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Extramural Research

Final Report: Environmentally-Mediated Endocrine Disruption in Estuarine Crustaceans: A 3-Taxon Multi-Generational Study of Sediment-Associated EDC Effects from the Genetic to Population Levels

EPA Grant Number: R827397
Title: Environmentally-Mediated Endocrine Disruption in Estuarine Crustaceans: A 3-Taxon Multi-Generational Study of Sediment-Associated EDC Effects from the Genetic to Population Levels
Investigators: Chandler, G. Thomas , Ferry, John L. , Fulton, Michael H. , Quattro, Joseph M. , Scott, Geoffrey I. , Wirth, Edward F.
Institution: University of South Carolina at Columbia
EPA Project Officer: Turner, Vivian
Project Period: April 1, 1999 through March 31, 2002
Project Amount: $1,265,102
RFA: Endocrine Disruptors (1999)
Research Category: Economics and Decision Sciences , Endocrine Disruptors

Description:

Objective:

The objective of this research project was to construct a coupled abiotic contaminant transformation:reproductive impact model for known or suspected endocrine-active chemicals (EACs) contrasted in three ecologically important estuarine crustaceans: grass shrimp, amphipods, and benthic copepods. This model assessed potential mechanisms by which environmental processing of contaminants (e.g., UV photolysis) affect their apparent toxicological properties in the environment and how biochemical manifestations of toxicants are expressed and ultimately impact crustacean population fitness and maintenance at the genetic, reproductive, and population levels. This approach evaluated strengths of linkages between molecular and biochemical disruption and subsequent outcomes at the reproductive and population level. The hypothesis tested was that contaminants such as endosulfan (ES), fipronil (FP), chlorpyrifos, atrazine (ATZ), and polycyclic aromatic hydrocarbon (PAH) photoactivation products disrupt normal neuroendocrine/hormonal pathways, inducing mediated effects at molecular-to-cellular-to-organism-to-population levels of biological organization in crustaceans, and that these effects are manifested in a fashion that allows development of robust ecological risk assessment models.

Summary/Accomplishments (Outputs/Outcomes):

The common crustacean yolk protein vitellin (VTN) and the molting hormone 20-hydroxyecdysone (20HE) were explored as potential biochemical endpoints of endocrine—or at least reproductive—disruption in our crustacean models. Fluorescence-based enzyme-linked immunosorbent assays (ELISAs) and microscopy-based techniques were successfully developed and validated for VTN and 20HE quantification, and in vivo yolk localization. Coupled with chronic toxicity tests, these biochemical endpoints represented potential biomarkers for EAC exposure. Descriptions and applications of these assays for assessing endocrine toxicity in Crustacea are outlined throughout this report.

Ecdysteroid ELISA

A highly sensitive, fluorescent ELISA was developed for nonradiometric quantification of 20HE—the biologically active metabolite of the crustacean molting hormone ecdysone. This assay utilizes polyclonal antibodies raised specifically to 20HE, and is capable of detecting 20HE in a single adult copepod (Amphiascus tenuiremis), individual copepod egg sacks, and amphipod (Leptocheirus plumulosus) embryos. Furthermore, the assay is capable of differentiating 20HE titers between early and late embryo developmental stages for both crustacean species. Based on the assay’s sensitivity and crossreactivity, we used 20HE as an endpoint for detecting endocrine-mediated reproductive effects in our model crustaceans.

