United States EPA-600A-81-011
Environmental Protection March 1981
Agency
vvEPA Research and
Development
Results:
Interlaboratory Comparison
Bioconcentrafion Tests
Using Eastern Oysters
Prepared for
Office of Pesticides
and Toxic Substances
Prepared by
Environmental Research
Laboratory
Gulf Breeze FL 32561
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RESULTS: INTERLABORATORY COMPARISON-
BIOCONCENTRATION TESTS USING THE EASTERN OYSTER,
by
Steven C. Schimmel
and
Richard L. Garnas
Environmental Research Laboratory
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
GULF BREEZE, FLORIDA 32561
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory,
Gulf Breeze, Florida, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily reflect
the views and policies of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
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Abstract
This report summarizes the results of an inter!aboratory compari-
son for bioconcentration (BCF) testing using the eastern oyster
(Crassostrea virqinica) and the organic chemicals p,p'-DDE, 1,2,4-
trichlorobenzene, and pentachlorophenol. The mean BCF's and high to
low BCF ratios for p,p'-DDE, 1,2,4 trichlorobenzene, and pentachloro-
phenol were 52,600 (3.4), 168 (2.3), and 64 (2.4), respectively. The
test method (ASTM Draft 9) used for all three chemicals and four par-
ticipating laboratories resulted in data with a mean to low BCF ratio
of 2.7, which represented a "worse case" situation for an estimate of
variability. Considering the varying degrees of experience between
participants in conducting BCF tests, the wide geographic distribution
between participants, and the different dilution water characteris-
tics, the results were remarkably reproducible.
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CONTENTS
Abstract „ i i i
Tab! es vi
1. Introduction 1
2. Bi concentration Test Results 4
3. Discussion. 10
Appendices
A. Analytical Chemistry Performance Evaluation 11
EL Results of Chemical Performance Evaluation 14
C. Procedures for the Chemical Analysis of DDE, TCP, and TCB in Water
and Tissues 16
D. Final Reports of Laboratories Participating in the Bioconcentratiorr
Laboratory Comparison Studies 90'
E. Method for Sampling Oysters.... 197
F. Method for Lipid Analysis 198
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TABLES
No.
1. Calculated steady-state bioconcentration factors (BCF) for
p,p'-DDE, Pentachlorophenol (POP) and 1,2,4-Trichloro-
benzene (TCB), using the eastern oyster, Crassostrea
virginica
2. Performance of each laboratory in adhering to diluent and test
water condition requirements specified in ASTM bioconcentration
test procedures 6
3. Performance of each laboratory in adhering to ASTM bioconcen-
tration test procedures regarding eastern oyster (Crassostrea
virginica) size, loading, acclimation time, and mortality
during acclimation and testing 7
4. Analyses of lipids (dry-weight) in oysters obtained from lab-
oratories participating in the "round-robin" tests 8
VI
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INTRODUCTION
Under Section 4 of the Toxic Substances Control Act (TSCA), the
Administrator of the Environmental Protection Agency (EPA) can require
environmental effects testing of a chemical substance if (1) the
manufacture, use, distribution, or disposal of that substance may
present an unreasonable risk of injury to the environment; or (2) the
substance will be produced in substantial quantities, is expected to
enter the environment, and there are insufficient data to predict the
effects of the chemical substance on the environment.
When the Administrator issues a test rule for the performance of
environmental effects testing, he must also provide test standards to
be used in the development of test data. Before test standards are
proposed, steps should be taken by the Agency to insure that data
developed by each test standard are adequate and reliable.
The performance of inter!aboratory, or "round-robin," tests is one
way to validate individual test standards and determine the degree of
variability among data developed by different laboratories that use the
same methodology.
Because the Office of Toxic Substances' (OTS) Oyster Bioconcen-
tration Test Standard was not completed at the time of the contract
award, contractors were instructed to follow the American Society for
Testing and Materials (ASTM) "Standard Practice for Conducting Bio-
concentration Tests with Fishes and Saltwater Bivalve Molluscs"
(Draft 9). This method is similar to that in the now completed OTS
Test Standard.
This report is intended to summarize and review the final reports
of the laboratories that participated in the "round-robin tests." In
evaluating the bioconcentration data, we examined laboratory notebooks,
progress reports, and final reports to determine how closely each lab-
oratory adhered to the specific procedures outlined in the ASTM test
method.
Three contract laboratories and one EPA laboratory participated in
the "round-robin" tests. Each contract laboratory, in order to par-
ticipate, had to satisfy a number of technical criteria stipulated in
the scope of work. Two major criteria were: (1) competence in gas
chromatographic analysis of the selected chemicals, and (2) availa-
bility of suitable natural seawater for conducting the test under
flow-through conditions.
Test chemicals used in the bioconcentration "round-robin" tests
were l,l-dichloro-2,2-bis (p-chlorophenyl) ethylene (DDE); 1,2,4-
trichlorobenzene (TCB); and pentachlorophenol (PCP). Selection of
these chemicals depended upon a number of factors: (1) each possessed
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substantially different bioconcentration potentials as predicted by
their octanol/water partition coefficients; (2) ease of chemical
analysis; and (3) low mammalian toxicity.
The test species selected for the bioconcentration study was the
eastern oyster, Crassqstrea virginica. This species is important eco-
nomically as a human food source and ecologically in its wide distri-
bution on the Atlantic and Gulf Coasts; and it is valuable in
monitoring programs as a pollution indicator due to its ability to
bioconcentrate many organic chemicals.
A major source of variability in the interpretation of bioconcen-
tration tests results from the chemical analysis of tissues and water
for the respective chemicals. For this reason, we conducted an ana-
lytical chemistry performance evaluation for all participating labor-
atories (after awarding contracts) to identify any significant varia-
bility associated with chemical analyses in these studies.
Contaminated tissue and water samples were provided to each of the
participating laboratories (Appendix A). Samples were delivered by
messenger to each participant on the same day. Final reports were
required no later than 30 days following the delivery of the samples.
Only one participant (Lab 4) failed to satisfy this deadline.
The lead laboratory, the Environmental Research Laboratory (ERL),
Gulf Breeze, Florida, (Lab 1) conducted a large number of analyses for
each chemical and substrate to establish relative mean concentra-
tions, relative standard deviations, and corresponding 99% confidence
intervals (Appendix B). Results from participating laboratories are
tabulated as mean concentration and standard deviation. Those values
that grossly exceed the 99% confidence interval are starred. It is
clear from these data that one participant, Lab 2, had difficulty with
the chemical analysis. A survey of the reports submitted with the
performance evaluation results (Appendix C) revealed a remarkable
homogeneity of chemical methodology, except that Lab 2 selected not to
use a derivatization method for pentachlorophenol analysis and
subsequently encountered high background interference. The reasons for
poor performance by Lab 2 were not obvious from its report.
The participants received the results of the performance eval-
uation, and those deemed acceptable were allowed to proceed with
testing. Participants displaying poor performance were cautioned about
their respective methods and improvements were suggested. Lab 2 was
required to use a derivatization method for pentachlorophenol. The
final results of chemical analyses for the bioconcentration studies
are contained in the final reports (Appendix D).
In addition to the performance evaluation package, each partici-
pant was provided information on the test chemicals to choose properly
the test concentrations and sampling periods, as prescribed in the
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ASTM methods document. According to the ASTH method, concentrations of
the test chemicals in water should not elicit adverse effects on the
test organisms and, therefore, should not be greater than 0.1 times the
96-hour (h) EC50 (based on reduced shell deposition). Consequently,
contractors were provided with the oyster EC50 values for each chemical
(DDE, 14 yfj/i; PCP, 104 u/£; and TCB, >500 ug/i). The lowest value
allowed for the chemical in water (as required by the ASTM method) is
three times the analytical detection limit for each chemical tested by
the participating laboratory.
The sampling intervals for each chemical are dictated by the log-
arithm of the octanol/water partition coefficient (log P); the higher
the log P, the longer the time between samples and, therefore, the
longer the exposure. We provided each contractor with the estimated
log P for each chemical (5.69 for DDE, 2.23 for PCP, and 4.08 for
TCB).
In addition, each contract laboratory was requested to provide our
laboratory with control and experimental oysters (collected at steady
state) from each test to determine lipid content. This procedure was
suggested in the ASTM standard practice for compounds that are lipo-
philic.
The ASTM document specifies certain required test methods or con-
ditions in order for a test to be considered satisfactory. These con-
ditions are always associated with the word "must." For example, Sec-
tion 10.9.3 states (in part) that "a group of organisms must not be
used for a test if the individuals appear to be diseased or otherwise
stressed or if more than 3% die during the 48 h immediately before the
beginning of the test." Suggestions for good test practices are gen-
erally phrased in words such as "should," rather than "must." For
example, Section 10.3.2 states (in part) that "in any single test, all
mollusks should be from the same year class and the valve height of
the largest mollusk should be no more than 1.5 that of the smallest
mollusk." Each laboratory's final report was carefully scrutinized to
determine if the "musts" in the ASTM document were fulfilled. If not,
an attempt was made to determine if the test results were affected.
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BIOCONCENTRATION TEST RESULTS
Pentachlorophenol. Trichlorobenzene and DDE Bloconcentration Factors
The calculated steady-state bioconcentration factors (BCFs) for
the three chemicals tested by the four participating laboratories are
listed in Table 1. Steady-state BCFs were provided for each chemical
by all laboratories.
Pentachlorophenol (PCP)—Results of the PCP bioconcentration
studies indicated that the steady-state BCFs varied from 34 to 1,294.
The obvious disparity of Lab 4's BCF (1,294) with the other three
prompted a request that Lab 4 verify their calculations. Lab 4
reported that no error could be found and that its data were accurate.
We deleted Lab 4's value from our discussion of the results because it
was nearly 16 times higher than that of any other laboratory and sig-
nificantly higher than any value we could find in the literature.
Labs 1,2, and 3 provided PCP bioconcentration factors of 82, 34,
and 76, giving a mean of 64 ± 26. The ratio of the highest BCF value
divided by the lowest (the high to low ratio, H/L ratio) for the three
labs was 2.4. This value represents the "worst case" estimate of the
test variability for PCP bioconcentration.
1,2,4-Trichlorobenzene (TCB)—The results of the TCB bioconcen-
tration tests, (Table 1) indicate that the mean BCF for the four lab-
oratories was 168 ± 65; the H/L ratio, 2.3.
DDE—The results of the DDE bioconcentration tests (Table 1) show
that the steady-state BCFs ranged from 27,200 to 91,800, indicating
that the mean BCF for this insecticide was 52,600 ± 27,700 and the H/L
ratio, 3.4.
Analysis of the Adherence of Laboratories to the ASTM Methods and
Scope of Work.
Final reports from each laboratory involved in the bioconcentra-
tions "round-robin" tests were carefully reviewed to determine if the
"must" requirements of the ASTM document and the contract's Scope of
Work were implemented. Tables 2, 3 and 4 address these requirements
and their implementation.
Each laboratory, except Lab 4, provided sufficient information to
make an adequate appraisal of diluent water quality and test water
conditions used in the bioconcentration tests (Table 2). The only
information provided by Lab 4 was the temperature of the testwater.
For diluent water quality, Labs 1, 2 and 3 each measured salinity, pH,
and dissolved oxygen (D.O.) and those values were within prescribed
limits. Particulates were measured by Labs 1 and 3, but not by 2 and
4; only Lab 3 measured total organic carbon (TOC). Total organo-
chlorines were measured by Lab 1 only.
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Table 1. Calculated steady-state bioconcentration factors (BCF) for p.p'-DDE,
Pentachlorophenol (PCP) and 1,2, 4-Trichlorobenzene (TCB), using the
eastern oyster, Crassostrea virginica.
LABORATORY p.p'-DDE
1 42,652
2 48,602
3 91,826
4 27,191
Y = 52,568
Std. dev. = 27,684
H/L ratio = 3.4
PCP
82
34
76
1,294*
64
26
2.4
TCB
147
115
264
148
168
65
2.3
*Data not considered in y, std. dev. and H/L ratio.
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en
Table 2. Performance of each laboratory in adhering to diluent and test water
condition requirements specified in ASTM bioconcentration test proce-
dures. "Yes" designates compliance with the requirements; "No" desig-
nates that the required dilution water observations were not made, or
that test water conditions were not within required limits.
LABORATORY
Salinity
1 Yes
2 Yes
3 Yes
4 No
DILUENT WATER QUALITY
pH D.O. Particulates TOC
or
COD
Yes Yes Yes No
Yes Yes No Mo
Yes Yes Yes Yes
No No No No
Total
Organochlorine
COD
Yes
No
No
No
TEST WATER CONDITIONS
Salinity
012 g/kg)
1 Yes
2 Yes
3 No*
4 No
Temperature pH
(>8<28°C) (<0.8 unit)
Yes Yes
Yes Yes
Yes Yes
Yes No
D.O.
(>6Q%
saturation)
Yes
Yes
Yes
No
*Acceptable salinity only for the Pentachlorophenol test.
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Table 3. Performance of each laboratory in adhering to ASTM bioconcentration test procedures regarding
eastern oyster (Crassostrea virginica) size, loading, acclimation time, and mortality1 during
acclimation and testing. "Yes" indicates compliance with the requirements; "No" indicates non-
compliance or data were not reported.
Oyster size
Laboratory (largest < 1.5 x smallest)
1 Yes
2 Yes
3 Yes
4 Yes
Mortality*
Acclimation
Yes
Yes
Yes
Yes
Test
Yes
Yes
No
Yes
Acclimation
Time*
(>4 days)
Yes
Yes
Yes
Yes
Loading*
(>U/oyster/h)
Yes
Yes
Yes
No**
*A "must" requirement in the ASTM procedure.
**Loading was acceptable only for the pentachlorophenol test.
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Table 4. Analyses of lipids (dry-weight) in oysters obtained from
laboratories participating in the "round-robin" tests.
Oysters were collected at steady-state and analyzed indi-
vidually. .
Laboratory
1
2
3
4
N*
5
8
8
8
Neutral
2.4
3.7
6.0**
3.6
Mean Percentage Lipids
Polar
-
4.1
6.0
5.2
Total
-
7.8
12.0**
8.8
00
*N = number of individual oysters measured to determine mean percentage
lipid
** = significantly (<* = 0.05) higher in percentage lipid than labs 2 and
4, using ANOVA and Duncan's multiple range test
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Measurement of test-water conditions varied among laboratories
(Table 2). Salinity was within prescribed limits (>12 g/kg) for
Labs 1 and 2, but Lab 3 maintained salinity above 12 g/kg only during
the PCP test. As stated earlier, Lab 4 reported only test tempera-
tures in their final report. The report did state, however, that "open
ocean seawater" was used for their test; from this, one could assume
that salinity was >12 g/kg. The remaining test water conditions
(namely, temperature, pH, and D.O.) were measured by Lab 1, 2, and 3
and were within the prescribed limits.
Assuming that total organochlorine pesticides (other than the test
compounds) were sufficiently low that the oysters were not adversely af-
fected, we would expect that only the low salinity data from Lab 3 might
adversely affect the test results. Salinity in two of their tests drop
as low as 2 g/kg (Appendix D-2). Pumping rates of eastern oysters exposed
to these low salinities were undoubtedly reduced; however, Lab 3 made no
mention of reduced pumping rates or reduced deposition of feces or pseudo-
feces. The high steady-state BCFs associated with Lab 3 tend to indicate
that the oysters were unaffected.
Lipid Analyses at Steady-State
Each contract laboratory was requested to provide ERL, Gulf Breeze
with oysters collected at steady-state from each test so that we could
determine the oyster lipid content. Oysters were received frozen and
remained so until lipid analyses were conducted. Eight individual
analyses were made on oysters from each laboratory to determine polar,
neutral and total percentage-lipid (dry weight). Results of these
analyses are listed in Table 4. Lab 1 (ERL, Gulf Breeze) lipid anal-
yses were incomplete because different procedures were used to deter-
mine lipid than those used for the other laboratories (see Appendix F).
Also, Lab 1's oysters were used to identify potential toxicant-related
effects on lipid content of which none was statistically different;
therefore, were not used for comparison with other labs.
A comparison of the percent lipid values of labs 2, 3 and 4 (Table
4) with their respective steady-state BCF (Table 1) demonstrates a po-
tential relationship. Lab 3's oysters contained significantly higher
neutral and total lipids than those of Labs 2 and 4; correspondingly,
oysters of Lab 3 were also higher than those of the other two labora-
tories in the steady-state BCFs for all three chemicals tested. We did
not attempt to adjust the steady-state BCFs for lipid content, because
accurate wet weight could not be determined for the oysters. However,
recommendations for changes in the ASTM procedure to utilize fully
lipid data for BCF normalization follow in the Discussion section of
this report.
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DISCUSSION
The results of the bioconcentration "round-robin" tests indicate
that the ASTM method can generate reproducible results. The mean H/L
ratio for all three chemicals and four laboratories (excluding Lab 4
for POP) was 2.7, and represents the "worse case" situation for an
estimate of variability. Because replicate tests were not conducted,
no statistical techniques could be applied to the data. The mean H/L
ratio of 2.7, however, indicates good reproducibility, considering the
varying degrees of experience among laboratories in conducting biocon-
centration tests, wide geographic distribution among laboratories, and
different dilution water characteristics.
