The Precision of the ASTM Bioconcentration Test
(U.S.) Environmental Research Lab.
Duluth, MN
Oct 80
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
KTIS
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EPA-600/3-81-022
United States FEBRUARY 1981
Environmental Protection
Agency . D09l-!6!'«
&EPA Research and
Development
THE PRECISION OF THE ASTM
BIOCONCENTRATION TEST
Prepared for
OFFICE OF TOXIC SUBSTANCES
Prepared by
Environmental Research
Laboratory
Duluth MN 55804
AND
Center for Lake Superior
Environmental Studies
Superior, WI 54880
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
DEPORT NO.
'PA-600/3-81-022-
3. RECIPIENT'S ACCESSION NO.
-AUTHORS Patricia Kosian, Armond Lemke, Karen Studders,
id Gilman Veith
fTTITLE AND SUBTITLE
The Precision of the ASTM Bioconcentration Test
5. REPORT DATE
February 1981 Issuing Date,
8. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO.
S. PERFORMING ORGANIZATION NAME ANO ADDRESS
10. PROGRAM ELEMENT NO.
Environmental Research Laboratory-Duluth, U.S. EPA
620] Congdon Boulevard, Duluth, Minnesota 55804
11. CONTRACT/GRANT NO.
•«. SPONSORING AGENCY NAME ANO ADDRESS
13. TYPE OF REPORT ANO PERIOD COVERED
Environmental Research Laboratory-Duluth, U.S. EPA,
6201 Congdon Boulevard, Duluth, Minnesota 55804
f. SPONSORING AGENCY CODE
IS. SUPPLEMENTARY NOTES
JATA8STRACT
The ASTM method for measuring the bioconcentration factor (BCF) of chemicals
was evaluated using 1,2,4-trichlorobenzene (TCB), hexachlorobenzene (HCB), and
,p'-DDE (DDE). Four replicate, 28-day exposures of the chemicals to fathead
'innows 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 calculation of the BCF by dividing the 28 day residues by the mean
water concentration is inadequate. Two alternate methods of calculating the BCF are
d iscussed.
17.
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Unclassified
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itr
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Unclassified
22. PRICE
EPA Form 2220-1 ("•»• 4-77) pnevious eoi TPON is OBSOLETE
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EPA 600/3-81-022
February 1981
The Precision of Che ASTM Bioconcencracion Tesc
by
Patricia Kosian^, Arroond Lerake , Karen Scudders^, and Oilman Veith*
-Environmental Research Laboracory-Duluch
U.S. Environmental Protection Agency
6201 Congdon Boulevard
Duluth, Minnesota 55804 ' ' . '
and
^Center for Lake Superior Environmental Studies
University of Wisconsin-Superior
Superior, Wisconsin 54880
October, 1980
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INTRODUCTION.
The purpose of chis study was to evaluate the bioconcentrat ion factor
(BCF) X test method suggested by ASTM (1) through the participation in an
interlaboratory 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 exposure data. Hexachlorobenzene CHCB), p,p'-DDE (DDE) and
1,2,4-trichlorobenzene (TCB) were selected for the round-robin evaluation
because they were anticipated to exhibit varying depuration rate constants
and bioconcentrat ion factors while minimizing complications of metabolism and
of the need for using dissimilar analytical methods.
Discussions of the bioconcent rat ion process hav-? been published (2, 3,
A, 5). Bioconcentrat ion is defined as the direct uptake of a chemical into
aquatic organisms through the gill or other membranes. The bioconcentrat ion
factor is the rat-io 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. C2) proposed that the uptake process can be modeled by the
first order relationship:
d£E. s KICW - K2cF
dt
where Cw and Cp are the chemical concentrations in the water and fish,
respectively, and Kj and K2 are che uptake and depuration rate constants,
respectively. Since steady-state is defined 'as the point where dC/dt = 0, it
is clear that chemicals which have small depuration rate constants, i.e.