Vitellin/Vitellogenin ELISA

Vitellogenin (VTG) has been widely used as a biomarker of estrogenic exposure in fish, leading to the development of standardized assays for VTG quantification. Alternatively, standardized quantitative assays for invertebrate, particularly crustacean, lipovitellin are lacking. For VTN ELISA development, polyclonal antibodies were raised against purified amphipod (L. plumulosus) VTN and tested for crossreactivity with copepod and grass shrimp VTN. These polyclonal antibodies are specific for purified L. plumulosus VTN as determined by immunoblotting and immunofluorescence microscopy, and are strongly crossreactive with partially purified copepod (A. tenuiremis) and grass shrimp (Palaemonetes pugio) VTN. Thus, these anti-VTN antibodies were used to develop and validate an indirect, competitive ELISA for the quantification of VTN in all three crustaceans. In routine assays, the working range of the VTN ELISA was 31.25-1,000 ng VTN/mL (75-25 percent maximum antibody binding [B/B0]) with a 50 percent B/B0 intra- and interassay variation of 3.9 percent (N = 9) and 12.5 percent (N = 26), respectively. This ELISA is capable of detecting as low as 2 ng-VTN/mL, and can accurately detect VTN in as few as four copepods. The ELISA significantly discriminated positive (gravid female) and negative (male) grass shrimp, amphipod, and copepod samples.

Semiquantification of Embryo Yolk

Confocal laser-scanning microscopy (CLSM) represents a powerful but largely unexplored, ecotoxicological tool for rapidly assessing in vivo effects of toxicants on marine invertebrate embryo quality and development. We developed a new semiquantitative CLSM approach for assessing relative yolk quantity in marine invertebrate embryos (harpacticoid copepods) produced by parents reared from hatching to adult in waterborne or sediment-associated contaminants. This method is based on fluorogenic labeling of embryo yolk using the fluorescent yolk-specific probe BODIPY® 505/515 (Molecular Probes) and subsequent statistical analysis of areal pixel intensities over multiple Z-series CLSM sections using a General Linear Model (GLM)-Nested ANOVA procedure.

Grass Shrimp (P. pugio) Exposures

UV-A Irradiation and ES. Although much is known regarding photoperiodic effects on crustacean egg production, the effects of UV light on reproduction has not been investigated. Likewise, little is known concerning the interaction between UV and xenobiotic exposure on crustacean reproductive cycles. In this study, male and female grass shrimp were exposed to sublethal concentrations of ES (0.2 µg/L and 0.4 µg/L ES) under both white fluorescent (WF) and UV-A (315-400 nm) light conditions for 50 days in laboratory bioassays. Female endocrine (VTG, ecdysteroids, and cholesterol), reproductive (percent gravid, clutch size), and embryo (days to hatch, hatching success, and hatching survival) responses were assessed. UV-exposure alone caused a significant (> fourfold) increase in total P. pugio female egg production over the course of 50 days. Exposure to ES and UV significantly lowered the percentage of gravid females relative to UV controls, whereas ES-exposed shrimp under WF lighting did not exhibit these trends. Although higher VTG concentrations and lower ecdysteroid titers were correlated with increased female egg production, cholesterol titers only exhibited a dose-dependent change when exposed to ES. Embryos from females exposed to UV had significantly lower ecdysteroid titers and shorter hatching times, but there were no differences in embryo VTG concentrations, hatching success, or hatching survival. ES simply countered UV-mediated stimulatory reproductive effects and either: (1) induced shunting of female energy away from reproduction to xenobiotic metabolism; or (2) interacted with UV-triggered neurotransmitter signaling.

