Development of Species-Specific Ah Receptor-Responsive Third Generation CALUX Cell Lines with Enhanced Responsiveness and Improved Detection Limits.

The Ah receptor (AhR)-responsive CALUX (chemically activated luciferase expression) cell bioassay is commonly used for rapid screening of samples for the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), dioxin-like compounds, and AhR agonists/antagonists. By increasing the number of AhR DNA recognition sites (dioxin responsive elements), we previously generated a novel third generation (G3) recombinant AhR-responsive mouse CALUX cell line (H1L7.5c3) with a significantly enhanced response to DLCs compared to existing AhR-CALUX cell bioassays. However, the elevated background luciferase activity of these cells and the absence of comparable G3 cell lines derived from other species have limited their utility for screening purposes. Here, we describe the development and characterization of species-specific G3 recombinant AhR-responsive CALUX cell lines (rat, human, and guinea pig) that exhibit significantly improved limit of detection and dramatically increased TCDD induction response. The low background luciferase activity, low minimal detection limit (0.1 pM TCDD) and enhanced induction response of the rat G3 cell line (H4L7.5c2) over the H1L7.5c3 mouse G3 cells, identifies them as a more optimal cell line for screening purposes. The utility of the new G3 CALUX cell lines were demonstrated by screening sediment extracts and a small chemical compound library for the presence of AhR agonists. The improved limit of detection and increased response of these new G3 CALUX cell lines will facilitate species-specific analysis of DLCs and AhR agonists in samples with low levels of contamination and/or in small sample volumes.

Comparative toxicity of pyrethroid insecticides to two estuarine crustacean species, Americamysis bahia and Palaemonetes pugio.

Pyrethroid insecticides are widely used on agricultural crops, as well as for nurseries, golf courses, urban structural and landscaping sites, residential home and garden pest control, and mosquito abatement. Evaluation of sensitive marine and estuarine species is essential for the development of toxicity testing and risk-assessment protocols. Two estuarine crustacean species, Americamysis bahia (mysids) and Palaemonetes pugio (grass shrimp), were tested with the commonly used pyrethroid compounds, lambda-cyhalothrin, permethrin, cypermethrin, deltamethrin, and phenothrin. Sensitivities of adult and larval grass shrimp and 7-day-old mysids were compared using standard 96-h LC50 bioassay protocols. Adult and larval grass shrimp were more sensitive than the mysids to all the pyrethroids tested. Larval grass shrimp were approximately 18-fold more sensitive to lambda-cyhalothrin than the mysids. Larval grass shrimp were similar in sensitivity to adult grass shrimp for cypermethrin, deltamethrin, and phenothrin, but larvae were approximately twice as sensitive to lambda-cyhalothrin and permethrin as adult shrimp. Acute toxicity to estuarine crustaceans occurred at low nanogram per liter concentrations of some pyrethroids, illustrating the need for careful regulation of the use of pyrethroid compounds in the coastal zone.

Surface charge controls the fate of Au nanorods in saline estuaries.

This work reports the distribution of negatively charged, gold core nanoparticles in a model marine estuary as a function of time. A single dose of purified polystyrene sulfonate (PSS)-coated gold nanorods was added to a series of three replicate estuarine mesocosms to emulate an abrupt nanoparticle release event to a tidal creek of a Spartina -dominated estuary. The mesocosms contained several phases that were monitored: seawater, natural sediments, mature cordgrass, juvenile northern quahog clam, mud snails, and grass shrimp. Aqueous nanorod concentrations rose rapidly upon initial dosing and then fell to stable levels over the course of approximately 50 h, after which they remained stable for the remainder of the experiment (41 days total). The concentration of nanorods rose in all other phases during the initial phase of the experiment; however, some organisms demonstrated depuration over extended periods of time (100+ h) before removal from the dosed tanks. Clams and biofilm samples were also removed from the contaminated tanks post-exposure to monitor their depuration in pristine seawater. The highest net uptake of gold (mass normalized) occurred in the biofilm phase during the first 24 h, after which it was stable (to the 95% level of confidence) throughout the remainder of the exposure experiment. The results are compared against a previous study of positively charged nanoparticles of the same size to parameterize the role of surface charge in determining nanoparticle fate in complex aquatic environments.

