. Neth. 14, 123}130. Wester"eld, M. (1995). ¹he Zebra,sh Book: A Guide for the ¸aboratory ;se of Zebra,sh Danio rerio. Eugene, OR: Univ. of Oregon Press. Westerlund, L., Billsson, K., Andersson, P. L., Tysklind, M., and Olsson, P.-E. (2000). Early life-stage mortality in zebra"sh (Danio rerio) following maternal exposure to polychlorinated biphenyls and estrogen. Environ. ¹oxicol. Chem. 19, 1582}1588. Wiegand, C., Krause, E., Steinberg, C., and P#ugmacher, S. (2000). Metabolism of the S-triazine herbizide atrazine in early life stages of the zebra"sh, Danio rerio. Mar. Environ. Res. 50, 73.
Effects of Sevin (Carbaryl Insecticide) on Early Life Stages of Zebrafish (Danio rerio) Nancy E. Todd and Maryke Van Leeuwen Department of Biology, Manhattanville College, 2900 Purchase Street, Purchase, New York 10577 Received August 24, 2001
Sevin brand carbaryl insecticide is one of the most commonly used insecticides in the United States, with great potential for leaching into ground- and surface water reserves. Its possible teratogenic e4ects were tested on zebra5sh eggs in four dilutions of decreasing concentration. The average mortality rate was low, indicating that Sevin does not directly kill embryos at these concentrations. Eggs and embryos were consistently smaller than the control starting at 24 h after spawning until hatching. Embryos in the highest concentration took up to twice as long to hatch as the control. This delayed hatching time increases vulnerability to predation. In addition, as minnows are lower on the food chain, bioaccumulation of Sevin in tissues may increase in larger predators, a4ecting their metabolism and reproduction. 2002 Elsevier Science (USA) Key Words: zebra5sh; teratogen; insecticide; development.
INTRODUCTION
Aquatic ecosystems that run through agricultural or industrial areas have high probability of being contaminated by runo! and groundwater leaching by a variety of chemicals. In this study, Sevin brand carbaryl insecticide was diluted to serve as a proxy for trace amounts in runo! to test its teratogenic e!ects on the development of Danio rerio embryos. Sevin is very harmful and potent to nontarget species. It is one of the three most commonly used insecticides in the United States, with 10}15 million pounds being used annually (Cox, 1993). Sevin has been registered for use on 100 di!erent crops, animals, ornamental plants, and indoor areas since 1958. It contains 21.3% 1-naphthyl-N-methylcarbamate (approximately 2 lb per gallon of solution), and is slightly colored, mostly odorless, and a crystalline solid, but can be found in a concentrated liquid (which is used in this study). According to Carpenter et al. (1961) the melting point is 1423C, vapor pressure is less then 0.005 mm of Hg at To whom correspondence should be addressed. E-mail: toddn@ mville.edu. Fax: (914) 323-5480.
263C, and density is 1.232 at 203C. Sevin is only 0.01% soluble in water while it is 40% soluble in organic solvents (Carpenter et al., 1961). Sevin is slightly soluble in hydrocarbons, more soluble in chlorinated hydrocarbons and alcohols, moderately soluble in ketones, and very soluble in dimethylformamide, pyridine, diethanolamine, and dimethylsulfoxide (Carpenter et al., 1961). Sevin as a chemical is a cholinesterase inhibitor that is free of phosphorus (Carpenter et al., 1961). It also inhibits the action of the enzyme acetylcholinesterase (AChE), which controls the chemical reaction that transforms acetylcholine into choline involved in the transmission of nerve impulses across neuromuscular junctions (Cox, 1993). Inhibition of AChE causes the loss of normal muscle control. In low concentrations, Sevin has physiological and behavioral e!ects in "sh along with a decrease in amino acid levels in muscle, damage to gills and liver cells, kidney lesions, and slowing of "n regeneration (Cox, 1993). In higher concentrations, Sevin causes the inhibition of the synthesis of proteins in "sh livers, decreased respiration rate, alterations in feeding and social behavior, and decreased growth rate (Cox, 1993). Fish embryos have shown reduced pigmentation, slowed development, vision abnormalities, and reduction of the size of the embryo (Cox, 1993). The Environmental Protection Agency (EPA) has identi"ed carbaryl-based pesticides having great potential to leach into groundwater. Carbaryl has been found in groundwater in California, Missouri, New York, Rhode Island, Virginia, and Wisconsin, and in one-"fth of rivers in New Jersey (Cox, 1993). Previous research has examined the e!ects of a variety of pesticides and other chemicals on zebra"sh development. These studies can be divided into several areas: acute toxicity studies, physiological studies on adult "sh, reproductive behavior studies involving numbers and viability of eggs spawned by adult females, and developmental abnormalities in eggs and larvae. While chemicals used as pesticides have been tested in their pure forms on zebra"sh development, the goal of this research is to examine the e!ects of commonly used, commercially available products that
267 0147-6513/02 $35.00 2002 Elsevier Science (USA) All rights reserved.
