Peter B. Key

Assessment of risk reduction strategies for the management of agricultural nonpoint source pesticide runoff in estuarine ecosystems.

Agricultural nonpoint source (NPS) runoff may result in significant discharges of pesticides, suspended sediments, and fertilizers into estuarine habitats adjacent to agricultural areas or downstream from agricultural watersheds. Exposure of estuarine fin fish and shellfish to toxic levels of pesticides may occur, resulting in significant declines in field populations. Integrated pest management (IPM), best management practices (BMP), and retention ponds (RP) are risk management tools that have been proposed to reduce the contaminant risk from agricultural NPS runoff into estuarine ecosystems. Field studies were conducted at three sites within coastal estuarine ecosystems of South Carolina (SC) from 1985 to 1990 that varied in terms of the amount and degree of risk reduction strategies employed. An intensively managed (IPM, BMP, and RP) agricultural treatment site (TRT) was studied for pesticide runoff impacts. From 1985 to 1987, there were minimal (some IPM and BMP) management activities at TRT, but from 1988 to 1990, TRT was managed using an intensive risk reduction strategy. A second unmanaged agricultural growing area, Kiawah (KWA), was also studied and compared with TRT in terms of pesticide runoff and the resulting impacts on grass shrimp (Palaemonetes pugio) and mummichogs (Fundulus heteroclitus). A third, non-agricultural, reference site (CTL) was used for comparing results from the managed and unmanaged agricultural sites. In situ toxicity tests and field samples of the grass shrimp populations were conducted at each site and compared in terms of survival and the effectiveness of current risk reduction strategies. Significant runoff of insecticides (azinphosmethyl, endosulfan, and fenvalerate) along with several fish kills were observed at TRT prior to the implementation of rigorous risk reduction methods. A significant reduction of in stream pesticide concentrations (up to 90%) was observed at TRT following the implementation of strict NPS runoff controls, which greatly reduced impacts on estuarine fish and shellfish. At the unmanaged KWA, continued impacts due to the runoff of these insecticides were observed, along with several fish kills. Additional monitoring indicated that gravid female grass shrimp populations from KWA had elevated levels of P-glycoprotein (P-gp), a multidrug resistance protein, which may transport various pesticides across cellular membranes. Comparison of field results with laboratory toxicity tests established that pesticide exposure was the primary cause of observed field impacts at each site. These findings clearly indicate the value of an integrated risk reduction strategy (BMP, IPM, and RP) for minimizing impacts from NPS agricultural pesticide runoff.

Characterization of cholinesterase activity in tissues of the grass shrimp (Palaemonetes pugio)

Inhibition of cholinesterases in estuarine organisms is a useful indicator of organophosphorus insecticide exposure. Recent research has indicated that more than one cholinesterase may be present in tissues of estuarine organisms and that these cholinesterases vary in their sensitivity to organophosphorus insecticides. Baseline cholinesterase activity for adult grass shrimp (Palaemonetes pugio) was determined with four substrates (acetylthiocholine iodide, acetyl-β-methylthiocholine iodide, propionylthiocholine iodide, S-butyrylthiocholine iodide) and three cholinesterase inhibitors (eserine sulfate, tetraisopropyl pyrophosphoramide, 1,5-bis(4-allyldimethyl-ammoniumphenyl) pentan-3-one dibromide). Cholinesterase activity was the highest with acetylthiocholine iodide at 57.42 nmol/mg P/min and the lowest with acetyl-β-methylthiocholine iodide at 6.17 nmol/mg P/min. The four substrates tested were not inhibited by tetraisopropyl pyrophosphoramide. Eserine sulfate significantly inhibited cholinesterase activity against all substrates except S-butyrylthiocholine iodide. The results of substrate specificity and cholinesterase inhibition in grass shrimp indicate that acetylthiocholine iodide is the most appropriate substrate for assessing cholinesterase inhibition in grass shrimp.

A Review of Grass Shrimp, Palaemonetes spp., as a Bioindicator of Anthropogenic Impacts

Coastal systems are ecologically important environments due to their diversity and productivity, but they can also serve as sinks for pollutants transported via runoff and atmospheric deposition. Grass shrimp, of the genus Palaemonetes, are a common inhabitant of East and Gulf coast estuaries of the United States and are proposed in this paper as a bioindicator of human impacts on estuarine systems. Using grass shrimp as such would involve their entire life cycle and consist of biomonitoring studies, in situ and laboratory toxicity tests, and development of biomarkers of exposure. The current interest in developing ecological indicators has put a renewed relevance on grass shrimp research. This paper reviews ecologically based studies, toxicity testing, and sublethal assessments in grass shrimp and shows how they have laid the groundwork for this genus to be an indicator species. It is concluded that correlating traditional grass shrimp assays with ecological monitoring and biomarkers of exposure is a goa...

