Research Grants/Fellowships/SBIR

2000 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 , NOAA / GLERL
Current 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, 1999 through March 31, 2000
Project Amount: $1,265,102
RFA: Endocrine Disruptors (1999) RFA Text |  Recipients Lists
Research Category: Economics and Decision Sciences , Endocrine Disruptors , Health , Safer Chemicals



The objective of this study is to construct a coupled abiotic-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, and how molecular/cellular manifestations of EDCs are expressed and ultimately impact crustacean population fitness and maintenance at the genetic, reproductive, and population levels. This approach will allow 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 and PAH photo-activation products disrupt normal neuro-endocrine/hormone pathways inducing mediated effects at molecular cellular organism population 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:

Year 1 research has begun to assess the ecotoxicological effects of EDCs from a molecular to population-level perspective using our three crustacean models?Palaemonetes pugio (grass shrimp), Leptocheirus plumulosus (amphipod), and Amphiascus tenuiremis (infaunal copepod). Thus far, two classes of compounds have been/are being evaluated for their EDC potential including: (1) organochlorine cyclodiene insecticide endosulfan, which has been responsible for more fish kills than all other pesticides combined, and has been implicated as a potential EDC in in vitro lab tests; and (2) steroidally-shaped PAHs with (6- hydroxychrysene and 1-hydroxypyrene) and without (chrysene, pyrene) UV photo-activation. Specific progress to date is as follows:

Phototransformation Analytical Chemistry Progress to Date. The central hypothesis of the photooxidation group is that photochemcial transformations of endocrine disrupters over some sediments may result in their oxidative transformation to more biologically active forms. To that end, in the first year of our research, we have been investigating the surface mediated oxidation of endosulfan (a weakly estrogenic insecticide) and chrysene (a polycyclic aromatic hydrocarbon common in South Carolina estuaries) to endosulfan sulfate, or 6-hydroxychrysene, respectively, both strong endocrine disrupters. Several milestones have been passed in this initial year of work:

  • Establishment of Xe-arc solar simulator for high and low light intensities, and construction of the appropriate reactors for examining the photochemistry of sediment suspensions.

  • Development of local facility with the analytical techniques needed for: (a) actinometric light intensity measurement, (b) analysis of trace (ppb) quantities of endosulfan and chrysene, and (c) concurrent analysis of the large number of apparent photooxidation products.

  • Development of techniques for the preparation of chrysene-coated surfaces for use as model sediments.

  • Preparation of purified endosulfan (alpha and beta) from crude stock material.

  • Preliminary measurements of target oxidation rates and product identification/quantification (vide infra).
To date, analysis of endosulfan "spiked" and homogeneous experiments have not identified any appreciable difference in the percent total degradation of / -endosulfan between UV-irradiated and non-irradiated experiments. However, heterogeneous catalysis of / -endosulfan may serve as a mechanism for the formation of endosulfan sulfate and a suite of related oxidation products (Figure 1). We now hypothesize that: (1) the formation of endosulfan sulfate occurs only through the heterogeneous catalyzed oxidation of / -endosulfan, and (2) that the formation of the suite of related degradation products occurs only after hydrolysis and the formation of endosulfan diol.

PAH Photochemistry. The loss of chrysene in both homogeneous and heterogeneous solutions due to photodegradation processes was monitored by GC/MS. During irradiation, the sample developed a yellow color with time indicative of chrysene phototransformation. No color change was observed in samples that were kept in the dark. It has been observed that when photolyses were carried out on laponite (spiked), the photodegradation rate increased nearly 32 percent ([KDABCO K]/K=0.32) compared to direct photolysis. Photolysis on laponite and silica gel (coated) produced phthalate acid and 2-formyl benzoic acid. Both compounds were identified by comparison compared with authentic standards by GC-MS. These products of chrysene photolysis have not been reported before. The photodegradation of chrysene on laponite was found to be first order in chrysene (Figure 2).

Figure 1. Positively identified endosulfan degradation products.