Several problems in the ASTM bioconcentration test procedure were
evident to us and to some of the contract labs. One difficulty was the
precise determination of steady-state during the test. Currently, the
procedure recommends the use of Analysis of Variance (ANOVA) to deter-
mine when steady-state has been attained (i.e., to determine when there
is no significant difference in residues from one sampling interval to
the next). The difficulty encountered was the occurrence of a wide
range of chemical residues in oysters during a single sampling period.
This wide variability about the mean caused difficulty in reliably
estimating steady-state. We have no firm recommendations for solving
this problem during this critical portion of the test (critical in that
one is unsure exactly when to start depuration), since the oyster's
biological variability is the basic cause. The only recommendation we
can support is to extend the exposure portion of the test one sampling
period beyond the minimum period to ensure that steady-state has been
reached.
Another difficulty encountered by contract laboratories was the
lack of accessible satistical models for analyzing bioconcentration
data. We at ERL, Gulf Breeze analyzed all data for uniformity
(NONLIN); but, in general, models such as BIOFAC and NONLIM are not
universally available and often require a professional statistician
for interpretation. We recommend that OTS, in concert with the re-
search laboratories, provide specific statistical packages and that
they be made available to contract laboratories.
The ASTM bioconcentration method could be improved by including
specific techniques for lipid analyses. We found substantial varia-
tion in the water content of oysters submitted by the various contract
laboratories due to sampling and storage differences and could not
normalized BCF values, which are based on wet weight, by using lipid
values, which are based on dry weight. We recommend that standardized
oyster sampling procedures be used (Appendix E). When oysters are
sampled for lipid analysis at steady-state, the wet weight should be
determined at the time of sampling; oysters should be freeze-dried and
reweighed before total lipids are determined (see Appendix F).
10
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APPENDIX A
ANALYTICAL CHEMISTRY PERFORMANCE EVALUATION
11
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APPENDIX A
November 7, 1979
U.S. Environmental Protection Agency
Environmental Research Laboratory, Gulf Breeze
OTS Bioconcentration Round Robin
Analytical Chemistry Performance Evaluation
Samples Enclosed:
1. 1,2,4-Trichlorobenzene—test material-20 gm.
2. Pentachlorophenol—test material-20 gm.
3. p,p'-DDE—test material-2xl gm.
4. 1,2,4-Trichlorobenzene—water spike (acetonitrile)-5 ml.
5. Pentachlorophenol—water spike (acetonitril<-.)-5 ml.
6. p,p'-DDE—water spike (acetonitrile)-5 ml.
7. Oyster Tissue Extract—blank (acetonitrile)-2x100 ml.
8. 1,2,4-Trichlorobenzene—tissue extract (acetonitrile)-J.OO ml.
9. Pentachlorophenol—tissue extract (acetonitrile)-100 ml.
10. p,p'-DDE—tissue extract (acetonitrile)-100 ml.
CAUTION; Refrigerate samples when not in use. Prior to use, allow bottle
to reach room temperature. Agitate bottle prior to sampling.
Analytical Standard Preparation: Prepare all analytical standards for
this study from the test material provided.
t
Water Sample Preparation: Add 1000 ml of test seawater to a 2000 ml separatory
funnel. Add exactly 500 microliters of appropriate water spike concentrate
to the separatory funnel and mix well. The sample is ready for extraction and
analysis. A blank 1000 ml seawater sample should be analyzed concurrently
for background.
Tissue Sample Preparation: Add 75 ml of aqueous 2% sodium sulfate (weight/
volume) to a 125 ml separatory funnel. Add exactly 10 milliliters of
appropriate oyster tissue extract to the separatory funnel and mix well.
The sample is ready for extraction, cleanup, and analysis. Blank oyster
tissue extract samples should be analyzed concurrently for background.
Reporting Results: Report,,in detail, the exact procedures used in this
study for extraction, cleanup, and analysis. Do not correct for background
or fortified recoveries. For water samples, report results in micrograms
per liter to the number of significant figures that your method will allow.
12
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Page 2
For tissue samples, report results in micrograms per gram, whereby 10
milliliters of extract is equivalent to 5 grams of wet tissue, and to the
number of significant figures that your method will allow. If replicate
samples are analyzed, report each result. In the final report, include
all original chromatograms (especially blanks), computer reports,
personnel data sheets, and calculations.
i
Report in detail the exact procedures to be used for water and tissue
analysis in the proposed bioconcentration studies with 1,2,4-trichloro-
benzene; pentachlorophenol; and p,p'-DDE. Include results of spike
samples and limits of detection. Further questions and final reports
should be addressed to:
Mr. Steven Schimmel
U.S. Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, Florida 32561
904-932-5311
Final reports for this performance evaluation must be received by us
no later than December 7, 1979. Actual bioconcentration tests may begin
after notification of successful completion of this performance evaluation
by Mr. Schimmel.
13
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APPENDIX B
CHEMICAL PERFORMANCE EVALUATION
14
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APPENDIX B
OYSTER BIOCONCENTRATION ROUND ROBIN: RESULTS OF CHEMICAL PERFORMANCE EVALUATION
Pentachlorophenol
SUBSTRATE WATER
Intended 4.0
Concentration ug/i
Lab
Std
Lab
Std
Lab
Std
Lab
Std
1
T
. Dev.
N
99%
C.I.
2
y
. Dev.
N
3
T
. Dev.
N
4
T
. Dev.
N
3.8
0.92
21
2.0-5.6
4.3
0.39
5
3.4
0.12
3
3.0
0.071
2
TISSUE
0.50
ug/g
0.35
0.062
14
0.23-0.47
0.63*
0.070
5
0.37
0.017
3
0.31
0
2
Trichlorobenzene p,p'
WATER TISSUE WATER
5.0 1.0 1.0
ug/A ug/g ug/i
4.7 0.67 0.91
0.048 0.034 0.030
13 13 13
3.8-5.6 0.60-0.74 0.85-0.97
57.* 0.65 1.7*
4.4 0.019 0.17
44 4
3.9 0.74 0.7
0.12 0.046 0.14
33 3
4.2 0.51 0.85
0 0.064 0.014
23 2
'-DDE
TISSUE
1.0
ug/g
0.86
0.046
15
0.77-0.95
0.96
0.017
4
1.0
0.0058
3
0.66*
0.021
2
15
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APPENDIX C
PROCEDURES FOR THE CHEMICAL
ANALYSIS OF DDE, TCP, AND TCB IN
WATER AND TISSUES
16
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CONTENTS
APPENDIX C
PROCEDURES FOR THE CHEMICAL ANALYSIS OF DDE,
TCP, AND TCB IN WATER AND TISSUES
1. Methods for the Analysis of 1,2,4-Trichlorobenzene 18
2. Determination of Pentachlorophenol in Marine Biota and Seawater
by Gas-Liquid Chromatography and High-Pressure Liquid
Chromatography 24
3. Pesticide Analytical Manual for BCF Contracting Agencies 28
4. Analytical Chemistry Performance Evaluation
(Materials and Methods) , 48
5. Analytical Chemistry Performance Evaluation
(Results and Discussion) 52
6. Scope of Work 58
7. Analytical Chemistry Performance Evaluation
(Methodology for Analysis) 63
17
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METHODS FOR THE ANALYSES OF
1,2,4-TRICHLOROBENZENE
A. EXTRACTION
1. TISSUE: One to eight grams of tissue were weighed into a 150 mm x 25 mm
(O.D.) screw top test tube and extracted four times with 5 mi acetonitrils using
a model PT10-ST Willems Polytron. After each extraction the test tube was centri-
fuged and the liquid layer decanted into a 120 mi oil sample bottle containing
10 mi of pet ether. IMPORTANT: The petroleum ether should be added to the
acetonitrils prior to the addition of the sodium sulfate solution. Finally add
75 mi of 2% (w/v) aqueous sodium sulfate solution after all acetonitril extracts
have been added to the sample bottle. The bottle was shaken for one minute and
the layers allowed to separate. The upper pet ether layer was pipetted into a
25 m£ concentration tube. Repeat this step two more times. The pet ether was
concentrated to about one mi on a nitrogen evaporator with a water bath maintained
at 35°C.
2. WATER: One liter seawater samples were extracted two times with 2-100 mi
portions of pet ether, dried by passing through a stainless steel funnel containing
a small plug of glass wool and collected in a 20 mi Kuderna-Danish concentration.
IMPORTANT: 1,2,4-TRICHLOROBENZENE is volatile, therefore, pet ether must be
added to the sample container prior to the addition of seawater samples. Spiked
seawater samples were treated in this manner prior to the fortification with
trichlorobenzene.
B. CLEANUP
After concentration, the sample was transferred to a 9 mm chromaflex column
containing 3.0 grams of Florisil topped with 2.0 grams of anhydrous sodium sulfate
and rinsed with 10 mi hexane. 1,2,4-trichlorobenzene was eluted from the column
with 20 m£ of hexane.
18
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C. GAS CHROMATOGRAPHIC PARAMETERS
•
A Hewlett Packard model 5710A. gas chromatograph with an electron-capture
detector was used for the analysis. The operating parameters were: oven
temp. 125°C; detector temp. 30Q°C; injector temp. 200°C; carrier gas, helium
at 30 raJi/min. ; alternation 32. The column was 182 cm x 2 mm (I.D.) glass
packed with 2% SP2100 used with the computer controlled sampler.
D. DETECTION LIMIT
Residues from oyster tissue were calculated on a wet weight basis. The
lower limit of detection for 1,2,4-trichlorobenzene in tissue and water is
0.010 ppm and 0.002 ppb respectively. The lower limit is based on a dilution
of 1 mi per gram of tissue used and 2 mi, dilution for a one liter seawater
sample. The standard concentration is 0.040 ng/yi and 5 microliters is routinely
used for each injection. IMPORTANT: All standards should be made up by spiking
into a solvent, not into open glassware.
19
-------
E. TABLE OF RECOVERIES FROM TISSUE AND SEAWATER
fortified (ppm) recovered (ppm) % rec.
OYSTER
10
10
5.0
5.0
2.0
2.0
1.0
1.0
5.0
5.0
5.0
fortified (ppb)
0.08
0.12
0.20
0.50
1.0
0.040
0.080
0.080
0.40
0.40
1.0
8.3
8.5
4.1
4.0
1.7
1.6
0.81
0.87
5.0
5.1
4.9
recovered (ppb)
0.075
0.12
0.18
0.46
0.99
0.043
0.082
0.081
0.33
0.32
0.83
83
85
81
80
85
82
81
87
100
102
97
% rec
94
100
90
92
99
108
103
101
83
80
83
SEAWATER
20
-------
I
_J
_:.._ i _ '_. . i
• i
'I
-
- I-
,_... I . .
:_. i .....
I ..
21
-------
t-
U_ _ .
f
... -1
•'?•
i'
-.CO—
68
•T.--±E
. . i : •_ . '
r ]•••• -._.....;-,-:«——-,
|-— ••" -~r-i—---i—! :
' ._ .____._•_.__ L. i.. '.! _. ... i
_-:.... . . : i |I".T (.-,-! .
"~Tr.7.7i"7T"t'
-------
FDA No. 195
WITHDRAWN 10-26-67
Technical Material
DATA SHEET
FOOD AND DRUG ADMINISTRATION
Reference Standards Section, Residue Chemistry Branch,
Division of Pesticides, Bureau of Science, Washington, D.C. 20204
FDA No. 195
Lab Code: 66-45
Name: TCB(1,2,4)
Systematic Name: 1,2,4-Trichlorobenzene
Empirical Formula: Cg^C^ M.W. 181.46 State: Liquid
Structure:
Cl
Stability: Stable
Toxicity: May be irritating to eyes and mucous membranes
Manufacturer's Assay: Boiling Range: 213 - 214 °C
Rev Sept 1969
23
-------
Published in Journal of Agricultural & Food Chemistry, May-June 1979, Pages 554-557, by the American Chemical Society
Determination of Pentachlorophenol in Marine Biota and Sea Water by Gas-Liquid
Chromatography and High-Pressure Liquid Chromatography
Linda F. Faas* and James C. Moore
A method is described for measuring pentachlorophenol (POP) in samples from the estuarine environment
Gas-liquid Chromatography (GLC) is used to determine PCP residues in tissues as low as 0.01 ppm by
formation of the ethyl diazohydrocarbon derivative, followed by Florisil cleanup. Application of the
method to exposed organisms indicates that PCP accumulates in mullet (Mugil cephalus}, grass shrimp
(Palaemonetes pugio), and eastern oysters (Crassostrea uirginica). Sea water concentrations as low
as 0.002 ppb may be detected by formation of the amyl diazohydrocarbon derivative. Formation of the
amyl derivatives of PCP and several related compounds gives GLC separation not possible with the
methyl or ethyl derivatives. Parameters are outlined for high-pressure liquid Chromatography (LC)
determination of the free phenol without cleanup. Ultraviolet detection limits for PCP by LC are 5.0
ppm in tissues and 2.0 ppb in seawater.
Pentachlorophenol (PCP) is a herbicide, fungicide/ was estimated to be 20 million pounds (Hoos, 1978).
bactericide, and insecticide that, together with its salts, PCP has been found in drinking water (Abrams et al.,
has a broad spectrum of industrial, agricultural, and 1975), rivers, lakes, and streams (Buhler et al., 1973;
domestic applications (Benvenue and Beckman, 1967). Fountaine et al., 1976; Pierce and Victor, 1978; Rudling,
U.S. production of PCP in 1977 was expected to be 80 1970), sewage effluents (Abrams et al., 1975; Buhler et al.,
million pounds (Cirelli, 1978), and annual Canadian usage 1973), aquatic biota (Pierce and Victor, 1978; Rudling,
1970; Zitko et al., 1974), and even in man (Barthel et al.,
1969; Rivers, 1972).
U.S. Environmental Protection Agency, Environmental The toxicity of PCP to aquatic organisms (Adelman et
Research Laboratory, Sabine Island, Gulf Breeze, Florida al.. 1976; Benvenue and Beckman, 1967; Schimmel et al.,
32561. 1978) and its effects on settling communities (Tagatz et
24
-------
POP Determination in Marine Biota
J. Agric. Food Chem., Vol. 27, No. 3, 1979 555
al., 1977) have been investigated. Also. PCP contamination
has -Seen implicated in several fish kills (Pierce and Victor,
1978: Schimmel et al., 1978).
These data indicate that PCP poses a potential threat
to 'he estuarine environment. Therefore, a sensitive re-
liable method is required for routine analysts of PCP in
marine biota and seawater. A variety of analytical methods
for analysis of PCP have been reported (Barthel et al.,
1969; Benvenue and Beckman, 1967; Buhler et al., 1973;
Fountaine et al., 1975; Pierce and Victor, 1978; Rivers,
1972; Rudling, 1970) but do not describe the determination
of PCP in marine biota and sea water.
This paper describes the extraction of PCP from sea
water and fish (Mugil cephalus), shrimp (Palaemonetes
pugio), and oyster (Crassostrea virginica) tissues by
formation of the ethyl and amyl diazohydrocarbon de-
rivatives for GLC analysis, with Florisil cleanup for tissue
samples. A technique for high-pressure liquid chroma-
tography (LC) determination of PCP in tissues and sea
water without derivatization or cleanup is outlined. Tables
of retention times of the amyl derivatives illustrate the
GLC separation of PCP from several related compounds.
EXPERIMENTAL SECTION
Apparatus. We employed Varian Models 1400 and
2100 gas chromatographs equipped with SH electron-
capture detectors and 182 cm x 2 mm i.d. glass columns
parked with 2% SP2100 on 100/120 mesh Supelcoport,
0.75% SP2250:0.97% SP2401 on 100-120 mesh Supel-
coport or 5% QF-1 on 80-100 mesh Gas-Chrom Q. Op-
erating parameters were: oven temperature, 180-190 °C;
injector temperature. 210 °C; detector temperature, 200
°C; nitrogen carrier gas. 25 mL/min. Identity of the PCP
eihyl and amyl derivatives was confirmed by gas chro-
matography-mass spectrometry (GC-MS), using a Fin-
nigan Model 1015 mass spectrometer. The Waters LC
system consisted of a Model U6-K injector, Model 440 UV
absorbance detector operated at 254 nm and 0.01 aufs
using a Model 6000A pump. A Waters /iBondapak CN
column with dimensions of 30 cm x 3.9 mm i.d. was used
isocratically with 2.5% (v/v) isopropyl alcohol in isooctane
at 2.0 mL/min. Initially, it was difficult to obtain re-
producible peaks for PCP with LC. After repeated in-
jections, however, conditions stabilized. Therefore, column
conditioning may be necessary to achieve the LC results
reported here. The tissue blender was a Willems Polytron
Model 10-ST (Brinkman Instruments, Westbury, NY).
Reagents. All solvents were Nanograde (Burdick and
Jackson Laboratories. Inc.). The A/-nitrosoguanidine
precursors used to generate diazohydrocarbons were ob-
tained from Aldrich Chemical Company. (CAUTION:
Extreme care must be exercised in handling /V-nitroso-
guanidine precursors that are potent mutagens, skin ir-
ritants, and carcinogen suspects.) The reagent grade
anhydrous sodium sulfate was obtained from Baker
Chemical Co. Florisil, PR Grade, 60-100 mesh (Floridin
Company) was activated overnight at 1.10 °C before use.
Standards. Pentachlorophenol (PCP). 2,4,5-tri-
chlorophenol, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T),
2,4-dichlorophenoxyacetic acid (2,4-D), 2-methoxy-3,6-
dichlorobenzoic acid (dicamba), and 2-(2,4,5-trichloro-
phenoxy)propionic acid (silvex) standards were obtained
from the Pesticide Reference Standard Section, U.S.
