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2002 Progress 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 Period Covered by this Report: April 1, 2001 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 is to construct a coupled abiotic-endocrine disrupting chemical (EDC) transformation/reproductive impact model for known or suspected EDCs contrasted in three ecologically important estuarine species-grass shrimp, amphipods, and benthic copepods. This model will assess mechanisms by which environmental processing of EDCs affect their apparent toxicological properties in the environment, how molecular/cellular manifestations of EDCs are expressed, and how they ultimately impact crustacean population fitness and maintenance at the genetic, reproductive, and population levels. This approach will allow the U.S. Environmental Protection Agency (EPA) to evaluate strengths of linkages between EDC molecular/cellular and genetic disruption, and subsequent outcomes at the population level. The hypothesis being tested is that contaminants such as endosulfan, fipronil, chlorpyrifos, and polycyclic aromatic hydrocarbons (PAH) photo-activation products disrupt normal neuro-endocrine/hormonal pathways, inducing mediated effects at molecularcellularorganismpopulation levels of biological organization in crustaceans; and that these effects are manifested in a fashion that allows development of predictable ecological risk assessment models.

Progress Summary:

Our research to date has assessed the ecotoxicological effects of endosulfan (Year 1), chlorpyrifos (Year 1 and 2), chrysene (Year 1), pyrene (Year 1), fipronil (Year 2 and 3), atrazine (Year 3), and tebufenozide (Year 3) from a molecular to population-level perspective using one or more of our three crustacean models: Palaemonetes pugio (grass shrimp), Leptocheirus plumulosus (amphipod), and Amphiascus tenuiremis (infaunal copepod). Thus far, three classes of compounds have been evaluated for their reproductive toxicity and EDC potential including: (1) organophosphate insecticides (chlorpyrifos); (2) organochlorine insecticides (endosulfan and fipronil); (3) herbicides (atrazine); (4) ecdysteroid-mimicking bisacylhydrazines (tebufenozide); and (5) steroidally structured PAHs with (6-hydroxychrysene) and without (chrysene) UV photo-activation. Specific progress to date in Year 3 of this project was built upon Years 1 and 2, and is as follows:

Ecotoxicology Progress for Year 3

ELISA and Microscopy-Based Assays for Assessing Endocrine Toxicity. In Years 1, 2, and 3, 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 these crustaceans. Fluorescence-based microplate assays (ELISAs) were successfully developed and validated (Year 2) for the quantification of these two biochemical endpoints, and represent potential screening tools for EDC exposure assessments. Furthermore, semi-quantitative confocal laser-scanning microscopy (CLSM) techniques were developed (Years 1 and 2) to specifically evaluate crustacean embryo vitellin in vivo using the fluorescent vitellin-specific probe BODIPY® 505/515 (Molecular Probes). We found that CLSM represents a powerful, easy, but largely unexplored, ecotoxicological tool for rapidly assessing in vivo effects of EDCs on crustacean embryo quality and development. Applications of these assays for assessing endocrine toxicity in crustacea are outlined below.

Copepod EDC Exposures. Microplate-based bioassays were completed in Year 3, with Amphiascus tenuiremis exposed to fipronil. The following results were seen with regard to endocrine toxicity:

Fipronil. Stage 1 A. tenuiremis copepodites were reared individually to adults (12 days) in 0 ng/L and 600 ng/L and analyzed for lipovitellin VTN concentrations prior to mating. There were no significant differences in virgin male VTN titers exposed to 0 ng/L or 600 ng/L. In contrast, virgin females from the 600 ng/L fipronil treatment exhibited significantly higher VTN concentrations than control females. In a similar experiment (see below), fipronil-exposed females extruded eggs (when mated with control males) just as well as control females, and clutch sizes in both treatments were not significantly different. Fipronil-induced VTN expression appears to have no affect on female reproductive capability, but most likely serves as a protective mechanism to shunt off bioavailable fipronil. Additionally, we initially hypothesized that fipronil-induced male infertility (see below) may be due to female-like VTN induction. These results suggest that VTN induction in males is not a factor governing their inability to successfully inseminate females, and fipronil most likely affects hormonally-regulated spermatogenic pathways.