K« -> 0, will require long exposure t irnes in order co observe steady-scac-e .
1
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Consequently, one method to estimate the BCF using the ratio of CF/CW ac
the end of an arbitrary exposure period may only be an accurate measure of
the bioconcentration factor for chemicals with appreciable 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 throughout the exposure.
Branson et al. (2) proposed that this problem can be alleviated by
defining the bioconcentration factor as the ratio of the rate constants,
Kj/K2« This eliminates the need to expose fish until steady-state is
achieved, but it introduces the uncertainity 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 bioconcentracion 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)CWCl-e~k2c).
If BCF 2. Ki/K2, then CF/CW = BCF(l-e~k2c). This is similar to
the equation proposed by Ernst (3). Therefore, if the values of Cp/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.
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EXPERIMENTAL PROCEDURES
Exposure System
The case syscem consisted of a syringe injeccor exposure system
described by DeFoe (6). The three chemicals were tested simultaneously in
quadruplicate by preparing acetone stock solutions containing appropriate
amounts of HCB, DDE, and TCB to produce exposure concentrations of approxi-
mately 0.2, 0.15, and 20 ug/1, respectively, when 8 ul of acetone was
injected into each liter of dilution water* The control tanks received an
equal quantity of acetone.
The test water was unfiltered Lake Superior water heated to 21°C_+
0.5°C and contained greater than 90% DO saturation. Hardness and pH measured
at the initiation of the test were 45-47 mg/1 (as CaCOj) and 7.8, respec-
tively. Other chemical characteristics of Lake Superior water may be found
in Biesinger and Christensen (7).
The test organism used in the bioconcentration cest was the fathead
minnow (Pimephales promelas).obtained from the Environmental Research
Laboratory-Duluth culture. Juvenile fish weighing 0.1-0.15 g were fed a
daily diet of frozen brine shrimp CSan Francisco Bay Brand) supplimented- wic.h
dry trout chow pellets (Glenoce Mills). The trout chow was purchased for
experimental work and was previously shown to be free of. significant
quantities of pesticides. Handling, holding and acclimation procedures for
the fish were followed according to the ASTM guidelines for conducting
bioconcentration tests (1).
After chemical analyses verified stable exposure concentrations in the
test chambers, 40 fish were transferred to each tank containing 27 liters of
water. The flow rate of incoming water was 250 ml per minute. Samples of 4
fish from each tank were randomly removed on day 0, 2, 4. 8. 16, and 28 of
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che uptake phase and on day 35, 49, and 56 of the depuration phase. The fish
were removed from che canks and placed in a beaker of ice wacer. Afcer all
movement ceased, chey were blotted dry, weighed, and frozen in solvenc-rinsed
glass vials.
The water samples were siphoned direccly from che tank into a 500 ml
volumetric flask (250 ml for control canks) co which 25 ml of hexane had
previously been added. A 1.5 inch teflon coated stirring bar was placed in
che flask and the sample was vorcex mixed for 1 hr. The cwo phases were
allowed to separate for 0.5 hr, and a 1.5 ml aliquot of hexane was
transferred Co a gas chromotography injection vial for analysis.
The accuracy of che analytical mechods was checked by determining che
percent recovery of known amounts of I,2,4-crichlorobenzene,
hexachlorobenzene, and p,p'-DDE in che wacer. The percent recovery for che
water analysis was 95.4%, 98.6%, and 102.8%, for TCB, .HCB, and DDE,
respectively (N=6). The. water concentrations were corrected for percent
recovery.
Analytical Methods
Composite whole fish samples were homogenized with 40 gm of granular
anhydrous Na2SO^ (Mailinckrodt Inc.). The powdered homogenate was
transferred co a 300' ml chromacographic column and eluted with 250 ml of
redistilled hexane into a 250 ml volumetric flask. Because of the high
concentration, no cleanup procedure was performed. After the necessary
dilutions were made, quantitation of TCB, HCB, and DDE was performed by gas
chromatography.