Environmental Protection Agency, Washington, DC.
Tetrachlorohydroquinone, p-nitrophenol, 2,4,6-tri-
chlorophenol, and 2,3,4,6-tetrachlorophenol were obtained
•from Chem Service, Inc., West Chester, PA.
Primary standards were prepared hy diluting 100 mg to
100 mL with benzene. Working standards for GLC were
Table I. Recoveries of PCP from Fortified Oyster,
Shrimp, and Fish Tissues" and from Sea Water
fish
(edible tissue)
oyster
shrimp
(whole body)
fish
(whole body)
fortifica-
tion level,
ppm
0.05
0.05
0.05
15.06
deriva-
tive
formed
ethyl
ethyl
ethyl
no. of
sam-
ples
5
4
5
4
percentage
recov.
X ± SD
90.6 t
91.5 t
91.8 t
95. 0±
13.3
3.8
6.1
6.5
5.0*
1.0C
0.01
ethyl
amyl
15
5
5
89.4 t 5.7
95.8 i 3.1
97.2 ± 7.8
0 Tissue samples were 3.0-10.6 g. b Determined by LC
as the free phenol. c Extracted from 100 mL of sea water
with 2-50-mL portions of 1:1 (v/v) diethyl ether/petrole-
um ether.
prepared by diluting the primary standard solutions with
petroleum ether. The PCP fortification standard was
prepared by diluting the primary standard to 10 ng/^L
with acetone. Working standards for LC were prepared
in 2.5% (v/v) isopropyl alcohol in isooctane. All standards
were stored in amber bottles closed with Teflon-lined screw
caps.
Procedures. One to eight grams of tissues were weighed
into 150 x 25 mm o.d. screw-cap test tubes and extracted
four times with 5-mL portions of acetonitrile for 30 3 using
a Willems Polytron tissue homogenizer. The test tube and
contents were centrifuged after each extraction, and the
acetonitrile was transferred to a 120-mL oil sample bottle.
Seventy milliliters of 2% (w/v) aqueous sodium sulfate was
added to the combined extracts. The pH was adjusted to
2-3 with 6 N HC1. (Aqueous sodium sulfate and 6 N HC1
were prepared with distilled water that had been acidified
and extracted with hexane to remove interfering coex-
tractives.) This mixture was extracted with one 10-mL
portion and then with two 5-mL portions of hexane. The
bottle, sealed with a Teflon-lined screw cap, was shaken
1 min, the solvent phases were allowed to separate, and
the upper hexane layer was pipetted into a 25-mL con-
centrator tube. The combined extracts were then con-
centrated to 0.5 mL by evaporation under a gentle stream
of nitrogen while in a water bath maintained at a tem-
perature of 40-50 °C. The ethyl derivative was formed
by adding 10-12 drops of diazoethane, as described by
Stanley (1966). Fresh diazohydrocarbons were prepared
weekly and stored at -4 °C. (CAUTION: Gloves and a
high-draft hood must be used in handling diazohydro-
carbons, which are toxic and potentially explosive carci-
nogens.) This mixture was allowed to stand at room
temperature for 20 min, and the excess diazoethane was
evaporated with a gentle stream of nitrogen. A 9-mm
Chromaflex column (Kontes Glass Company, Vineland,
NJ) was filled with 3.0 g of Florisil topped with 2.0 g of
anhydrous sodium sulfate and washed with 10 mL of
hexane. The concentrate was quantitatively transferred
to the column with three 0.5-mL portions of hexane and
the ethyl derivative was eluted with 20 mL of 5% (v/v)
diethyl ether in hexane. The volume was adjusted to the
appropriate concentration for analysis by GLC.
For water samples, 1 L of sea water was extracted twice
with 100-mL portions of 1:1 (v/v) diethyl ether/petroleum
ether in a 2-L separatory funnel. The extract, collected
in a Kuderna-Danish concentrator, was evaporated to 5
mL on a steam table with a Snyder column. The con-
centrator tube was transferred to a nitrogen evaporator;
the extract was concentrated to 0.5 mL, derivatized with
-------
556 J. Agric. Food Chem.. Vol. 27. No. 3, 1979
Faas. Moore
1
4
1 4 3 • 1 •
TIME d+*m)
Figure 1. (A) LC chromatograms of the extracts of oyster, fish, and shrimp tissues fortified with 15 ppm PCP. Shaded area represents
chromatogram of blank tissue extracts. Fish tissue extract was cleaned with a basic methylene chloride extraction; oyster and shrimp
tissues were not. Amount injected was 15 nL. (B) GLC chromatogram of combined four 1-L seawater extracts. Shaded area represents
combined blank extracts and PCP concentration is 0.002 ppb. Amount injected was 5 nL.
Table II. Measured Residues of PCP in Fish, Shrimp, and
Oysters Exposed to Several Measured Concentrations of
PCP in Flowing Sea Water for 96 h
species
fish (Mugil cephalus)
shrimp (Palaemonetes pugio)
i-
oyster (Crassest re a uirginica)
concn in
water,
ppb
46.0
85.0
157.0
32.0
54.0
76.0
249.0
2.8
26.0
tissue
residues,
ppm
0.29
6.7
8.8
0.050
0.10
0.23
0.43
0.18
0.86
diazoethane for GLC analysis, and analyzed without
Florisil cleanup. \
Interference with the PCP ethyl derivative was often
observed for PCP determinations in sea water at con-
centrations lower than 0.01 ppb. To reduce this back-
ground, sea water was extracted at pH 9 with methylene
chloride before extracting PCI' at pH 2-3. Also, vent-peak
tailing and interference from coextractives or impurities
in the diazohydrocarbons were decreased by washing the
extract with 5 mL of 20% (v/v) distilled water in metnanol
(Thompson, 1974). All glassware was washed with 1 N
KOH, distilled water, and Nanograde acetone (Thompson,
1974). Formation of the amyl derivative increased re-
tention time sufficiently to separate PCP from early
eluting peaks that were observed. The diazopentane was
prepared by the method of Shafik et al. (1973), and de-
rivatization of PCP was carried out in the same manner
as formation of the ethyl derivative.
For analysis by LC, sea water and tissues were extracted
as outlined above, without derivatization or Florisil
cleanup. However, for tissue analyses, a basic methylene
chloride extraction was sometimes necessary to reduce
background interference (primarily in fish extracts). The
aqueous sodium sulfate solution was adjusted to pH 9 and
the solution extracted once with 10 mL of methylene
chloride. The methylene chloride was discarded, the pH
of the remainder was adjusted to 2-3, and the solution was
extracted with hexane as above.
A known amount of the PCP fortification standard was
added to sea water and tissue samples just prior to analyses
to determine recoveries. The standard for comparison was
Table III. Retention Times of Ethyl and Amyl Derivatives of Several Phenols and Acids Relative to Aldrin on Three
Different GLC Columns
compound
2,4,6-trichlorophenol
p-nitrophenol
2,4,5-trichlorophenol
dicamba
2,3,4,6-tetrachlorophenol
2,4-D
silvex
pentachloro phenol
2,4, 5-T
tetrachlorohydroquinone
aldrin
2%SP
ethyl
0.14
0.17
0.20
0.27
0.28
0.38
0.57
0.55
0.62
0.65
1.00
2100
amyl
0.40
0.52
0.54
0.72
0.79
0.98
1.41
1.54
1.64
5.37
1 00
0.75% SP2250:0
ethyl
0.13
0.25
0.20
0.32
0.27
0.48
0.63
0.56
0.80
0.68
i nn
.97% SP 2401
amyl
0.34
0.65
0.51
0.85
0.73
1.24
1.58
1.52
2.11
5.30
i r\t\
5%
ethyl
0.17
0.55
0.28
0.51
0.34
0.79
0.96
0.64
1.20
0.83
1 f\f\
QF-1
amyl
0.40
1.38
0.64
1.19
0.79
1.82
2.06
1.47
2.70
4.45
* "* **
-------
J. Agric. Food Chem., Vol. 27, No. 3, 1979 557
prepared from the fortification standard.
RESULTS AND DISCUSSION
The GLC chromatograms of fish, shrimp, and oysters
that had been exposed to or fortified with PCP were clean
and showed no interfering peaks. The lower limit of
detection for PCP in tissues by the GLC method with ethyl
derivatization and Florisil cleanup was 0.01 ppm.
LC offers a rapid method for determination of PCP
residues in tissues and sea water without derivatization or
Florisil cleanup. Concentrations of PCP above 2.0 ppb in
sea water and 5.0 ppm in tissues can be quickly determined
by this method. LC chromatograms of PCP recovered
from fortified oyster, shrimp, and fish tissues are shown
in Figure 1A.
Table I lists the average percentage recovery of PCP
from fortified fish, shrimp, and oyster tissues and sea water
by the above extraction procedures. Residues measured
in tissues of exposed animals are shown in Table II. These
residues were determined by GLC after formation of the
ethyl derivative and Florisil cleanup.
Formation of the amyl derivative can be used to separate
PCP from impurities, coextractives and several related
compounds. Figure IB illustrates the composite extract
of 4 L of sea water. By using the amyl derivative and
techniques to determine concentrations of PCP less than
0.01 ppb in sea water, PCP can be resolved from back-
ground at 0.002 ppb.
From the retention times of the ethyl and amyl de-
rivatives of PCP and several related compounds listed in
Table III, it can be seen that the amyl derivatives increase
retention times sufficiently to give GLC separation not
possible with the ethyl derivatives. Although no one
column provides complete resolution, a combination of any
two columns will allow separation of all ten compounds.
The amyl derivative can be used for tissue as well as sea
water samples should PCP be present in combination with
any of these compounds.
Our method, successfully tested in the estuarine envi-
ronment, offers: routine application, minimum cleanup,
elimination of interference with coextractives and several
related compounds observed with other derivatives, and
improved sensitivity.
ACKNOWLEDGMENT
We thank Steven C. Schimmel, James M. Patrick, Jr.,
and Monte Treadway for collecting and exposing test
animals and Steven S. Foss and Terry Miller for tables and
illustrations.
LITERATURE CITED
Abrams, E. F., Derkics, D., Fonp, C. V., Gunman, D. K., Slimak,
K. M., Office of Toxic Substances, U.S. Environmental Pro-
tection Agency, EPA-560/3-75-002, 1975.
Adelman. I. R., Smith, L. L., Jr., Siesennop, G. D., J. Fish. Res.
Board Can. 33, 203-208 (1976).
Barthel, W. F., Curley, A., Thrasher, C. L., Sedlak, V. A.,J. Assoc.
Off. Anal. Chem. 52(2), 294-298 (1969).
Benvenue, A., Beckman, H., Residue Rev. 19, 83-134 (1967).
Buhler, D. R., Rasmusson, M. E., Nakaue, H. S., Environ. Sci.
Technol. 7(10), 929-934 (1973).
Cirelli, D., Environ. Sci. Res. 11, 13-18 (1978).
Fountaine, J. E., Joshipura, P. B., Keliher, P. N., Water Res. 10,
185-188 (1976).
Fountaine, J. E., Joshipura, P. B., Keliher, P. N., Anal. Chem.
47(1). 157-159 (1975).
Hoos, R., Environ. Sci. Res. 11, 3-11 (1978).
Pierce, R. H., Jr., Victor, D. M., Environ. Sci. Res. LI, 41-52 (1978).
Rivers, J. B., Bull. Environ. Contam. Toxicol. 8(5), 294-296 (1972).
Rudling, J., Water Res. 4, 533-537 (1970).
Schimmel, S. C., Patrick, J. M., Jr., Faas, L. F., Environ. Sci. Res.
11, 147-156 (1978).
Shafik, T. A., Bradway, D. E., Enos, H. F., Yobs, A. R., J. Agric.
Food Chem. 21(4), 625-629 (1973).
Stanley, C. W., J. Agric. Food Chem. 14(3), 321-323 (1966).
Tagatz. M. E., Ivey, J. C., Moore, J. C., Tobia, M., J. Toxicol.
Environ. Health 3, 501-506 (1977).
Thompson, J. R., Ed., "Analysis of Pesticide Residues in Human
and Environmental Samples", prepared by Pesticide and Toxic
Substances Effects Laboratory, National Environmental
Research Center, U.S. EPA, Research Triangle Park, NC, 1974.
Zitko, V., Hutzinger, 0., Choi, P. M. K., Bull. Environ. Contam.
Toxicol. 12(6), 649-653 (1974).
Received for review October 2,1978. Accepted January 16,1979.
Gulf Breeze Contribution No. 350. Mention of a commercial
product does not constitute endorsement by the U.S. Environ-
mental Protection Agency. Presented at the Division of Pesticide
Chemistry, 176th National Meeting of the American Chemical
Society, Miami Beach, FL, Sept 1978.
27
-------
PESTICIDE ANALYTICAL MANUAL
FOR
BCF CONTRACTING AGENCIES
by
Alfred J. Wilson, Jr.
U. S. Bureau of Commercial Fisheries
Biological Laboratory
Gulf Breeze, Florida 32561
28
-------
This compilation of procedures was prepared Co promote uniformity
among contracting laboratories in the analysis of organochlorine
pesticide residues in the marine environment.
The methods are adaptations of techniques described in the
literature of pesticide residue methodology. We have success-
fully employed these methods for the past three years and have
obtained adequate recovery rates with a minimum of time and
effort.
I have tried to describe the procedures in detail so that: a
technician with a tnyi-Lmum of experience should be able to
perform the analysis with little difficulty.
It is beyond the scope of this manual to discuss the theory and
operation of Gas-Liquid chromatography. A brief section is
devoted to operational hints which the beginner will find use-
ful.
29
-------
EQUIPMENT
The following list of equipment is based on the analysis of
six samples at a time.
Sample Preparation:
Osterizer with six cutting heads
6 1-pint Mason jars
Sample Extraction:
\
Six place Soxhlet apparatus complete with extraction
assemblies for 123 mm x 43 mm Whatman extraction shells
6 Whatman extraction shells, 123 mm x 43 mm, single
thickness
6 3-ball Snyder columns (Kontes Glass Company, Catalog
number K-50300)
Sample Clean-up:
6 250-ml separatory funnels with teflon stopcocks
12 crystallizing dishes, 70 mm x 50 mm
12 chromatographic tubes, 400 mm x 20 mm
12 powder funnels, 100 mm diameter
12 500-ml Erlenmeyer flasks with 24/40 joint
6 250-ml vacuum flasks
12 funnels, 65 mm diameter
12 25-ml glass stoppered graduated cylinders
Miscellaneous Equipment:
Slide warming table (Ranson)
Freezer
Water bath, eight hole
Fume hood
Glass wool 30
-------
Miscellaneous Equipment (contd.):
2 Vacuum pumps
Wash bottles •
Pipettes, volumetric, transfer (1, 2, 3, 4, 5, 10 ml)
Volumetric flasks (25, 50, 100, 250, 500 ml)
Separatory funnel, 2000 ml
2 oz. Amber* bottles with Teflon lined cap's for standards
(A. H. Thomas)
Pesticide standards: Obtain reference grade material direct .
from manufacturers or from U. S. Public Health Service,
Pesticides Repository, P. 0. Box 49&, Perrine, Florida 33157.
REAGENTS
Sodium sulfate, anhydrous, powder (J. T. Baker #3898)
Quso - G30 Unreductionized (Philadelphia Quartz Co.)
Sand, Sea (Fisher Scientific #S-25)
Petroleum ether, ethyl ether and acetonitrile (Burdick and
Jackson redistilled solvents)
Acetonitrile: Saturate in a separator/ funnel with petroleum ether.
Vent pressure by opening stopcock. •
6% eluting mixture: dilute 60 ml of ethyl ether to 1000 ml with
petroleum ether.
15% eluting mixture: dilute 150 ml of ethyl ether to 1000 ml with
petroleum ether.
Florisil PR Grade (Floridin Co., 3 Pcnn Center, Pittsburgh, Pa.
15235): for use, heat at least 5 hours at 130°C. Store in desiccator
at room temperature. If stored more than 2 days after heating, it
must be reheated and cooled to room temperature before use.
31
-------
REAGENTS (contd.)
Celite 545 (Johns-Manvilie Co.)
Magnesium oxide (Fisher Scientific, Sea Sorb #43): Treat as
follows: Prepare a distilled water slurry of about 500 grams,
heat on boiling water bath about 30 minutes and filter with
suction. Dry overnight at 130°C., let cool and pulverize
with Osterizer. Store in closed jar.
Magnesium oxide - Celite mixture: Mix treated MgO with Celite
1:1 by weight.
SAMPLE PREPARATION
Cleanliness is essential during all operations. Gas-liquid
chromatography permits the detection of organochlorine pesticides
at the 10"^ gram level and less. All glassware and utensils
must be thoroughly cleaned and rinsed with acetone' to prevent
cross contamination of samples.
Samples for bioassay should be prepared as soon as possible after
collection. If necessary, they may be held over for 1 or 2 days
maintained as you would fresh food; they need to be refrigerated
but not frozen. They may be frozen for longer storage but care
must be taken on thawing them, so that all of the drained tissue
fluids are saved and blended with the sample. MoHusks should be
shucked before freezing.
Oysters, Clams, and Mussels
1. Open by cracking hinge. Remove free water by shaking out 3
times after shell has been opened but before the adductor
muscle is cut. Then, shuck 12 individuals into clean Mason jar.
