CHAPTER 2 EFFECTS OF THREE FUNGICIDES ON HATCHING SUCCESS, SURVIVAL, AND DEVELOPMENT OF AMPHIBIAN LARVAE.

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1 CHAPTER 2 11 EFFECTS OF THREE FUNGICIDES ON HATCHING SUCCESS, SURVIVAL, AND DEVELOPMENT OF AMPHIBIAN LARVAE. Abstract-- A number of factors have been suggested to explain recently observed amphibian declines, including pesticide exposure. Effects of chronic exposure to three common fungicides (chlorothalonil, fosetyl-al and mancozeb) on hatching success and larval survival and development of amphibians were evaluated in two separate experiments. Commercial formulation Daconil@Ultrex, Chipco@Aliette Signature and Fore@fungicides were used. Three test concentrations for each experiment were determined from preliminary LC50 tests with southern leopard frog (Rana utricularia) tadpoles. Wood frog (Rana sylvatica) and spotted salamander (Ambystoma maculatum) eggs were exposed to fungicides in static tests in 5-L aquaria. Also, wood frog and gray treefrog (Hyla chrysoscelis) tadpoles were exposed to fungicides from approximately two weeks post-hatching to metamorphosis in 60-L aquaria. In this experiment, fungicides were reapplied once every two weeks with water renewal. Hatching success of wood frogs was significantly decreased in 0.5 mgll offore@. Wood frog hatchlings in Fore@concentrations of 0.5 mgll also exhibited a significantly greater number of deformities. High concentrations offore@ (0.5 mgll) significantly decreased survival of wood frog and gray treefrog tadpoles. High concentrations ofchipco@ Aliette Signature (28.3 mgll) also significantly decreased survival of gray treefrog tadpoles. Metamorphosed wood frogs exposed to Fore@concentrations 2: 0.1 mgll throughout development exhibited a greater number of deformities. Effects on hatching success, larval survival, and occurrence of deformities may contribute to amphibian population

2 trends and will likely influence persistence of populations if exposure to these 12 concentrations occur in natural environments. INTRODUCTION Amphibian species that require relatively stagnant or slow-flowing water for breeding and larval development may be at risk for pesticide exposure in agricultural and urban areas when pesticides are applied directly to or reach breeding areas via runoff[1]. In natural environments, chronic or repeated exposures to acutely sublethal levels of pesticides are more likely than single-event, short-term exposure to acutely lethal levels. Environmental levels of chemicals and principal pathways of exposure should be taken into account when designing laboratory studies [2, 3]. Traditionally, laboratory toxicity tests have used standard lab species and extrapolated results to estimate effects in natural environments. Tests on resident species may be more appropriate when using toxicity data to assess the potential for contaminants to cause adverse effects measured in field studies [4]. Because species vary in their response to different toxins [3], several species representing diverse native taxa should be tested. Wood frogs (Rana sylvatica) breed in shallow, often temporary, bodies of water throughout northern North America and are described as explosive breeders. All eggs are laid within a few days in early spring, after which adult frogs retreat from the breeding area [5-7]. Average development of a wood frog from egg to metamorphosis emcompasses 45 days [7]. The spotted salamander (Ambystoma maculatum) also breeds in early spring. Spotted salamander breeding is often correlated with a moderate amount of rainfall and threshold temperatures in the range of 5 - lode