Males reared in 0, 220, 360, and 600 ng/L and that successfully inseminated virgin females showed no difference in ecdysteriod titers relative to controls. In contrast, those males linked to female reproductive failures showed significantly higher ecdysteroid levels at 220 ng/L, while unsuccessful males exposed to 360 ng/L exhibited significantly lower ecdysteroid titers. The biological relevance of this finding is unclear, given that higher fipronil exposures (600 ng/L) did not significantly elevate ecdysone levels in reproductive-failing males. On the other hand, male/female pairs that were reproductively unsuccessful in all fipronil treatments exhibited similar trends in ecdysteroid induction or suppression, suggesting a hormonal role in reproductive failure.

Grass Shrimp EDC Exposures. Aqueous laboratory bioassays were completed in Years 2 and 3, with Palaemonetes pugio exposed to: (1) UV or non-UV irradiated endosulfan; (2) chlorpyrifos; or (3) fipronil. The following results were seen with regard to endocrine toxicity:

UV-A and Endosulfan. Male and female grass shrimp, Palaemonetes pugio, were exposed to sublethal concentrations of endosulfan (200 ng/l and 400 ng/l ES) under both white fluorescent (WF) and UV-A (315-400 nm) light conditions for 50 days in laboratory bioassays. Female endocrine (vitellogenin, 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 (> 4-fold) 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 vitellogenin 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 vitellogenin concentrations, hatching success, or hatching survival. These results indicate that UV-A exposure has a pronounced effect on grass shrimp (P. pugio) reproduction, and is likely mediated through 5-hydroxytrptamine (5-HT)-related neuroendocrine pathways.

Chlorpyrifos and Fipronil. Male and female grass shrimp, P. pugio, were exposed to sublethal concentrations of chlorpyrifos (0, 100, and 200 ng/L) or fipronil (0, 100, or 200 ng/L) under white fluorescent light conditions for 45 days in laboratory bioassays. Chlorpyrifos treatments were not significantly toxic, whereas fipronil significantly reduced adult survival at 200 ng/L (19.6 percent relative to controls). Gravid female P. pugio body weight and length were not significantly different across all chlorpyrifos and fipronil treatments. Furthermore, chronic exposure to chlorpyrifos or fipronil at 100 or 200 ng/L did not significantly affect total gravid female production based on total survival over 45 days. Chlorpyrifos or fipronil did not significantly increase or lower gravid female vitellogenin concentrations. Although chlorpyrifos or fipronil did not significantly affect gravid female cholesterol titers, there was a significant increase in ecdysteroid titers when exposed to both 100 and 200 ng/L nominal chlorpyrifos. Enhanced ecdysteroid titers were not observed under fipronil exposure. Overall, we found that chronic, low-level exposure to chlorpyrifos or fipronil did not have any significant effects on gravid female grass shrimp reproduction. Likewise, neither chlorpyrifos nor fipronil had a significant affect on female vitellogenin concentrations. Although there were increased ecdysteroid titers in gravid females exposed to chlorpyrifos, cholesterol titers and growth were not affected. As a result, ecdysteroid titers, alone, did not lend significant insight into a chlorpyrifos-associated endocrine effect. Thus, elevated ecdysteroid titers may be a function of subtle downstream chlorpyrifos effects on neurotransmitter signaling. Likewise, fipronil appears to have subtle, but not significant, effects on cholesterol. However, these effects, like chlorpyrifos, do not manifest themselves on growth (length and weight). In conclusion, it appears as though, at least under cool fluorescent, laboratory light conditions, that chlorpyrifos and fipronil did not interact with endocrine pathways leading to vitellogenesis and/or steroidogenesis. The reproductive effects of chlorpyrifos and fipronil should be investigated under UV exposure, an environmentally realistic parameter that can increase bioassay sensitivity.