Differences in response of two model estuarine crustaceans after lethal and sublethal exposures to chlorpyrifos.

This study assessed the in vitro and in vivo effects of an acetylcholinesterase enzyme inhibitor (chlorpyrifos) in two estuarine crustaceans: grass shrimp (Palaemonetes pugio) and mysid (Americamysis bahia). The differences in response were quantified after lethal and sublethal exposures to chlorpyrifos and in vitro assays with chlorpyrifos-oxon. Results from the in vitro experiments indicated that the target enzyme, acetylcholinesterase (AChE), in the two species was similar in sensitivity to chlorpyrifos inhibition with IC50s of 0.98 nM and 0.89 nM for grass shrimp and mysids, respectively. In vivo experiments showed that mysids were significantly more sensitive to chlorpyrifos-induced AChE inhibition after 24 h of exposure. The in vivo EC50s for AChE inhibition were 1.23 µg L(-1) for grass shrimp and 0.027 µg L(-1) for mysids. Median lethal concentrations (24h LC50 values) were 1.06 µg L(-1) for grass shrimp and 0.068 µg L(-1) for mysids. The results suggest that differences in the response of these two crustaceans are likely related to differences in uptake and metabolism rather than target site sensitivity.

A long-term monitoring study of chlorophyll, microbial contaminants, and pesticides in a coastal residential stormwater pond and its adjacent tidal creek.

Stormwater ponds are commonly used in residential and commercial areas to control flooding. The accumulation of urban contaminants in stormwater ponds can lead to water-quality problems including nutrient enrichment, chemical contamination, and bacterial contamination. This study presents 5 years of monitoring data assessing water quality of a residential subdivision pond and adjacent tidal creek in coastal South Carolina, USA. The stormwater pond is eutrophic, as described by elevated concentrations of chlorophyll and phosphorus, and experiences periodic cyanobacterial blooms. A maximum monthly average chlorophyll concentration of 318.75 µg/L was measured in the stormwater pond and 227.63 µg/L in the tidal creek. Fecal coliform bacteria (FCB) levels were measured in both the pond and the tidal creek that exceeded health and safety standards for safe recreational use. A maximum monthly average FCB level of 1,247 CFU/100 mL was measured in the stormwater pond and 12,850 CFU/100 mL in the tidal creek. In addition, the presence of antibiotic resistant bacteria and pathogenic bacteria were detected. Low concentrations of herbicides (atrazine and 2,4-D: ), a fungicide (chlorothalonil), and insecticides (pyrethroids and imidacloprid) were measured. Seasonal trends were identified, with the winter months having the lowest concentrations of chlorophyll and FCB. Statistical differences between the stormwater pond and the tidal creek were also noted within seasons. The tidal creek had higher FCB levels than the stormwater pond in the spring and summer, whereas the stormwater pond had higher chlorophyll levels than the tidal creek in the summer and fall seasons. Chlorophyll and FCB levels in the stormwater pond were significantly correlated with monthly average temperature and total rainfall. Pesticide concentrations were also significantly correlated with temperature and rainfall. Pesticide concentrations in the stormwater pond were significantly correlated with pesticide concentrations in the adjacent tidal creek. Chlorophyll and FCB levels in the tidal creek, however, were not significantly correlated with levels in the pond. While stormwater ponds are beneficial in controlling flooding, they may pose environmental and human health risks due to biological and chemical contamination. Management to reduce residential runoff may improve water quality in coastal stormwater ponds and their adjacent estuarine ecosystems.

Effects of the synthetic pyrethroid insecticide, permethrin, on two estuarine fish species.