268
TODD AND VAN LEEUWEN
contain these chemicals as ingredients on zebra"sh embryo development to hatching. Previous studies have examined the acute toxicity of various pesticide chemicals to eggs and early-life-stage development (Elonen et al., 1998; Gallo et al., 1995; Groth et al., 1993; Henry et al., 1997; Samson and Shenker, 2000; Westerlund et al., 2000; Wiegand et al., 2000). The research reported here focuses speci"cally on egg development under exposure to Sevin. So, the aforementioned studies are not summarized. Sastry and Siddiqui (1982) looked at the e!ects of Sevin on metabolism in freshwater snakehead "sh blood, liver, muscles, kidneys, intestine, brain, and gills. No mortality was recorded, but at 30 and 60 days after exposure, the "sh were hyperglycemic and hyperlactemic. Sevin increased the blood sugar levels and decreased liver and muscle glycogen. The liver showed an increase in lactic acid. Fa grvus-Van Ree and Payne (1997) studied the e!ects of toxaphene, a mixture of polychlorinated terpenes, on the reproduction of zebra"sh. While their study focused on adult females, they also found signi"cant mortality and deformation in embryos from fertilized eggs laid by the females at concentrations of 2.2 g toxaphene/g daily. Ensenbach and Nagel (1995) mixed lindane and 3,4-dichloroaniline with Rhine River water and tap water to examine the e!ects of complex chemical mixtures in di!erent zebra"sh life stages. Egg hatching was not a!ected by lindane or 3,4-dichloroaniline, but survival of juvenile "sh in both chemicals in river and tap water was a!ected to varying degrees. In addition, growth appeared to be reduced in the mixtures, but was di$cult to quantify. Zebra"sh are used as a proxy for the species of Cyprinidae inhabiting the Hudson River. This family is by far the largest in the New York region with about 48 di!erent species (Decker et al., 1985). Minnows provide food for many larger "sh, and their consumption can lead to bioaccumulation of chemicals in tissues of larger species. Zebra"sh are related to the daces (Clinostomus spp.), and the most common in New York is the redside dace (Clinostomus elongatus), seen in many of the tributaries that lead into the Hudson River (Decker et al., 1985). The blacknose dace (Rhinichthys atratulus), longnose dace (Rhinichthys cataractae), and northern redbelly dace (Phoxinus eos) are also common (Decker et al., 1985). MATERIALS AND METHODS
Breeding and Egg Collection A 5-gal breeding tank was established with a 3:2 ratio of females to males for optimal breeding conditions. Fish were fed #aked food once a day and supplemented with bloodworms three times a day. Adults were kept in treated (aged) tap water and not exposed to Sevin. Eggs were removed by siphoning the marbles between 2 and 6 h after spawning.
The eggs were split into "ve equal groups consisting of a control and four dilutions. Dilutions A stock solution of 16 ml of Sevin to 3000 ml of aged water was established and used to mix four dilutions: Dilution 4 (1/1000 stock to aged tapwater), Dilution 3 (1/750 stock to aged tapwater), Dilution 2 (1/500 stock to aged tapwater), and Dilution 1 (1/250 stock to aged tapwater). The control was aged tapwater. Egg Observation and Data Collection Eggs were suspended in dark, nylon pantyhose in 3 liters of solution to keep them separate from each other. Each solution was aerated and maintained at room temperature. Every 24 h the eggs were removed from the solutions for observation. The eggs were staged, measured, checked for viability, and placed back into solution. When hatching started to occur, eggs were removed from containers and placed in Petri dishes with appropriate dilutions. Each egg was observed every 24 h until all eggs were hatched. The maximum hatching time was 144 h, and this marked the end of the experiment. This method allowed us to track the development of each individual egg until hatching. Eggs were staged using the reference stages listed in Kimmel et al. (1995) and by information in Wester"eld (1995). Not all stages were represented in the eggs in this study, as observations were done every 24 h. Eggs were measured using a microscope with an ocular micrometer. Diameter measurements are of the embryo only, not the entire egg. Length measurements are of the entire length of the hatched embryo. All eggs from trials 1}4 (total of 26 eggs) were combined into a single data set for analysis. RESULTS
Viability The highest overall mortality rate (31%) was in Dilution 1. The lowest mortality rate (15%) occurred in Dilutions 3 and 4. The control had a mortality rate of 19% and Dilution 2 had a rate of 23%. Dilutions 3 and 4 initially declined at 24 and 48 h, respectively, but then remained constant for the duration of the experiment. Dilutions 1 and 2 declined steadily starting at 24 h until the end of the 144 h (Table 1, Fig. 1). Embryo Size Average diameter and length are plotted in line charts to illustrate growth from 0 to 144 h. Diameter measures and length measures for each dilution are plotted on the same
EFFECTS OF SEVIN ON ZEBRAFISH
TABLE 1 Average Egg Viability for Control and All Dilutions 0h
24 h
48 h
72 h
96 h
269
72 h and in Dilution 4 (0.0031) at 96 h (diameter). Overall, there was good consistency in size among all eggs.