3 [8, 9]. Mean incubation periods of days were observed in spotted salamander 13 eggs in field studies in New Jersey and Ohio, USA [8, 10]. Mean incubation periods in some laboratory settings are much lower and range from days (S. Julian and J. Howard, personal communication). Spotted salamander larvae metamorphose approximately 2-4 months following hatching [5]. The gray treefrog (Hyla chrysoscelis) breeds throughout spring and summer in temporary pools and semipermanent ponds in the eastern United States and southeastern Canada [5, 6, 11]. Gray treefrogs exhibit strong breeding site fidelity and rarely migrate between breeding ponds [11]. Ptacek [12] observed average larval development periods of38 and 44 days in two experiments with laboratory-raised H. chrysoscelis. Spring breeding and larval development can coincide with periods of heavy pesticide application and run-off. Chlorothalonil, fosetyl-al, and mancozeb fungicides are used on turfgrass in large recreational areas such as golf courses and in agricultural areas and can reach amphibian breeding sites. Effects of chronic exposure of amphibian larvae to chlorothalonil and fosetyl-al have not been previously studied. One recent study in the Sierra Nevada Mountains, California found that adult frogs had chlorothalonil in tissue at detectable levels (33.3 ng/g) [13]. Harris et al. [14] evaluated the toxicity of six pesticides including DG, a fungicide containing 76-80% mancozeb, using continuous and discontinuous tests on northern leopard frog (Rana pipiens) and green frog (Rana clamitans) tadpoles. The Dithane@ DG 96-h LC50 (concentration lethal to 50% of exposed individuals) contained mg/l mancozeb. Tadpoles were also tested in discontinuous tests with 4-d exposure and 7.5-d non-exposure cycles. Dithane@ DG LC50 concentrations for 13-d continuous and 16-d discontinuous

4 exposures contained and 0.20 mgll mancozeb, respectively. Deformity 14 frequencies were used to calculate EC50s (effective concentrations that resulted in 50% observations). The EC50 concentration contained 0.04 mgll ofmancozeb. In the current study, two laboratory experiments were conducted to investigate possible effects on amphibian embryos and larvae chronically exposed to commercial formulations of fungicides containing chlorothalonil, fosetyl-al, and mancozeb. In the first experiment, wood frog and spotted salamander eggs were exposed to fungicides in static tests. In the second experiment, wood frog and gray treefrog larvae were exposed to fungicides with water renewal and repeated dosage occurring every two weeks. Repeated dosages mimicked repeated applications in natural environments. The objectives ofthe current study were to investigate the effects of chronic exposure to three common fungicides on hatching success, and larval survival and development, of diverse amphibian taxa. Chronic, repeated fungicide exposure at levels of 1-50% of 48-hour LC50 concentrations (the concentration lethal to 50% oftest organisms) was expected to significantly decrease survival of amphibian eggs and larvae. Sublethal effects including effects on growth, time to metamorphosis, and number of gross deformities, were expected to increase with dosage. MATERIALS AND METHODS (Batch , ISK Biosciences Corporation, Mentor, Ohio), Signature (Batch F , Rhone-Poulenc Ag Company, Research Triangle Park, North Carolina), and L4429, Rohm and Haas Company,

5 Philadelphia, Pennsylvannia) commercial fonnulation fungicides were used for all 15 experiments. Ultrex (82% chlorothalonil) is a chloronitrile fungicide, Chipco@ Aliette Signature (80% fosetyl-al) is an organophosphate fungicide, and Fore@ (80% mancozeb) is a ethylenebisdithiocarbamate (EBDC) fungicide. Fungicide solutions were made with dechlorinated water. The average (:!: ISD) water temperature of egg exposure aquaria was 20.0 :J: 0.3 C. The average (:!: 1SD) water temperature of larval exposure aquaria was 21.5 :J: 0.2 C. Larval aquaria ph ranged from Water conductivity oflarval aquaria consistently measured 0.2 Ils/cm. Southern leopard frog (Rana utricularia) tadpoles were ordered from Carolina Biological Supply, Burlington, North Carolina in early spring of Wood frog and spotted salamander eggs were field collected for all experiments from breeding sites near Frostburg, Maryland in the spring of Gray treefrog eggs were ordered from Charles D. Sullivan Company, Inc., Nashville, Tennessee in late summer To effect more natural conditions, uncontaminated sediment was collected from two nearby ponds with no history of pesticide application and where amphibians were breeding successfully. Approximately 0.25 kg sediment and 1 kg sediment (wet weight) was added to the bottom of egg exposure aquaria and larvae exposure aquaria, respectively. Three test concentrations for fungicides for each experiment were determined from preliminary 48-h LC50 tests with southern leopard frog tadpoles at approximate Gosner stage 26 [15]. In the LC50 tests, tadpoles were exposed to five different concentrations of fungicides in 5-L aquaria containing dechlorinated water. Ten tadpoles were randomly assigned to each test aquarium. Fungicide concentrations represented several orders of magnitude, and were based loosely on those reported for fish on Material