Limited toxicity data are available for estuarine and marine species and the widely used pyrethroid insecticide, permethrin. This study determined acute effects of permethrin on survival, lipid peroxidation, acetylcholinesterase activity, and splenocyte proliferation for two fish species found in South Carolina estuaries; juvenile red drum (Sciaenops ocellatus) and adult mummichog (Fundulus heteroclitus). Juvenile S. ocellatus were significantly more sensitive than adult F. heteroclitus to permethrin exposure, with a 96-h LC50 value of 8 µg/L determined for red drum compared to 23 µg/L for mummichog. Lipid peroxidation activity of the liver increased in permethrin-treated fish compared to control animals after 24 h and decreased after 96 h. Permethrin had no effect on acetylcholinesterase activity of the brain at the concentrations tested. Permethrin exposure significantly inhibited splenocyte proliferation, indicating an immunosuppressive effect. Most of the effects of permethrin on fish cellular stress enzymes and survival occurred at concentrations much higher than those typically measured in the environment. However, inhibition of splenocyte proliferation in juvenile red drum occurred at approximately twice that of measured permethrin concentrations in surface water. These findings may prove useful to the future management and regulation of pyrethroid insecticide use near estuarine habitats.

Acute toxic effects of endosulfan sulfate on three life stages of grass shrimp, Palaemonetes pugio.

In this study, the toxicity of endosulfan sulfate, the primary degradation product of the insecticide endosulfan, was determined in three life stages of the grass shrimp (Palaemonetes pugio). After 96 h exposure to endosulfan sulfate, the grass shrimp adult LC50 was 0.86 microg/L (95% CI 0.56-1.31), the grass shrimp larvae LC50 was 1.64 microg/L (95% CI 1.09-2.47) and the grass shrimp embryo LC50 was 45.85 microg/L (95% CI 23.72-88.61 microg/L). This was compared to the previously published grass shrimp 96-h LC50s for endosulfan. The toxicity of the two compounds was similar for the grass shrimp life stages with adults more sensitive than larvae and embryos. The presence of sediment in 24h endosulfan sulfate-exposures raised LC50s for both adult and larval grass shrimp but not significantly. The USEPA expected environmental concentrations (EEC) for total endosulfan and endosulfan sulfate and the calculations of risk quotients (RQ) based on the more sensitive adult grass shrimp 96-h LC50 clearly show that environmental concentrations equal to acute EECs would prove detrimental to grass shrimp or other similarly sensitive aquatic organisms. These results indicate that given the persistence and toxicity of endosulfan sulfate, future risk assessments should consider the toxicity potential of the parent compound as well as this degradation product.

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.

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.

Lethal and sublethal effects of the pyrethroid, bifenthrin, on grass shrimp (Palaemonetes pugio) and sheepshead minnow (Cyprinodon variegatus).

This study investigated the lethal and sublethal effects of the pyrethroid insecticide bifenthrin on adult and larval grass shrimp, Palaemonetes pugio, and adult sheepshead minnows, Cyprinodon variegatus. The effects were determined by conducting 96-h aqueous static renewal tests and 24-h static tests with sediment. Oxidative stress biomarkers, lipid peroxidation, glutathione, and catalase were also assessed. The 96-h aqueous LC50 value for adult shrimp was 0.020 microg/L (95% CI: 0.015-0.025 microg/L) and for larval shrimp was 0.013 microg/L (95% CI: 0.011-0.016 microg/L). The 96-h aqueous LC50 for adult sheepshead minnow was 19.806 microg/L (95% CI: 11.886-47.250 microg/L). The 24-h sediment LC50 for adult shrimp was 0.339 microg/L (95% CI: 0.291-0.381 microg/L) and for larval shrimp was 0.210 microg/L (95% CI: 0.096-0.393 microg/L). The oxidative stress assays showed some increasing trends toward physiological stress with increased bifenthrin concentrations but they were largely inconclusive. Given the sensitivity of grass shrimp to this compound in laboratory bioassays, additional work will be needed to determine if these exposure levels are environmentally relevant.

Toxicity comparison of the shoreline cleaners Accell Clean® and PES-51® in two life stages of the grass shrimp, Palaemonetes pugio.