120 h 144 h
Stages Control Dilution Dilution Dilution Dilution
Control Dilution Dilution Dilution Dilution
4 3 2 1
4 3 2 1
A. Raw numbers (of 26 total for 26 24 22 26 25 23 26 23 23 26 24 22 26 23 22 B. Percentage 100 92 100 96 100 88 100 92 100 88
each dilution) 21 21 21 22 22 22 22 22 22 22 21 20 21 20 20
viability 85 81 88 85 88 85 85 81 85 81
81 85 85 81 77
81 85 85 77 77
21 22 22 20 18
81 85 85 77 69
chart. The break indicates the hatching time for each dilution (Fig. 2). The descriptive statistics are summarized in Table 2. The highest variance (0.012) occurred in the control at 120 h (length) and in Dilution 3 (0.011) at 72 h (diameter). The lowest variance (0.000052) occurred in the control at
Compared with the control, eggs in Dilution 1 were delayed in stage development, with the initial delays beginning at 24 h. The control started hatching at 72 h, but eggs in Dilution 1 did not hatch until 144 h after spawning. At 24 h, the majority of the controls were at 18}21 somites, Dilution 4 eggs at 18 somites, Dilution 3 at 18}14 somites, Dilution 2 at 14 somites, and Dilution 1 at 10 somites. At 48 h the majority of control eggs were at long and high pec, Dilution 4 eggs at high pec, Dilution 3 at prim-22, Dilution 2 at prim-16, and Dilution 1 at prim-6. At 72 h the majority of the controls were at stages hatched pec "n and pec "n, while Dilution 4 eggs were at long pec and hatched pec "n, Dilution 3 at high pec and pec "n, Dilution 2 at high pec, and Dilution 1 at prim-22. At 96 h the controls had all hatched and had a protruding mouth, Dilution 4 eggs had hatched with a protruding mouth and hatched pec "n, Dilution 3 were at pec "n, Dilution 2 at long pec, and Dilution 1 at high and long pec. At 120 h all the controls still had a protruding mouth along with all of Dilution 4 "sh.
FIG. 1. Viability percentages from 0 to 144 h indicated the smallest decrease in egg viability of 15% for Dilutions 3 and 4, and the largest decrease of 31% for the highest dilution, No. 1.
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TODD AND VAN LEEUWEN
FIG. 2. Mean diameter and length from 0 to 144 h. The values are based on the average from all eggs.
The majority of Dilution 3 eggs had hatched and had a protruding mouth; some had not hatched but had a protruding mouth. Dilution 2 and Dilution 3 eggs were similar, while Dilution 1 eggs had not hatched and were at pec "n and protruding mouth. At the "nal 144 h, all eggs had hatched and had reached the "nal stage of protruding mouth. DISCUSSION
Based on the appearance of stages in development, Sevin a!ected the developing embryos by retarding stage progression. Eggs in Dilution 1 were delayed the longest, about 48}72 h, followed by Dilutions 4, 3, and 2 which were delayed 24}48 h. The threshold concentration of Sevin at which developmental and growth retardation occurred was Dilution 2. The control eggs were the "rst to hatch at 72 h, which is the normal hatching time of zebra"sh. Hatching of eggs in Dilutions 4 and 3 was delayed until 96}120 h. Dilution 2 eggs took 120 h to hatch after the control and Dilution 1 eggs took the longest to hatch at 144 h after the control.