6 Safety Data Sheets. The southern leopard frog LC50 tests served as range-finding 16 tests to determine standard experimental concentrations for all species evaluated in the experiments. Egg exposure experiments In egg exposure experiments, three test concentrations (0.1 x LC50, 0.25 x LC50, and 0.5 x LC50) and a control (0 x LC50) were evaluated. Egg exposure experiments were conducted as static tests in 5-L aquaria with one initial fungicide treatment. Wood frog and spotted salamander eggs were tested in consecutive trials in the spring of Aggregates of approximately wood frog eggs and spotted salamander eggs were counted and randomly assigned to 72 and 48 aquaria, respectively. The number of egg masses that could be collected at an early developmental stage limited the number of aquaria used in the spotted salamander egg exposure experiment. The experimental setup was a completely randomized block design comprised of three blocks for wood frog eggs and two blocks for spotted salamander eggs. All fungicides were evaluated in the same experiment. In the wood frog experiment, each 24-aquaria block contained two replicates of each test concentration for each fungicide. Each block also contained two control aquaria for two fungicides and an additional two replicates for 0.25 x LC50 concentrations of one fungicide. Each fungicide had a total of four control replicates, six 0.1 x LC50, six 0.5 x LC50 replicates, and eight 0.25 x LC50 replicates. In the spotted salamander egg exposure experiment, each 24 aquaria-block contained two replicates of each test concentration for each fungicide. Each block also contained two control aquaria each for two fungicides and an additional two replicates for 0.25 x LC50 concentrations of

7 one fungicide. Each fungicide had a total of four 0.1 x LC50 and four 0.5 x LC50 17 replicates. Daconi1@U1trex and Chipco@Aliette Signature had six 0.25 x LC50 replicates each, whereas Fore@had four. Daconi1@Ultrex and Fore@had two control aquaria each, whereas Chipco@Aliette Signature had four. Eggs were acclimatized for 4 h before fungicides were added. Larvae were removed from aquaria two to three days after the last observed hatching in control aquaria. Hatching rates were determined for each aquarium using consensus counts, i.e. larvae were repeatedly counted until a consensus number was achieved. Larvae and any remaining unhatched eggs were examined using a dissection microscope or magnifying glass. Numbers and descriptions of gross deformities were recorded. Larvae exposure experiments In larvae exposure experiments, three test concentrations (0.01 x LC50, 0.1 x LC50, 0.5 x LC50) and a control (0 x LC50) were evaluated. Larval experiments were conducted as static tests with pulsed fungicide dosage occurring with 75% water renewal every two weeks. Wood frog eggs were hatched in the laboratory in the spring of Gray treefrog eggs were hatched in the laboratory in late summer A total of 10 tadpoles was randomly assigned to each aquarium in a 48-aquaria completely randomized design composed of four experimental blocks. Each 12-aquaria block contained one replicate of each test concentration for each fungicide. Each block also contained one additional 0.1 x LC50 concentration for one fungicide and two control aquaria. Each fungicide had a total of four 0.01 x LC50 and four 0.5 x LC50 replicates. Daconil@Ultrex and Chipco@Aliette Signature had five 0.1 x LC50

