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-
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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.
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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
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