The lipid content of each tissue sample was determined gravimetrically
using a drying period of 15 minutes ac 110°C. The lipid content ranged from
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6.1% co 9.8% with a mean value of 8.44 +_ 0.8 with N=23.
The gas chromacographic analyses were performed on a 5730A
Hewlett-Packard gas chromatograph. with an auto sampler and a Hewlett-Packard
e
3354B lab automation data system. The gas chromatograph was equipped with a
^Ni electron capture detector held at 300°C. The injection port
temperature was 2500C. The 6 ft 2 mr x 3 mm (OD) glass column was packed wich
1.5% SP-2250/1.95% SP-2401 on 100/120 mesh supelcoport (Supelco Inc.). The
carrier gas was 5% methane in argon wich a flow rate of 40 ml per minute.
The water samples were analyzed at a programmed column temperature of
140°C co 190°C ac 4"C per rainuce. The cissue samples were, anlayzed at a
programmed column temperature of 100° to 220°C at 8°C per minute for TCB and
HCB and at 200°C isothermal for DDE. The percenc recovery for spiked tissue
samples were 102% wich N=3 for TCB 99% with N=3 for HCB and 111% with N=3 for
DDE. The tissue concencracions were correcced for recovery.
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RESULTS
The results of Che biocoricentration tests using TCB are presented in
Table I. The data show that the water concentrations decreased) approximately
20% from the initial concentrations during the 28 day exposures. TCB was not
detectable in the initial test fish but had accumulated to 15.8-18.4 ug/g
after two days of exposure. After the initial rapid uptake, the
concentrations increased slowly to maximum concentrations of 1-9.0-26.3 ug/g
in the four exposures. The ratio of .Cp/Cw for TCB in Exposure 1 ranged
from 1,080 after two days to 1,640 after 28 days. The Cp/Cw in Exposure
2 ranged from 1,170 after two days to 1,770 after 28 days. The Cp/Cw in
Exposure 3 ranged from 1,070 after two days to 1,800 after 28 days. The •
Cp/Cw in Exposure 4 ranged from 1,070 after 2 days to 1,730 after 28
days. TCB was eliminated rapidly from fish during the depuration phase of
the study. All fish contained 0.60 ug/g .or less seven days after the
chemical ceased to be added. After 21 days in clean water the fish contained
approximately 0.1 ug/g.
The results of the bioconcentration tests using HCB are presented in
Table 2. The water concentration decreased approximatley 15% during the
exposure and the mean'concentrations were all approximately 0.15 ug/1. HCB
concentrations in the test fish were initially about 0.09 ug/g. After two
days of exposure, the HCB concentration increased ten-fold to approximately 1
Mg/g. .In contrast to the TCB exposures, HCB residues continually increased
. during the 28-day tests to maximum concentrations ranging from 4.55 to 6.22
ug/g in the four tests. The Cp/Cw in Exposure 1 ranged from 5,700 after
cwo days to 33,000 after 28 days. The Cp/Cw in Exposure 2 ranged from
7,400 after two days to 32,000 after 28 days. The ranges of two day and 28
day CF/CW in Exposures 3 and 4 were 5,700 to 39,000 and 5,400 to 37.000.
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TABLE I. Summary of Analytical Results (ran 1,2,4-Tr ichlorobeniene Bloconcentratlon Tests
Exposure 1
Exposure 2
Exposure 3
Exposure 4
Test
Day
0
2
4
8
16
28
35
49
56
Water
14. 6a
14.6
13.9
13.3
12.3
11.3
0.1
<0.l
<0.1
Fish
<0.0lb
I5.ed
15.4
16.1
19.0
18.5
0.25
MA
0.03
Llpld Cr
<*) cw
6.06C
7.99 .1,080
7.55 1,100
MA 1,200
9.28 1.540
9.22 1.640
8.77
6.63
5.82
Water
13.8
13.8
13.7
13.5
12.9
12.4
O.I
<0.01
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TABLE 2. Summary of Analytical Results trom Hexachlorobenzene Bio'concentratIon Tests
Exposure 1
Test
Day
0
2
4
8
16
28
35
49
56
Water
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respectively. HCB concentrations decreased from approximately 5-6 ug/g co
approximately 1-2 ug/g during the 28 days depuration study.
