2005 Progress 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 Period Covered by this Report: May 16, 2005 through November 15, 2006
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 objective of this research project is to develop a new integrated lifecycle toxicity testing system for persistent bioaccumulative toxicants (PBTs) that will provide concurrent data on PBT bioaccumulation, endocrine activity, reproductive effects, and population-level impacts using meiobenthic copepods, 96-well microplate culture (per a new American Society for Testing and Materials [ASTM] and Organization for Economic Cooperation and Development standard), and Leslie-matrix population growth modeling. We are focusing on environmentally realistic mixtures of persistent halogenated compounds found commonly in coastal South Carolina and comparing endocrine/reproductive health of field versus laboratory-exposed copepod populations as a function of lipid:carbon normalized body burdens (bioaccumulation factors, biota-sediment accumulation facts) and hence developing a Critical Body Residue (CBR) approach for predicting how chronic PBT exposure will impact these ecologically important crustaceans at the population maintenance level.

Progress Summary:

Persistent organic pesticides (POPs) and polybrominated diphenyl ethers (PBDEs) are lingering problems in the marine environment. Performing bioaccumulation experiments on the marine meiobenthic copepod Amphiascus tenuiremis with these compounds in aqueous solution is virtually impossible because of the extremely hydrophobic nature of many of these compounds. A novel technique was developed during Year 1 of the project that utilizes high-performance liquid chromatography (HPLC)-grade silica gel (5-10 μm MGD) as an artificial sediment substrate—spiked with a POP and PBDE mixture—as an exposure vehicle to A. tenuiremis for a full lifecycle in 96-well microplate-based bioassay.

This approach is novel and circumvents a POP/PBDE problem inherent to microplate volumes of creating sufficiently high exposure concentrations of these compounds, which have extremely low aqueous solubilities. This approach has the spin-off benefit of providing a means for testing toxicological effects and bioaccumulation of hydrophobic contaminants, both in mixtures and individually, at concentrations similar to those seen in sediment porewaters where these meiobenthic copepods live and without the limitations of poor organism observability inherent to sediments. Thus far, individual copepods have been exposed from hatching to maturity in HPLC-grade silica substrates at three mixture-designed series with high survival success (Figure 1) for all POPs tested so far except lindane. The acute toxicity of lindane may require dropping it from our matrix for full lifecycle/reproduction testing; if so, we will substitute at least one other POP such as endosulfan or fipronil (desthionyl or the sulfide degradation products). Another benefit of HPLC-grade silica substrate is that it is optically clear even though it is in the muddy-sediment median grain size spectrum; thus embryo-to-adult copepods still can be observed in microplates as they develop using inverted light or fluorescence microscopy (Chandler, Klauber, Ferguson, in preparation).

Figure 1. Control-Normalized Copepod Survival Rates by POP

Table 1 shows predicted body burdens per copepod of the four halogenated POPs investigated up to now (BDE 47, BDE 99, chlordane, and lindane) plus three additional compounds that we are evaluating in summer 2006. Body burdens were predicted using a simple model assuming 7 percent lipid mass per copepod (determined empirically), a lipid density of 0.8 g/mL, and a total body mass of 1.25 μg dry weight. Calculations assume ideal interactions between water and lipid, where lipid has similar contaminant loading properties to octanol. Predicted burdens were then calculated from lipid volumes per copepod (109.4 pL 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 are reported here on an individual copepod basis as our ultimate objective is ability to measure and link critical body burdens to the individualized ASTM E2317-04 microplate standard method. Thus far, limits of quantification for CBRs have been compound dependent. Approximately 13-14 pg body burdens are necessary to quantify BDE 47, BDE 99, chlordane, and lindane via negative chemical-ionization mass spectrometry.

Table 1. Predicted Body Burdens per Copepod


log Kow

Aqueous Solubility (μg/L)

Cin lipid (pg/pL)

Predicted Body Burdens (pg/cope)
at 100 μg/L Exposure

BDE 47





BDE 99















p,p - DDD





3,5 - Dichlorophenol










Future Activities:

Limits of detection are presently being established for p,p-DDD; 3,5-dichlorophenol; and endosulfan. A field-based PBDE- and chlordane-exposed population has been discovered in New Market Creek, Charleston, South Carolina. Copepods and surrounding sediments have been collected. In Year 2, full lifecycle microplate bioassays of these compounds will be completed and linked to CBRs using the analytical methods and quality assurance/quality control steps developed for the bioassay in Year 1.

Journal Articles:

No journal articles submitted with this report: View all 5 publications for this project

Supplemental Keywords:

chlorinated organics, EDC, water pollution, population dynamics, invertebrate, toxicity, meiobenthic copepods, sediments, endocrine disruptors, amphipods, ecological impacts, lifecycle toxicity, risk assessment,, 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, bioindicator, ecological effects, ecological exposure, risk assessment, biomarkers, food web, assays, endocrine disrupting chemicals, sediment, sexual development, endocrine disrupting chemical, Leslie matrix population growth model, EDCs, exposure, animal models, toxicity, ecological impacts, amphipods, benthic copepods, estrogen response, hormone production, ecological risk assessment model, estuarine crustaceans, bioaccumulation, lifecycle toxicity

Progress and Final Reports:

Original Abstract
  • 2006 Progress Report
  • Final Report