Oil spills are a significant source of coastal pollution. Shoreline cleaners, used to remove oil from surfaces during spill response and remediation, may also act as toxins. Adult and larval grass shrimp, Palaemonetes pugio, were tested for lethal and sublethal impacts from two shoreline cleaners, Accell Clean SWA® and PES-51®, alone and in combination with crude oil using Chemically Enhanced Water Accommodated Fractions (CEWAFs). Median lethal toxicity values determined for the individual cleaners were similar. However, when tested in mixture with oil as CEWAFs, Accell Clean SWA resulted in greater hydrocarbon concentrations in the water column and greater toxicity than PES-51. Increased glutathione levels were observed for adult shrimp exposed to Accell Clean SWA, and glutathione was elevated in shrimp exposed to both CEWAFs. Larval shrimp development was delayed after exposure to both CEWAFs. These findings may have implications for managing and mitigating oil spills.

Agricultural pesticides and selected degradation products in five tidal regions and the main stem of Chesapeake Bay, USA.

Nutrients, sediment, and toxics from water sources and the surrounding airshed are major problems contributing to poor water quality in many regions of the Chesapeake Bay, an important estuary located in the mid-Atlantic region of the United States. During the early spring of 2000, surface water samples were collected for pesticide analysis from 18 stations spanning the Chesapeake Bay. In a separate effort from July to September of 2004, 61 stations within several tidal regions were characterized with respect to 21 pesticides and 11 of their degradation products. Three regions were located on the agricultural Delmarva Peninsula: The Chester, Nanticoke, and Pocomoke Rivers. Two regions were located on the more urban western shore: The Rhode and South Rivers and the Lower Mobjack Bay, including the Back and Poquoson Rivers. In both studies, herbicides and their degradation products were the most frequently detected chemicals. In 2000, atrazine and metolachlor were found at all 18 stations. In 2004, the highest parent herbicide concentrations were found in the upstream region of Chester River. The highest concentration for any analyte in these studies was for the ethane sulfonic acid of metolachlor (MESA) at 2,900 ng/L in the Nanticoke River. The degradation product MESA also had the greatest concentration of any analyte in the Pocomoke River (2,100 ng/L) and in the Chester River (1,200 ng/L). In the agricultural tributaries, herbicide degradation product concentrations were more strongly correlated with salinity than the parent herbicides. In the two nonagricultural watersheds on the western shore, no gradient in herbicide concentrations was observed, indicating the pesticide source to these areas was water from the Bay main stem.

Effects of salinity on oil dispersant toxicity in the eastern mud snail, Ilyanassa obsoleta.

Chemical dispersants can be a beneficial method for breaking up oil slicks; however, their use in mitigation could pose potential toxic effects on the marine ecosystem. Dispersants may be transported to lower salinity habitats, where toxicity data for aquatic species have not been established. This study examined the effect of salinity on oil dispersant toxicity in the eastern mud snail, Ilyanassa obsoleta, using two dispersants authorized for oil spill response, Corexit® 9500A and Finasol® OSR 52. Median lethal toxicity values (LC50) and sublethal effects were examined at 10, 20, and 30 ppt salinity in adult and larval mud snails. Two biomarkers (lipid peroxidation and acetylcholinesterase) were used to measure sublethal effects. The 96-h static renewal LC50 values indicated significant differences in toxicity between dispersants and salinities. Larval snails were significantly more sensitive than adult snails to both dispersants, and both life stages were significantly more sensitive to Finasol than to Corexit. Larval snails were more sensitive to dispersants at lower salinity, but adult snails were more sensitive at higher salinities. Dispersants increased lipid peroxidation and decreased acetylcholinesterase activity. These results demonstrate that dispersant toxicity varies among compounds and organism life stages, and that physicochemical properties of the environment, such as salinity, can affect the potential toxicity to estuarine species.

A survey of trace element distribution in tissues of the dwarf sperm whale (Kogia sima) stranded along the South Carolina coast from 1990-2011.

