2006 Progress Report: An Integration of Copepod-Based BAFs, Lifecycle Toxicity Testing, and Endocrine Disruption Methodologies for Rapid Population-Level Risk Assessment of Persistent Bioaccumulative ToxicantsEPA 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, 2006 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 is to develop a new integrated lifecycle toxicity testing system for persistent bioaccumulative toxicants (PBTs) that will provide concurrent data on PBT bioaccumulation, reproductive/endocrine level effects, and population-level impacts using meiobenthic copepods, 96-well microplate cultures (per new American Society for Testing and Materials [ASTM] and Organization for Economic Cooperation and Development [OECD] standards), and Leslie matrix population growth modeling. We have focused on persistent halogenated compounds found commonly in coastal South Carolina, and compared reproductive health and population growth across test copepod populations as a function of lipid-normalized body burdens (AFs). This allowed development of a Critical Body Residue (CBR) approach for predicting how chronic PBT exposure will impact these ecologically important microcrustaceans at the population maintenance level.
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 due to the extremely hydrophobic nature of many of these compounds. A novel technique was developed in the first grant year that utilized 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 a 96-well microplate-based bioassay. In this second and final grant year, extensive multigenerational microplate culturing (copepod hatching stage through two broods) experiments were completed with the POPs lindane (LD), dichlorodiphenyl dichloroethane (DDD), and fipronil sulfide (FS). 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 an 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 CBRs 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 are as follows:
No toxic responses were seen for any of the DDD seawater (30S) exposure concentrations of 11.8, 22.5, 45.0, and 90.0 μg/L. At 35 days, 90.0-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, 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 establish 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 bioconcentration factor (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 established the first-ever 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—copepod lifecycle toxicity tests were conducted using A. tenuiremis at concentrations of 0.09, 0.18, 0.36, and 0.72 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 0.18, 0.36, and 0.72 μg/L), delayed copepodite development (at 0.72 μg/L), delayed total development (at 0.18, 0.36, and 0.72 μg/L), decreased reproductive success (at 0.36 and 0.72 μg/L), and decreased mean viable offspring (at 0.72 μg/L). A sublethal CBR relating to reproductive effects of fipronil sulfide was computed. Using a trimmed Spearman-Karber analysis, an EC50 of 0.16 μg/L (0.12 to 0.21 μg/L 95% CIE) was calculated, which corresponded to CBRs (averaged across all sexes) and sex-specific lipid-normalized CBRs (averaged from gravid female or adult male lipid-normalized body burdens) of 2.8 pg FS/μg lipid (95% CIE: 2.2 to 3.6 pg FS/μg lipid) and 0.38 pg FS/μg dry weight (95% CIE: 0.27 to 0.52 pg/μg dry weight), respectively. 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. “Concentration in lipid” values and predicted body burdens are reported on an individual copepod basis, since our ultimate objective is to be able to measure and link critical body burdens to the individualized ASTM E2317-04 microplate standard method.
William W. Klauber, M.Sc. Environmental Health Sciences (August 1, 2007); Kate Washburn, M.Sc. Environmental Health Sciences (continuing); Elizabeth Smith, B.Sc. Marine Science, (December 15, 2007)
Final Leslie matrix population growth modeling to predict fipronil sulfide lipid-normalized CBRs at a 50% projected three-generation population growth suppression will be conducted.