Chlorpyrifos and FP. Organophosphate (OP) insecticides are widely used throughout the United States as agricultural and urban pesticides and are among the highest priority pesticides for review under the 1996 Food Quality Protection Act. OPs exert their toxicity through inhibition of acetylcholinesterase, an enzyme involved in terminating nerve impulse transmission at cholinergic synapses. Chlorpyrifos is a potent OP recently restricted to agricultural applications. Phenylpyrazole insecticides are rapidly gaining use throughout the United States, replacing compounds such as chlorpyrifos, and generally target gamma-aminobutyric acid (GABA)-gated chloride channels in arthropods. As one of the first phenylpyrazoles introduced for pest control, FP has a high affinity for insect GABA-receptors and currently is registered in the United States for residential fire ant control, turf grass management, and rice cultivation. In this study, male and female grass shrimp were exposed to chlorpyrifos (0, 0.1, and 0.2 µg/L) or FP (0, 0.1, or 0.2 µg/L) under WF light conditions for 45 days in laboratory bioassays. The occurrence of gravid females, body weight and length, cholesterol, ecdysteroids, and VTG in gravid females was determined to elucidate possible endocrine responses to chronic chlorpyrifos or FP exposure. Chlorpyrifos treatments were not significantly toxic, but FP significantly decreased adult survival at 0.2 µg/L (19.6 percent decrease relative to controls). Gravid female body weight and length were not different across all chlorpyrifos and FP treatments. In addition, chlorpyrifos or FP at 0.1 or 0.2 µg/L did not significantly affect total gravid female production based on total survival for more than 45 days. Chlorpyrifos or FP did not significantly increase or decrease gravid female VTG concentrations. Although chlorpyrifos or FP did not affect gravid female cholesterol titers, there were significant increases in ecdysteroid titers after exposure to 0.1 and 0.2 µg/L nominal chlorpyrifos. Enhanced ecdysteroid titers were not observed under FP exposure. Overall, we observed no biologically significant chlorpyrifos or FP effects on female grass shrimp egg production and related reproductive parameters (VTG, cholesterol, and ecdysteroids). Although ecdysteroid titers were higher in gravid females exposed to chlorpyrifos, we observed no significant effects on body weight and length.

Development of a Microplate-Based Marine Copepod Life-Cycle Toxicity Test

Few rapid (< 30 days) screening tools are available to assess contaminant effects on invertebrate reproduction. In the early phases of this project, we developed a microplate-based copepod toxicity test that focused on rapid rearing of exposed or unexposed male and female copepods (A. tenuiremis) through the development of their reproductive organs to mating, fertilization, egg extrusion, and hatch. (This assay may be easily extended if multiple clutch or posthatch assessments are desired per individual female). This microplate method evaluates teratogenesis, growth, development rates, sex ratio change, sex-specific fertility, and reproductive success with > 90 percent control survival rates. Throughout all phases of this project, this microplate-based bioassay was tested and standardized using a suite of chemicals (see example data below), and currently is under review by the American Society for Testing and Materials and the Organisation for Economic Co-operation and Development as a standard guide for aquatic toxicity testing and screening of potential endocrine disrupting chemicals.

A Methodological Summary of This Toxicity Test. In each of one or more treatments and a control(s), individually isolated A. tenuiremis are maintained and reared in 144 or more individual microwell (300 µL) test chambers from immediately after birth (less than 24 hours old) or at the first juvenile stage until sexual maturity and production of progeny. Microwells are dispersed among three low-binding, hydrophilic microplates per treatment. The solutions in the microwell test chambers during maturation are renewed every 3 days, and copepods are fed a 1:1 mixed algal cell suspension (107 cells/mL) every 6 days. In each of one or more control treatments, at least 144 isolated copepods (one copepod per microwell) are maintained in seawater containing the carrier solvent or a selected concentration of test material. Copepod survival and the number of days it takes for development from a first stage nauplius or juvenile copepodite to a reproductively mature copepod are checked and recorded daily. Once copepods have matured to adults, the gender of each copepod is determined and individual male:female mating pairs are allowed to mate for 12 days in new, isolated microwell test chambers with seawater containing control solvent or test material. The solutions in the microwell test chambers during reproduction are renewed and fed as mentioned above. After single pairing of adult male and female copepods in each control and treatment solution, each mating pair is checked daily for the following data: number of days for a female to extrude the first brood, number of days between first brood extrusion and hatching of first-generation nauplii, number of hatched and surviving nauplii, number of unhatched or necrotic eggs, and the number of females to produce viable offspring over the entire mating period. The test is terminated 12 days past the median time of first brood release in the control treatment(s) to allow for delays in first brood release by copepods exposed to the test material. This microplate-based life-cycle toxicity test has a duration of approximately 24 days.

