JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH Part B—Pesticides, Food Contaminants, and Agricultural Wastes Vol. B38, No. 5, pp. 539–549, 2003
Lethal and Sub-lethal Effects of the Fungicide Chlorothalonil on Three Life Stages of the Grass Shrimp, Palaemonetes pugio Peter B. Key,1,* Shelli L. Meyer,2 and Katy W. Chung1 1
Center for Coastal Environmental Health and Biomolecular Research, National Ocean Service, Charleston, South Carolina, USA 2 Graceland University, Lamoni, Iowa, USA
ABSTRACT Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) is the second most widely used fungicide in the United States. Due to the widespread use of chlorothalonil, it is important to investigate the effects chlorothalonil may have on estuarine species such as the grass shrimp, Palaemonetes pugio. This study examined the toxicity of chlorothalonil to three life-history stages (embryo, larvae, adult) of the grass shrimp. Also, molting frequency, growth response and metamorphosis from a larval life cycle pulsed exposure assay were examined as sub-lethal indicators of chlorothalonil exposure. Results showed embryos were the least sensitive with a 96-h Median Lethal Concentration (LC50) of 396.0 mg/L (95% Confidence Interval [CI] 331.3 – 472.4 mg/L). The adult 96-h LC50 was 152.9 mg/L (95% CI 120.3 – 194.5 mg/L). Larvae were the most sensitive to chlorothalonil exposure with a 96-h LC50 of 49.5 mg/L (95% CI 44.4 – 55.27 mg/L). In the life cycle pulsed exposure assay, all surviving larvae in the treatments required significantly more molts to reach postlarvae than the control. Other measured parameters showed differences between treatments and control but there was no statistical significance. This research demonstrated that chlorothalonil is highly toxic to grass shrimp and that larval grass shrimp would be the most
*Correspondence: Peter B. Key, Center for Coastal Environmental Health and Biomolecular Research, National Ocean Service, 219 Fort Johnson Road, Charleston, SC, USA; E-mail: Pete.
[email protected]. 539 DOI: 10.1081/PFC-120023512 Copyright D 2003 by Marcel Dekker, Inc.
0360-1234 (Print); 1532-4109 (Online) www.dekker.com
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Key, Meyer, and Chung appropriate life stage to use for chlorothalonil risk assessments since that stage is the most sensitive. Key Words: Chlorothalonil; Fungicide; Grass shrimp; Palaemonetes pugio; Sub-lethal effects; Pulsed exposure assay.
INTRODUCTION Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) is the second most widely used fungicide in the United States. It has a wide range of uses including agriculture, golf course maintenance and paint products. Usage per year has surpassed 14 million pounds with application rates ranging from 0.75 lbs. to >22lbs active ingredient/A.[1] The USEPA[1] reports chlorothalonil as having a relatively short aquatic half-life of 44h while Caux et al.[2] give a range from 7.7h at 15°C in fresh water to up to 10 days in marine water. Chlorothalonil has been found in surface waters of several US states.[3] Scott et al.[4] found chlorothalonil in 84.6% of samples taken in Florida Bay in 1996 and 1997 with a peak concentration of 2.7 ng/L. Chlorothalonil affects fungi by combining with glutathione, a peptide in plant and animal tissue important in the oxidation-reduction process and the activation of enzymes.[1] This leaves enzymes that require glutathione unable to function and interferes with the enzyme role in cellular respiration. Without cellular respiration, the fungus is unable to produce energy, thus leading to chlorothalonil’s toxic effects.[3] Chlorothalonil may also bind with and inhibit glyceraldehyde-3-phosphate dehydrase that will in turn inhibit glycolysis.[2] Chlorothalonil is acutely toxic to fish, crab larvae, shrimp, frogs, and water fleas with LC50s between 100 and 1000ppb.[1,3] Understanding the effect chemicals such as chlorothalonil have on non-target organisms is important for maintaining the ecosystems in which they live. The grass shrimp, Palaemonetes pugio, has a major role in the transfer of energy in an estuarine ecosystem and therefore has been used extensively as a model estuarine invertebrate in toxicity testing.[5,6] Due to the widespread use of chlorothalonil, it is important to investigate the effects chlorothalonil may have on estuarine species such as P. pugio. This study examined the toxicity of chlorothalonil to three life-history stages (embryo, larvae, adult) of the grass shrimp. Also, molting frequency, growth response and metamorphosis from a larval life cycle pulsed exposure assay were examined as sub-lethal indicators of chlorothalonil exposure.