Figure 2. Chrysene photodegradation on laponite (coated). Iave= 2.6 x 10-5 eins/min, pH: 8.3, laponite loading: 1 g, initial chrysene concentration 40 mM, formyl benzoate qualified but not present at quantifiable concentration.

Copepod/Amphipod Progress to Date. In Year 1, an in vivo lipovitellin-based assay of copepod and amphipod individual egg/embryo quality was developed using low-intensity dual-channel Laser-Scanning Confocal Microscopy (LSCM) and the boron-containing fluorogenic vitellin probe BODIPY 505/515. Eggs/embryos of the sediment-dwelling harpacticoid copepod Amphiascus tenuiremis are small (~20 µm thick) with a clear chorion and yolk vitellin tightly packaged into vesicles that are easily stained, sectioned, visualized, and quantified by LSCM?non-destructively. Amphiascus eggs are extruded in reasonably planar masses of 5-7 eggs/mass that can be dissected from females immediately after fertilization/extrusion, stained with BODIPY (a nontoxic vital stain), placed into sterile seawater in 96-well microplates and an EDC of interest, and tracked through development to hatching. OR, laboratory and field-exposed gravid females can be collected, fixed in glutaraldehyde, stained with BODIPY, have their egg-sacs removed and staged, and then quantified for comparative differences in individual egg/embryo quality using the highly accurate LSCM digital measurement capabilities and direct BODIPY intensity values from the LSCM photomultiplier tube. OR, <24 h old larvae (nauplii) can be reared in contaminated sediments or in microplates through the entire lifecycle and collected/processed as above once they have mated and produced eggs. This latter approach was used in Year 1 of this study to assess the effects of the common sediment-associated PAH chrysene on lipovitellin content of A. tenuiremis reared from nauplius to adult in the presence/absence of ultraviolet light. The effects of UV and non-UV irradiated chrysene on Amphiascus egg/embryo lipovitellin levels were assessed using LSCM and the BODIPY fluorochrome stain as follows:

  • 200 gravid females were placed in <38 µm sterile muddy sediments for 24 h. Approximately half hatched their embryos (net >800 nauplii), which were sieved and sorted into random 50-member lots.

  • Muddy sediments from pristine North Inlet, SC (<63 µm grain diameter; 13% solids; <50 ng/g PAH) were spiked (or not spiked for controls) with chrysene at 500 and 2500 ng/g-dry sediment by evaporating chrysene onto glass jar walls and rolling sediments for 10 days.

  • 12-mL spiked and control sediments were pipetted into quadruplicate 100-mL glass beakers filled with 50-mL seawater and fitted at the 50-mL mark with 1-cm windows covered by 52 µm mesh. Each beaker was placed under 2-mL/min single pass seawater flow, and cultured concurrently for 26 days in the presence/absence of ultraviolet light (surface intensity = 25 percent of mean solstice peak value in SC). Copepod larvae/juveniles were fed 106-7 freeze-killed phytoplankton cells every third day, with rations increased an order of magnitude on day 6.

  • After 26 days, all surviving gravid females were sieved from sediments, fixed in EM-grade glutaraldehyde, staged/selected for <24 h old embryos, stained for 60 min with 100 µM BODIPY 505/515, and excited at 488 nm using the Argon/Krypton mixed laserset on the LSCM. A short pass 550 nm emission filter was used for all PMT recordings of BODIPY. A transmitted light detector was used to define egg/embryo 2-D boundaries for areal BODIPY intensity delineations. A minimum of 3-5 egg/embryos per egg sac were sampled, with 3-8 females per treatment assayed. (Note: 10 or more females per treatment is the ideal.). In the highest 2500 ng/g UV-exposed sediments, only 2 females survived but none were gravid; so no egg quality assessment could be performed.