Copepod (A. tenuiremis) Exposures

FP. FP is a novel GABA receptor-specific phenylpyrazole insecticide commonly used near estuarine environments for rice production, turf-grass management, and residential insect control. In this study, we evaluated the acute, developmental, and reproductive toxicity of FP to the estuarine harpacticoid copepod A. tenuiremis. FP was highly toxic to A. tenuiremis (adult 96 hours LC50 = 6.8 µg/L) and was more toxic to male copepods (96 hours LC50 = 3.5 µg/L) than nongravid female copepods (96 hours LC50 = 13.0 µg/L). Using a microplate-based toxicity test, we successfully reared single juvenile (Stage-I copepodite) individuals of A. tenuiremis to adulthood in 200-µL microwells and concurrently assessed developmental and reproductive effects (after paired virginal matings) of environmentally relevant aqueous FP concentrations (0.16, 0.22, and 0.42 µg/L measured). Throughout the entire life cycle, copepod survival in all treatments was greater than 90 percent. However, FP at 0.22 µg/L and higher significantly delayed male and female development from Stage-I copepodite to adult by approximately 2 days. More importantly, FP significantly halted female egg extrusion by 71 percent in the 0.22 µg/L FP treatment and nearly eliminated reproduction (94 percent failure) in the 0.42 µg/L FP treatment. A three-generation Leslie matrix-based population growth model of FP reproductive and life-cycle impacts predicted a 62 percent decline in A. tenuiremis population size relative to controls at only 0.16 µg/L. Interestingly, acute FP neurotoxicity seems unlikely as male and female copepods appeared healthy and were reproductively active at all FP concentrations tested. In fact, multiple spermatophore sacs on microwell bottoms—an observation indicative of active copulation—were noted in all treatments throughout the monitored mating period (9 days). However, FP-induced reproductive malfunctions such as alterations in male spermatogenesis likely account for low reproductive success at greater than 0.2 µg/L.

Sex-specific reproductive and transgenerational effects of the GABA-disrupting insecticide FP also were determined for the copepod, A. tenuiremis, using a microplate life-cycle bioassay. More than 340 individual Stage-I juvenile copepodites were reared to adult in 12 days in only 200 µL of control (CTL) or 0.63 µg FP/L seawater solution. Individual virgin male:female pairs were crossmated for all possible combinations within and across rearing treatments and allowed to mate for 12 days in CTL or 0.63 µg FP/L solution. Environmentally realistic FP concentrations (0.63 µg/L) showed no significant lethality to any mating combination, but had strong, sex-linked impacts on copepod reproduction. At this concentration, FP evoked 73 and 89 percent inhibition of reproduction when FP-reared males were mated with either control- or FP-reared females in FP solution, respectively. In contrast, when CTL-reared males were mated with FP-reared females in FP solution, there was no reduction in reproductive success. When FP-reared males were mated with either female group in FP-free solution, these mating pairs displayed a 3-day delay in time to brood sac extrusion and ultimately did reproduce. Sublethal FP concentrations had greater reproductive impacts on male A. tenuiremis than females, suggesting that sex can play a significant role in determining risk of FP to estuarine copepods.