Few studies report trace elements in dwarf sperm whale (Kogia sima). As high trophic level predators, marine mammals are exposed through diet to environmental contaminants including metals from anthropogenic sources. Inputs of Hg, Pb, and Cd are of particular concern due to toxicity and potential for atmospheric dispersion and subsequent biomagnification. Liver and kidney tissues of stranded K. sima from coastal South Carolina, USA, were analyzed for 22 trace elements. Age-related correlations with tissue concentrations were found for some metals. Mean molar ratio of Hg:Se varied with age with higher ratios found in adult males. Maximum concentrations of Cd and Hg in both tissues exceeded historical FDA levels of concern, but none exceeded the minimum 100µg/g Hg threshold for hepatic damage. Tissue concentrations of some metals associated with contamination were low, suggesting that anthropogenic input may not be a significant source of some metals for these pelagic marine mammals.

Relationship between uptake capacity and differential toxicity of the herbicide atrazine in selected microalgal species.

Microalgal species vary in their sensitivity to the triazine herbicide, atrazine. This study examined both atrazine uptake and cellular characteristics of microalgae to determine if either can be used to predict algal sensitivity. Standard toxicity tests were performed on five microalgal species, each representing a different algal division or habitat. Test species listed in order of increasing sensitivity were: Isochrysis galbana, Dunaliella tertiolecta, Phaeodactylum tricornutum, Pseudokirchneriella subcapitata, and Synechococcus sp. Each species was exposed to 14C-atrazine at its growth rate EC50 concentration (44-91 microg/L). At five time-points over 96 h, samples were filtered to collect algae and washed with unlabeled atrazine to displace labeled atrazine loosely absorbed to the cell surface. Radioactivity present on filters and in the growth medium was measured by liquid scintillation counting. Relationships between algal species-sensitivity to atrazine and compound uptake, cell dry weight, cell volume, and cell surface area were determined by linear regression analysis. Cell size measurements (based on dry weight, biovolume, and surface area) were significantly correlated with atrazine uptake (R2 > 0.45, P-value < 0.05). There was a significant correlation between atrazine uptake and species-sensitivity to atrazine (R2 = 0.5413 , P-value = 0.0012). These results indicate that smaller cells with greater surface area to volume ratios will incorporate more atrazine, and in general, will be more sensitive to atrazine exposure. However, I. galbana, with small cell size and relatively high atrazine uptake was the least sensitive species tested. This species and others may have mechanisms to compensate for atrazine stress that make predicting responses of microalgal communities difficult.

Environmental effects and fate of the insecticide bifenthrin in a salt-marsh mesocosm.

Bifenthrin is a widely used synthetic pyrethroid insecticide that is often applied to crops, turf, and residential structures for the control of insects. Like other insecticides, bifenthrin has the potential to contaminate bodies of water that are adjacent to the application site via spray drift and runoff during storm events. The objective of this study was to examine the lethal and sublethal effects of bifenthrin on grass shrimp, Palaemonetes pugio, and sheepshead minnow, Cyprinodon variegatus in a 28 d mesocosm experiment under estuarine conditions. Endpoints included mortality and growth and the oxidative stress biomarkers of lipid peroxidation, glutathione, and catalase. In the mesocosm experiment, 24 h and 96 h caged shrimp LC50s were 0.061 and 0.051 µg L(-1), respectively. The uncaged grass shrimp 28 d LC50 was 0.062 µg L(-1). Fifty percent mortality was not reached in the uncaged sheepshead minnow. Bifenthrin did not have a significant effect on the growth of the shrimp, but there was an increasing impact on fish growth. However, it is uncertain as to whether this pattern is a direct effect of the chemical or if it is due to increased food availability resulting from mortality in prey species. The oxidative stress assays were largely inconclusive. Bifenthrin was eliminated rapidly from the water column and readily partitioned to sediments. The LC50s for adult and larval P. pugio were below published Estimated Environmental Concentration (EEC) values and were within the range of bifenthrin concentrations that have been measured in rivers, channels, and creeks.

Comparative risk assessment of permethrin, chlorothalonil, and diuron to coastal aquatic species.