2. Blend for 1 minute or until sample is well homogenized.
3. Place 30-5 grams of the homogenate in a tared, numbered, 1-pint
standard Mason jar and record all weights on data sheet.
4. Cap with Ostcrizer Cutting Assembly and place in freezer to
about 1/2 hour - DO NOT FREEZE. Use separate cutting head for
each sample to save time.
5. Remove from freezer and add exactly three times the sample's
weight of the desiccant mix.
32
-------
c
C
The desiccant mix is made up of about 10% Quso (precipitated
silica) and 90% anhydrous sodium sulfate. It is convenient
to make the mix by weighing out exactly 9 grams of Quso
and adding enough Na2SO, to bring the Mix to the required
weight of precisely 3X the amount of sample homogenate,
(2X for fish samples, up to 9X for plankton).
9
6. Mix desiccant and sample in jar thoroughly with a spatula
being sure to :.ncorporate all tissue adhering to sides and
bottom of j£.r.
7. Recap with Cutting Assembly and place in freezer. Allow
mixture to freeze (about 1 hour).
8. Remove from freezer and grind until mixture is relatively free
flowing. Cover -jar with can or plastic beaker while grinding
as a safety measure.
9. Refreezing and regrinding may be necessary to obtain a free
flowing mixture.
10. . Remove Cutting Assembly and seal with standard Mason jar cover.
Store sample in freezer.
Fish
Fish samples: 15 fish are blotted dry and passed through a meat
grinder three times, mixing between grinding. A 35-gram
(approximately) aliquot is added to a tared, number 1-pint
Mason jar and the sample prepared as for oysters, except that
exactly 2 times the sample weight of desiccant mix is added.
Small fish (15) - total weight less than 60 grams; blot fish
dry and freeze. Remove from freezer, separate fish and place'
in tared, numbered Mason jar. Add exactly 2 times the sample
weight of desiccant mix and grind immediately. Cover jar with
can or plastic beaker xjhile grinding. Return sample to freezer
for 1 hour or longer: remove sample and regrind. Refreezing
and regrinding may be necessary to obtain a free flowing sample,
Do not use high speed until sample is partially homogenized.
Snails °
Crack shell and remove entire body. Proceed with Step 2 under oysters.
33
-------
Lobster and Large Crabs
Remove and discard dorsal carapace of cephalothorax after
cutting free any attached soft tissues. Proceed as with fish.
Plankton
Drain sample on reagent grade filter paper; remove jelly fish,
ctenophores, obvious small fish and pieces of trash, and trans-
fer to clean glass dish such as a watch glass. Mix well with
spatula, remove and weigh a small sample (5 gms. or less). Dry
this sample at 90°C. for 24 hours and weigh for determination of
moisture content. Proceed with the remainder of the sample as
for oysters, starting with Step 3. Because of varying amount
of moisture, residues in plankton are recorded on a dry weight
basis and therefore appear to be relatively higher.
If 30 gram, samples cannot be obtained, a minimum of 10 grams can
be analyzed. When preparing samples of less than 30 grams,
adjust the weight of desiccant mix added so that sample plus
desiccant will be about 100 grams.
(Example:
Sample weight
Desiccant factor
Desiccant weight
Gross weight
SAMPLE EXTRACTION
i. Write the sample number in pencil on the extraction shell.
2. Weigh shell and record on sample data sheet.
3. Using a clean powder funnel, transfer prepared sample from Mason
jar to shell and record weight of sample plus shell on sample
data sheet.
4. Place about 1/4" thick a wad.of glass wool on top of each shell.
34
10.4 grams (wet weight)
-------
5. Number a 300 ml extraction flask with sample number, add
1/4 teaspoon full of sand and approximately 250 nil of
petroleum ether.
6. Place shell containing sample in Soxhlet apparatus and join
the corresponding numbered extraction flaski Turn on heater
and water and extract sample for 4 hours. Heaters should be
adjusted to permit solvent to completely cycle once every
6 to 7 minutes.
7. To obtain.weight of sample extracted, divide net weight of
sample by a number one greater than the Na2$0/ factor used
to prepare.sample.
Example (oyster sample):
Weight of prepared sample -h shell 238.3 grams
Weight of shell 113.5 "
Weight of prepared sample 124.8 "
Na2S04 factor is 3 thus: 124-8 = 31.2 grams extracted.
4
SAMPLE CLEAN-UP
Note 3. A. Acetonitrile - petroleum ether partition
1. Turn off heaters on extractors after 4 hours.
2. After boiling has completely stopped, remove extraction flask
from Soxhlet apparatus and discard shell containing sample.
•j
3. Add more sand to flask, attach Snyder column and evaporate
to about 10 ml on steam table in a furae hood. Wash Snyder
column with about 5 ml of petroleum ether and let drain
into extraction flask.
4. Remove Snyder column and transfer entire extract to a 250 ml
separatory funnel equippped with teflon scopcock. Dilute '
extract to 25 ml. Before analysis, add 25 ml of petroleum
ether to each 250 ml separatory funnel and etch at the 25 ml
level.
35
-------
f
5. Add 50 ml of acetonitrile saturated x*ith petroleum ether,
and shake for 1 minute,
6. Drain lower layer (Acetonitrile) into a, crystallizing dish,
7. Repeat Step 5.
8. Drain second 50 ml lower layer into the same crystallizing
dish as in Step 6« Discard upper ether layer remaining
in separatory funnel. *
9. Evaporate acetonitrile extract just to dryness in hood on
a slide warming table (approximately 40 °C.) with draft
over top of. dish.
B. Florisil column
1. Prepare 400 mm x 20 msi chromatographic tube with 4 inches
(after settling) of Florisil, topped with 1/2 inch of
Na^SO, . Tap or vibrate column to settle material. Before
analysis, check each lot of Florisil with a mixture of
pesticide standards to assure that separation is correct,
complete and the recoveries are quantitative. All
monitored pesticides should elute from the column with
200 al of the 67, eluting mixture, except dieldrin and
endrin which will elute with 200 ml of the 15% eluting
mixture. These volumes may have to be adjusted from
batch to batch of Florisil.
2. Wash column with 35 tal of petroleum ether and discard wash.
3. Place a 500 ml Erlenmayer flask equipped with 24/40 joint
under column and transfer entire extract from, crystallizing
dish to column with petroleum ether. When all of extract
has past through Na^^O^ layer, elute column with 200 ml of
67* ethyl ether in petroleum ether.
4, When the last of this elute has passed through Na£SO^ layer,
remove receiving flask and replace with another. Then elute
column with 200 ml of 15% ethyl ether in petroleum ether.
5. After the column has been eluted, add sand to each receiving
flask and fit Sayder column. Evaporate to approximately
10 ml on a steam table. Rinse Snyder column, with about
5 ml of petroleur.. ether.
36
-------
c
6. Quantitatively transfer 6% eluate to a 25 ml glass-
stoppered graduated cylinder with petroleum ether and
dilute to the 25 ml mark. The 6% extract is now suitable
for gas chromatography.
7. The 15% extract needs further clean-up on an MgO - Celite
column. Attach a 400 mm x 20 mm chromatograph tube to
a 250 ml vacuum flask with a rubber stopper. Attach
vacuum hose to side arm. With full vacuum, add 10 grams
of a 1:1 raixtura of MgO - Celite. Bleed vacuum line ..
•
and wash column with 35 ml petroleum ether so'that the
elutipn rate is approximately 15 - 20 ml/miri.% It is not
necessary to discard wash. Quantitatively transfer the
15% eluate to this column. When last of sample just
reaches top of MgO - Celite mixture, elute column with
100 ml petroleum ether. Quantitatively return eluate
to 500 ml Erlenmeyer flask and evaporate on steam table
as in Step 5.
8. Quantitatively transfer MgO - Celite eluate to a glass-
stoppered graduated cylinder with petroleum, ether and
dilute to the 25 ml mark. The 15% extract is now ready
for gas chroma to graphy.
GAS LIQUID CHROMATOGRAPHY
Be thoroughly familiar with the operation and theory of your gas
chromatograph before attempting to evaluate your extraction and
clean-up procedures. Read and understand the manufacturer's
instruction manual.
The following list of suggestions will be helpful to those using
the Model 610D Varian Aerograph Gas Chromatograph.
N'ote U. 1. If only one column is used, obtain 3% DC 200 on Gas Chrom
Q 80/100 mesh from Applied Science Laboratories, State
College, Pennsylvania. Pack this material in a 5' x 1/8"
Pyrex column and condition for 24 hours at 225°C. The
second recommended column packing material is 5% QF-1
on Gas Chrom Q 80/100 mesh. This material is also packed
in 5'. x 1/8" Pyrex column, but must be conditioned 3 days
at 225°C. The latter column provides different retention
37
-------
Nota 5,
times for pesticides and thus aids in. identification.
Use silane treated glass wool (Applied Science
Laboratories) to cap ends of column.
2. Bake all silicone "0" rings and injector septa overnight
at 2QO°C. before use.
3. Replace injector septa and clean glass injector inserts
with solvent at the end of each working day.
4. Replace 13X molecular sieve in gas line filter when
nitrogen cylinder is changed.
5. Oil oven fan every 30 days.
6. Remove EC detector from column effluent overnight and
store in heated EG gold cylinder. (This cuts down on
the column bleed to the tritium foil, thus prevents
frequent cleaning.)
7. Standards made up at the following concentrations (nanograa
per microliter) will provide an on scale chroiaatograza of
each standard at 1QX to 32X attenuations, depending upon
detector sensitivity. Inject 5 microliters. See appendix
for sample chromatograms.
8.
Standard A
Lindane
Heptachlor
Aldrin
Heptachlor epoxide
p,p' DDE
p,p' DDD (IDE)
PsP' DDT
Standard 3
0.008 ng/ul
0.02 "
0.02 "
0.02 "
0.04 "
0.08 "
0.12 "
Dieldrin
Endrin
0.05 ng/ul
0.05 "
Standard C
Methoxychlor 0.3 ng/ul
At chart speeds of 30 inches/hour, the ideal retention, distance
for DDT is about 120 mm. This is obtained with the above
column packing materials at oven temperature around 1S5°G. and
KT2 flow of 40 ml/min. These parameters may vary depending on
the batch of column packing material obtained.
38
-------
9. Check all operating parameters (temperature, gas flow,
and standing current) frequently until you are familiar
with identifying malfunctions in these areas.
10. Quantitate by the following equation:
'1
W = Weight of sample in grams
V = Volume of extract in niilliliters
w = Weight of pesticide in standard sample injected
(nanogram)
v = Volume of extract injected (microliters)
d-^ = Peak height in mm. for w
d- - Peak height in mm. for v
11. To insure reliability, inject a standard solution after at
least every third sample.
12, When sample concentration is high and peak is off scale on
recorder, dilute extract by an appropriate factor to bring
peak height on scale.
13. Inject 5 microliters of standards and sample with a 10
microliter Hamilton syringe equipped with guide.
14. Confirmation of analysis is most easily obtained by the use
of two columns of different polarity such as DC-200 and QF-1.
Thin-layer chromatography and other chemical techniques may
also be used.
15. The o,p' isomers of DDT, although not frequently found, are
difficult to separata on the abova column packings'.
(1) p,p' DDE and o,p' ODD and (2) P,p' ODD and o,p' DDT are
not resolved on a DC-200 column. o,p' ODD and o,p' DDT are
not resolved or. a QF-1 colurji. As experience is gained, the
analyst should experiment with mixtures of these column packing
materials in an attempt to separate these isomers. With our
particular lot of these packings, we have found that a 1:1
mixture of 3% DC-200 and 5% QF-1 on Gas Chrom Q 80/100 mesh
will separate these isomers.
39
-------
APPENDIX
References:
i
Burchfield, H. P. and Donald E. Johnson.
Guide to the Analysis of Pesticide Residues, Volume I and II..
U. S. Department of Health, Education and Welfare Public
Health Service, Office of Pesticides, Washington, D. C» 2020L.
'. .~r
Mills, P. A., J. Onley and R. Gaither.
Rapid Method for Chlorinated Pesticide Residues in Nonfatty
Foods. Journal of the Association of Official Analytical
Chemist, 46:186 (1963).
Johnson, L*
Collaborative Study of a Method for Multiple Chlorinated
Pesticide Residues in Fatty Foods. Journal of the Association
of Official Analytical Chemist, 48:668 (1965.)
40
-------
ANALYSIS. REPORT
ENVIRONMENTAL PROTECTION AGENCY
GULF BREEZE, FLORIDA, 32561
SAMPLE NO.:
DATE:
COLLECTOR:
DESCRIPTION:.
LOCALITY:
DATE PREPARED:.
Gross Weigh t:__
Tare Weight:
GROSS SAMPLE WEIGHT:
Tare:
SAMPLE WEIGHT:
Sample weight:
Na,SO. Factor:
^4
Weight:
SAMPLE WEIGHT EXTRACTED:
Tare :
Cross Weight:
BATE ANALYZED:
ANALYST:
METHOD: So:chlet~Soxhlet/Partition—Soxhlet/Column—Micro—Semimicro—Sriperatory
funnel—Seperatory funnel /Column- -Other
STANDARD SAMPLE INTERNAL
i
i
i
i
REMARKS:
41
-------
:•• x I/si" 3f, UC 200 oa Gas Ciro^ 0 oO/10C mi^
A - ti^r. L-2 tier.; 1 vX
J.v.'.rt 3;:ciJ: 30 i;mhis/;xour
C-.v.;: -a-ap. : 1?1° C.
I-jeer:-- ta:v.p.: 210° C.
D-ucctor tcuip.: 210J C.
:•:« Clew rnts: ijO r.l/r.ir..
Q
a
O
,J,i
a
5
CJ O
n v.
4J — <
f/5 T3
•Z. C
O -4
C U
42
-------
liUCHATCOaAM a2
\.
Variau Airo^r.vv; MoJul 610D
5' :c I/a" Qe-l or. Ga^ Clr.rcn Q 6C/10G
Attanuatior.: I5X
Chart sp&irf: 30 inchcj/hcur
Ovip. tcui?. : 133" C.
Injector tii?.: 210° C.
Octactor te:sp.: 210'° C.
N., flow rate: iiO ir.l/ir.i:i.
u
o
u
(3
.u
O.
JU
4-
O
H
G
43
-------
r
Sa=a conditions as Chro-nato^raa: #
C
u
•o
44
-------
c
ADDENDUM
Note 1.
For the preparation of in house samples, the use of G30
Unreductionized Quso may be omitted from the procedure. The use of Quso
permits the shipment of prepared samples wrapped in aluminum foil via
surface mail. Quso keeps the prepared sample in a free flowing condition
and laboratory tests indicate no loss or degradation of the compounds
monitored for periods up to 30 days at room temperature.
Note 2.
An extraction shell is no longer used. Place a thick wad of
glass wool in the bottom of the Soxhlet extractor, add a known weight
of sample in the extractor and top with another wad of glass wool.
Note 3.
For organochlorine pesticides that do not partition favorably
into acetonitrile such as rairex and the PCB's the following procedure is
used: Extracts are concentrated to approximately 10 ml and transferred
in 3 to 4 ml portions to a 400 mm x 20 mm chromatographic column containing
76 mm of unactivated Florisil. After each portion settles in the column,
vacuum is applied to evaporate the solvent. This is repeated after each
addition and after three 5 ml petroleum ether rinses of the extraction
flask. Vacuum is disconnected after all solvent is evaporated and the
residue is eluted from the column with 70 ml of a 9:1 mixture of acetoni-
trile and distilled water. The eluate is evaporated just to dryness in. a
crystallizing dish in hood on a slide warming table (40° C) with draft
over top of the dish.
Note 4.
2%* OV-101 and a mixed 0.75%. GV-17: 0.97%, OV-210 all on Gas
Chrom Q 100/120 in 152 cm x 2 mm (ID) glass columns are now being used.
These columns are used in Varian Model 1200, 1400 and 2100 gas chromatographs.
Note 5.
Standard concentrations are one half the values indicated. Mirex
standard contains 0.1 ng/ul.
Sediment Samples
Marine sediments may be scooped up in shallow water by hand but
preferably they are collected with a coring device so that the relative
depth of the layers sampled can be determined. The samples should be well
45
-------
c
mixed with a spatula after having removed obvious debris and spread in a
reasonable uniform layer not more than 1/4" thick on aluminum foil. The
samples should be air dried for 24 to 72 hours without resort to head or
vacuum. When dry, enough of the material should be blended in the Osterizer
to yield approximately 30 grams, of sample. The pulverized sample is added
to the Soxhlet extractor and extracted with a 9:1 mixture of petroleum
ether and acetone. The extract is concentrated to approximately 10 ml and
transferred directly to a Florisil column, page 8, Section B.
Host marine sediments contain large quantities of sulfur compounds
which cause interference when extracts are analysed by gas chromatography.
One ml of metallic mercury added to about 5 ml of the final extract and
agitated will precipitate some of these compounds prior to GC analyses.
46
-------
Gulf Coast Research Laboratory
EAST BEACH
OCEAN SPRINGS. MISSISSIPPI 39564
CONTBOU-ZD BY THC BOAHO OP TRUSTEES
INBT1TUTIONI OF HlOHIW LEARNING
STATC or MISSISSIPPI
[CROBIOLOGY SECTION
December 7, 1979
Mr. Steven C. Schimmel
U.S. Environmental Protection Agency
Environmental Research Laboratory-Gulf Breeze
Sabine Island
Gulf Breeze, Florida 32561
Dear Steve:
Please find enclosed our quality assurance package
pertinent to USEPA/GCRL Contract 68-03-2860, "Bioconcen-
tration Test: Inter-Laboratory Comparison Using the
Eastern Oyster."
