United States
                                  Environmental Protection
                                  Environmental Research
                                  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
                                    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).
                                   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:
                                  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	

  ASTM, CF/CW at 28 days
for HCB and' DDE were clearly not at
  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.
  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
  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

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