Analysis of pesticide runoff from mid-Texas estuaries and risk assessment implications for marine phytoplankton

During 1993, estuarine surface water samples were collected from the mid-Texas coast (Corpus Christi to Port Lavaca, TX). Agricultural watershed areas as well as tidal creeks immediately downstream were chosen as sampling sites along with adjoining bay sampling stations. Collections were made throughout the growing season (February to October 1993) before and after periods of significant (>1.25 cm) rainfall. All samples were initially screened for the presence of pesticides using enzyme-linked immunosorbent assay (ELISA) test kits (EnviroGard™) for triazine herbicides and carbamate insecticides. All samples were extracted and then analyzed using gas chromatography (GC) for quantification of atrazine. Only samples testing positive for carbamate insecticides via ELISA were further extracted for GC analysis to quantify aldicarb and carbofuran. Additionally, laboratory toxicity tests using phytoplankton were examined from published, peer-reviewed literature and compared with the atrazine field levels found in Texas. Results of ELISA screening indicated the presence of triazine herbicides in nearly all samples (>93%). GC analysis further confirmed the presence of atrazine concentrations ranging from <0.01–62.5 μg/L. Screening tests also found detectable levels of carbamate insecticides (aldicarb and carbofuran) that were also confirmed and quantified by GC. Comparison of measured concentrations of atrazine compared with published toxicity tests results indicated that there was a potential environmental risk for marine/estuarine phytoplankton in surface waters of Texas estuaries, particularly when the chronic nature of atrazine exposure is considered.

The sensitivity of grass shrimp, Palaemonetes pugio, embryos to organophosphate pesticide induced acetylcholinesterase inhibition.

Grass shrimp, Palaemonetes pugio, are common inhabitants of salt marshes along the Atlantic and Gulf coasts of North America. Grass shrimp embryos are brooded externally on the abdomen of adult females for about 2 weeks prior to hatching. In South Carolina, the spring spawning period for grass shrimp coincides with the period of peak pesticide application on crops grown along the South Carolina coast. Thus, grass shrimp of all developmental stages are at risk of exposure to pesticides present in nonpoint source agricultural runoff. Organophosphate (OP) insecticides are commonly applied agricultural chemicals which produce toxicity by inhibiting the nervous system enzyme, acetylcholinesterase (AChE). The purpose of this study was to examine the development of AChE activity in grass shrimp embryos and to assess their sensitivity to OP-induced AChE inhibition. Embryos were exposed for 24 h to either chlorpyrifos or malathion. All exposure concentrations were nominal and ranged from 0 to 2.00 µg l(-1) for chlorpyrifos and from 0 to 120.00 µg l(-1) for malathion. Quantifiable levels of AChE activity first appeared at Stage V of development and increased as embryonic development progressed. AChE inhibition by the OPs was assessed in Stage VI and Stage VII embryos. Both stages of embryos were more sensitive to chlorpyrifos than malathion. The 24-h Effective Concentration (EC(50)) values for chlorpyrifos were 0.49 µg l(-1) (95% C.I.=0.33-0.77 µg l(-1)) and 0.36 µg l(-1) (95% C.I.=0.33-0.38 µg l(-1)) for Stage VI and Stage VII embryos, respectively. In comparison, malathion 24-h EC(50) values were 55.53 µg l(-1) (95% C.I.=22.08-80.73 µg l(-1)) for Stage VI embryos and 29.93 µg l(-1) (95% C.I.=25.22-44.22 µg l(-1)) for Stage VII embryos. For both OPs, there were no significant differences in the EC(50) values calculated for Stage VI and Stage VII embryos; however, AChE inhibition was significantly (P</=0.05) greater in Stage VII embryos at the two highest exposure concentrations for each insecticide. A comparison of the results of these embryo tests with those found for adult and larval toxicity tests indicated that embryos were at least as sensitive to both the OPs as larval and adult grass shrimp. Embryo bioassays provide a number of important advantages over traditional laboratory toxicity tests including reduced laboratory space requirements, large numbers of embryos from a few ovigerous females, and small volumes of waste.

Toxicity and physiological effects of brominated flame retardant PBDE-47 on two life stages of grass shrimp, Palaemonetes pugio.

This study examined the effects of a polybrominated diphenyl ether (PBDE) compound, PBDE-47, on adult and larval stages of the estuarine grass shrimp (Palaemonetes pugio). The 96-h LC50 test resulted in an estimate of 23.60 microg/L (95% confidence interval=14.51-38.37 microg/L) for larval shrimp. Adult shrimp had a higher 96-h LC50 of 78.07 microg/L (95% CI=65.1-93.63 microg/L). Four physiological biomarkers glutathione (GSH), lipid peroxidation (LPx), cholesterol (CHL) and acetylcholinesterase (AChE) were then assessed to study the sublethal effects of PBDE-47 exposure. GSH, LPx and AChE levels in both adults and larvae were not affected by PBDE-47 at concentrations up to 50 microg/L for 96 h. CHL levels were elevated in adults and larvae at the lowest exposure concentrations tested, but significant differences were found only in adult exposures. Effects associated with PBDE-47 aqueous exposures were observed at levels well above those reported in the environment.