For our PCP tissue samples (Table 3), the data are
reported in y/Kg rather than y/g as requested in your
November 7 letter. I apologize for this oversight but
the table was already prepared by the time I realized
my error, and I felt that the benefit gained in having
the table re-typed would not nearly offset the resulting
secretarial wrath. Original chromatograms and our pro-
posed sampling schedule are appended. Due to an auto-
mobile accident involving the technician primarily
involved in the processing of these samples, the rough
calculations have not been organized into a readily
understandable form. He should return to work on the
llth or 12th, and I hope to forward these calculations
to you by the end of the week.
If there is any additional information that you may
need in this regard, or if you or Dick have any questions
about the information presented, please let me know.
Sincerely,
William W. Walker, Ph.D.
Microbiologist
WWW:mt
cc
Ends. 47
-------
USEPA/GCRL Contract 68-03-2860
"Bioconcentration Test: Inter-Laboratory Comparison Using the Eastern Oyster"
Analytical Chemistry Performance Evaluation
December 7, 1979
Materials and Methods
A. Water
I. DDE
Bay water utilized in these extractions was pumped from the
Mississippi Sound estuary system and collected directly from the
headbox which feeds the oyster tanks to be used in the upcoming
bioconcentration tests. Triplicate 1-L samples were amended with
500 ul DDE water spike (provided by EPA), triplicate 1-L samples
were amended with 500 yl prepared spike (2.0 y/ml DDE and 0.4
Y/ml aldrin), and duplicate 1-L samples received no amendment
and served as blanks. Aldrin was added to the known DDE spike
simply to check the recovery of a second chlorinated hydrocarbon
insecticide through our procedure.
200 ml hexane and 200 ml acetone, both pesticidequality
solvents, were added to each 1-L water sample in a 2-L separatory
funnel, mixed well by shaking, and the organic and aqueous layers
allowed to separate. The hexane (and acetone) layer was removed
to a 1-L separatory funnel, and the aqueous fraction extracted
a second time with 400 ml hexane: acetone (1:1 v/v) and a third
and fourth time with 100 ml hexane only. The combined hexane
extract (600 ml total added) was then washed six times with
200 ml distilled water (1200 ml total) to assure acetone removal
and dried by passage through anhydrous soldium sulfate.
48
-------
The volume of each dried extract was measured and recorded,
and each extract evaporated to near-dryness with a Brinkman-
Buchi Rotavapor-R equipped with a Haake FK refrigeration system.
Each extract was then made up to 10 ml volume, loaded into a
Varian series 8000 autosampler, and analyzed by gas chromato-
graphic methods (Tracer MT-222 G.C. equipped with a Ni63
electron-capture detector). A 6-foot column containing 1.5%
OV-17 + 1.95% QF-1 on Chromosorb W, HP, 100/120 mesh was used.
Detector, inlet, and oven temperature were 285, 235, and 185 C,
respectively. Carrier gas (N2) flow was 100 ml/min.
II. Trichlorobenzene
The extraction procedure used for TCB was identical to
that for DDE with the following exceptions. TCB known spike
was added to bay water as 500 ul of a 0.8368 y/ml TCB solution.
Following evaporation to near dryness, each extract was brought
to 5 ml volume (and diluted 1:5 prior to analysis). Regarding
G.C. conditions, a 6-foot column containing 6% QF-1 + 4% SE-30
on Chromosorb W, HP, 100/120 mesh was used, and respective
detector, inlet, and oven temperatures were 285, 235, and 165 C.
Carrier gas (N2) flow was 50 ml/min.
III. Pentachlorophenol
The extraction procedure used for PCP was identical to that
for DDE with the following exceptions. Each water sample was
acidified to pH 2-3 with HC1 prior to extraction. PCP known
spike was added to bay water as 500 ul of a 0.10 y/ml PCP
solution. Following evaporation to near-dryness, each extract
(and the G.C. standard) was methylated by the addition of an
excess (approx. 1 ml) of etherial diazomethane. Oiazomethane
49
-------
was produced from Diazald according to the procedure described
ft
by Aldrich Chemical Company on the Diazald label. Excess
diazomethane was driven off with a gentle N2 stream, and the
methylated PCP extract was brought to 10 ml volume in hexane.
G.C. conditions were identical to those for DDE.
B. Tissue
I. DDE
Oysters used in these extractions were collected from
Biloxi Bay and were in the 4-6 cm valve height size range.
Oysters were maintained at the Laboratory's Oyster Hatchery
facility at Point Cadet, Biloxi, Mississippi until such time
as they were transferred to the Toxicology Laboratory on the
main campus of GCRL in Ocean Springs. Oysters were maintained
in flowing bay water for two weeks prior to sampling for these
quality assurance tests. It may be of interest to note that
during this holding period, the production of feces and pseudo-
feces was both obvious and prolific, indicating an adequate food
supply and good water quality.
Extractions were performed in triplicate for known and
unknown DDE analyses, and in duplicate for blanks. Each tissue
sample (for known DDE concentration^) consisted of approximately
5 gm oyster tissue (actual weights varied slightly from 5 gm,
were recorded, and DDE concentrations calculated on a per gram
basis), rinsed with bay water and blotted dry. Each tissue
sample was placed in a 150 ml beaker, amended with 20 ml
pesticidequality acetonitrile, and homogenized well using a
Tekmar SDT tissue homogenizer. An additional 20 ml acetonitrile
and the known spikes (500 ul 2.0 y/ml DDE and 500 ul 0.4
50
-------
aldrin) were then added and the homogenination step repeated.
Each sample was centrifuged for 4 minutes at 10,000 rpm and
10 C using an IEC B-20A refrigerated centrifuge, the super-
natant liquid decanted into a 250 ml separatory funnel
containing 75 ml 2% aqueous sodium sulfate, and the contents
mixed. At this point, 10 ml of unknown DDE extract (provided
by EPA) was added to each of three and 10 ml of oyster tissue
blank (also provided by EPA) to each of two 250 ml separatory
funnels containing sodium sulfate and mixed. 40 ml pesticide-
quality hexane was then added to each of the above 10 separatory
funnels, mixed well, and the hexane and aqueous layer allowed
to separate. The aqueous phase was re-extracted two additional
times with 40 ml hexane and the combined (120 ml) hexane extract
dried by passage through anhydrous sodium sulfate.
The volume of each dried extract was measured and recorded,
and each extract evaporated to near-dryness. Each sample was
then quantitatively transferred to a pre-washed Florisil column
(18 gm Florisil topped with sodium sulfate pre-washed with 5%
diethyl ether in hexane) and eluted with 2-100 ml additions of
5% diethyl ether in hexane. Each cleaned extract was then
evaporated to near-dryness, brought to 10 ml volume with hexane,
and analyzed as described in A-I.
II. Trichlorobenzene
The tissue extraction procedure for TCB was identical to
that for DDE except for the alterations described in section A-II.
III. Pentachlorophenol
The tissue extraction procedure for PCP was identical to
that for DDE except for the alterations described in section A-III.
51
-------
Further, the derivitized PCP was cleaned using 3 gm Fieri si 1
pre-washed with 10 ml 5% diethyl ether in hexane and eleuted
with 2-20 ml additions of 5% diethyl ether in hexane.
Results and Discussion
A. DDE
The recovery of p,p'-DDE from bay water and oyster tissue is shown
in Table 1. Extractable DDE from unknown (provided by EPA) spikes of
water and tissue was consistent across the triplicate replications,
with respective means for water and tissue of 0.963 y/L and 1.006 y/gm.
These values were not corrected for the somewhat high recoveries of
DDE in the known spikes prepared by our own personnel (108.3 and
112.7 percent for water and tissue, respectively). No DDE was
detected in our water or either tissue blank.
B. Trichlorobenzene
The recovery of 1,2,4-Trichlorobenzene from bay water and oyster
tissue is indicated in Table 2. Mean recoveries from the unknown
water and tissue spikes were 3.92 y/L and 0.739 y/gm, respectively,
again, not corrected for known spike recoveries (102.0 and 106.1
percent for water and tissue, respectively). As was the case for
DDE, agreement across replications was good, and no TCB was detected
in any of the three blanks.
C. Pentachlorophenol
The recovery of Pentachlorophenol from bay water and oyster
tissue is shown in Table 3. It should be noted at the onset of
PCP discussion that PCP was detected in our dilution water blanks,
in our oyster blanks, and in the oyster tissue blanks provided by
EPA. Second column confirmation has not been done to make certain
52
-------
Table 1. Recovery of p,p'-DDE from bay water and oyster tissue, GCRL.
(Analytical Chemistry Performance Evaluation, Contract
68-03-2860)
Concentration (y/L or gm) Blank Percent Recovery
Unknown Spike Concentration Known Spike
Replicate (Provided by EPA) (y/L or gm) (Prepared by GCRL)
1 0.823 0 111.
2 0.976 0 106.
3 1.090 106.
x 0.963 0 108.
EPA GCRL
1 0.988 0 0 115.
2 1.010 0 0 112.
3 1.020 111.
x 1.006 0 0 112.
3
6
9
3
0
0
0
7
Note: The concentrations listed for the unknown spike have not been
corrected for recovery of the known spike.
53
-------
Table 2. Recovery of 1,2,4-Trichlorobenzene from bay water and
oyster tissue, GCRL. (Analytical Chemistry Performance
Evaluation, Contract 68-03-2860)
Concentration (y/L or gm) Blank Percent Recovery
Unknown Spike Concentration Known Spike
Replicate (Provided by EPA) (y/L or gm). (Prepared by GCRL)
1
2
3
X
1
2
3
X
3.82 0 108.8
3.97 0 95.2
3.96
3.92 0 102.0
EPA GCRL
0.696 0 0 105.9
0.788 0 0 107.4
0.732 105.0
0.739 0 0 106.1
Note: The concentrations listed for the unknown spike have not been
corrected for recovery of the known spike.
54
-------
Table 3. Recovery of Peritachlorophenol from bay water and oyster
tissue, GCRL. (Analytical Chemistry Performance
Evaluation, Contract 68-03-2860).
Concentration (Y/L or Kg)
Unknown Spike
(Provided by EPA)
Blank
Recovery of
Known Spike^
Concentration (Prepared by GCRL)
Replicate ,a
1 3.30
2 3.53
3 3.53
x 3.45
1 377.7
2 346.3
3 374.9
x 366.3
2b (Y/L
3.16 0.
3.39 0.
3.39
3.31 0.
EPA
363.5 15.0
332.1 13.4
360.7
352.1 14.2
or Kg)
117
157
137
GCRL
25.3
23.2
24.3
Y/L (Kg) Percent0
0.210 na
0.165 na
0.180 na
0.185 96.0
28.7
27.9
27.6
28.1
a Concentration in unknown spike not corrected for recovery in known
spike or blank.
Concentration in unknown spike corrected for blank concentration
(by subtraction) but not for recovery in known spike.
C (0.185 - 0.137)/0.05 = 0.96 X 100 = 96%
55
-------
that the peak in question is indeed PCP, nor has the possibility of
\»
PCP-contaminated glassware, reagents, etc. been ruled out. These
possibilities will be investigated, but for the purpose of this
discussion, it is assumed that the peaks in question are PCP, and
the calculations done accordingly.
In the unknown water spike (Table 3, top, columns 2 & 3), PCP
recovery was consistent across replications, with a mean recovery
of 3.31 Y/L after blank correction. In our own prepared spike,
O.OSy PCP was added, and a 96 percent extraction efficiency was
reflected for three replicates. The PCP concentration of 0.137
Y/L detected in our dilution water, however, is almost triple the
0.05 Y/L prepared PCP concentration, and calculations of recovery
percentage are essentially meaningless for all practical purposes.
We assumed that the PCP detected in our dilution water originated
from bulkhead pilings recently installed in our small craft harbor
(about 300 yards from the source of our dilution water). To see if
this was indeed the case, we re-sampled dilution water pumped into
the Toxicology Building, sampled water from the small craft harbor
per se, and sampled water from our Oyster Hatchery facility located
approximately 2 miles by water from the main campus of GCRL and
analyzed for PCP. Resultant PCP concentrations were 0.209, 0.204,
and 0.199 Y/L, respectively for Toxicology, Harbor, and Oyster
Hatchery water, indicating that, if what we are seeing really is
PCP, contamination is probably both uniform and widespread in the
immediate geographic area.
In tissue (Table 3, bottom), PCP recovery was quite high, but
consistent across the three replications. After correction for
56
-------
blank concentration, PCP concentration in the unknown tissue extract
was 352.1 Y/Kg. PCP recoveries in the EPA and GCRL tissue blanks
were 14.2 and 24.3 y/Kg, respectively, and essentially precluded
any meaningful determination of PCP recovery from GCRL-prepared
tissue spikes.
57
-------
U. S. Environmental Protection Agency Contract 68-03-2860
"Bioconcentration Test: Inter-Laboratory Comparison Using the Eastern Oyster"
Scope of Work
Test Chemicals
1. £,£'-DDE - calculated time to steady state 513.78 hrs (21 days, 9.78 hrs).
2. 1,2,4-Trichlorobenzene - calculated time to steady state 110.72 hrs
(4 days, 14.72 hrs).
3. Pentachlorophenol - calculated time to steady state 19 hrs.
General Considerations
1. Test chemicals will be for the most part analyzed (in tissue) on the
basis of wet weight (oyster meats removed from shell, rinsed with dilution
water, blotted, and weighed) . However, dry-weight •••^••••^^•••^••••fepBHt
shall be performed: (a) at the beginning of the uptake phase, (b) at steady
state, and (c) upon completion of depuration for each test material.
2. Prior to actual testing, we will spike one liter of our dilution water
with both an unknown (supplied by EPA) and a known (prepared by Toxicology
personnel) amount of each test material. These water samples will be extracted
and analyzed to ascertain the efficiency of our extraction procedure. Then,
once testing begins, water from each test chamber will be analyzed weekly along
with control water fortified with' the test material in question at a concentra-
tion approximating that in the test chamber. The recovery (extraction efficiency)
from the fortified control in each week's analysis must not deviate more than one
standard deviation unit from that produced in the extraction of the unknown
amount of test chemical conducted previously. If deviation is greater than one
standard unit, the analysis (weekly) must be repeated.
3. The concentrations of test materials should be no greater than 10 percent
of the 96-hour EC5Q to the eastern oyster biased on shell deposition. Based on
PCP, p_,p_'-DDE, and trichlorobenzene ECso values of 104, 14, and 500 + ppb,
respective water concentrations in the test chambers should be 10.4, 1.4 and
50 ppb.
4. Tests are begun by placing the oysters in the test chambers after the
test solution has been flowing long enough for the concentration of test material
to become constant. Constancy will be ascertained by comparing two sets of water
samples taken at least 24 hours apart. Test oysters will be maintained in flowing
dilution water (no chemicals) for a minimum of four days to certify good health.
5. Salinity, temperature, dissolved oxygen, and pH in (a) the headbox and
(b) each test and control chamber will be determined at least twice daily (early
morning and late afternoon). As needed, artificial sea salts will be utilized
58
-------
p,p'-DDE Bioconcentration Test ( S = 513.78 hours)
Sampling Procedure: All sampling times are 8:00 a.m.
DAY
1
2
3
4
6
9
12
14
16
19
21
23
25
(0 hr)
(24 hrs)
(48 hrs)
(72 hrs)
(120 hrs)
(192 hrs)
(264 hrs)
(312X3)
(3U> *»** )
(432- k*> )
(480 hrs,
(528 hrs,
(576 hrs,
-
-
-
-
-
-
-
-
-
-
steady state
S + 2 days)
S + 4 days =
test
test
test
test
test
test
test
test
test
test
)
0)
30
35
40
46
(696 hrs)
(816 hrs)
(936 hrs)
(1080 hrs)
- test
- test
- test
- test
tissue and water, control tissue and water
tissue and water
tissue and water
tissue and water
tissue and water
water only
tissue and water, control tissue and water
water only
tissue and water
water only
- test tissue and water
- test tissue and water
- test tissue and water, control tissue
and water
tissue and water
tissue and water
tissue and water
tissue and water, control tissue and water
59
-------
to maintain the salinity of the dilution water at or above 12 ppt. Dilution
water will be intensely aerated in the headbox to insure that the concentration
of dissolved oxygen will not fall below 60 percent of saturation. Temperature
should not deviate more than 2 C from 22 C at any time during the test.
60
-------
Trichlorobenzene Bioconcentration Test (S = 110.72 hours)
Sampling Procedure:
DAY 1
6:00 a.m. (0 hr) - test tissue and water, control tissue and water
1:00 p.m. (7 hrs) - test tissue and water
7:00 p.m. (13 hrs) - test tissue and water
DAY 2
10:00 a.m. (28 hrs) - test tissue and water
DAY 3
1:30 p.m. (55.5 hrs) - test tissue and water, control tissue and water*
DAY 5
9:00 p.m. (Ill hrs, steady state)
test tissue and water,
control tissue and water
DAY 7
9:00 p.m. (159 hrs, S + 2 days) - test tissue and water
DAY 9
9:00 p.m.' (207 hrs, S + 4 days = U) - test tissue and water,
control tissue and water
DAY 11
8:45 a.m. (242.75 hrs)
test tissue and water
DAY 12
4:30 a.m. (262.5 hrs)
test tissue and water
DAY 13
8:15 a.m. (290.25 hrs)
test tissue and water
DAY 14
12:00 noon (318 hrs)
61
test tissue and water,
control tissue and water
-------
Pentachlorophenol Bioconcentration Test ( S = 19 hours )
Sampling Procedure:
DAY 1
1:00 p.m.