8 replicates each, whereas six. and two control 18 replicates each, whereas Signature had four. At the beginning of the experiment, wood frog tadpoles were at approximate Gosner stage 26 and gray treefrog tadpoles were at approximate Gosner stage 24 [14]. Tadpoles were fed tadpole chow ad libitum so that some food was visible in aquaria at all times. Aquaria received 12 h of artificial light daily and were filtered twice weekly. Tadpole survival was observed daily. Tadpoles were weighed at the beginning of the experiment and again when hind limbs emerged. Weights were recorded as average wet weight per aquarium. Time to metamorphosis was recorded as the number of days to front limb emergence from day 1 of the experiment. Experiments ended when front limbs of tadpoles emerged. Water analysis Water samples were taken from 0.5 x LC50 concentration larval aquaria 24 h after dosage for each fungicide. Samples were sent to University of Guelph Laboratory Services, Guelph, Ontario, for pesticide analysis using mass spectrometry. Statistics LC50 values were calculated using probit analysis [2] and SAS@V7 software [16]. Data were checked for normality using probability plots. Prior to analysis, percent hatching success, percent deformities, and percent survival data were transformed using arc-sine square root transformation [17]. In most cases, data were not normal and in some cases, variances were heterogeneous. Because parametric ANOV A assumptions were violated, nonparametric analyses were used. Minitab 12.1 software [18] was used for nonparametric and chi-square analyses. Kruskal-Wallis analysis of variance by

9 ranks was used to test for differences between different concentration exposure 19 treatment groups (a = 0.05) for all egg experiment data and growth and larval development period data for larvae experiments. Dunn procedure pair-wise multiple comparisons were used when Kruskal-Wallis results were significant [17]. Because there were so many pair-wise comparisons, differences could exist by chance. The experiment-wise errorrate was adjusted (.501 k(k -1).JN(N + 1)/12(1lnu + liny)) to take into account all of the comparisons (L. Revennaugh, personal communication). Chi-square analysis was used for larvae experiment survival and deformity data [17]. Multiple chi-square analysis comparisons were done by comparing responses in different concentrations against responses in controls for each fungicide (a = 0.05). RESULTS Range-finding LC50 tests Median lethal concentrations (LC50) were determined for all commercial fungicides from range-finding toxicity tests on southern leopard frog tadpoles. The 48- h LC50 for Daconil@Ultrex was mg/l. The 48-h LC50 for Chipco@Aliette Signature was mg/l. The 48-h LC50 for Fore@was 1.00 mg/l Egg exposure experiments Hatching success of wood frogs varied in different concentrations offore@ and was significantly lower than other dosages in 0.5 mg/l (p = 0.000: Fig. 1). Test chemicals did not significantly affect hatching success of spotted salamanders (Appendix I, Table 2). Wood frog hatchlings exhibited a significantly greater number

10 'C 70 CI).c u 60 1G :J: 50 -c CI) u ~ 40 CI) Q. 30 a a a b Control Fore Concentration (mg/l) Fig. 1. Median percent of wood frog (Rana sylvatica) eggs hatched in Fore@ concentrations (control is 0.00 mg/l). Value at the base of each bar is number of replicates. Treatments with the same letter at the top of the bar are not significantly different from each other ata = Error bar represents range among replicates.

11 21 'C CI) E~ E 60 CI) E: CI) u ~ CI) Q. 40 a 30 b a a Control Fore Concentration (mg/l) Fig. 2. Median percent defonnities observed in wood frog (Rana sylvatica) hatchlings in replicates offore@ concentrations ranging from 0.00 mgll (control) to 0.50 mgll. Values at the base of each bar are number of replicates for each treatment; four control replicates were used. Treatments with the same letter at the top of the bar are not significantly different from each other at a = Error bar represents range among replicates.

12 of deformities in 0.5 mgll (p = 0.000: Fig. 2). Deformities were 22 characterized as lordosis (curvature of the tailor lower vertebral column), skin blisters, abnormally-shaped abdomens, and edema (distension of the body with fluid). Some hatchlings exhibited more than one deformity. Lordosis was the most common wood frog deformity and was observed in 98% of deformed hatchlings from eggs exposed to 0.5 mg/l offore@. Skin blisters were observed in 96%, edema in 20%, and abnormally-shaped abdomens in 7% of deformed hatchlings from eggs exposed to 0.5mg/L Fore@. Lordosis was observed in a small percentage of both wood frog (1.3%) and spotted salamander (2.0%) control hatchlings and abnormally-shaped abdomens were observed in a small percentage of spotted salamander control hatchlings (0.6%). Test chemicals did not significantly affect hatching success of spotted salamanders (Appendix I, Table 2). Test chemicals did not significantly affect the occurrence of deformities in spotted salamander hatchlings (Appendix I, Table 6), however there was a notable difference between the median percentage of deformed hatchlings in Fore concentrations:::: 0.25 mg/l offore@(100%) and the median percentage of deformed hatchlings in control replicates (2.6%). Two control replicates and four replicates of each test concentration were tested in the spotted salamander egg experiment. Fleiss' procedure for estimating necessary sample sizes to detect differences between proportions [17, 19] indicated that sample sizes were not adequate to detect the 97.4% differences in proportions of deformed hatchlings in the spotted salamander egg experiment. Samples sizes of 6 aquaria for each treatment concentration and 3 control aquaria were needed to detect these differences with 80% power.