The results of the bioconcentration tests using DDE are presented in
Table 3. The water concentrations in the DDE exposure also decreased about
15% during the exposures and the mean concentrations were all approximately
0.13 ug/1. DDE concentrations in the test fish were initially 0.10 ug/g and
these residues increased about 9-fold to approximately 0.9 ug/g after two
days of exposure. Like HCB exposures, the fish in the DDE tests continually
accumulated DDE throughout the test and a steady-state condition was not
observed. The Cp/Cw in Exposure 1 ranged from 6,200 after two days to
48,00 after 28 days. In Exposure -2, the CF/CW ranged from 6,600 after
two days to 46,000 after .28 days. The range of two day and 28 day Cp/Cw
in Exposures 3 and 4 were 7,700 to 57,000 and 5,500 to 50,000, respectively.
DDE concentrations declined approximately 2 ug/g in che four tests during the
depuration study.
The results of .these tests are presented graphically in Figure 1.
Figure l(a) illustrates that the TCB exposure produced the only resemblence
of steady-state for the three chemicals. Figures l(b) and l(c) clearly show
that the 28 day bioconcentration factor underestimates the BCF for the
bioaccumulative chemicals. In comparing the uptake curves in Figure 1, it
must be recognized that the mean water concentrations in the TCB exposures
were approximately 100 times those in the HCB and DDE exposures and chat che
absolute residue concentrations are not a measure of the bioaccumulation
potential of the chemicals. Moreover,, the apparent variability in
concentrations in Figure 1 refleccs variations in che water concentrations in
addition to the analytical and biological variations.
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TABLE 3. Summary of Analytical Results from p.p'-DDE Bioconcentration Tests
Exposure I
Exposure 2
Exposure 3
Exposure 4
Test
Gay
0
2
4
8
16
28
,35
49
56
Water
(ug/l)
0.15
0.15
0.14
0.13
0.11
0. 12
0.02
<0.01
<0.01
Fish
(ug/q)
0.10
0.94
1.19
2.39
3.81
5.74
5.25
NA
3.48
Llpld Cc
(?) *£
6.06
7.99 6,200
7.55 8,500
NA 18.000
9.28 35,000
9.22 48,000
8.77
6.63
5.82
Water
(ug/i)
0.13
0. 13
0.18
0.18
0.16
0. 14
<0.01
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The data are presented on a more comparative basis in Figure 2 in which
Che ratio Cp/Cw is plotted versus time of exposure. This plot corrects
for the different exposure concentrations within the four tests with each
chemical. Moreover, the relative accumulation between chemicals is
apparent. This figure gives a visual summary of the precision of the
exposures on a scale which should encompass most chemicals tested by this
method.
Figure 3 presents the data from the depuration phase of- this study. The
data show that the DDE concentration in fish remained'essentially constant
during the 54 days in clean water. HCB was eliminated more rapidly than. DDE
while the residues of TCB declined rapidly to near the detection limit within
one week. These data on elimination rates are inversely related to the BCF
which is expected from the bioconcentration kinetics. If BCF at steady-state
is the uptake rate divided by the depuration rate, chemicals with very small
depuration rates should have large BCF if the uptake rate is comparable to
other lipophilic chemicals.
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DISCUSSION
This scudy demonstrates thac che proposed method provides a reproducible
test for measuring the bioconcentrat ion factor. Using the 28 day BCF values
for the four tests, Che 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 che range was 46,000 to 56,000.