Atrazine. ATZ is one of the most widely used herbicides in the United States. ATZ concentrations in coastal environments typically range from 0.09-46 µg/L, with rare but measured concentrations near 60 µg/L at edge-of-field conditions. The objectives of this study were to assess developmental and reproductive effects of ATZ over multiple generation exposures of the copepod A. tenuiremis. Copepods were exposed to sublethal ATZ concentrations (2.5, 25, and 250 µg/L) using a microplate life-cycle bioassay. Individual Stage-I copepodites (C1, n = 60/treatment) were reared through two generations (F0 and F1) to sexual maturity and individually mated in microwells containing 200 µL of ATZ solution. Copepod survival across all treatments and generations was less than 95 percent. ATZ did not significantly affect development times to reproductive maturity, time to female egg extrusion, or time to egg hatch. However, reproductive failures increased across generations with increasing ATZ concentrations. Reproductive failures in the 0, 2.5, 25, and 250 µg/L ATZ treatments were 11, 11, 20, and 24 percent for the F0 and 4, 9, 26, and 38 percent for the F1 generation, respectively. Compared to controls, total nauplii production per female was reduced by approximately 22 percent in F0 females exposed to 250 µg/L ATZ, and by approximately 23, 27, and 23 percent in F1 females exposed to 2.5, 25, and 250 µg/L ATZ treatments, respectively. The combined effect of reproductive failure and reduced offspring production significantly reduced total population growth in the F1 generation, even at ATZ concentrations lower than that considered safe for seawater chronic exposure (26 µg/L). Interestingly, a small proportion of the F1 copepods exposed to ATZ (2, 2, and 6 percent for the 2.5, 25, and 250 µg/L treatments, respectively) showed distinct malformations of the urosome and/or caudal rami. These malformations also were observed in ATZ-reared F2 generation in the 2.5 and 25 µg/L treatments (3 percent incidence for both treatments). None of these malformations have been observed in less than 1,000 control copepods in similar microplate bioassays in our laboratory. Individuals showing malformations were unable to swim, and the majority did not survive to sexual maturity; those that survived to adulthood were unable to produce viable offspring.

Chrysene. We chose the common PAH, chrysene, as a model toxicant to investigate the utility of our lipovitellin-based CLSM egg/embryo quality screening tool. Chrysene has a chemical structure that is steroidal in nature, particularly when photo-oxidized by UV light to 6-hydroxychrysene. This photo-oxidation product exhibits antiandrogenic properties in vitro in mammalian models, and may have endocrine-active properties in marine invertebrates. In this study, we hypothesized that vitellogenesis may be affected in female copepods (A. tenuiremis), and that these effects could be detected in day-old embryos via fluorogenic labeling with the yolk-specific probe BODIPY® 505/515, and direct CLSM photomultiplier-based measurement. This fluorescent yolk-labeling method was able to detect statistically significant differences in yolk concentrations in marine copepod eggs/embryos from females exposed to UV and chrysene-contaminated sediments. Yolk intensities in embryos from females cultured throughout their life cycles in clean sediments were statistically identical with or without UV exposure. UV exposure had no discernible effect on embryo yolk content. In contrast, yolk intensities in embryos of females cultured throughout their life cycle in chrysene-contaminated sediments were significantly higher in the non-UV exposed 2,500 ng-chrysene/g-sediment (65.7 percent higher) and UV-exposed 500 ng-chrysene/g-sediment (76.6 percent higher) treatments.

Environmental Chemistry Studies

ES. The sulfite ester insecticide ES (alpha and beta isomers) is known to undergo competing hydrolysis and oxidation in the environment. The partitioning between those pathways is of particular concern because the oxidation pathway leads to a known endocrine disrupter, ES sulfate. Although photolysis has no observable effect on the fate of ES, we have found that various surfaces are selective in preferentially catalyzing the hydrolysis of beta- to alpha-ES (light and dark). In this study, we investigated the effect of suspended solids on the oxidation and hydrolysis of ES (alpha and beta) and its degradation products: ES diol, ES sulfate, ES ether, and ES lactone in 0.001M NaHCO3 buffer (pH 8.15). Suspensions of sea sand, TiO2, alpha-Fe2O3, alpha-FeOOH, Laponite®, and SiO2 all catalyzed the hydrolysis of ES to the less toxic ES diol, whereas suspended creek sediment (Bread and Butter Creek, SC, 4 percent OC) inhibited ES hydrolysis. Heterogeneous and homogeneous rate constants of ES hydrolysis were measured and indicate that beta-ES hydrolyzes faster than alpha-ES. This observation was explained by a more stable transition state for beta-ES that was confirmed with ab initio molecular orbital calculations (STO-6G) on the anionic intermediates of ES hydrolysis. Rates of ES hydrolysis over the different surfaces corresponded to their tritium-exchange site density and suggest a mechanism involving surface coordination prior to nucleophilic attack. The oxidation of alpha- and beta-ES to the persistent pollutant ES sulfate was not observed in this study. The effect of stereoselective hydrolysis on ES in the environment was measured in three model estuarine mesocosms spiked with alpha-ES, beta-ES, and a mixture of alpha- and beta-ES (technical grade). In our model estuaries, beta-ES hydrolyzed rapidly with little available for biodegradation to ES sulfate. Our results indicate that approximately 75 percent of total ES to ES sulfate conversion in estuaries is from biodegradation of the alpha stereoisomer. In addition, ES hydroxyether was identified as a product of ES in the mesocosm water column, and ES gamma-hydroxyacid, the alkaline hydolysis product of ES lactone, was identified as a terminal product of ES degradation (carboxylate pKa = 4.3).