2:00 p.m.
3:30 p.m.
5:45 p.m.
10:30 p.m.
(0 hrs)
(1 hr)
(1.5 hrs) -
test tissue and water, control tissue and water
test tissue and water
test tissue and water
(4.75 hrs) - test tissue and water
(9.5 hrs) - test tissue and water, control tissue and water*
(19 hrs, steady state)
test tissue and water,
control tissue and water*
(67 hrs, S + 2 days)
test tissue and water,
control tissue and water
DAY 6
8:00 a.m.
12:45 p.m.
5:30 p.m.
10:15 p.m.
(115 hrs, S + 4 days = U) -
(119.75 hrs)
(124.5 hrs)
(129.25 hrs)
test tissue and water, control
tissue and water
test tissue and water
test tissue and water
test tissue and water
(134 hrs) - test tissue and water, control tissue and water
* Control tissue and water may be dropped from sampling scheme for these times.
Introduction of test chemical stopped at U = S + 4 days.
62
-------
MARINE BIOASSAY LABORATORIES
OTS Bioconcentration Round Robin
Analytical Chemistry Performance Evaluation
Methodology for analysis of p,p'-DDE and 1,2,^-Trichlorobenzene:
i
1). Water Samples
500 /ul of water spike concentrate added to 1 liter of seawater
which was extracted 3 times with 7 ml of hexane. 1 ml of 1 rag/1 solution
of aldrin was added to the extract as internal standard in case p,p'-DDE
analysis and 0.1 ml of 10 mg/1 solution of 1,2,3-Trichlorobenzene as
internal standard in case of 1,2,4-Trichlorobenzene. The extracts made
to 25 ml with hexane prior to gas chromatographic determination.
2). Tissue Samples
10 ml of tissue sample extract was added to 75 ml of 2% Na~SOu
solution and extracted with 5 ml, ^ ml, and 3 ml of hexane. The
centrifuged hexane extracts were combined and concentrated to 0.3 ml
using a Kontes concentrator. The concentrate was placed onto a
chromatographic column containing 1.7 gm of activated florisil and
eluted with 12 ml of hexane containing 1% methanol. The eluate was then
made up to 25 ml with hexane. To 5 ml of eluate was added 200 /il of 1 mg/1
solution of aldrin as internal standard for p,p'-DDE and 100/ul of 10 mg/1
solution of 1,2,3-Trichlorobenzene was added to eluate as internal standard
for 1,2,4-Trichlorobenzene prior to gas chromatographic determination.
Gas Chromatographic determination
Column - 6' 1.5% OV17 / 1.95% QF1
Nitrogen flow rate - 80 ml/min
Detector - Electron Capture
Temperature p,p'-DDE - 205°C
1,2,4-Trichlorobenzene - 120°C
63
-------
% MARINE BIOASSAY LABORATORIES
I
Results:
Treatment p,p'-DDE 1,2,4-Trichlorobenzene
Water extract O.SG^g/l
0.84
Tissue extract 0.67 ^ug/g 0.48
0.64/ig/g 0.58/ug/g*
Water spike 102 % 95 %
Tissue spike 101 % 97 %
Water detection limit 0.002 Aig/1 0.002 Aig/1
Tissue detection limit O.OOloig/g 0.001 /ug/g
*crystallization occurred in extract supplied.
Initial extraction and clean up steps will be identical
to those used in performance evaluation. Concentration using a Kontes
concentrator will be used if necessary prior to gas chromatographic
det erminat ion.
64
-------
MARINE BIOASSAY LABORATORIES
£
Methodology for analysis of pentachlorophenol
1). Water Sample Extraction
500 oil of water spike concentrate added to 1 liter of seawater.
1 ml of concentrated sulfuric acid is added and then extracted twice
with 12 ml of hexane. To the combined hexane extracts was added 100/ul
of 10 ppm (mg/1) aldrin solution as internal standard.
2). Tissue Sample Extraction
10 ml of tissue sample extract added to 75 ml of 2% Na SO .
0.5 ml of 40% NaOH is added and extracted twice with 10 ml of hexane,
discarding the solvent layer. The aqueous layer is acidified with
1 ml of concentrated sulfuric acid prior to extraction with two portions
of 12 ml of hexane. Tb the combined hexane.extractions was added 100/ul
of 100 mg/1 aldrin solution as internal standard.
3). Methylation
N-methyl-N-nitroso-N'-nitro-guanidine was added to 4 ml of
20% sodium hydroxide and 5 ml hexane till a constant yellow color is
obtained in the hexane layer. To 1 ml of sample extract is added 0.2
ml of the hexane solution. After 20 minutes 1 ml of 20% water/methanol
•solution added to quench the reaction.
Gas Chromatography
Column:
Nitrogen flow rate:
Detector:
Temperature:
6f 1.5% OV17 / 1.95% QF
80 ml/min
Electron Capture
Q
pentachlorophenol - 205 C
65
-------
MARINE BIOASSAY LABORATORIES
Results:
Treatment Pentachlorophenol
Water extract 2.89yug/l
2.95 Aig/1
Tissue extract 0.31/ug/l
0.31/ig/l
Water spike 93%
Tissue spike 30%
Water detection limit 0.01/ug/l
Tissue detection limit O.OlyUg/1
66
-------
II
^^^m m ^w » • • ^^
BIONOMICS
MARINE RESEARCH LABORATORY
Route 6, Box 1002
Pensacola, Florida 32507
(904) 492-0515
5 December 1979
MX. Steven C. Schimmel
U.S. EPA
Environmental Research Laboratory
Sabine Island
Gulf Breeze, Florida 32561
Dear Steve:
This concerns our project L16, contract 68-03-2859.
Enclosed are the results of the analytical chemistry per-
formance evaluation. A separate write-up is provided for each
chemical (1,2,4-trichlorobenzene, p,p'-DDE, and pentachlorophenol)
and original chromatographs are attached to the appropriate
write-up.
As requested, no correction was made for percentage recovery.
Please note too, that no correction was made for the "background"
concentrations of pentachlorophenol detected in blank seawater
and oyster tissue samples.
We are ready to begin testing and look forward to your '
contacting us next Monday, 10 December.
Please call me, Pete Shuba, or Tom Maziarz if there are
questions or if you need additional information.
Sincerely, ,
^
iod Parrish
Director
tamlv
„ij_u V
RP: jl
Enclosures
cc: Shuba
Maziarz
Project file
67
-------
Methodology for Pentachlorophenol Analyses
Project L16, Contract 68-03-2859
PERFORMANCE EVALUATION SAMPLES
Water
Samples were prepared according to U.S. Environmental
Protection Agency (EPA) instructions contained in an unsigned
letter from the Gulf Breeze, Florida, Laboratory dated 7 November
197S. Each sample was acidified (pK 1-2) with concentrated sulfuric
acid and then partitioned with one 100-milliiiter (mi) portion of hex-
ane. After phase separation, the hexane fraction was collected in a
100-m£ volumetric flask, the volume brought to 100 mi, and the sample
analyzed by electron capture gas chromatography.
A. Spike recovery samples showed the method to be 98%±12%
efficient (Appendix A).
B. Analyses of the pretest "unknown" water sample showed
the mean measured concentration to be 4.3 ug/£±0.6 yg/Jl (Appendix B).
Tissue
Tissue samples were prepared according to the letter of
instructions cited above. Each sample was made basic (pH 8-9)
with sodium hydroxide, and was partitioned with two 20-mJl portions
of hexane. The samples were then acidified (pH 1-2) with hydro-
chloric acid and again partitioned with hexane (one 20-m£ portion).
After phase separation, the hexane was collected in a 25-mi con-
centrator tube, the volume adjusted to 20 mi, and the sample was
*
analyzed by electron capture chromatography.
A. Spike recovery samples showed the method to be 77%±9%
efficient (Appendix C).
68
-------
B. Analyses of the pretest "unknown" tissue sample showed
the mean measured concentration to be 0.63 ug/g±0.08 ug/g
(Appendix D).
Gas chromatograph conditions
The instrument was a Perkin Elmer Sigma 2 equipped with a
63Ni electron capture detector. Lower limit of detection was 0.04
micrograms (pg) per liter (i) based on a I-i water sample and
0.04 ug/g based on a 5-g tissue sample.
Column: 1% SP124ODA on 100/120 Suppelcoport 6-foot (ft) x 2-mm glass
Carrier gas: Argon/methane (10%) at 20 m£/min
Oven-isothermal: 195°C
Injector: 300°C
Detector: 300°C
Attenuation: 3 2
Amp Range: 3
Chart Speed: 0.33 cm/min
69
-------
Appendix A
WATER SPIKE RECOVERY SAMPLES
Standard (Std.) concentration = 0.1 ng/u£ = 0.5 ng/injection (inj)
Std. peak height = 93.0 mm
Std. retention (ret.) time = 8.5 mm
Spike concentration = 2.0 ug/£
Replicate
1
2
3
4
5
Blank
Blank
Peak height
(mm)
99.5
97.3
80.8
100.0
78.0
9.2
6.5
Ret . time
(mm)
9.0
8.5
8.5
8.5
8.5
8.5
8.5
Measured cone.
(ug/£)
2.1
2.1
1.7
2.2
1.7
0.2
0.1
Recovery
(%)
105
105
85
110
85
n/a
n/a
Average recovery = 98%±12-%
Calculations
Std. Sample
Mass x Peak x Dilution
Concentration = Inj . Height Factor
Std. Original
Peak x Sample
Height Size
1 = (0.5 ng) (99.5 mia) (4,000) = 199,000 ng = 2,140 ng = 2.1 ppb
(93.0 mm)(1 £)93 £ £
2 = (0.5 ng) (97.3 mm) (4,000) = 2,093 ng =2.1 ppb
(93 .0 mm) (1 £)£
3 = (0.5 ng) (808 mm) (4,000) = 1,738 ng =1.7 ppb
(99.0 mm)(1 £) £
4 = (0.5 ng)(100.0 mm)(4,OQO)= 2,151 ng =2.2 ppb
(93.0 mm)(1 £) I
5 = (0.5 ng) (78.0 mm) (4,000) = 1.677 ng = 1.7 ppb
(93.0 mm)(1 £) £
70
-------
Blank * = (Q.5 ng) (9.2 mm) (4,000) = 198 ng = 0.2 ppb
(93.0 mm) (1 I) Ji
Blank 2 - (0.5 ng) (6.5 mm) (4,000) = 140 ng = 0.1 ppb
(93.0 mm) (1 a.) £ ^
71
-------
Appendix B
PRETEST "UNKNOWN" WATER SAMPLES
Std. concentration =0.1 ng/y£ =0.5 ng/inj
Std. peak height = 141.5 (averaged)
Std. ret. time - Q.9 cm
Replicate Peak height Ret. time Measured cone
(mm)
1 67
2 67
3 53
4 58
5 61
Blank 7
(cm)
8.5
9.0
8.5
9.0
8.5
8.5
(uq/i)
4.7
4.7
3.8
4.1
4.3
0.5
Mean measured concentration =4.3 yg/£±0.6 ug/£
Calculations
Std. Sample
Mass x Peak x Dilution
Concentration = Inj. Height Factor
Std. Original
Peak x Sample
Height Size
1 = (0.5 ng) (67 mm) (20,000) = 4,735 ng = 4.7 yg/£
(141.5 mm) (1 £,) I
2 = (0.5 ng) (67 mm) (20,000) = 4,735 ng = 4.7 ug/£
(141.5 mm)(1 i) I
3 = (0.5 ng) (53 mm) (20,000) = 3,746 ng = 3.8 yg/i
(141.5 mm)(1 I) 4
4 = (0.5 ng) (58 mm) (20,000) = 4,311 ng = 4.3 yg/JZ,
(141.5 mm) (1 SL) SL
5 = (0.5 ng) (61 mm) (20,000) = 4,311 ng = 4.3 ug/£
(141.5 mm)(1 £) £
• lank = (0.5 ng) (7 mm) (20,000) = 49.5 ng =0.5 ug/£
(141.5 mm)(1 I) I
72
-------
Appendix C
TISSUE SPIKE RECOVERY SAMPLES
Std. concentration = 0.1 ng/u£ =0.5 ng/inj
Std. peak height = 153.0 mm
Std. ret. time = 9.5 mm
Spike concentration = 2.0 ug = 0.4 ug/g
Replicate Peak height Ret. time Measured cone.
(mm) (mm) (ug/g)
1 133.0 1.0 0.35
2 125.0 1.0 0.33
3 85.5 1.0 0.22
4 125.5 1.0 0.33
5 116.0 1.0 0.30
Blank 3.0 1.0 0.008
Average recovery = 77%±11%
Recovery
88
83
55
83
75
n/a
Calculations
1
2
3
4
5
,k
Std. Sample
Mass x Peak x Dilution
Concentration = In^. Height Factor
Std. Original
Peak x Sample
Height Size
= (0.5 ng) (133 mm) (4,000) = 348 g = 0.35 ug/g
(153.0 mm) (5.0 g) g
= (0.5 ng) (125.0 mm) (4,000)= 327 ng = 0.33 ug/g
(153.0 mm) (5.0 g) g
= (0.5 ng) (85.5 mm) (4,000) = 224 ng = 0.22 ug/g
(153 mm) (5.0 g) g
= (0.5 ng) (125.5 mm) (4,000)= 328 ng = 0.33 ug/g
(153 mm) (5.0 g) g
= (0.5 nq) (116.0 mm) (4,000)= 303 ng = 0.30 ug/g
(153 mm) (5.0 g) g
= (0.5 nq) (3.0 mm) (4, 000) = 8 ng = 0.008 ug/g
(153 mm)(5.0 g) g
73
-------
Appendix D
PRETEST "UNKNOWN" TISSUE SAMPLES
Std. concentration = 0.1 ng/yJi = 0.5 ng/inj
Std. peak height = 99.5 mm (averaged)
Std. ret. time = 1.1 cm
Replicate Peak height Ret. time Measured cone
1
2
3
4
5
Blank
(mm)
147.2
163.0
169.0
169.0
129.0
23.2
(cm)
1.0
1.0
1.05
1.0
1.05
1.0
(ug/g)
0.59
0.66
0.68
0.68
0.52
0.09
Mean measured concentration = 0.63 ug/g±.08 ug/g
Calculations
Std. Sample
Mass x Peak x Dilution
Concentration = Inj. Height Factor
Std. Original
Peak x Sample
Height Size
1 = (0.5 ng) (147.2 mm) (4,000) = 592 ng = 0.59 ug/g
(99.4 mm)(5.0 g) g
2 = (0.5 ng)(163.0 mm)(4,000) = 655 ng = 0.66 ug/g
(99.5 mm)(5.0 g) g
3 = (0.5 ng)(169.0 mm)(4,000) = 679 ng =0.68 ug/g
(99.5 mm)(5.0 g) g
4 = (0.5 ng)(169.0 mm)(4,000) = 679 ng =0.68 ug/g
(99.5 mm)(5.0 g) g
5 = (0.5 ng)(129.0 mm)(4,000) = 519 ng =0.52 ug/g
(99.5 mm)(5.0 g) g
ilank = (0.5 ng)(23.2 mm)(4,000) = 93 ng =0.09 ug/g
(99.5 mm)(5.0 g) g
74
-------
EXPERIMENTAL SAMPLES
Water
Each 100-m£ water sample will be measured volumetrically
and placed in an amber glass bottle with a Teflon®-lined screw
cap. The sample will be quantitatively transferred to a 250-m£
separatory funnel with one 20-m£ hexane rinse, acidified (pH 1-2)
with sulfuric acid, and agitated for 2 minutes. After phase
separation, the hexane fraction will be collected, the volume
adjusted to 20 m£, and the sample will be analyzed by electron
capture gas chromatography under the conditions stated in
PERFORMANCE EVALUATION SAMPLES, Water.
Tissue
Each sample of oyster tissue will be weighed (wet weight)
and will be homogenized with 0.4N sodium hydroxide by using
a Polytron® tissue grinder. The aqueous phase will be passed
through filter paper into a 25Q-m& separatory funnel and will
be partitioned with 20 m£ of hexane for "clean up". This
hexane fraction will be discarded. The sample will then be
acidified (pH 1-2) with concentrated sulfuric acid and re-extracted
with one 20-m£ portion of hexane. After phase separation, the
hexane fraction will be collected, the volume adjusted to 20 m£,
and the sample will be analyzed by electron capture gas chroma-
tography under the conditions stated in PERFORMANCE EVALUATION
SAMPLES, Tissue.
75
-------
Methodology for 1,2,4-Trichlorobenzene Analyses
Project L16, Contract 68-03-2859
PERFORMANCE EVALUATION SAMPLES
Water
Samples were prepared according to U.S. Environmental
Protection Agency (EPA) instructions contained in an unsigned
letter from the Gulf Breeze, Florida, Laboratory dated 7 November
1979. Each sample was then partitioned with one 100-milliliter
(m£) portion of hexane and shaken for 2 minutes (min). The hexane
fraction was collected in a 100-mJl volumetric flask, the volume
brought to 100 m£, and the sample analyzed by electron capture
gas chromatography.