Application of Confocal Laser-Scanning Microscopy (CLSM). 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 ultraviolet light (UV) to 6-hydroxychrysene. This photo-oxidation product exhibits anti-androgenic 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 (Amphiascus 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.

The fluorescent yolk labeling method was able to stain and detect statistically significant differences in yolk concentrations in A. tenuiremis eggs from females exposed to UV and/or chrysene (CHRY)-contaminated sediments. Control yolk intensities in less than 24-hour-old embryos of females cultured throughout their lifecycle in clean sediments were statistically identical with or without UV exposure. In contrast, yolk intensities in 24-hour-old embryos of females cultured throughout their lifecycle in CHRY-contaminated sediments were significantly higher in the non-UV exposed 2,500 ng CHRY/g-sed (67 percent higher) and UV-exposed 500 ng CHRY/g-sed (76 percent higher) treatments. Females exposed to 500 ng CHRY/g-sed without UV exhibited yolk intensities that were significantly lower (18 percent) than UV-exposed females at the same CHRY concentration, but significantly higher (45 percent higher) than both UV and non-UV exposed controls. A five-fold increase in CHRY concentration (2,500 ng CHRY/g-sed) in the absence of UV also enhanced yolk deposition to eggs, but yolk levels were modestly, but significantly, lower (6 percent) than eggs from the UV-exposed 500 ng CHRY/g-sed treatment. Chrysene exposure during maturation to female reproductive maturity significantly enhanced yolk deposition to eggs/embryos and was strongly enhanced by UV irradiation. Although the direct mechanism of CHRY-induced yolk deposition is unknown, CHRY may exhibit hormonal properties that mimic endogenous crustacean hormones such as ecdysteroids.

Microplate-Based Life-Cycle Reproductive and Endocrine Toxicity Tests. In Year 1, we developed techniques for the complete life-cycle culture of individual copepods (A. tenuiremis) from egg hatching or Stage I copepodite to F1 egg extrusion in 200 µL microwell plates in only 3 weeks. In Year 2, we improved this 96-well rapid screening tool to provide: (1) consistently less than 95 percent control survival; (2) a 12-15 day run time; and (3) a focus upon reproductive/developmental time windows and endpoints. Few rapid screening tools are available to assess EDC effects on invertebrate reproduction, and no rapid assays have been developed for sediment infauna. This approach provides a unique reproductive life-cycle screening assay focusesd on the rapid rearing of exposed/unexposed males and females through the development of their reproductive organs to mating, fertilization, egg extrusion, and hatch. Furthermore, the assay may be easily extended if multiple clutch or posthatch assessments are desired per individual female. Also, as A. tenuiremis is normally an infaunal, sediment-ingesting copepod, this model allows assessment of both waterborne and sediment-associated EDC effects. To this end, we tested for chemical effects on A. tenuiremis juvenile development, successful maturation to reproductive adult, disruption of fertilization, egg quality, and egg development/hatching.

Fipronil. In Year 2, a definitive microplate assay of the recently registered GABA-blocking insecticide, fipronil, showed significant delays in development, 95 percent reproductive failure, and a significant shift in sex ratios at 600 ng/L. The lowest concentration studied (110 ng/L), yielded significant reproductive failures in mated pairs, but no sex ratio changes. In Year 3, another microplate assay was performed in which Stage-I copepodite juveniles were reared individually to mature adults in 0 (carrier control), 0.36, 0.6, or 1.5 ng/mL fipronil. Individual juveniles were tracked daily for developmental delays, anomalies in secondary sex characteristics, and time to reproductive adult. On day 12, individual pairs of virgin males and virgin females from within each treatment/control group were transferred to clean seawater microwells and allowed to crossmate for 5 days. Each pair was then scored as to whether or not they produced viable embryos, and how many embryos were produced per female (i.e., clutch size). Fipronil caused large and significant reproductive failures (i.e., zero egg production) for pairs reared up in 0.36, 0.6, and 1.5 ng/mL, but fipronil caused no significant differences in numbers of viable embryos produced per female for those few females that were able to reproduce. Fipronil also caused a significant reversal in sex ratios from the normal 60 percent female:40 percent male to 40:60 female:male, and caused significant delays in the number of days from mating to egg extrusion in all treatments. Fipronil caused no mortality significantly different from controls (< 10 percent) at these concentrations. The sex ratio changes, developmental and reproductive effects of fipronil on projected population size was predicted using the forementioned Leslie matrix stage-structured model and an empirically-parameterized Monte Carlo multigenerational simulation. This model predicted that after only three generations, exposures as low as 0.2 ppb fipronil would cause an approximately 80 percent reduction in the population size of A. tenuiremis.