Egg Lipovitellin Assay Findings. Lipovitellin intensities in <24 h old embryos of A. tenuiremis females cultured throughout their lifecycle in PAH-free sediments were almost identical under exposure and non-exposure to UV light; thus, UV per se had no effect on lipovitellin production/deposition into eggs. Lipovitellin intensities in <24 h old embryos of A. tenuiremis females cultured throughout their lifecycle in chrysene-contaminated sediments were significantly higher (76.6 percent) in UV-exposed 500 ng/g sediments, and also in non-UV exposed 2500 ng/g sediments (65.7 percent). Non-UV exposed 500 ng/g females exhibited lipovitellin levels that were significantly lower than UV-exposed females (18 percent), but significantly higher (45 percent) than both UV and non-UV exposed chrysene-free controls. Interestingly, a five fold increase in chrysene concentration in the absence of UV also enhanced vitellin production/deposition to eggs; but the enhancement was significantly less than the 500 ng/g UV exposure. The ecological significance of this finding is unclear given that UV exposure at this chrysene level wiped out most test fauna, and UV is abundant in the intertidal zone where these copepods live.

These results suggest that UV exposure of sediments contaminated with moderate levels of chrysene may lead to strongly enhanced lipovitellin production and deposition into A. tenuiremis eggs. Whether this enhanced deposition leads to improved larval survival and/or fitness needs to be determined next. However, stimulated lipovitellin deposition to egg/embryos may confer a maladaptive advantage to the mother. There are a number of meiofaunal studies that have shown that petroleum hydrocarbons enhance egg production numbers by benthic copepods (i.e., clutch size increases), but no studies have assessed whether enhanced numbers are also accompanied by enhanced egg quality. As lipovitellin is a major constituent of copepod eggs, increased production could lead directly to increased egg numbers. Chrysene had no significant effect on mean clutch sizes in this study.

Rapid 96-Well Microplate Assay for EDC Effects. Few rapid screening tools are available to assess EDC effects on invertebrate reproduction, and no rapid assays have been developed for sediment infauna. To this end, we have developed an infaunal copepod reproductive life-cycle assay for water-borne EDCs that allows tests for effects on larval development, successful maturation to reproductive adult, disruption of fertilization, egg quality, egg development/hatching, and genetic makeup of parents and offspring using individually exposed Amphiascus tenuiremis. As many EDCs are lipophilic and persistent, they often accumulate in sediments and pose the highest risk to sediment-dwelling infaunal invertebrates. Infaunal copepods are the second most abundant metazoan group in estuarine/marine sediments, and are extremely important in food webs and carbon/nutrient cycling. Amphiascus tenuiremis can be easily cultured from egg to adult in sediments or in 96-well hydrogel?-coated microplates. In microplates, A. tenuiremis passes through 6 larval naupliar stages in 11 days, and five copepodite stages in 9 days. In this assay, individual first stage nauplii or copepodites are isolated and cultured to adulthood (21 or 14 days, respectively) in 200 ml aliquots of water-borne chemicals having suspected endocrine-disrupting activity. Individuals are monitored daily via inverted stereomicroscope for survival, molting success, sex ratio outcomes, and time to adult. After reaching the adult stage, copepod cross-matings can be manipulated between individuals within and across treatments and controls under "clean" seawater conditions. A paradigm of harpacticoid copepod biology is that, for egg fertilization to be successful, mating usually must occur immediately after the virgin female's last copepodite molt to adulthood and before her exoskeleton is chitinized. At least for A. tenuiremis, this paradigm does not hold true because: (1) isolated females can be fertilized successfully up to 13 days post adulthood, and (2) females will not extrude eggs unless paired with a reproductively mature male. Thus, rigidly controlled matings can be executed with high success without the need to focus on the fifth copepodite to adult time-window. This feature of A. tenuiremis' biology provides for a unique reproductive life-cycle screening assay focused on 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 post-hatch assessments are desired per individual female. High rates of control survival for A. tenuiremis and the easy determination of endpoints useful in the prediction of population-level effects demonstrate the utility of this rapid microplate assay for screening potential endocrine disrupting chemicals. As A. tenuiremis is normally an infaunal, sediment-ingesting copepod, this model allows assessment of both water-borne and sediment-associated EDC effects. Also, nauplii cannot swim so visualization of larval effects is much easier than for most other copepods. This screening assay can be manipulated to focus on the full life-cycle or only the reproductive-system maturation time windows. Newly extruded egg sacs also can be detached from the female and cultured to hatching in 96-well microplates with >90 percent success; thereby allowing direct microscopical evaluation of development, microplate reader assays of fluorogenic probe systems (e.g., AChE, vitellin, mitochondrial activity), and genetic analyses of isolated brood cohorts.