A. Spike recovery samples showed the method to be 96%±6%
efficient (Appendix A).
B. Analyses of the pretest "unknown" water sample showed the
mean measured concentration to be 57.5 ug/2,±6.2 \ig/£ (Appendix B) .
Tissue
Tissue samples were prepared according to the letter of
instructions cited above. Each sample was then partitioned with
one 20-m3, portion of petroleum ether. The ether fraction was
passed through a bed of anhydrous sodium sulfate into a round-
bottom flask. The sodium sulfate bed was then rinsed with two
20-m£'portions of petroleum ether. The sample was concentrated
on a rotary evaporator at ambient temperature to a volume of
approximately 10 m£, quantitatively transferred to a 25-m£ con-
centrator tube, and the volume was further evaporated to 1 m£ by
using a Kontes tube heater at 30 degrees Celsius (°C) and a stream
of purified nitrogen.
76
-------
The 1 mS, of concentrate was transferred to a 9-millimeter
(mm) chromaflex florisil column topped with approximately 5 grams
(g) of sodium sulfate. The transfer was made quantitatively by
using two 5-mfi, hexane rinses. The florisil column had been
previously rinsed with 20 m£ of hexane. After the extract had
filtered into the column> the column was eluted with one 20-m&
portion of 15% ethyl ether/petroleum ether, volume/volume.
The eluate was collected in a 25-mJl concentrator tube, the
volume adjusted to 20-mJl with petroleum ether, and the sample
was analyzed by electron capture gas chromatography.
A. Spike recovery samples showed the method to be 83±6± efficient
(Appendix C).
B. Analyses of the pretest "unknown" tissue sample showed the
mean measured concentration to be 0.64 ug/g±0.04 ug/g (Appendix D).
Gas chromatograph conditions
The instrument was a Perkin Elmer Sigma 2 equipped with a
63Ni electron capture detector. Lower limit of detection in water
was 0.05 micrograms (yg) per liter (£) based on a 1-Jl water sample;
lower limit of detection in tissue was 0.1 ug/g based on a 5-g
tissue sample.
Column: 3% OV-101 on 80/100 mesh Gaschrom Q 6-foot (ft) x 2-mm glass
Carrier gas: Argon/methane (10%) at 20 mA/min
Oven-isothermal: 100°C
Injector: 250°C
Detector: 250°C
Attenuation: 4
Amp Range: 3
77
-------
Appendix A
WATER SPIKE RECOVERY SAMPLES
Standard (Std.) = 0.5 nanograms (ng)/u£ = 2.5 ng/5 u£ injection (inj)
Std. retention (ret.) time =1.1 centimeter (cm) =3.33 min
Std. peak height = 1.22 cm (averaged)
Spike concentration = 10 ug/£
Replicate
A
B
C
D
Blank
Peak height
(mm)
120.8
115.2
111.0
121.0
n/d
Ret . time
(cm)
1.1
1.0
1.1
1.0
— — —
Measured cone .
(ug/£)
9.9
9.4
9.1
9.9
___
Recovery
(%)
99
94
91
99
-— —
Average recovery = 96% ± 6%
Calculations
Std. Sample
Mass x Peak x Dilution
Concentration = Inj. . Height Eactor
Std. Original
Peak x Sample
Height Size
A = (2.5 ng) (120.8 mm) (4,000) = 9,902 ng =9.9 v
(122 iron) (1 A) 11
B = (2.5 ng) (115.2 mm) (4,000) = 9,443 ng = 9.4 ug/2,
(122 mm)(1 £) I
C = (2.5 ng) (111.0 mm) (4,000) = 9,098 ng = 9.1 yg/JZ,
(122 ram) (1 9.) «.
D = (2.5 ng) (121.0 mm) (4,000) = 9,918 ng =9.9 yg/fc
(122 mm) (1 £)
Note: Samples were analyzed concurrently with water performance
evaluation samples; original chromatogram is attached to
Appendix B.
78
-------
Appendix B
PRETEST "UNKNOWN" WATER SAMPLES
Std. =0.5 ng/uA =2.5 ng/5 y£ inj
Std. ret. time = 1.1 cm= 3.33 min
Std. peak height = 122 mm (averaged)
Replicate Peak height Ret. time Measured cone
1
2
3
4
Blank
(mm)
132.0
149.8
149.0
130.0
n/d
(cm)
1.1
1.1
1.05
1.05
___
(yg/i)
54.1
61.4
61.1
53.3
— — —
Mean measured concentration 57.5 ug/J, ±6.2 ug/2,
Calculations
Std. Sample
Mass x Peak x Dilution
Concentration = Inj. Height Factor
Std. Original
Peak x Sample
Height Size
1 = (2.5 ng) (132.0 mm) (20,000) = 54,098 ng = 54.1 pg/2,
(122.0 mm) (1.0 2,)I
2 = (2.5 ng) (149.3 mm) (20,000) = 61,393 ng = 61.4 ug/£
(122.0 mm) (1.0 I) SL
3 = (2.5 ng) (149.0-mm) (20,000) = 61,066 ng = 61.1 ug/2,
(122.0 mm)(1.0 £) £
4 = (2.5 ng) (130.0 mm) (20,000) = 53,279 ng = 53.3 u
(122.0 mm) (1.0 1) SL
79
-------
Appendix C
TISSUE SPIKE RECOVERY SAMPLES
Std. =0.5 ng/y£ =2.5 ng/5 u£ inj
Std. ret. time = 1.05 cm = 3.33 min
Std. peak height = 1.07 mm (averaged)
Spike concentration = 3.75 ug/g
Replicate
1
2
3
4
Blank
Peak height
(mm)
173.0
172.5
155.3
163.3
n/d
Ret. time Measured cone.
(cm) (ug/g)
1.03
1.05
1.05
1.02
— — —
3.23
3.22
2.90
3.04
Recovery
(%)
86
86
77
81
-— —
Average recovery = 83% ± 6%
Calculations
Std. Sample
Mass x Peak x Dilution
Concentration « Inj. Height Factor
Std. Original
Peak x Sample
Height Size
1 = (2.5 ng) (173.0 mm) (4,000) = 3,225 ng =3.23 ug/g
(107.3 mm)(5.0 g) g
2 = (2.5 ng) (122.5 mm) (4,000) = 3,215 ng =3.22 ug/g
(107 .3 mm) (5.0 g) g
3 = (2.5 ng) (155.3 mm) (4,000) = 2,895 ng =2.90 ug/g
(107.3 mm)(5.0 g)g
4 = (2.5 ng) (163.3 mm) (4,000) = 3,044 ng =3.04 ug/g
(107.3 mm)(5.0 g) g
80
-------
Appendix D
PRETEST "UNKNOWN" TISSUE SAMPLES
Std. =0.25 ng/u£ =1.25 ng/5 u£ inj
Std. ret. time = 1.1 cm = 3.33 min
Std. peak height = 63.8 mm (averaged)
Replicate Peak height Ret. time Measured cone
1
2
3
4
Blank
Blank
(mm)
41.6
39.9
40.0
42.5
n/d
n/d
(cm)
1.1
1.1
1.1
1.15
— — —
(ug/g)
0.65
0.63
0.63
0.67
— — —
Mean measured concentration 0.64 ug/g ± 0.04 ug/g
Calculations
Std. Sample
Mass x Peak x Dilution
Concentration = Inj. Height Factor
Std. Original
Peak x Sample
Height Size
1 = (1.25 ng)(41.6 mm) (4,000) = 652 ppb = 0.65 ug/g
(63.8 mm)(5.0 g)
2 = (1.25 ng) (39.9 mm) (4,000) = 625 ppb =0.63 ug/g
(63.8 mm)(5.0 g)
3 = (1.25 ng) (40.0 mm) (4,000) = 627 ppb =0.63 ug/g
(63.8 mm) (5.0 g)
4 = (1.25 ng) (42.5 mm) (4,000) = 666 ppb = 0.67 ug/g
(63.8 mm)(5.0 g)
81
-------
EXPERIMENTAL SAMPLES
Water
Each 100-mJl water sample will be measured volumetrically
and placed in an amber glass bottle with a Teflon®-lined screw
cap. It will be partitioned with one 20-m£ portion of hexane and
analyzed by electron capture gas chromatography.
Tissue
Each sample of oyster tissue will be weighed (wet weight)
and will be homogenized with acetonitrile (10 mi) by using
a Polytron® tissue grinder. The sample will be centrifuged and
the supernatent passed through filter paper into a 250-m£ separatory
funnel. The process will be repeated and the filter paper rinsed
with 10 mJi of acetonitrile. The extract will be combined with
150 mSL of 2% sodium sulfate-in-water. The sample will then be
back-extracted with one 20-m£ portion of petroleum ether. After
separation, the ether will be passed through a bed of sodium
sulfate (approximately 20 g) and into a round-bottom flask.
The sodium sulfate bed will be rinsed with two 20-m£ portions
of petroleum ether.
The remainder of the analytical procedure will follow that
outlined under PERFORMANCE EVALUATION SAMPLES, Tissue.
82
-------
Methodology for p,p'-DDE Analyses
Project L16, Contract 68-03-2859
PERFORMANCE EVALUATION SAMPLES
Water
Samples were prepared according to U.S. Environmental
Protection Agency (EPA) instructions contained in an unsigned
letter from the Gulf Breeze, Florida, Laboratory dated 7 November
1979. Each sample was extracted twice with 100-milliliter (ra£)
portions of methylene chloride. Each time the solvent layer was
allowed to separate and was passed through a bed of anhydrous
sodium sulfate (approximately 20 grams [ g]) into a round-bottom
flask. The sodium sulfate bed was rinsed with an additional
100-m£ of the solvent after the last extraction. The combined
extract was concentrated to approximately 5 mJl on a rotary evapora-
tor at ambient temperature. The remaining methylene chloride
was quantitatively transferred to a 15-m£ centrifuge tube with
two 5-!ti£ washes of hexane. The tube was placed in a Kontes tube
heater (temperature 35 degrees Celsius [ °C] and the solvents
evaporated to 5 mi under a stream of purified nitrogen. The
remaining sample was diluted to 10 m£ with hexane and analyzed
by electron capture gas chromatography.
A. Spike recovery samples showed the method to be 96%±6%
efficient (Appendix A).
B. Analyses of the pretest "unknown" water sample showed the
mean measured concentration to be 1.66 ug/£+C.18 ug/£ (Appendix B),
Tissue
Tissue samples were prepared according to the letter of
instructions cited above. Each sample was then extracted twice
83
-------
catenae. Each time the
solvent layer was allowed to separate and was passed through a bed
of anhydrous sodium sulfate (approximately 20 g) into a round-
bottom flask. The sodium sulfate bed was rinsed with two 20-mi
portions of methylene chloride after the last extraction.
The combined extract was concentrated to approximately 1 mJt
on a rotary evaporator at ambient temperature. The remaining
extract was quantitatively transferred with approximately 10 ml
of hexane to a 9-mm chromoflex florisil column topped with
approximately 5 g of sodium sulfate. The column had been pre-
».
viously washed with 20 mi of hexane.
After the extract had filtered into the column, the column
was eluted with one 20-m£ portion of 6% ethyl ether/petroleum
ether. Volume of the collected fraction was adjusted to 20 m£
and the sample was analyzed by electron capture gas chromatography.
A. Spike recovery samples showed the method to be 85%±5%
efficient (Appendix C).
B. Analyses of the pretest "unknown" tissue sample showed the
mean measured concentration to be 0.96 yg/g±0.02 yg/g (Appendix D).
Gas chromatograph conditions
The instrument was a Perkin Elmer Sigma 2 equipped with a
63Ni electron capture detector. Lower limit of detection was 0.02
micrograms (yg) per liter {£,) based on a l-£ water sample and 0.02 yg/g
based on a 5-g tissue sample.
Column: 3% OV-101 on 80/100 mesh Gaschrom Q 6-foot (ft) x 2-mm glass
Carrier gas: Argon/methane (10%) at 20 m£/minute (min)
Oven-isothermal: 200°C
Injector: 250°C
Detector: 250°C
Attenuation1 16
Amp Range: 4 84
-------
Appendix A
WATER SPIKE RECOVERY SAMPLES
Standard (Std.) = 0.146 nanograms (ng)/u£ =0.73 ng/5 u£ injection (inj)
Std. retention (ret.) time = 1.30 centimeter (cm)= 3.9 min
Std. peak height =95.7 millimeter (mm) (averaged)
Spike concentration =0.75
Replicate
A
B
C
D
E
Peak height
(mm)
90.0
93.2
92.3
97.0
100.0
Ret . time
(cm)
1.31
1.30
1.33
1.30
1.30
Measured cone.
(ug/£)
0.687
0.711
0.704
0.740
0.763
Recovery
(%)
92
95
94
99
102
Average recovery = 96%±6%
Calculations
Std. Sample
Mass x Peak x Dilution
Concentration = Inj. Height Factor
Std. Original
Peak x Sample
Height Size
A = (0.73 ng) (90.0 mm) (1,000) = 686.5 ng = 0.687 ug/2,
(.95.7 mm) (1 I) a
B = (0.73 ng) (93.2 mm) (1,000) = 710.9 ng = 0.711
(95.7 mm)(1 I) i
C = (0.73 ng)(92.3 mm)(1,000) = 704.1 ng = 0.704
(95.7 mm)(1 I) i
D = (0.73 ng) (97.0 mm) (1,000) = 739.9 ng = 0.740 ug/2.
(95.7 mm)(1 i) i
E = (0.73 ng) (100.0 mm) (1,000)= 762.8 ng = 0.763 ug/5,
(95.7 mm)(1 i) i
85
-------
Appendix B
PRETEST "UNKNOWN" WATER SAMPLES
Std. = 0.146 ng/yJl = 0.73 ng/5 y£ inj
Std. ret. time = 1.30 cm = 3.9 min
Std. peak height = 96.2 mm (averaged)
Replicate
1
2
3
4
Blank
Peak height
(mm)
106.4 (averaged)
122.5 (averaged)
100.0
107.0
n/d
Ret . time
(cm)
1
1
1
1
.32
.30
.30
.33
— — —
Measured cone.
(ug/£)
1.62
1.86
1.52
1.62
— __
Mean measured concentration 1.66 yg/£±0.18 yg/£
Calculations
Std. Sample
Mass x Peak x Dilution
Concentration = Inj. Height Factor
Std. Original
Peak x Sample
Height Size
1 = (0.73 ng) (106.4 mm) (2,000) = 1,615 ng = 1.62 yg/S,
(96.2 mm)(1 £) £
2 = (0.73 ng) (122.5 mm) (2,000) = 1,859 ng = 1.86 yg/2,
(96.2 mm)(1 I) I
3 = (0.73 ng) (100.0 mm) (2,000) = 1,513 ng = 1.52 yg/X,
(96.2 mm)(1 A) I
4 = (0.73 ng) (1-07.0 mm) (2, OOP) = 1,624 ng = 1.62 yg/£
(96.2 mm) (1 JL) I
86
-------
Appendix C
TISSUE SPIKE RECOVERY SAMPLES
Std. = 0.146 ng/u& = 0.73 ng/5 y£ inj
Std. ret. time = 1.33 cm= 3.9 min
Std. peak height = 99.0 cm
Spike concentration =0.15 ug/g, 5.0 g samples
Replicate
A
B
C
D
Blank
Blank
Peak height
(mm)
85.0
83.0
87.0
87.0
2.0
n/d
Ret . time
(cm)
1.30
1.35
1.40
1.35
1.35
Measured cone .
(ug/q)
0.13
0.12
0.13
0.13
0.003
Recovery
87
80
87
87
n/a
Average recovery = 85%±5%
Calculations
Std. Sample
Mass x Peak x Dilution
Concentration = Inj. Height Factor
Std. Original
Peak x Sample
Height Size
A = (0.73 ng) (85.0 mm) (1,000) = 125.4 ng =0.13 ug/g
(99.0 mm)(5.0 g) g
B = (0.73 ng)(83.0 mm)(1,000) = 122.4 ng =0.12 ug/g
(99.0 mm)(5.0 g) g
C = (0.73 ng) (87.0 mm) (1,000) = 128.3 ng =0.13 ug/g
(99.0 mm)(5.0 g) g
D = (Q.73 ng) (87.0 mm) (1,000) = 128.3 ng =0.13 ug/g
(99.0 mm)(5.0 g) g
1 = (0.73 ng)(2.0 mm)(1,000) = 2.95 ng = 0.003 ug/g
(99.0 mm)(5.0 g) g
:h sample spiked with 0.75 ug p,p'-DDE which equals 0.15 ug/g for a 5.0 g
iple.
• 87
-------
Appendix D
PRETEST "UNKNOWN" TISSUE SAMPLES
Std. = 0.146 ng/u£ =0.73 ng/5 yi inj
Std. ret. time = 1.35 cm = 4.1 min
Std. peak height = 92.5 mm (averaged)
Replicate Peak height Ret. time Measured cone
1
2
3
4
5
Blank
Blank
(mm)
145.5
96.0
149.0
153.0
152.2
n/d
n/d
(cm)
1.35
1.35
1.33
1.33
1.30
— — —
(ug/g)
0.975
0.606a
0.941
0.966
0.961
___
Mean measured concentration = 0.96 yg/g±0.02 ug/g
included in calculation. Sample visibly bumped" during
evaporation and part of sample was lost.