We also used this microplate assay to determine whether fipronil reproductive toxicity was sex-linked. In Year 3, a cross-mating experiment was performed with the benthic copepod A. tenuiremis to determine whether males or females were more sensitive to fipronil exposure. We exposed Stage I copepodites to 0 and 600 ng/L fipronil for 12 days. After reaching sexual maturity, females and males were paired in the following conditions for an additional 12 days: (1) control female x control male; (2) control female x 600 ng/L male; (3) 600 ng/L female x control male; and (4) 600 ng/L female x 600 ng/L male. All four pairing conditions occurred in both 0 and 600 ng/L fipronil solutions to determine if copepods recovered when fipronil-exposed individuals were placed into clean water. All four mating conditions in the carrier control treatment had >80 percent reproductive success, but mating condition (2) and (4) exhibited a significant delay of approximately 3 days from pairing to extrusion of an egg sac, when compared to mating conditions (1) and (3). When mated in 600 ng/L fipronil, mating conditions (1) and (3) had less than 80 percent reproductive success, while mating conditions (2) and (4) had significantly depressed reproductive success with means values of 32.2 percent and 20 percent, respectively. In addition, a significant delay (approximately 3 days) from pairing to extrusion of an egg sac was seen in mating condition 2, when compared to mating conditions 1 and 3. These results suggest that males were more sensitive to fipronil exposure than females; but 600 ng/L fipronil-exposed males recover when transferred to clean seawater. Thus, this microplate approach was able to show not only that fipronil suppressed reproductive success in A. tenuiremis, but that this suppression was sex-linked to infertility of fipronil-reared males. In addition, no significant difference was found between any of the mating conditions and their respective control for either number of days from extrusion to hatch of nauplii or brood size (number of unhatched eggs plus number of nauplii). This has been seen in past fipronil exposure experiments, suggesting that once a mated pair has produced a viable clutch, fipronil has little affect on their reproductive success.

Tebufenozide. One of the newest classes of insect growth regulating (IGR) "designer pesticides" are the bisacylhydrazine insecticides, which affect target pest species by mimicking the insect molting hormone, 20-hydroxyecdysone (20HE)-an essential endocrine signaling molecule for insect development and reproduction. Due to their selectivity for lepidopteran pests, the bisacylhydrazines are assumed to possess low toxicity to nontarget organisms, but limited research has been conducted that evaluates adverse effects to aquatic crustacean species. A 96-well microplate life-cycle screening bioassay was conducted with the meiobenthic copepod A. tenuiremis to evaluate potential endocrine modulating effects of tebufenozide. Initially, 48 Stage-I copepodites were exposed in solutions containing 0, 50, 500, and 2,000 µg/L tebufenozide and monitored daily for development. Following adult maturation, individual males and females within each treatment were mated and monitored for reproductive effects. Control organisms exhibited a male:female sex ratio of 57:43, compared to ratios of 43:57, 44:56, and 44:56 for the 50, 500, 2,000 µg/L treatments, respectively. In addition, significant developmental delays were observed with copepodites requiring 10.1 ± 1.1 days (500 µg/L) and 9.8 ± 1.0 days (2000 µg/L) to reach adult maturity, compared to 8.9 ± 1.1 days in control organisms. Tebufenozide caused no significant mating or reproductive effects; however, offspring production was reduced by <28 percent.