13 23 Larvae exposure experiments Survival of wood frog tadpoles was significantly decreased in 0.5 mg/l (p = :Fig.3). Survival of gray treefrog tadpoles was significantly decreased in 0.1 mg/l and 0.5 mg/l Fore@ (p = 0.041, P = 0.000, respectively :Fig. 3) and 28.3 mg/l Chipco@ Aliette Signature (p = :Fig. 4). The median growth of wood frog in different treatment groups ranged from g (Appendix II, Table 9). The median growth of gray tree frog tadpoles in different treatment groups ranged from g (Appendix II, Table 10). Median periods of larval development in different treatments were days for wood frogs (Appendix II, Table 13) and days (Appendix II, Table 14) for gray treefrogs. Growth and larval development periods of wood frogs and gray treefrogs were not significantly affected by treatments. No treatments resulted in significantly greater occurrences of deformities in wood or gray treefrog tadpoles, however wood frog metamorphs from Fore@ concentrations::: 0.1 mg/l exhibited a greater number of deformities (p = :Fig.5). All deformities were hind limb deformities where the motility of one or both hind limbs was restricted by a locked femur-tibiofibula joint (knee joint). The same deformity was observed in four gray treefrog metamorphs exposed to Fore@ concentrations::: 0.1 mg/l, however these observations were not statistically significant (Appendix II, Table 22). Water analysis Water analysis revealed a chlorothalonil concentration of mg/l in a sample (n = 1) taken from a larval experiment 60-L aquarium 24 h after dosage. The

14 1 a) 0.9 't:j Q) 0.8.~ 0.7 ::::J 0.6 CJ) c: ~ Q. 0.3 e 0.2 2: *" Q. 0.1 a Control 0.01 J Fore Concentration (mg/l) b) "C 0.8 C1) > 0.7 'E ::::J 0.6 * CJ) c :e D- 0.3 e Q * 17?Q ~A 0 C2J Control Fore Concentration (mg/l) Fig. 3. Survival of a) wood frog (Raila sy/vatica) tadpoles and b) gray treefrog (Ry/a chrysoscelis) tadpoles in Fore :If concentrations ranging from 0.00 mgll (control) to 0.50 mg/l. Value at the base of each bar is number of individuals that survived in that treatment * denotes proportions that differed significantly from expected proportions (controls).

15 25 "Q).- > ~ ~ rn c o ~ oq, ~ D o * 23'. Control Chipco Aliette Concentration (mg/l) Fig. 4. Survival of gray treefrog (Hyla chrysoscelis) tadpoles in Chipco@ Aliette concentrations ranging from 0.00 mg/l (control) to mg/l. Value at the base of each bar is number of individuals that survived in that treatment. * denotes proportions that differed significantly from expected proportions (controls).

16 'i 0.8 E 0.7 ~ 0.6 c r: 0.5 o :e o n o Control 0.01 * 0.10 *...~~.i,.~-"*..~...iy.i I( tj~~ ~ ;1,..: j{'~ 0.50 Fore Concentration (mg/l) Fig. 5. Deformities in wood frog (Rana sylvatica) metamorphs exposed to Fore@ fungicide throughout larval development. Control aquaria contained 0.00 mg/l of Fore@. Value at the base of each bar is number of deformed metamorphs in that treatment. * denotes proportions that differed significantly from expected proportions (controls).