In Year 1, a definitive assay of the EDC pyriproxyfen (400 and 800 µg/L) showed significant delays in development and a shift in sex ratios, but only mild lethality. As pyriproxyfen is a known EDC, these results provide evidence that this bioassay is sensitive and can detect developmental and reproductive effects pertinent to population-level protection of these abundant and ecologically-important crustaceans. Other suspected EDCs are currently being evaluated (e.g., fipronil, tebufenozide) in aqueous and sediment-associated phases. Also, embryos of the amphipod model Leptocheirus have been cultured this way and have exhibited significant delays in hatching under pyriproxyfen exposures of 400-800 µg/L.

Grass Shrimp EDC Progress to Date. Several bioassays have been completed with adult and embryonic Palaemonetes pugio exposed to UV or non-UV irradiated endosulfan in seawater in the laboratory. The following results were seen with regard to reproductive and tissue lipid-based endpoints:

  • Reproduction was decreased by endosulfan (0, 200, or 400 ng/L) in a dose dependent manner in terms of the rate at which females became gravid and the numbers of females which became gravid. Numbers were reduced 18 percent in non-UV enhanced light and by approximately 40 percent (200 and 400 ng/L) in UV-enhanced exposures.

  • Clutch size was not affected.

  • There were no significant differences among treatments in terms of hatching success or time until hatch.

  • Total lipids (TL) were reduced in a dose dependent manner based on endosulfan exposure but were not affected by presence/absence of UV light (Figure 3).

  • Lipid class composition was altered by endosulfan treatment and light source. In embryos, there was a marked increase in the percent of wax esters or sterol esters in the UV-enhanced treatments. This component increased in a dose dependent manner. In females, PL generally increased with endosulfan dose and TAG seemed to decrease in a dose dependent manner. In males, PL decreased with increasing endosulfan exposure.

EDC Eco-Genetics Progress to Date. 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 GABA gene. During this first year of research, we have concentrated our efforts on sample collection, DNA extraction, and development of appropriate PCR primers. At this time, sampling for genetic evaluation of field populations is at least 90 percent complete, and extraction of DNA from these samples is approximately 20 percent complete. Progress also has been made in the following interrelated groups of goals:

  1. Collection of 966 shrimp from 21 locations throughout the species range.
  2. Positive species identification and gender determination of the collected shrimp.
  3. Endosulfan LC50 test conducted on 757 grass shrimp.
  4. Began DNA extraction from individual shrimp.
  5. Developed two sets of primers to target the cytochrome oxidase b gene.
  6. Began development of primers to target the GABA receptor.
  7. Preliminary analysis of available data nationwide.

Collection of 966 shrimp from 21 locations throughout its range. Collections were made from both personal efforts and collaborative efforts with researchers throughout the North American range of Palaemonetes pugio. Collection sites represent both "pristine" waterways and those with a history of cyclodiene exposure.

Positive species identification and gender determination of collected shrimp. Species identification and gender determination were carried out on shrimp collected from the field as well as those individuals used in the toxicology test. Species identification was carried out using the criteria outlined by Holthius (1952) and gender determination was carried out using the appendix masculina as the primary determinant.

Endosulfan LC50 test carried out on 757 grass shrimp. Shrimp (n=757) from an area with no recent history of cyclodiene exposure were used to conduct a 12-hour endosulfan exposure LC50 test. The shrimp from this test will be analyzed for variation at the GABA receptor and used to identify resistant genotypes. A cyclodiene resistance marker will be identified from this information.

Figure 3. Effect of endosulfan exposure on total lipids.

Began DNA extraction from individual shrimp. Extraction of DNA from collected shrimp is complete for approximately 20 percent of samples. Extractions were originally conducted using the Qiagen DNAEasy extraction kit; however, LiCl treatments have been added to increase PCR efficacy.