MATERIALS AND METHODS Collection and Maintenance Grass shrimp were collected from Leadenwah Creek (N 32°36’12@ ; W 80°07’00@), a pristine tidal tributary of the North Edisto River Estuary, SC.[7] Shrimp were acclimated in 76-L tanks at 25°C, 20 parts per thousand (%) salinity and 16-h light:8-h dark cycle and fed a mixture of Tetramin1 Fish Flakes and newly hatched Artemia. Gravid females were placed in brooding traps to allow larvae (zoea) to hatch and
Effects of Chlorothalonil on Grass Shrimp
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escape without interference. Larvae from at least 10 females were pooled for all tests. Embryos (Stage VI—defined oval eye, body movement, rapid heartbeat) were excised from the gravid females. Chemical The chemical used for all assays was chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) obtained from Chem Service (West Chester, PA, USA). Purity for the insecticide was 98%. Pesticide grade acetone was used as a carrier (0.1%) in all tests. Acetone was added to the control groups equal to the amount of carrier solvent used for the toxicity tests. 96-h Bioassay The five nominal concentrations of chlorothalonil used for the adult shrimp test were 31.3, 62.5, 125.0, 250.0 and 500.0 mg/L. There were three replicates for each treatment and the control. Each 4L-glass jar held two liters of 20% seawater and 10 adult shrimp were randomly placed in each. The jars were incubated for 96 h with aeration on a 16-h light:8-h dark cycle in a 25°C environmental chamber. Average water quality parameters were temperature 25.6°C, pH 8.0, salinity 20.7% and dissolved oxygen 6.2 mg/L. Every 24 hours dead shrimp were removed, and the test solutions were renewed. Adult shrimp were not fed during the test. A second 96-h test was conducted on larval grass shrimp. The same number of replicates and concentrations as in the adult tests were used. Ten newly hatched larvae were randomly placed into 600-mL glass beakers containing 400 mL of test solution. The larvae were exposed at 25°C and a 16-h light:8-h dark cycle in an environmental chamber. Average water quality parameters were temperature 25.3°C, pH 8.1, salinity 20.1% and dissolved oxygen 6.1 mg/L. Every 24 hours, dead larvae were removed and the test solution was changed. The larvae were fed Artemia every day after changing the test solution. Stage five embryos of grass shrimp were also tested in a 96-h bioassay. The embryos were removed from the female by gently sweeping a probe back and forth along the pleopods. Embryos were then placed one to a well of a 24-well plate for each nominal concentration of 62.5, 125.0, 250.0, 500.0, and 1000.0 mg/L and one control. The test was run in 0.22 mm filtered 20% seawater. Every 24-h the embryos were examined for mortality and the test solution was changed. The plates were kept on a rotating shaker at 80 rpm in an environmental chamber set at 27°C and 24 h of darkness.[8,9] Larval Life-Cycle Pulsed Exposure Assay A larval life cycle pulsed exposure assay was conducted from newly hatched larvae to the juvenile stage.[6] Chlorothalonil pulse exposures were 6h/day (simulating the length of a tidal cycle) every five days (simulating the average number of days between rain events in coastal SC). Each pulse exposure was administered in seawater with a salinity of 10%, simulating field observations of tidal creek salinity after rain events in SC.[7] At the end of each 6-h pulse exposure, animals were placed in clean 20%
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seawater. Seawater (20%) was changed every 24-h between exposures. In addition, a low salinity control (10%) received the same handling as the exposures to assess potential salinity effects. Chlorothalonil exposures were performed on Days 0, 5, 10, 15 and 20 of the test. In previous research, the average number of days to reach postlarval status in grass shrimp collected from Leadenwah Creek was between 20 and 22.[6,10] Newly hatched larvae were exposed in 4-L glass aquaria containing three acrylic cages. Cages were covered with 200-mm mesh NITEX1 screening and contained 10 compartments. One larva was randomly placed into each compartment for each concentration and controls. The nominal concentrations were 31.3, 62.5, 125, 250 , and 500 mg/L. Each exposure aquaria contained 750 mL of media. Tests were conducted in an environmental chamber at 25°C and a 16-h light:8-h dark cycle. Average water parameters in the 20% control were temperature 26.5°C, pH 8.1, salinity 20.3% and dissolved oxygen 6.5 mg/L. Average control water parameters at the end of each 10% pulse dose were 26.3°C, 8.1 pH, 10.8% salinity and 6.1 mg/L dissolved oxygen. Each day before changing the water, molts and dead larvae were counted, recorded and then removed. Larvae were fed Artemia once a day after each water change. When remaining larvae in all concentrations and control reached post-larval status (juvenile stage), the test was terminated. All larvae that reached post-larval status were oven dried for 48 hours at 60°C and the dry weight was determined. Statistics and Analysis Using the Trimmed Spearman-Karber Method,[11] the Median Lethal Concentration (LC50) for each test was calculated with a 95% confidence interval. The lowest concentration that had statistically significant mortality, the Lowest Observed Effect Concentration (LOEC), and the highest concentration with no statistically significant mortality, the No Observable Effect Concentration (NOEC), were determined. The Threshold Concentration, an estimated effects level, was calculated from the geometric mean of the LOEC and NOEC.[9] Analysis of variance (ANOVA) was used to determine if significant group differences (P0.05) existed for the following parameters: dry weight, days to postlarvae and molts to postlarvae. Data were
Table 1. Stage Adult 48-h 96-h Larvae 48-h 96-h Embryo 48-h 96-h
Toxicity values for three grass shrimp life stages exposed to chlorothalonil for 96-h. LC50 (mg/L)
95% CI (mg/L)
LOEC (mg/L)
NOEC (mg/L)
Threshold concentration (mg/L)
203.8 152.9
163.6 – 253.8 120.3 – 194.5
– 31.3
–