The greatest concern in estimating the BCF in the bioconcentration rest
is not the method of testing, but rather the method of calculating the BCF.
As stated previously, the use of the 28 days 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 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 cjirve-f itt ing program, CANDLES,
developed at ERL-D. Table 4 presents t'fie results of the analyses. These
results demonstrate that all three methods 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 che BIOFAC and
CANDLES program estimated that che sceady-scace 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 co che 35,000 escimated from the 28 day ratio of Cp/Cw,
che latter method is clearly inappropriate. For DDE, che BIOFAC method
established a steady-state BCF of 180,000 while CANDLES estimated 110,00
compared co che 28 day value of 50,000.
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TABLE 4. Comparison of BCF Values Computed by These Mechods
Method
ASTM, CF/CW at 28 days
BIOFAC
CANDLES
Estimated Bioconcentracion Factor
TCB
1,700
1,600
1,500
HCB
35,000
52,000
48,000
DDE
50,000
180,000
110,000
13
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REFERENCES
American Society for Testing and Materials. 1979. Proposed standard
practice for conducting bioconcentration tests with fishes and
•saltwater bivalve molluscs. Draft No. 10. Philadelphia, PA.
Branson, D. R., G. E. Blau, H. C. Alexander, and W. B. Neely. 1975.
Bioconcentration of 2,2',4,4'-tetrachiorobiphenyl in rainbow trout
as measured by an accelerated test. Trans. Am. Fish. Soc. 4:
785-792.
Ernst, W. 1977. Determination of the bioconcentration potential of
marine organisms - A steady state approach. Chemosphere 11:
731-740.
Hamelink, J. L., R. C. Waybrant, and R. C. Ball. 1971. A proposal:
Exchange equilibria control the- degree chlorinated hydrocarbons are
biologically magnified in lentic environments. Trans. Am. Fish.
Soc. -100(2): 207-214.
Veith, G. D., D. L. DeFoe, and B. V. Bergstedt. 1979. Measuring and
estimating the bioconcentration factor of chemicals in fish. J.
'Fish. Res. Board Can. 36: 1040-1048.
.DeFoe, D. L. 1975. Multichannel toxicant injection system for
flow-through bioassays. J. Fish. Res. Board Can. 32(4): 544-546.
Biesinger, K. E., and G. M. Christensen. 1972. Effects of various
metals on survival, growth, reproduction and metabolism of Daphnia
magna. J. Fish. Res. Board Can. 29(12): 1691-1700.
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LIST OF FIGURES
Figure l(a) . Uptake and depuration of 1,2,4-trichlorobenzene in fathead
. minnows (Pimephales promelas).
Figure Kb) Uptake and depuration of hexachlorobenzene in fathead minnows
(Pimephales promelas).
Figure l(c) Uptake and depuration of p.p'DDE in fathead minnows
(Pimephales promelas).
Figure 2 Uptake of TCB, HCB, and DDE in fathead minnows (Pimephales
promelas).
Figure 3 Depuration of TCB, HCB, and DDE in fathead minnows
(Pimephales promelas).
15
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Depuration
26
Exposure I
•———- Exposure 2
•- • Exposures
Exposure 4
24 32 4O
TIME (.days)
48
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IO
8
o
o:
O
o
O
LL
O
0
Uptake
j 1
Depuration
• Exposure I
^ Exposure 2
• Exposures
x Exposure 4
8
16 24 32
TIME (days)
40
48
56
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10
8
O
or
O
O
O 2
0
o
Uptake
Depuration
.X.
o Exposure I
_ Exposure 2
• Exposures
.* Exposure4
I I
I I
8
16 24 32 4O
TIME (days)
48
56
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CD
Q
*. TC8
0
68 10 12
TIME (days)
14
16
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5r
4-
HC8
'
o
9
-. TCS
0-
3O 34 38 42 46
TIME (days)
50
54
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