Developed two sets of primers to target the cytochrome oxidase b gene. The cytochrome oxidase b gene has been successfully amplified and sequenced; it will be used as an indicator of naturally occurring population genetic structure and gene flow at a locus not associated with cyclodiene resistance.

Began development of primers to target the GABA receptor. Several sets of primers have been developed to amplify the GABA receptor with varying levels of success. Currently, we are working to perfect these primers for efficient and reliable amplification of the GABA receptor locus.

Preliminary analysis of available data. Preliminary analysis of data suggests a phylogeographic split between the Gulf of Mexico and the Atlantic coast populations. This is consistent with data available for several other coastal marine organisms with a similar distribution range. A secondary result of the analysis has been that we are able to use the cytochrome oxidase b locus as another character to confirm species identification of the shrimp and minimize the potential for misidentification of the two most common, co-occurring species of Palaemonetes (pugio and vulgaris).

Future Activities:

Photochemistry Group. In the second year of this project, the chemistry group will address the following goals: (1) carry out the photooxidation of chrysene and endosulfan over known photocatalytically active surfaces such as amorphous FeOOH; (2) determine the products and kinetics of endosulfan and chrysene photodegradation in the known photosensitizer dissolved organic matter; (3) extend both of these studies across a matrix of ionic strengths designed to emulate the transition between freshwater and seawater within estuarine environments; and (4) begin similar studies of the pesticides fipronil (an increasingly-used organochlorine GABA disruptor) and the insect growth regulating EDC pyriproxifen.

Copepod/Amphipod Group. Although this study represents a first pass at using modern Laser Scanning Confocal Microscopy in ecotoxicological applications, these techniques hold promise as a new tool for rapid assessment of individual egg quality in microscopic test fauna. Larger crustacean fauna (e.g., grass shrimp and some amphipods like Leptocheirus) similarly have eggs that are small enough and clear enough for direct lipovitellin characterization via LSCM. Letocheirus embryo lipovitellin has been successfully quantified using these techniques. We also are presently developing antibody-based fluorescent probes for other EDC-linked endpoints (e.g., ecdysone and chitinase) in these crustacean models in addition to benthic copepods. Antibody-based fluorescent probes can be visualized/quantified by microplate readers and LSCM in ways similar to those presented here for BODIPY. The 96-well microplate assay will be further refined for whole egg/whole larvae assessments of lipid and vitellin change under EDC exposure for both copepod and amphipod models. Our ultimate goal with the copepod and amphipod work is to link our established reproduction bioassays (i.e., multiple population-level endpoints) with EDC-linked molecular endpoints to ascertain a better understanding of chemical "endocrine disruption" consequences at the ultimate resource protection level for sediment infauna?the population.

Grass Shrimp Toxicology Group. Three major tests are scheduled for the next 12 months. During the fall 2000, we will expose reproducing adult grass shrimp in controlled laboratory bioassays to the organophosphate insecticide chlorpyrifos or the organochlorine insecticide fipronil. During the spring 2001, we will begin a mesocosm exposure to chlorpyrifos or fipronil, as well as the last reproductive exposure. The final reproductive exposure will be to the insect growth regulator, pyriproxifen. After chronic testing is complete, laboratory analysis of all tissue residues will be completed within the remaining 12 months of the grant period.

Ecogenetics Group. Activities for the second year are directed entirely at completing the sequencing of the GABA receptor locus from collected individuals. Three major tasks are required for this objective. The first task is completion of DNA extraction from individual shrimp. The second task is the optimization of the amplification of the GABA receptor locus. The third task is to obtain sequence information at the GABA receptor locus in both laboratory animals and in field studies.

Journal Articles on this Report : 2 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 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
R827397 (2000)
R827397 (2002)
R827397 (Final)
R825279 (Final)
R826399E02 (Final)
  • Abstract from PubMed
  • Abstract: Wiley-Abstract
  • 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
    R827397 (2000)
    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
    2002 Progress Report
    Final Report