Using mummichog (Fundulus heteroclitus) arrays to monitor the effectiveness of remediation at a Superfund site in Charleston, South Carolina, USA

We previously developed a cDNA array for mummichogs (Fundulus heteroclitus), an estuarine minnow, that is targeted for identifying differentially expressed genes from exposure to polycyclic aromatic hydrocarbons and several metals, including chromium. A chromium-contaminated Superfund site at Shipyard Creek in Charleston, South Carolina, USA, is undergoing remediation, providing us a unique opportunity to study the utility of arrays for monitoring the effectiveness of site remediation. Mummichogs were captured in Shipyard Creek in Charleston prior to remediation (2000) and after remediation began (2003 and 2005). Simultaneously, mummichogs were collected from a reference site at the Winyah Bay National Estuarine Research Reserve (NERR) in Georgetown, South Carolina, USA. The hepatic gene expression pattern of fish captured at Shipyard Creek in 2000 showed wide differences from the fish captured at NERR in 2000. Interestingly, as remediation progressed the gene expression pattern of mummichogs captured at Shipyard Creek became increasingly similar to those captured at NERR. The arrays acted as multidimensional biomarkers as the number of differentially expressed genes dropped from 22 in 2000 to four in 2003, and the magnitude of differential expression dropped from 3.2-fold in 2000 to no gene demonstrating a difference over 1.5-fold in 2003. Furthermore, the arrays indicated changes in the bioavailability of chromium caused by hydraulic dredging in the summer of 2005. This research is, to our knowledge, the first report using arrays as biomarkers for a weight-of-evidence hazard assessment and demonstrates that arrays can be used as multidimensional biomarkers to monitor site mitigation because the gene expression profile is associated with chromium bioavailability and body burden.

Lethal and sub-lethal effects of the fungicide chlorothalonil on three life stages of the grass shrimp, Palaemonetes pugio.

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 µg/L (95% Confidence Interval [CI] 331.3–472.4 µg/L). The adult 96‐h LC50 was 152.9 µg/L (95% CI 120.3–194.5 µg/L). Larvae were the most sensitive to chlorothalonil exposure with a 96‐h LC50 of 49.5 µg/L (95% CI 44.4–55.27 µg/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 appropriate life stage to use for chlorothalonil risk assessments since that stage is the most sensitive.

Toxicity of the Mosquito Control Pesticide Scourge® to Adult and Larval Grass Shrimp (Palaemonetes pugio)

Abstract This study investigated the toxicity of various concentrations of technical resmethrin and Scourge® on adult and larval Palaemonetes pugio, a common grass shrimp species. Two types of tests were conducted for each of the resmethrin formulations using adult and larval grass shrimp life stages, a 96-h static renewal aqueous test without sediment, and a 24-h static nonrenewal aqueous test with sediment. For resmethrin, the 96-h aqueous LC50 value for adult shrimp was 0.53 μg/L (95% confidence interval (CI): 0.46–0.60 μg/L), and for larval shrimp was 0.35 μg/L (95% CI: 0.28–0.42 μg/L). In the presence of sediment, technical resmethrin produced a 24-h LC50 value for adult shrimp of 5.44 μg/L (95% CI: 4.52–6.55 μg/L), and for larval shrimp of 2.15 μg/L (95% CI: 1.35–3.43 μg/L). For Scourge®, the 96-h aqueous LC50 for adult shrimp was 2.08 μg/L (95% CI: 1.70–2.54 μg/L), and for larval shrimp was 0.36 μg/L (95% CI: 0.24–0.55 μg/L). The 24-h sediment test yielded an LC50 value of 16.12 μg/L (95% CI: 14.79–17.57 μg/L) for adult shrimp, and 14.16 μg/L (95% CI: 12.21–16.43 μg/L) for larvae. Adjusted LC50 values to reflect the 18% resmethrin concentration in Scourge® are 0.37 μg/L (adult), 0.07 μg/L (larvae) for the 96-h aqueous test, and 2.90 μg/L (adult), 2.6 μg/L (larvae) for the 24-h sediment test. Larval grass shrimp were more sensitive to technical resmethrin and Scourge® than the adult life stage. The results also demonstrate that synergized resmethrin is more toxic to P. pugio than the nonsynergized form, and that the presence of sediment decreases the toxicity of both resmethrin and Scourge®

Lethal and sublethal effects of simvastatin, irgarol, and PBDE-47 on the estuarine fish, Fundulus heteroclitus

This study investigated the effects of simvastatin, a lipid-regulating drug; irgarol, an antifouling biocide; and PBDE-47, a brominated flame retardant, on the estuarine fish, Fundulus heteroclitus. Sublethal effects (changes in glutathione (GSH), lipid peroxidation (LPx), acetylcholinesterase (AChE), and cholesterol (CHL) levels) and lethal effects (survival) were determined after individual exposure to the three compounds. There were no significant differences in GSH or CHL levels in fish exposed to any of the test compounds. LPx levels significantly decreased with increasing irgarol concentrations. AChE levels were significantly lower in fish exposed to simvastatin at the 1.25 mg/L concentration and significantly higher at the PBDE-47 concentration of 0.0125 mg/L. The LC50 values were 2.68, 3.22, and > 0.1 mg/L for simvastatin, irgarol and PBDE-47, respectively.