17 expected concentration of chlorothalonil from a mgil dosage ofdaconil@ 27 Ultrex fungicide was approximately mg/l. Water analysis revealed a fosetyl-al concentration of24 mg/l in a sample (n = 1) taken 24 h after a Chipco@ A1iette fungicide treatment containing approximately mg/l fosetyl-al. The slight differences between dosage and analysis concentrations for chlorothalonil and fosetyl-al may have been due to nonuniform mixing of the chemical in the commercial formulation or aquaria water, or experimental error in the dosing procedure. Water analysis also detected an ethiobisdithiocarbamate fungicide at a concentration of 0.05 mg/l in a water sample taken 24 h after a Fore@ fungicide treatment containing approximately 0.40 mg/l mancozeb. The lowered mancozeb concentration was hypothesized to be primarily due to a rapid degradation of mancozeb, however absorption to sediment could have also reduced concentrations. DISCUSSION Fungicides were not significantly toxic to spotted salamander eggs. However, sample sizes in the spotted salamander experiment were small and Fleiss' procedure for estimating necessary sample sizes indicated that greater numbers of replicates were needed to detect differences in proportions. Experiments should be repeated with a greater number of replicates to determine more accurately the toxicity of test chemicals to spotted salamander eggs. Fore@ dosages were toxic to all anuran species tested. Specific effects were reduced hatching success and greater occurrence of deformities in egg experiments, and reduced survival and greater occurrence of deformities in tadpole experiments.

18 Differences existed in the responses of individual organisms to exposure. 28 Differences in responses are common in toxicology studies and likely reflect natural variation that is attributable to different genetic makeup and condition of individuals [2]. In previous studies, mancozeb exposure has caused hind limb paralysis and mortality in rats, lung lesions in pigeons, and supressed immune systems and increased thyroid-stimulating hormone levels in humans [20-23]. Ethylenethiourea (ETD) is a degradation product of mancozeb, and is reported to cause cancer and birth defects in experimental animals [24]. The similarities between findings in this study and that of Harris et al. [14] provide strong evidence for toxicological effects on amphibian species with exposure to mg/l mancozeb in chronic or repeated exposure conditions. Harris et al. observed 0% hatching success of green frog (Rana c/amitans) eggs in Dithane@ DG concentrations containing ~ 7.8 mg/l of mancozeb. In this study, wood frog hatching success was significantly decreased in Fore concentrations containing ~ 0.4 mg/l mancozeb. Harris et al. noted a significantly greater number of deformities than expected in Dithane@ DG concentrations containing mg/l mancozeb. Similarly, wood frog hatchlings exhibited greater deformities in Fore@ concentrations containing 0.4 mg/l mancozeb in this study. Harris et al. observed mostly lateral and dorsal tail flexure deformities and a small percentage of abdominal edemas. These observations closely matched those of wood frog hatchlings in this study. Harris et al. did not note skin blisters or abdominal deformities not related to edema in tadpoles in their study. They exposed green frog tadpoles to Dithane@ DG continuously for 13 d and found a 13-d LC50 containing mg/l mancozeb. They also exposed green frog tadpoles to Dithane@ DG discontinuously for 16 d and found a

19 16-d LC50 containing 0.20 mg/l mancozeb. In this study, wood frog and gray 29 treefrog tadpole survival was significantly decreased by concentrations containing ~ 0.08 mg/l mancozeb when tadpoles were exposed from two weeks posthatching to metamorphosis. Harris et al. noted effects on growth of green frog tadpoles at Dithane@ DG concentrations containing ~ mancozeb in 16-d discontinuous exposure tests. In this study, wood frog and gray treefrog tadpole growth was not significantly decreased in Fore@ concentrations however, growth was measured as weight whereas growth was measured as length in Harris et al.'s study. Significant differences in growth rates may have been detected with length measurements. Both weight and length measurements are recommended for future studies. Mancozeb degrades rapidly to ETU in the presence of water and oxygen [24]. Water analysis in this study suggested that mancozeb test concentrations degraded by approximately 90% over a 24-h period. The lowered mancozeb concentration was likely due to a rapid degradation of mancozeb to ETU in experimental concentrations and/or some loss ofmancozeb from the water column to the sediment of aquaria. ETU was not measured in this study. Toxicological effects observed with mancozeb treatments may be attributed to ETU or a synergistic or additive effect of both mancozeb and ETU. Harris et al. collected water samples from orchard sites known to use mancozeb fungicides in southern Ontario, Canada. No mancozeb was detected in the samples, however ETU was not tested for. Harris et al. suggested that the environmentally relevant concentrations ofmancozeb were < 0.01 mgll. Given the findings of Harris et al. and the toxicological effects observed at mancozeb concentrations ~ 0.08 mgil in this study, it is possible that mancozeb application in