Atrazine. Atrazine is one of the most widely used herbicides in U.S. agricultural crop production, with concentrations ranging from less than 10 ng/L to 480 µg/L in estuaries of the Southeast. A 96-well microplate life-cycle screening bioassay was conducted to assess the reproductive and developmental effects of multiple generation exposures of the meiobenthic copepod A. tenuiremis to environmentally relevant atrazine concentrations. Stage 1 copepodites (C1) were followed to adulthood in individual wells containing 200 µL of test solution (0, 2, 20, and 200 µg/L). C1s were monitored daily and several endpoints were recorded. Upon reaching maturation, individual virgin copepods were mated pairwise in wells containing original test concentrations. Offspring from the first brood were reared to adults under an identical exposure regimen as the parent generation. Copepod survival across all treatments and generations were >95 percent. Although atrazine did not affect development to reproductive maturity, time to egg extrusion, or time to egg hatch, less than 6 percent of developing copepodites exhibited mild deformities in the eurosome (i.e., the tail) across all atrazine treatments. Total nauplii production per female pooled over two consecutive broods was reduced by 22 percent (relative to controls) in F0 females exposed to 200 µg/L atrazine and by 23 percent, 27 percent, and 32 percent in F1 females exposed to 2, 20, and 200 µg/L atrazine treatments, respectively (p <0.05). Reproductive failures increased across generations with increasing atrazine concentrations. Reproductive failures were 11, 11, 20 and 24 percent for the F0 and 4, 9, 26, and 38 percent for the F1 in the 0, 2, 20, and 200 µg/L treatments respectively. The combined effect of offspring production reduction and reproductive failure significantly reduced the total viable population production. Compared to controls, F0 total population production was reduced by 34, 42, and 143 nauplii, while F1 total population production was reduced by 87, 255, and 278 nauplii in the 2, 20, and 200 µg/L atrazine treatments, respectively. These findings suggest that multigenerational exposure to atrazine has significant effects on overall population growth, even at concentrations lower than the level considered safe for sea water chronic exposure (26 µg/L).

Environmental Chemistry Progress for Year 3

Endosulfan. The sulfite ester insecticide endosulfan ( and 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 arthropod endocrine disrupter, endosulfan sulfate. Although photolysis has no observable effect on the fate of endosulfan, we have found that various surfaces are selective in catalyzing hydrolysis of endosulfan preferentially to (light and dark). In Years 2 and 3, we investigated the effect of suspended solids on the oxidation and hydrolysis of endosulfan ( and isomers) and its degradation products: endosulfan diol, endosulfan sulfate, endosulfan ether, and endosulfan lactone in 0.001 M NaHCO3 buffer (pH 8.15). Suspensions of sea sand, TiO2, -Fe2O3, -FeOOH, Laponite®, and SiO2 all catalyzed the hydrolysis of endosulfan to the less toxic endosulfan diol. Suspended creek sediment (Bread and Butter Creek SC, 4 percent OC) inhibited endosulfan hydrolysis. Heterogeneous and homogeneous rate constants of endosulfan hydrolysis were measured. These results indicate that -endosulfan hydrolyzes faster than -endosulfan. This observation was explained by a more stable transition state for -endosulfan that was confirmed with ab initio molecular orbital calculations (STO-6G) on the anionic intermediates of endosulfan hydrolysis. Rates of endosulfan 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 -endosulfan and -endosulfan to the persistent pollutant endosulfan sulfate was not observed in this study. The effect of stereoselective hydrolysis on endosulfan in the environment was measured in three model estuarine mesocosms (aqueous volume approximately 300 L; Spartina and associated sediment dwellers), that were spiked with , , and a mixture of and endosulfan (technical grade). In our model estuaries, endosulfan hydrolyzed rapidly and comparatively little was available for biodegradation to endosulfan sulfate. Our results indicate that approximately 75 percent of total endosulfan to endosulfan sulfate conversion in estuaries is from biodegradation of the a stereoisomer. In addition, endosulfan hydroxyether was identified as a product of endosulfan in the mesocosm water column and endosulfan -hydroxy acid, the alkaline hydolysis product of endosulfan lactone, was identified as a terminal product of endosulfan degradation (carboxylate pKa = 4.3).