Fipronil. A multivariate model of the fate of FP was developed in the laboratory to predict its degradation rate and product profile in the presence of sunlight, natural organic matter, bicarbonate, and nitrate. There were several abiotic pathways available for FP degradation in this system, including direct photolysis and oxidation by hydroxyl radical, singlet oxygen, or hydrogen peroxide. However, product studies indicated that FP was quantitatively converted to desthio-FP, a product that is associated with direct photolysis alone. It was observed that natural organic matter acted to decrease the rate of FP degradation, either by competition for photons or transient oxidants. This model was applied to predict the fate of FP in modular estuarine salt-marsh mesocosms. At test initiation, technical-grade FP (0.5 µg/L) was directly spiked into three replicate mesocosms, and the loss of aqueous FP was monitored over 28 days. Aqueous FP concentrations rapidly decreased over 28 days, and kinetic modeling suggests that FP loss occurred in two distinct phases with an initial rate of loss (t < 96 hours) approximately fourfold higher. FP sulfide—the reduction product of FP—was not detected in aqueous samples, but rapidly formed in sediments as the major product of FP degradation. FP sulfone—the major biological oxidation product—was rapidly generated in the water column, but trace amounts were detected in sediments. FP desulfinyl—the direct photolysis product—was present in all samples, with rapid formation in the water column and steady increases in sediments over 28 days. In summary, FP rapidly partitioned into the sediment and rapidly reduced into FP sulfide (approximately 50-70 percent of total sediment concentrations) as a result of the anoxic estuarine (salt marsh) sediments.


Journal Articles on this Report : 26 Displayed | Download in RIS Format

Other project views: All 61 publications 26 publications in selected types All 26 journal articles