20 areas close to amphibian breeding sites could affect developmental success of 30 amphibian larvae negatively. Chronic or repeated exposure of amphibian larvae to mancozeb fungicides at levels matching those used in this study or greater would likely affect persistance of populations in natural environments. Test concentrations in this study were fractions of R. utricu/aria LC50 values. Without specific monitoring, it is often difficult to assess what fungicide concentrations, if any, amphibians are exposed to in natural breeding areas. It is unlikely that levels similar to the 28.3 mg/l Aliette Signature concentration that affected gray treefrog tadpole survival are found in natural waters, except in spill situations. Ultrex concentrations ranged from mg/l to mg/l and contained approximately to mg/l chlorothalonil. Chlorothalonil residues of 5.0 x X mg/l were found in surface water in August 1997 in Sequoia National Park, California, USA at 2000m elevation [13]. Chlorothalonil was also measured at a concentration of 6.5 mg/l in surface water collected in Michigan ( a concentration well above the R. utricu/aria LC50 value observed in the preliminary range-finding tests in this study. No toxic effects of chlorothalonil were observed from chronic exposure of amphibian eggs and larvae in this study, but effects may occur at higher levels or with longer periods of exposure. This study attempted to bridge the gap between field conditions and laboratory environments by including natural pond sediment in test aquaria and using native test species. The extrapolation of lab results to field observations also requires that ecologically relevant levels of pesticides be used in laboratory tests. Field monitoring is

21 needed to determine if levels of mancozeb fungicides are present at toxic levels in 31 natural areas. In addition, the potentially toxic breakdown product ofmancozeb, ETU, should also be monitored. Species-specific responses were observed in this study. If mancozeb or ETU does occur at measurable levels in areas experiencing amphibian declines, laboratory experiments should be conducted with native species of these areas to establish or provide evidence against links between mancozeb application and amphibian declines. REFERENCES 1. Cowman DF, Mazanti LE Ecotoxicology of "new generation" pesticides to amphibians. In Sparling DW, Linder G, and Bishop CA, eds, Ecotoxicology of Amphibians and Reptiles. Society of Environmental Toxicology and Chemistry (SETAC) Pensacola, FL, USA, pp Rand GM Fundamentals of Aquatic Toxicology: Effects, Environmental Fate, and Risk Assessment, 2 nd ed. Taylor and Francis, Washington, DC, USA. 3. Birge WJ, Westerman AG, Spromberg JA Comparative toxicology and risk assessment of amphibians. In Sparling DW, Linder G, and Bishop CA, eds, Ecotoxicology of Amphibians and Reptiles. Society of Environmental Toxicology and Chemistry (SETAC) Pensacola, FL, USA, pp Sparling DW, Bishop CA, Linder G The current status of amphibian and reptile ecotoxicological research. In Sparling DW, Linder G, and Bishop CA, eds,

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24 effects of occupational exposure to mancozeb. Archives of Environmental Health 34 51: Steenland K, Cedillo L, Tucker J, Hines C, Sorenson K, Deddens J, Cruz V Thyroid hormones and cytogenetic outcomes in backpack sprayers using ethylenebisdithiocarbamate (EBDC) fungicides in Mexico. Environmental Health Perspectives 105: Edwards IR, FerryDG and Temple WA Fungicides & related compounds, In Hayes WJ, Laws ER eds, Handbook of Pesticide Toxicology. Academic Press, New York, NY, USA pp 2-4.