This delayed development in stages is supported by statistical analysis. To compare the means of each dilution with that of the control, F tests for variance and two-tailed Student t tests were performed. The overall results of the F tests indicate that the control and the dilutions were not signi"cantly di!erent. The only signi"cant P values at 95% con"dence (P(0.05) were between the control and Dilution 4 at 48 h (diameter), the control and Dilutions 3 and 2 at 120 h (length), and the control and Dilutions 2 at 144 h (length) (Table 2). This indicates that the variances in the di!erent dilutions are comparable. To test whether the embryos in di!erent dilutions were signi"cantly di!erent in size from the controls, two-tailed t tests with equal or unequal variance (according to the F test) were performed. The results of the analysis show a signi"cant di!erence starting at 24 h (diameter) between the controls and Dilutions 2 and 1. From this period on, all the t tests were signi"cant for all dilutions and the controls except at 72 h (diameter) between the control and Dilutions 4 and 3 (Table 2). While all eggs were maintained at constant room temperature between 21 and 243C, this lower temperature may
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EFFECTS OF SEVIN ON ZEBRAFISH
TABLE 2 t Test and F Test Results for Embryo Size in Control and All Dilutions until Hatching Dilution 4
Dilution 3
Dilution 2
Dilution 1
A. Diameter measurements 0h Control F test t test
0.8641 0.8853
0.6950 0.8772
0.9099 0.4297
0.9375 0.8558
24 h Control F test t test
0.3992 0.9216
0.6817 0.1234
0.8812 0.0028
0.0892 0.0000
48 h Control F test t test
0.0212 0.0004
0.0817 0.0000
0.2174 0.0000
0.1581 0.0000
72 h Control F test t test
1 0.2418
1 0.1995
0.9999 0.0000
1 0.0000
B. Length measurements 72 h Control F test t test
0.5211 0.0005
96 h Control F test t test
0.5523 0.0000
0.3797 0.0000
120 h Control F test t test
0.0549 0.0000
0.0010 0.0000
0.0021 0.0000
144 h Control F test t test
0.1548 0.0000
0.4314 0.0000
0.0218 0.0000
0.1388 0.0000
have a!ected development. Work is currently in progress to test the e!ect of temperature on embryo development and hatching time. Other conditions in Hudson River water, such as pH and turbidity, would need to be examined so that additional controls can be established. CONCLUSION
Sevin has a signi"cant e!ect on embryo size from the time the eggs are laid until they hatch. Embryos in the highest concentration, Dilution 1, developed more slowly and hatched later than the controls and eggs in other dilutions. Embryos are smaller than the controls even at the lowest
concentration of Sevin. Since the mortality percentage for this experiment was relatively low (mean of approximately 20%), these dilutions do not directly kill the embryos. However, Sevin does apparently retard development, a!ect embryo size, and delay hatching. Under normal conditions of 28.53C with 5}10 embryos/ml, zebra"sh take 48}72 h to develop from time of spawning to hatching (Kimmel et al., 1995). This satis"es a rapid developmental rate and generation time. Canalization is the uniformity and variation between batches of embryos (Bolker, 1994). This characteristic is very important in model systems and studies, because if the batches of eggs vary greatly the results will not be accurate and conclusive. Zebra"sh again satisfy this condition by spawning eggs that are similar in genotype when they develop consistently under the same environmental conditions. Bolker (1994) states that life history is important because with short generation times the evolutionary history of a species changes from genetics to other traits. This usually means a small adult size, making it easier to work with the species in the laboratory, but this also causes a problem. If the adults are not usually small and become small, there is usually a loss of structure, morphological novelties, and an increase in variation between individuals, making the uniformity unstable (Bolker, 1994). Although this study was not carried out in hatched larvae, we can hypothesize that larvae exposed to Sevin will be smaller than controls, and may su!er consequences suggested by Bolker (1994). When embryos are exposed to Sevin or other materials that delay hatching, the embryos are in the eggs longer, increasing their vulnerability to predators. Eggs exposed to Dilution 1 of Sevin (strongest dilution) took twice as long to hatch, thus increasing their chance of being eaten by predators. Another factor is bioaccumulation. It is assumed that Sevin penetrates the tissue of the a!ected "sh and becomes incorporated into tissues of predators that eat these "sh. Although Sevin cannot be directly linked to killing the "sh, the results indicate that the higher the concentration of Sevin, the lower the percentage viability. What will happen to the food chain and to the other species of "sh that depend on minnows as their food source when the overall population decreases because of pesticide use? If Sevin is indeed part of the runo! entering the Hudson River, these e!ects on "sh development would have a great impact on the overall population, breeding cycles, size of developing and adult minnows, and the entire food web. ACKNOWLEDGMENTS This was an undergraduate senior thesis project in the Department of Biology at Manhattanville College, and we acknowledge Dr. Annemarie Bettica and the resources of the department for funding this project. We also thank Alan Troast and Jenneen DeFiore for aid with "sh care and support, and Gary P. Aronsen for reading this manuscript.
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