Final Report: An Integration of Copepod-Based BAFs, Lifecycle Toxicity Testing, and Endocrine Disruption Methodologies for Rapid Population-Level Risk Assessment of Persistent Bioaccumulative Toxicants

EPA Grant Number: GR832211
Title: An Integration of Copepod-Based BAFs, Lifecycle Toxicity Testing, and Endocrine Disruption Methodologies for Rapid Population-Level Risk Assessment of Persistent Bioaccumulative Toxicants
Investigators: Chandler, G. Thomas , Ferguson, P. Lee
Institution: University of South Carolina at Columbia
EPA Project Officer: Carleton, James N
Project Period: May 16, 2005 through November 15, 2007
Project Amount: $298,907
RFA: Greater Research Opportunities: Persistent, Bioaccumulative Chemicals (2004) RFA Text |  Recipients Lists
Research Category: Land and Waste Management , Safer Chemicals , Hazardous Waste/Remediation , Health Effects


The fundamental objective of this research was to develop a new integrated lifecycle toxicity testing system for persistent bioaccumulative toxicants that would provide concurrent data on PBT bioaccumulation, reproductive/endocrine level effects, and population-level impacts using meiobenthic copepods, 96-well microplate culture (per a new ASTM and OECD standard), and Leslie-matrix population growth modeling.  We have focused on persistent halogenated compounds found commonly in coastal SC, and compared reproductive health and  population growth across test copepod populations as a function of lipid-normalized body burdens (AFs); hence developing a Critical Body Residue approach for predicting how chronic PBT exposure will impact these ecologically-important microcrustaceans at the population maintenance level.


Our research plan will address these objectives through a series of experiments designed to provide a holistic picture of persistent chlorinated pesticide bioaccumulation and endocrine/reproductive effects on model ecologically-important crustaceans in estuarine environments. These experiments will include laboratory egg-to-egg lifecycle toxicity tests with DDT, chlordane and lindane (singles & mixtures) at concentrations 1X, 2X and 5X measured field values in our model Shipyard Creek system. Dose-response experiments will test the potential for PCPs to bioaccumulate, cause endocrine action, and directly cause adverse effects on sensitive estuarine infaunal crustaceans (meiobenthic copepods). Meiobenthic copepods are keystone species in food-webs and the trophic transfer of lipophilic contaminants from sediments to fish, shrimps and crabs.

Summary/Accomplishments (Outputs/Outcomes):