The precise application of risk assessment can lead to different conclusions about risk depending on how species are grouped in the assessment. We compared the use of different risk assessment methods for three different classes of pesticide, the herbicide diuron, the fungicide chlorothalonil, and the insecticide permethrin for marine and estuarine species. Permethrin was the most toxic pesticide to marine and estuarine crustaceans. Diuron was the most toxic pesticide to algae, and chlorothalonil was most toxic to early life stages of molluscs and other invertebrates. Toxicity data (96 h LC50/EC50 values) were analyzed using a probability distribution on the ranked toxicity values and 10th centile values were calculated based on different groups of species and for all species combined. Our results indicate that an assessment of risk based on smaller taxonomic groups can be informative, especially for pesticides of less specific modes of action such as chlorothalonil.

Pilot estuarine mesocosm study on the environmental fate of Silver nanomaterials leached from consumer products.

Although nanosilver consumer products (CPs) enjoy widespread availability, the environmental fate, leaching, and bioaccumulation behaviors of silver nanoparticles (AgNPs) from these products are not well understood. In this work, three nanosilver CPs, two AgNP standards, and an ionic silver (Ag(+)) standard were studied in estuarine mesocosms. The CPs exhibited long-term release of significant amounts of silver over a 60d residence time in the mesocosms, and ultimately released 82 - 99% of their total silver loads. Measurements of total silver as a function of time, by inductively coupled plasma mass spectrometry (ICP-MS), indicated that the silver was transferred from the water column and accumulated in the estuarine biota, including hard clams, grass shrimp, mud snails, cordgrass stalks and leaves, biofilms, intertidal sediment, and sand. The ICP-MS results and calculations of bioconcentration and trophic transfer factors indicated that significant amounts of silver were taken up by the organisms through trophic transfer. Silver was also adsorbed from the seawater into the biofilms, sediment, and sand, and from the sand into the clams.

Transfer of gold nanoparticles from the water column to the estuarine food web.

Within the next five years the manufacture of large quantities of nanomaterials may lead to unintended contamination of terrestrial and aquatic ecosystems. The unique physical, chemical and electronic properties of nanomaterials allow new modes of interaction with environmental systems that can have unexpected impacts. Here, we show that gold nanorods can readily pass from the water column to the marine food web in three laboratory-constructed estuarine mesocosms containing sea water, sediment, sea grass, microbes, biofilms, snails, clams, shrimp and fish. A single dose of gold nanorods (65 nm length x 15 nm diameter) was added to each mesocosm and their distribution in the aqueous and sediment phases monitored over 12 days. Nanorods partitioned between biofilms, sediments, plants, animals and sea water with a recovery of 84.4%. Clams and biofilms accumulated the most nanoparticles on a per mass basis, suggesting that gold nanorods can readily pass from the water column to the marine food web.

Effects of the anti-fouling herbicide Irgarol 1051 on two life stages of the grass shrimp, Palaemonetes pugio.

This study investigated lethal and sublethal effects (glutathione, lipid peroxidation, cholesterol, and acetylcholinesterase) of the anti-fouling herbicide Irgarol 1051 on larval and adult grass shrimp (Palaemonetes pugio). The 96-hour LC50 test for larvae resulted in an estimated LC50 of 1.52 mg/L (95% confidence interval [CI] 1.26-1.85 mg/L). The adult 96-h LC50 was 2.46 mg/L (95% CI = 2.07-2.93 mg/L). Glutathione, lipid peroxidation, cholesterol and acetylcholinesterase levels were not significantly affected in adult grass shrimp by exposure of up to 3.00 mg/L irgarol. Lipid peroxidation and acetylcholinesterase levels in the larvae were significantly higher than controls in the highest irgarol exposures of 1.0 and 2.0 mg/L, respectively. Cholesterol levels were significantly reduced in larvae in all four irgarol concentrations tested while glutathione levels were not significantly affected in larvae. Both lethal and sublethal effects associated with irgarol exposure were only observed at concentrations well above those reported in the environment.