Fipronil. In Year 3, we investigated the correlation between laboratory and mesocosm studies of the fate of the phenylpyrazole insecticide fipronil. A multivariate model of the fate of fipronil was developed in the laboratory to predict its degradation rate and product profile in the presence of sunlight, natural organic matter (OM), bicarbonate, and nitrate. There were several abiotic pathways available for fipronil degradation in this system, including direct photolysis and oxidation by hydroxyl radical, singlet oxygen, or hydrogen peroxide. However, product studies indicated that fipronil was quantitatively converted to desthio-fipronil, a product that is associated with direct photolysis alone. It was observed that natural OM acted to decrease the rate of fipronil degradation, either by competition for photons or transient oxidants. This model was applied to predict the fate of fipronil in a series modular estuarine mesocosms at the NOS Center for Coastal Environmental Health and Biomolecular Research in Charleston, SC. In these experiments, the loss of aqueous fipronil (single-dose experiment, with initial Fipronil concentrations of 355 and 5,000 ng/L, three replicates at each concentration) was monitored over 28 days. Although the direct photolysis product was detected, the mass balance was dominated by fipronil-sulfone and fipronil-sulfide, products that are a signature of fipronil biodegradation. Nonetheless, direct photolysis appeared to account for 3-14 percent of all fipronil loss in the model environments, at 10 percent ambient UV-B (limited by the structure of the mesocosm). All products were confirmed by comparison of retention times (GC-ECD) and mass spectra (GC-ITMS) to synthesized standards.

Chrysene. In Years 2 and 3, we investigated the photooxidation of chrysene in aqueous and suspended solutions. The rate of photooxidation (l:300-800nm) of the PAH, chrysene, was significantly enhanced in aqueous suspensions of the smectite clay, laponite, relative to its rate of photolysis in aqueous solution. The photodegradation of chrysene was tested at pH 8.30, at several different ionic strengths that correlate to freshwater and saltwater. The kinetics of chrysene loss were first order in chrysene. The photodegradation products 1,4-chrysenediol, 1,4-chrysenequinone, phthalic acid, and 2-formyl benzoic acid were positively identified and quantified against analytical standards. The mechanism of chrysene degradation was probed by comparing the effects of methanol, bicarbonate, diazabicyclooctane, and several halides on the rate of chrysene oxidation. We found that added molecules that react with singlet oxygen can reduce the rate of chrysene loss by >90 percent. Molecules that react with HO had no effect on the rate of chrysene photodegradation. The overall rate of chrysene loss was governed by salinity and the extent of surface coverage. The steady state concentration of singlet oxygen varied positively with chrysene loading, but was significantly lower at salinities comparable to the marine environment. The relative contribution of Cl-, Br-, and I- to the reduction in [1O2]ss were measured. On a mole to mole basis, I- in seawater is shown to be the most effective 1O2 quencher. These results suggest that photoprocessing of particle-bound PAHs may fall off as rapidly as they are transported into marine environments. A predictive kinetic model for the photodegradation of adsorbed chrysene was developed.

Crustacean Eco-Genetics Progress for Year 3

The genetic portion of this project focuses on the development of genetic markers indicative of cyclodiene organochlorine exposure; and, to measure the genetic effects of exposure at the population level and especially for the -aminobutryic (GABA) receptor gene. During the first year of research we concentrated our efforts on sample collection, DNA extraction and development of appropriate polymerase chain reaction (PCR) primers. At the end of Year 2, sampling for genetic evaluation of 966 grass shrimp from 21 locations throughout its range is complete and extraction of DNA from these samples is complete. Collection sites represented both "pristine" waterways and those with a history of cyclodiene exposure. Results for Year 3 are as follows:

Phylogeographic Analysis of Grass Shrimp Genetic Data. We have conducted a mitochondrial DNA survey across P. pugio, P. vulgaris, and P. intermedius using the mtDNA marker, cytb. There is a very strong split between the Atlantic populations and those in the Gulf of Mexico for both P. pugio and P. vulgaris. The split occurs along the eastern coast of Florida, near Cape Canaveral, and has been observed for other organisms. The Atlantic populations are nearly fixed for a single haplotype that is absent in the Gulf of Mexico, while the Gulf of Mexico has a much higher degree of differentiation. P. intermedius is not distinct from P. pugio. We hypothesize that P. intermedius and P. pugio have recently split, but an additional gene will help determine whether this pattern is reflective of polymorphism retention following a recent split, or a long standing divergence. The nuclear vitellogenin locus has been successfully amplified, and we will be testing it for phylogeographic patterns consistent with that of mtDNA. This split combined with incongruent LC50 values that were reported from South Carolina and the panhandle of Florida. As a result, this led us to design a common garden LC50 toxicity test for these two regions. Because the two regions do not share haplotypes, individuals could be genetically identified as to the place of origin following toxicity testing in a common tank. The results indicate little, if any, difference in pesticide resistance between the two areas. To further understand the mechanism contributing to the strong phylogeographic split between the Atlantic and Gulf, we have collected more samples within the region of the break and have begun sequencing them. Interestingly, there appears to be a zone of overlap near Merritt Island, Florida, where most of the individuals possess a Gulf of Mexico-like haplotype; however, a few individuals have Atlantic haplotypes. We are currently trying to characterize the GABA locus using several different methods. Additionally, due to difficulties in amplifying the GABA locus, we have started an "eco-genomics" project to identify candidate genes that may be under selection. An EST (expressed sequence tags) library is being constructed, allowing us to screen at 400 random clones. This information, in conjunction with the phylogeographic data, will help us understand the influence of environmental history on the population genetics of species.

Genetic Resistance of Copepods to Fipronil. Individuals from the fipronil-exposed A. tenuiremis sediment monocultures have been collected for genetic analysis to compare their GABAA beta-like receptor gene sequence to that of non-exposed individuals. At this time, we have successfully isolated a GABAA beta-like receptor fragment from cDNA, and are currently cloning and sequencing additional fragments to increase replication and to gain a more detailed picture of the gene. This information will allow us to use PCR techniques to amplify the GABAA beta-like receptor gene from individual A. tenuiremis and compare genetic variation between exposed and nonexposed populations.

Future Activities:

We will continue to clone and sequence additional fragments to increase replication and to gain a more detailed picture of the gene.


Journal Articles on this Report : 18 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)
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)
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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. R827397 (2000)
    R827397 (2002)
    R827397 (Final)
    R825279 (Final)
    R826399E02 (Final)
  • Abstract from PubMed
  • Abstract: Wiley
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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)
    R827397 (Final)
    not available
    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)
    not available
    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 Schizas NV, Chandler GT, Coull BC, Klosterhaus SL, Quattro IM. Differential survival of three mitochondrial lineages of a marine benthic copepod exposed to a pesticide mixture. Environmental Science & Technology 2001;35(3):535-538. R827397 (2002)
    R827397 (Final)
    R825279 (Final)
    R825439 (1999)
    R825439 (Final)
    R826399E02 (Final)
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  • Abstract: ES&T
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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. R827397 (2002)
    R827397 (Final)
    R825439 (1999)
    R825439 (Final)
    R826399E02 (Final)
  • Abstract: SpringerLink Abstract
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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)
    not available
    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)
    not available
    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)
    not available
    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)
    not available
    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)
    not available
    Journal Article Walse SS, Shimizu KD, Ferry JL. Surface-catalyzed transformations of aqueous endosulfan. Environmental Science & Technology 2002;36(22):4846-4853. R827397 (2002)
    R827397 (Final)
    R829397 (Final)
    not available
    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. R827397 (2000)
    R827397 (2002)
    R827397 (Final)
    not available
    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. R827397 (2002)
    R827397 (Final)
    not available
    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:

    sediments, porewater, pesticides, southeast, 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
    Final 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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