Extensive multi-generational microplate culturing (copepod hatching stage through two broods) experiments were completed with the POPs lindane, DDD and fipronil sulfide.  Identical tandem microplate experiments were run concurrently to yield sufficient copepod biomass for lipid-normalized body-burden measurements of these chemicals in copepods relative to aqueous exposure concentrations over 35-40 day periods.  Multiple sublethal (i.e., < 20% mortalities, not significantly different from controls) POP concentrations were used so that individual’s developmental and reproductive endpoints could be measured with high statistical confidence over individual lifetimes.  Lipid-normalized copepod body burdens were calculated to provide a basis for CBR’s of any POPs causing significant developmental or reproductive toxicity.  Life-stage to life-stage transition probabilities, fecundities and mortalities were used to construct empirically-based Leslie-matrix population growth models for Amphiascus for each POP causing significant sublethal toxicity.  Results were as follows: 
No toxic responses were seen for any of the DDD seawater (30S) exposure concentrations of 11.8, 22.5, 45, and 90 μg/L.  At 35-days, 90 μg/L exposures produced maximum lipid-normalized body burdens of 594 ± 54 and 775 ± 54 pg-DDD/μg-lipid for gravid females and males respectively.  Significant sublethal toxic responses (p < 0.05) were observed for lindane (LD) corresponding to delayed total development from nauplius to adult (60.0 ± 30.5 and 44.5 ± 7.31 pg-LD/μg lipid for gravid females and males), decreased mean viable offspring production (122 ± 58.6 pg-LD/μg lipid for gravid females), decreased fertilization success and increased total chronic mortality (510 and 607 pg-LD/μg lipid for gravid females and males).  A Trimmed Spearman Karber analysis was performed to predict an EC50 for decreased reproductive output of 28.2 μg-LD/L with a 95% CIE of 22.3 - 35.7 μg-LD/L.  From these values, sublethal lipid-normalized CBRs of 354 (281-448) pg-LD/μg lipid and 253 (192-331) pg-LD/μg lipid were calculated for males and gravid females, respectively, relative to decreased reproductive success.  Sex-specific lindane log BCF values of 3.92 ± 0.13 and 4.06 ± 0.17 for gravid females and males respectively were measured at these CBRs.  Lastly, a Leslie-matrix based modeling approach was used to predict lipid-normalized population growth CBRs (for a 50% projected population reduction after three generations) of  60 and 58 pg-LD/μg lipid for males and gravid females respectively. 
For the persistent and predominant sediment degradation product of fipronil -- fipronil sulfide (FS) -- copepod life-cycle toxicity tests were conducted using A. tenuiremis at measured concentrations of 0.10, 0.20, 0.39, and 0.85 μg/L.  Tandem microplate bioaccumulation experiments were conducted to establish dry weight and lipid normalized body burdens at each exposure concentration.  Significant toxic effects ranged from delayed naupliar development (at 0.10, 0.39, and 0.85 μg/L), delayed copepodite development (at 0.85 μg/L), delayed total development (at 0.20, 0.39, and 0.85 μg/L), decreased reproductive success (at 0.39, and 0.85 μg/L), and decreased mean viable offspring (at 0.85 μg/L). A sublethal Critical Body Residue (CBR) relating to reproductive success/failure under FS exposure was computed.  Using Trimmed Spearman-Karber analysis, a Repro-EC50 of 0.16 μg/L (0.12 to 0.21 μg/L 95% CIE) was calculated corresponding to adult CBRs (averaged across sexes) and lipid-normalized CBRs of 0.38 pg-FS/μg dry weight (95% CIE: 0.27 to 0.52 pg/μg dry weight) or 2.8 pg-FS/μg lipid (95% CIE: 2.2 to 3.6 pg-FS/μg lipid), respectively.
Leslie matrix modeling was used to integrate all observed life table parameters including mortality, developmental failures, sex ratio shifts, infertility and fecundity into a predictive model of population growth and age structure over time.  Copepod population size projections as a function of FS exposure were produced from empirical microplate data and regressed against corresponding tissue residues (as above) using best-fit regression (r2 > 0.99) to predict the median effective tissue concentration (i.e., lipid and non-lipid normalized population EC50) that would produce a population size 50% of the FS-free control.  Intrinsic population growth rates (λ) predicted from empirical transition and fecundity rates using the Leslie matrix declined with dose > 20% from 1.27 in the controls down to 0.98 at the highest FS exposure.  A λ  of unity means the population growth rate is just replacing itself over time.  A rate of 1.27 means the population is expected to double every 2.6 lifecycles, but at 1.27 pg-FS/μg-dry weight, more than 7 generations would be required to double population size.  Consequently, by the third generation, sharply depressed population sizes as a function of FS body burden were predicted by the model compared to controls.  The model predicted fewer than five females would be gravid in the population by the third generation of exposure to both 0.39 and 0.85 μg-FS . L-1 (i.e., 9.6 – 10.2 pg-FS/μg-lipid).  A 50% population growth suppression would be predicted to occur at an aqueous exposure concentration between 0.10 and 0.20 μg-FS/L.   The median population growth response, (i.e., EC50 ), for the Leslie matrix modeling results was defined a priori as the lipid-normalized body burden where matriarchal population size after 3 generations would be predicted by regression to be 50% of the control.  In this study that critical body burden corresponded to a lipid-normalized CBR of 1.6 pg-FS · μg-1 lipid, which yielded a third generation EC50 prediction of 968 copepods in the population.  This concentration is 1.75 times lower than the lipid-normalized CBR predicted to suppress reproductive success by 50% .  Other treatment factors such as stage-specific (e.g., naupliar) FS mortality and sex ratio shifts that were captured and integrated in the Leslie matrix model, but not in the reproductive success prediction, likely drove the CBR prediction to a lower effective tissue concentration.  Eigenanalysis of the copepod stage matrix for the control microplate populations showed highest “elasticities” linked to the higher proportions of adult virgin females able to mate and become gravid in controls, and to the correspondingly higher reproductive output (fecundity). Conversely, the two highest FS concentrations showed highest elasticities for the low proportions of adult virgin females able to mate and become gravid, and for their correspondingly lower reproductive outputs.  [“Elasticities” are simply the proportional sensitivities of λ to small changes in the Leslie matrix elements for each empirical stage matrix of the FS exposed populations.]
The BCF values and sublethal CBR’s reported here for lindane and FS in A. tenuiremis are highly novel, as lipid normalized sublethal CBRs have never been reported for either legacy or current use pesticides for any meiobenthic organism to date.  Non-normalized CBR estimates for a few meiobenthos and most macrobenthos have been typically mortality based and reported for other types of nonionic contaminants such as DDT (and metabolites), PAHs and PCBs. All POP body burdens in this project were predicted using a simple model assuming 13.7% lipid mass per gravid female copepod (determined empirically) and 15.2% for males, a lipid density of 0.8 g/mL, and sex-specific dry weight body masses of 1.4 μg per gravid female, and 0.6 μg per adult male.  Calculations assume ideal equilibrium interactions between water and lipid where lipid has similar contaminant loading properties as octanol.  Predicted burdens were then calculated from mean lipid volumes per copepod, micro-well water volumes (250μL), log KOW values for each compound, and concentrations of each POP in aqueous solution.  “Concentration in lipid” values and predicted body burdens were reported here on an individual copepod basis since our original objective was developing ability to measure and link critical body burdens to the individualized ASTM E2317-04 microplate standard method. 