Type Citation Project Document Sources
Journal Article Bejarano AC, Chandler GT. Reproductive and developmental effects of atrazine on the estuarine meiobenthic copepod Amphiascus tenuiremis. Environmental Toxicology and Chemistry 2003;22(12):3009-3016. R827397 (2002)
R827397 (Final)
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Journal Article Bejarano AC, Pennington PL, DeLorenzo ME, Chandler GT. Atrazine effects on meiobenthic assemblages of a modular estuarine mesocosm. Marine Pollution Bulletin 2005;50(11):1398-1404
abstract available  
R827397 (Final)
not available
Journal Article Block DS, Bejarano AC, Chandler GT. Ecdysteroid concentrations through various life-stages of the meiobenthic harpacticoid copepod, Amphiascus tenuiremis and the benthic estuarine amphipod, Leptocheirus plumulosus. General and Comparative Endocrinology 2003;132(1):151-160. R827397 (2002)
R827397 (Final)
not available
Journal Article Cary TL, Chandler GT, Volz DC, Walse SS, Ferry JL. Phenylpyrazole insecticide fipronil induces male infertility in the estuarine meiobenthic crustacean Amphiascus tenuiremis. Environmental Science & Technology 2004;38(2):522-528. R827397 (2002)
R827397 (Final)
  • Full-text: ACS Publications Full Text
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  • Other: ACS Publications PDF
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  • Journal Article Chandler GT, Green AS. Developmental stage-specific life-cycle bioassay for assessment of sediment-associated toxicant effects on benthic copepod production. Environmental Toxicology and Chemistry 2001;20(1):171-178.
    abstract available   full text available
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    R825279 (Final)
    R826399E02 (Final)
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  • Journal Article Chandler GT, Cary TL, Volz DC, Walse SS, Ferry JL, Klosterhaus SL. Fipronil effects on estuarine copepod (Amphiascus tenuiremis) development, fertility, and reproduction: A rapid life-cycle assay in 96-well microplate format. Environmental Toxicology and Chemistry 2004;23(1):117-124. R827397 (Final)
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    Journal Article Chandler GT, Cary TL, Bejarano AC, Pender J, Ferry JL. Population consequences of fipronil and degradates to copepods at field concentrations: An integration of life cycle testing with Leslie matrix population Modeling. Environmental Science & Technology 2004;38(23):6407-6414
    abstract available  
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    Journal Article Chandler GT, Volz DC. Semi quantitative confocal laser scanning microscopy applied to marine invertebrate ecotoxicology. Marine Biotechnology 2004;6(2):128-137. R827397 (2002)
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    Journal Article Chandler G, Volz DC, Cary TL. Standard guide for conducting renewal microplate-based life-cycle toxicity tests with marine meiobenthic copepods. ASTM Standards for Aquatic Toxicology and Hazard Evaluation. R827397 (2002)
    R827397 (Final)
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    Journal Article Griffitt RJ, Chandler GT, Greig TW, Quattro JM. Cathepsin B and glutathione peroxidase show differing transcriptional responses in the grass shrimp, Palaemonetes pugio following exposure to three xenobiotics. Environmental Science & Technology 2006;40(11):3640-3645
    abstract available  
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    Journal Article Kong L, Ferry JL. Effect of salinity on the photolysis of chrysene adsorbed to a smectite clay. Environmental Science & Technology 2003;37(21):4894-4900. R827397 (2002)
    R827397 (Final)
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  • Journal Article Kong L, Ferry JL. Photochemical oxidation of chrysene at the silica gel-water interface. Journal of Photochemistry and Photobiology A-Chemistry 2004;162(2-3):415-421. R827397 (Final)
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    Journal Article Schizas NV, Chandler GT, Coull BC, Klosterhaus SL, Quattro JM. Differential survival of three mitochondrial lineages of a marine benthic copepod exposed to a pesticide mixture. Environmental Science & Technology 2001;35(3):535-538.
    abstract available   full text available
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    R825439 (1999)
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  • Full-text: ResearchGate - Abstract & Full Text HMTL
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  • Abstract: ES&T-Abstract
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  • Journal Article Schizas N, Coull B, Chandler G, Quattro J. Sympatry of distinct mitochondrial DNA lineages in a copepod inhabiting estuarine creeks in the southeastern USA. Marine Biology 2002;140(3):585-594.
    abstract available  
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  • Journal Article Staton JL, Schizas NV, Chandler GT, Coull BC, Quattro JM. Ecotoxicology and population genetics: The emergence of "phylogeographic and evolutionary ecotoxicology". Ecotoxicology 2001;10(4):217-222. R827397 (2002)
    R827397 (Final)
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    Journal Article Staton JL, Schizas NV, Klosterhaus SL, Griffitt RJ, Chandler GT, Coull BC. Effect of salinity variation and pesticide exposure on an estuarine harpacticoid copepod, Microarthridion littorale (Poppe), in the southeastern US. Journal of Experimental Marine Biology and Ecology 2002;278(2):101-110.
    abstract available   full text available
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    Journal Article Volz DC, Wirth EF, Fulton MH, Scott GI, Block DS, Chandler GT. Endocrine-mediated effects of UV-A irradiation on grass shrimp (Palaemonetes pugio) reproduction. Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 2002;133(3):419-434. R827397 (2002)
    R827397 (Final)
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    Journal Article Volz DC, Kawaguchi T, Chandler GT. Purification and characterization of the common yolk protein, vitellin, from the estuarine amphipod Leptocheirus plumulosus. Preparative Biochemistry & Biotechnology 2002;32(2):103-119. R827397 (2002)
    R827397 (Final)
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    Journal Article Volz DC, Wirth EF, Fulton MH, Scott GI, Strozier E, Block DS, Ferry JL, Walse SS, Chandler GT. Effects of fipronil and chlorpyrifos on endocrine-related endpoints in female grass shrimp (Palaemonetes pugio). Bulletin of Environmental Contamination and Toxicology 2003;71(3):497-503. R827397 (2002)
    R827397 (Final)
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    Journal Article Volz DC, Chandler GT. An enzyme-linked immunosorbent assay for lipovitellin quantification in copepods: A screening tool for endocrine toxicity. Environmental Toxicology and Chemistry 2004;23(2):298-305. R827397 (2002)
    R827397 (Final)
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    Journal Article Walse SS, Shimizu KD, Ferry JL. Surface-catalyzed transformations of aqueous endosulfan. Environmental Science & Technology 2002;36(22):4846-4853.
    full text available
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    Journal Article Walse SS, Scott GI, Ferry JL. Stereoselective degradation of aqueous endosulfan in modular estuarine mesocosms: formation of endosulfan gamma-hydroxycarboxylate. Journal of Environmental Monitoring 2003;5(3):373-379. R827397 (Final)
    R829397 (Final)
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    Journal Article Walse SS, Morgan SL, Kong L, Ferry JL. Role of dissolved organic matter, nitrate, and bicarbonate in the photolysis of aqueous fipronil. Environmental Science & Technology 2004;38(14):3908-3915.
    full text available
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    Journal Article Wirth EF, Lund SA, Fulton MH, Scott GI. Determination of acute mortality in adults and sublethal embryo responses of Palaemonetes pugio to endosulfan and methoprene exposure. Aquatic Toxicology 2001;53(1):9-18.
    abstract available   full text available
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    Journal Article Wirth EF, Lund SA, Fulton MH, Scott GI. Reproductive alterations in adult grass shrimp, Palaemonetes pugio, following sublethal, chronic endosulfan exposure. Aquatic Toxicology 2002;59(1-2):93-99.
    abstract available   full text available
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    Journal Article Wirth EF, Pennington PL, Lawton JC, DeLorenzo ME, Bearden D, Shaddrix B, Sivertsen S, Fulton MH. The effects of the contemporary-use insecticide (fipronil) in an estuarine mesocosm. Environmental Pollution 2004;131(3):365-371. R827397 (2002)
    R827397 (Final)
    not available
    Supplemental Keywords:

