AEPA United States Environmental Protection Agency Environmental Research Laboratory Duluth MN 55804 Research and Development EPA-600/S3-81-022 Apr. 1981 Project Summary The Precison of the ASTM Bioconcentration Test Patricia Kosian, Armond Lemke, Karen Studders, and Gilman Veith The ASTM method for measuring the bioconcentration factor (BCF) of chemicals was evaluated using 1,2,4- trichlorobenzene (TCB), hexachloro- benzene (HCB), and p,p'-DDE (DDE). Four replicate, 28-day exposures of the chemicals to fathead minnows were used to determine the precision of the test method. Using the 28-day values, the mean (±S.D.) BCF for TCB, HCB. and DDE were 1,700 (±70), 35.000 (±3,300), and 50,000 (±4,800), respectively. The results showed that steady-state residues are not attained for highly bioaccumulative chemicals in the 28-day exposure, and the calcu- lation of the BCF by dividing the 28 day residues by the mean water con- centration is inadequate. Two alternate methods of calculating the BCF are discussed. This Project Summary was devel- oped by EPA's Environmental Research Laboratory, Duluth. MN, to announce key findings of the research project that is fully documented in a separate report of the same title (see Project Report ordering information at back). Introduction The purpose of this study was to evaluate the bioconcentration factor (BCF) X test method suggested by ASTM through the participation in an inter- laboratory round-robin testing program. Although an evaluation of the round- robin tests will be published elsewhere, this laboratory examined the precision of the method in four replicate tests using three chemicals as well as several different methods of estimating the bioconcentration factor from the expo- sure data. Hexachlorobenzene (HCB), p,p'-DOE (DDE) and 1,2,4-trichlorobenzene (TCB) were selected for the round-robin evaluation because they were antici- pated to exhibit varying depuration rate constants and bioconcentration factors while minimizing complications of me- tabolism and of the need for using dissimilar analytical methods. Discussions of the bioconcentration process have been published. Biocon- centration is defined as the direct uptake of a chemical into aquatic orga- nisms through the gill or other mem- branes. The bioconcentration factor is the ratio of the chemical residue in the fish tissue and the concentration of the chemical in the water after a steady- state is observed. Branson et al. proposed that the uptake process can be modeled by the first order relationship: dt where Cw and CF are the chemical concentrations in the water and fish, respectively, and Ki and K2 are the uptake and depuration rate constants, respectively. Since steady-state is de- fined as the point where dC/dt = 0, it is clear that chemicals which have small depuration rate constants (i.e. Kz~0) will require long exposure times in order to observe steady-state. Consequently, one method to estimate the BCF using the ratio of CF/CW at the end of an arbitrary exposure period may only be an accurate measure of the bioconcen- ------- tration factor for chemicals with appre- ciable depuration rates where steady- state is reached quickly. For chemicals which are not depurated rapidly, the ratio may underestimate the steady- state bioconcentration factor because the residues continue to increase through- out the exposure. Branson et al. proposed that this problem can be alleviated by defining the bioconcentration factor as the ratio of the rate constants, Ki/K2. This elimi- nates the need to expose fish until steady-state is achieved, but it intro- duces the uncertainty of extrapolating beyond the exposure data and the tendency to amplify variability in the analytical measurements by dividing by a small number. The computer program for this model provided by Dow Chemical called BIOFAC was used as a second method to estimate the bioconcentration factor in this study. A third method used to estimate the bioconcentration factor was similar to the BIOFAC in that it assumed the uptake was a first order rate process. Integrating the uptake equation gives CF = (K,/K2)C«(1-e"k2l). If BCF - Ki/K2, then CF/CW = BCF(1-e-k2'). This is similar to the equation proposed by Ernst. Therefore, if the values of CF/CW are measured for varying time periods, t, a non-linear least squares analysis can be used to fit the data and estimate the steady-state BCF. This least squares analysis of the data was compared to the other methods of estimating the bioconcentration factor. Table 1. Comparison of BCF Values Computed by These Methods \ Estimated Bioconcentration Factor Method TCB HCB DDE ASTM, CF/CW at 28 days BIOFAC CANDLES 1,700 1.600 1,500 35.000 52.000 48.000 50,000 180.000 110.000 for HCB and' DDE were clearly not at steady-state. To compare different methods of estimating BCF from a given set of uptake and depuration data, the data were also analyzed using a modified BIOFAC computer program and a non- linear curve-fining program (CANDLES), developed at ERL-D. Table 1 presents the results of the analyses. These results demonstrate that all three meth- ods of estimating the BCF give essentially the same values for TCB, which can be expected since this chemical was tested to near steady-state. However, both the BIOFAC and CANDLES program esti- mated that the steady-state BCF for HCB and DDE are substantially higher than is estimated from the 28-day value. In the case of HCB, the BCF estimated from BIOFAC was 52,000 and that from CANDLES was 48,000. Compared to the 35,000 estimated from the 28-day ratio of CF/CW, the latter method is clearly inappropriate. For DDE, the BIOFAC method established a steady-state BCF of 180,000 while CANDLES estimated 110,000 compared to the 28-day value of 50,000. Conclusions This study demonstrates that the proposed method provides a reproduc- ible test for measuring the bioconcen- tration factor. Using the 28-day BCF values for the four tests, the mean (±S.D.) BCF for TCB was 1,700 (±70) and the range was 1,600 to 1,800 in the four tests. The mean (±S.D.) BCF for HCB was 35,000 (±3,300) and the range was 32,000 to 39,000. The mean (±S.D.) BCF for DDE was 50,000(±4,800) and the range was 46,000 to 56,000. The greatest concern in estimating the BCF in the bioconcentration test is not the method of testing, but rather the method of calculating the BCF. As stated previously, the use of the 28-day BCF can only be an adequate measure of the bioaccumulation potential when the 28-day BCF is representative of steady-state residues. The 28-day BCF Recommendations The method of measuring the biocon- centration factor is relatively simple and provides adequate precision. The method of calculating the bioconcentration factor from the data generated must be modified to account for the fact that steady-state conditions may not be achieved in a 28-day exposure. ------- Patricia Kosian and Karen Studders are with the Center for Lake Superior Environmental Studies. University of Wisconsin, Superior, Wl 54880 and Armond Lemke and Gilman Veith are EPA authors with the Environmental Research Laboratory, Duluth. MN 55804. Armond Lemke is the EPA Project Officer (see below). The complete report, entitled "The Precision of the ASTM Bioconcentration Test," (Order No. PB 81-168 932; Cost: $5.00. subject to change) will be available only from: National Technical Information Service 5285 Port Royal Road Springfield, VA 22161 Telephone: 703-487-4650 The EPA Project Officer can be contacted at: Environmental Research Laboratory U.S. Environmental Protection Agency Duluth, MN 55804 » US. OOVEBWMENT HWrrmQ OFFICE: 1M1 -757-012/7087 ------- United States Environmental Protection Agency Center for Environmental Research Information Cincinnati OH 45268 Postage and Fees Paid Environmental Protection Agency EPA 335 Official Business Penalty for Private Use $300 REGION 5 LIBRARY 230 S DEARBORN SXRtET CHICAGO IL 60604 ------- |