Expected Results:

The proposed work will, for the first time:

  1. address the potential for PCPs to bioaccumulate in trophically and ecologically important meiobenthic copepods;
  2. relate critical body residues of PCPs in field and lab populations to full lifecycle toxicity test endpoints of highest value to risk assessment (i.e., reproduction, population growth, population maintenance);
  3. and comprehensively evaluate endocrine disrupting potential of PCPs in crustaceans with regard to two hormonal systems intimately linked to growth and reproduction (i.e., ecdysone (molting) and vitellogenesis).

As copepod life-cycle testing is now before the OECD for adoption as a new endocrine disruptor screening tool, the information from this project will be of high value to OECD and environmental managers/agencies worldwide. Further, this work will generate new scientific knowledge related to the behavior of PCPs at the biochemical to whole organism to population levels.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other project views: All 5 publications 1 publications in selected types All 1 journal articles
Type Citation Project Document Sources
Journal Article Ferguson PL, Chandler GT, Templeton RC, DeMarco A, Scrivens WA, Englehart BA. Influence of sediment-amendment with single-walled carbon nanotubes and diesel soot on bioaccumulation of hydrophobic organic contaminants by benthic invertebrates. Environmental Science & Technology 2008;42(10):3879-3885. GR832211 (Final)
  • Abstract from PubMed
  • Supplemental Keywords:

    chlorinated organics, water pollution, population modeling, invertebrate, sediments, endocrine disruption, risk assessment, meiofauna, benthos, RFA, Health, Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, Toxicology, Environmental Chemistry, Ecosystem/Assessment/Indicators, Health Risk Assessment, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Risk Assessments, Ecological Effects - Environmental Exposure & Risk, Biology, Endocrine Disruptors - Human Health, ecological effects, risk assessment, bioindicator, ecological exposure, assays, biomarkers, food web, EDCs, endocrine disrupting chemical, endocrine disrupting chemicals, exposure, sediment, sexual development, Leslie matrix population growth model, animal models, ecological impacts, toxicity, amphipods, benthic copepods, estrogen response, hormone production, ecological risk assessment model, bioaccumulation, estuarine crustaceans, human health risk

    Progress and Final Reports:

    Original Abstract
  • 2005 Progress Report
  • 2006 Progress Report