    crustacean, copepod, amphipod, grass shrimp, endocrine-active chemicals, pesticides, UV irradiation, reproduction, southeastern United States, fipronil, estuarine, mesocosm, sediments, organic pollutants, sexual development, estrogen, progesterone, polymerase chain reaction, PCR, porewater, Southeast, U.S. EPA Region 4, agriculture, environmental chemistry., RFA, Health, Scientific Discipline, PHYSICAL ASPECTS, Toxics, Geographic Area, Water, Ecosystem Protection/Environmental Exposure & Risk, POLLUTANTS/TOXICS, Contaminated Sediments, exploratory research environmental biology, Environmental Chemistry, Ecosystem/Assessment/Indicators, Health Risk Assessment, Chemicals, HAPS, pesticides, State, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Risk Assessments, Ecological Effects - Environmental Exposure & Risk, Southeast, Physical Processes, Children's Health, Biology, Endocrine Disruptors - Human Health, ecological effects, risk assessment, South Florida, bioindicator, ecological exposure, assays, biomarkers, hydrocarbon, endocrine disrupting chemical, endocrine disrupting chemicals, exposure, sediment, sexual development, contaminated sediment, animal models, ecological impacts, human exposure, toxicity, amphipods, benthic copepods, estrogen response, South Carolina (SC), A 3-Taxon, Florida, hormone production, ecological risk assessment model, grass shrimp, Endosulfan Sulfate, estuarine crustaceans, human health risk

    Progress and Final Reports:
    Original Abstract
    2000 Progress Report
    2002 Progress Report

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    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.

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