Calculations
Std. Sample
Mass x Peak x Dilution
Concentration = Inj. Height Factor
Std.Original
Peak x Sample
Height Size
1 = (0.73 ng) (145.5 mm) (4,000) = 975.4 ng = 0.975 ug/g
(92.5 mm)(5.0 g) g
2 = (0.73 ng) (96.0 mm) (4,000) = 606.1 ng = 0.606 ug/g
(92.5 mm)(5.0 g) g
3 = (0.73 ng) (149.0 mm) (4,000) = 940.7 ng = 0.941 ug/g
(92.5 mm)(5.0 g) g
4 = (0.73 ng)(153.0 mm)(4,000) = 966.0 ng = 0.966 ug/g
(92.5 mm)(5.0 g) g
5 = (0.73 ng)(152.2 mm)(4,000) = 960.9 ng = 0.961 ug/g
(92.5 mm)(5.0 g) g
-------
EXPERIMENTAL SAMPLES
Water
Each 100-m£ water sample will be measured volumetrically
and placed in an amber glass bottle with a Teflon®-lined screw
cap.
Extraction and analysis methodology will be the same as
stated in PERFORMANCE EVALUATION SAMPLES, Water, except that each
sample will be extracted with two 20-m£ portions of methylene
chloride instead of two 100-mJl portions.
Tissue
Each sample of oyster tissue will be weighed (wet weight)
and will be homogenized with acetonitrile (10 m£) by using
a Polytron® tissue grinder. The sample will be centrifuged and
the supernatent passed through filter paper into a 250-m£ separatory
funnel. The process will be repeated and the filter paper rinsed
with 10 mA of acetonitrile. The extract will be combined with
150 mJi of 2% sodium sulfate-in-water. The sample will then be
back-extracted with methylene chloride.
The remainder of the analytical procedure will follow that
outlined under PERFORMANCE EVALUATION SAMPLES, Tissue.
89
-------
APPENDIX D
FINAL REPORTS OF LABORATORIES
PARTICIPATING IN THE BI CONCENTRATION
INTERLABORATORY COMPARISON STUDIES
90
-------
APPENDIX D-l
BIOACCUMULATION AND DEPURATION OF
THREE CHEMICALS BY EASTERN
OYSTERS (CRASSOSTREA VIRGINICA)
TOOL Heitmuller, Tom Maziarz, and Rod Parrish
EG&G, Bionomics Marine Research Laboratory
Route 6, Box 1002
Pensacola, Florida 32507
Contract 68-03-2859
Project Officer:
Mr. Steven C. Sch'immel
U.S. Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, Florida
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
GULF BREEZE, FLORIDA 32561
91
-------
ABSTRACT
Eastern., oyters (Crassostrea virginica) were exposed to
>ach of three chemicals—p,p'-DDEr pentachlorophenol, and
.,2,4-trichlorobenzene—in flowing, natural seawater. Individual
>ysters were sampled from the exposed populations at selected
jitervals and their whole-body tissues were chemically analyzed.
ifter equilibrium between tissue concentrations and water
:oncentrations had been reached (so-called plateau or steady-
,tate), the oysters were placed in flowing, natural seawater
:o which no test material was added. Individual oysters were
ampled at selected intervals and the rate of depuration was
.etermined by chemical analyses.
The ex-tent and rate of bioaccumulation varied widely among
he three tests* The highest BCF, 71,00OX, was calculated
or DDE? the BCF for TCB was 14IX and that for PCP was 37X»
ysters quickly bioaccumulated both TCB and PCP and appeared
o have reached, a, steady-state condition by 9 and 12 hours
f exposure, respectively. Bioaccumulation of DDE increased
radually throughout the 28-day exposure period.
The test materials were depurated by oysters in a manner
imilar to that by which they were bioaccumulated. Oysters
apidly depurated TCB and PCP (>90% after 24 hours), while
DE was gradually depurated over the 28-day period (93% on
ay 28) .
92
-------
CONTENTS
Abstract. . ........ • ........ ..... . 92
Figures .................... ...... ... 94
>les ...... ...... . . . ......... . 95
jbreviations and Symbols .......... ..... .. . . 96
Acknowledgements ................. ...... . 97
1. Introduction. .......... . ...... 98
2. Conclusions and Recommendations. . ...... 99
3. Materials and Methods. ______ . . . „ ......... 100
Test material ....... ........ 100
Test animals ................. 100
Test water ..» ........... ... 101
Test conditions. ........ ....... 101
Chemical analyses
4 . Results . ar»r? Discussion
DDE Test ....
PCP Test .
TCB Test
Summary. ......
leference ............ ....... - ...... HI
Appendix A ......... ....... ....... 124
93
-------
FIGURES
lumber
Accumulation of p,p'-DDE by eastern oysters 114
(Crassostrea virginica) .
Depuration of p,p'-DDE by eastern oysters 115
(Crassostrea virginica) following 28 days
of exposure to a mean measured concentration
of 0..3 ug/i.
Accumulation of pentachlorophenol by 118
eastern oysters (Crassostrea virginica) .
Depuration of pentachlorophenol by eastern
oysters (Crassostrea virginica) . following
144 hours of exposure to a mean measured
concentration of 10 ug/i.
Accumulation of 1,2,4-trichlorobenzene by
.eastern oysters (Crassostrea virginica)
Depuration of 1,2,4-trichlorobeirzene by 123
eastern oysters (Crassostrea virginica)
following 128 hours of exposure to a
measured concentration of 3.3 ug/i.
94
-------
TABLES
lumber
Measured concentrations of p,p'-DDE in 112
seawater during- the uptake phase of a bio-
accumulation study with eastern oysters
(Crassostrea virginica).
Measured concentrations of p,p'-DDE in
whole body tissue of eastern oysters
(Crassostrea virginica) during a bio-
accmnnl.ation study conducted in flowing,
unfiltered natural seawater.
Measured concentrations of pentachlorophenol
in seawater during the uptake phase of a
bioaccumulation study with eastern oysters
(Crassostrea virginica).
Measured concentrations of pentachlorophenol
in whole-body tissue of eastern oysters
(Crassostrea virginica) during a bioaccumu-
lation study conducted in flowing, unfiltered
natural seawater*
Measured concentrations of 1,2,4-trichloro- 120
benzene in seawater during the uptake phase
of a bioaccumulation study with eastern
oysters (Crassostrea virginica).
Measured concentrations of 1,2,4-trichloro- 121
benzene in whole-body tissue of eastern
oysters (Crassostrea virginica) during a
bioaccumulation study conducted in flowing,
unfiltered natural seawater.
95
-------
ABBREVIATIONS AND SYMBOLS
ABBREVIATIONS
EPA —
BMRL . —
DDE
PCP —
TCB
P- value —
BCF
gm; g
ppm
2. -"•— •
ppb
cm
m
PVC
ASTM
mi
null
v/v
cc
w/w
mv
ng
— Environmental Protection Agency
— Bionomics Marine Research Laboratory
— p,p'-DDE
— pentachlorophenol
— 1,2^4-trichlorobenzene
— octanol-water partition coefficient
— bioconcentration factor
— gram
— parts per million
— liter
— microgram
— parts per billion
— centimeter
— meter
— polyvinylchloride (
— American Society for Testing and Materials
— milliliter
— millimeter
— volume/volume
'•— cubic centimeter
—'• weight/weight
— millivolt
— nanogram
SYMBOLS
°C
°/
oo
— greater than
— less than
— degrees Celsius
— percent
— greater than or equal to
— less than or equal to
— times
— parts per thousand
96
-------
ACKNOWLEDGEMENTS
We thank Ms. Cathy Stewart for her assistance in the
preparation and conduct of the tests. Mr. Bob Bentley
prepared the graphs for this report, Dr. Pete Shuba
reviewed the manuscript/ and Ms. Jane Lewis typed the
report. We appreciate their assistance.
97
-------
SECTION 1
INTRODUCTION
In fulfillment of U.S. Environmental Protection Agency
(EPA) Contract 68-03-2859, EG&G, Bionomics Marine Research
Laboratory (BMRL), Pensacola, Florida/ participated in a
round-robin test program to determine the rate of bioaccumu-
lation and depuration of three test materials by eastern
oysters (Crassostrea virginica). The program was initiated
by EPA to help evaluate the efficacy of a proposed method
for conducting bioaccumulation studies. The materials
tested were p,p'-DDE (DDE), pentachlorophenol (PCP) and
1,2,4-trichlorobenzene (TCB).
Data from the three tests conducted at BMRL are main-
tained on file there.
98
-------
SECTION 2
CONCLUSIONS AND RECOMMENDATIONS
The results of the bioaccumulation studies with eastern
oysters and three chemicals indicated that the methods
described in Proposed Standard Practice for Conducting
Bioaccumulation ..Tests with Pishes and Saltwater Bivalve
Molluscs (ASTM, 1979) were generally applicable to oyster
testing.. We found that by translating the octanol-water
partition coefficient (P-value) of a test material into
the biochemical activity expected to occur within the tissues
of exposed oysters we were able to effectively predict the
general dynamics of the bioaccumulation response of the
oysters.
Although the method includes a scheme for estimating
bioaccumulation potential, we found some exceptions to the
suggested duration of exposure.and sampling schedules. The
reported P-value for 1,2,4-trichlorobenzene was 4.08 and
according to the method guidelines approximately 5 days was
required before this chemical reached steady-state in
oyster tissue. Our test results indicated that steady-state
was reached after only 9 hours of exposure. Based on these
results, we recommend more intensive sampling during the
early stages of the uptake phase in tests with materials
having P-values <5.00:
Computation of uptake and depuration rate constants
requires a sophisticated computer system not readily avail-
able to many researchers. Therefore we recommend that the
computation, of the uptake and depuration rate constants
remain optional and that the bioconcentration factor (BCF)
and graphs of uptake and depuration be required in all reports
99
-------
SECTION 3
MATERIALS AND METHODS
TEST MATERIAL
The samples of the three test materials were delivered to
BMRL by the Project Officer/ Mr. Steven C. Schimmel, EPA, on
8 November 1979. The sample of pentachlorophenol was a fine
crystalline material contained in two small, amber glass bottles
labeled "BIONOMICS MARINE RESEARCH LABORATORY, TEST MATERIAL,
PENTACHLOROPHENOL, 20 gm. " The sample of p,p'-DDE was a fine
white powder, contained in two small, amber glass bottles labeled
"BIONOMICS MARINE RESEARCH LABORATORY, TEST MATERIAL, p,p'-DDE,
1 gm." The sample of 1,2,4-trichlorobenzene was a clear, color-
less liquid contained in a small amber glass bottle labeled
"BIONOMICS MARINE RESEARCH LABORATORY, TEST MATERIAL, 1,2,4
TRICHLOROBENZENE,.. 20 gm." The samples were stored under
refrigeration (approximately 4 degrees Celsius [°C]), as specified
in an unsigned EPA letter dated 7 November 1979.
Stock solutions of each test material were prepared with
reagent grade acetone as the solvent/carrier. New stock solutions
were prepared weekly and stored in amber glass bottles with Teflon®-
lined caps.
For water samples, concentrations are expressed as micrograms
(yg) of test material per liter (I) of seawater or as parts per
billion (ppb). Concentrations measured in oyster tissue are
expressed as yg of test material per gram (g) of tissue or as parts
per million (ppm).
TEST ANIMALS
Adult oysters were purchased directly from oystermen's boats
on Apalachicola Bay, Florida, on 20 October 1979, and on the
same day were brought to BMRL where they were placed in flowing,
natural seawater. At no time were the oysters chilled or placed
in storage. The oysters were maintained at BMRL in large fiber-
glass tanks that received continuously flowing, unfiltered natural
seawater of ambient salinity and temperature until they were
selected for testing. During the period from receipt of oysters
to the start of the DDE and TCB exposures (13 December 1979), the
salinity of the seawater ranged from 20-35 parts per thousand
(°/oo) and the temperature ranged from 13-24°C. From receipt of
100
-------
the oysters to start of the PCP exposure (20 February 1980),
the salinity ranged from 20-35 °/oo and the temperature ranged
from 10-24°C.
Oysters selected for the DDE and TCB tests were approximate-
ly 5-8 centimeters (cm) , umbo to distal valve edge. Oysters
for the PCP test were approximately 6-8 cm, umbo to distal valve
edge. All the oysters appeared to be in good condition and were
growing well while they were being maintained.
TEST WATER
Water used to maintain and test the oysters was natural,
unfiltered seawater of ambient salinity and temperature which was
pumped from Big Lagoon, a Gulf of Mexico estuary adjacent to BMRL.
The pump intake was about 80 meters (m) offshore at a depth of
approximately 3m. The water was pumped through hard polyvinyl-
chloride (PVC) pipes into an elevated fiberglass reservoir and
flowed by gravity through PVC pipes into constant-level headboxes.
From there it was delivered to the holding tanks or test aquaria
by glass siphons.
The chemical composition of BMRL seawater is characterized
in Appendix A.
TEST CONDITIONS
The EPA Project Officer provided basic information about each
of the test materials to aid in planning the study. The reported
96-hour ECSO's (the concentration effective in causing a 50 percent
reduction of new shell growth after 96 hours of exposure) and the
log P-values (log of the octanol-water partition coefficient)
were, respectively, 14 ppb and 5.69 for DDE, 104 ppb and 2.23 for
PCP, and >500 ppb and 4.08 for TCB.
Methods for.each test followed those described in Proposed
Standard Practice for Conducting Bioaccumulation Tests with
Fishes and Saltwater Bivalve Molluscs (ASTM, 1979) which basically
states that animals exposed to a concentration of test material
are periodically sampled for chemical analysis of the test material
in their tissues. The exposure is continued until the amount of
test material bioaccumulated by the test animals reaches steady- .
state. If steady-state is not attained, the maximum exposure
period is 28 days, A bioconcentration factor (BCF) is calculated
by dividing the concentration of test material in the animals by
the concentration of the chemical in the water in which they
were exposed. Also, the method proposes a guide to estimate time
of exposure for an animal to attain steady-state based on the test
chemical's log-P value and subsequently recommends a tissue and
water sampling schedule appropriate for the estimated duration
of uptake. After attaining steady-state (or being exposed for a
101
-------
maximum of 28 days), animals are transferred to test material-free
seawater for depuration.
The three tests were conducted separately and for different
durations. However, the test system was the sarae for each. The
control and exposure glass aquaria (91-cm long x 61-cm wide x 15-cm
high) each received 120 Vhour of seawater via a calibrated glass
siphon. A syringe pump metered appropriate volumes of stock
solution or acetone into a glass mixing baffle that delivered the
seawater from the siphon into the aquarium. The volume of test
concentration or control seawater in each aquarium was approximate-
ly 70 ir resulting in about 41 volume replacements per 24 hours.
The test system was activated three days before each test to allow
time for system equilibration. Water samples were taken for
chemical analysis on pretest days -2 and -1.
For each test, two separate groups of 120 oysters were
impartially selected from the initial population. One group was
added to the exposure aquarium and the other to a control aquarium
that received a volume of reagent grade acetone (1.15 milliliters
[m&] per hour) which was equivalent to that volume added to the
exposure aquarium, but no test material. Individual oysters were
sampled from the initial population for .chemical analysis
of background concentrations of the test material.
During the uptake phase of a test, 4 oysters and 2 100-mJl
water samples were taken, from both the control and exposure
aquaria at the selected sampling periods. For each sampling
period, all 4 of the exposed oysters were shucked and the
individual meats (whole-body) were placed in separate glass jars
with Teflon-lined caps for chemical analysis of test material in
the tissue. The two water samples were processed for chemical
analysis of test material in exposure water. Only 2 of the 4
control oysters were retained for analyses, one for background con-
centrations of test material in oyster tissue and one .that was
fortified with a known amount of test material in order to determine
the percentage recovery during the analyses. Similarly, one
water sample from the control was for background analysis of test
material and one other was fortified with a known concentration of
test material in order to determine recovery efficiency for water
analyses. Immediately after sampling, the samples were either
extracted and prepared for analysis or stored in a freezer for
later chemical analysis. The results of the chemical analyses of
oyster tissue and test water were not corrected for background
concentrations or percentage recovery.
Four additional oysters were taken from the exposure aquarium
near the termination of the exposure period of each test for lipid
analysis. Four control oysters were also taken during the TCB
test; control oysters were not sampled for lipid analysis during
the DDE and PGP tests. These samples were provided to the EPA Gulf
Breeze Environmental Research Laboratory as directed by the Project
Officer. The oysters were shucked and the meats were placed in
individual, labeled glass culture tubes and stored in a freezer
until transported to the EPA lab.
102
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Following the exposure phase of a test, the remaining oysters
from both the control and the exposure were placed in separate,
clean aquaria that received flowing seawater but no test material
or acetone. Oysters from both treatments were sampled for chemical
analysis of test material in tissue in general accord with the
recommended schedule for depuration. The sampling procedure was
the same as described for the uptake phase except that no water
samples were taken.
Oysters could obtain plankton and other particulate matter
from the unfiltered seawater in which they were tested; there was
no supplemental feeding.
The salinity and temperature were measured daily in the control
aquarium. Dissolved oxygen and pH were measured periodically in
both the control and exposure aquaria during the uptake and depura-
tion phases.
DDE Test
Oysters were exposed to a nominal concentration of 1.0 ppb
DDE for 28 days. Samples for chemical analysis of DDE in water
and oyster tissue were taken on days 1.75, 3.5, 7, 14, 21 and
28 of exposure. Following exposure, samples for chemical analysis
of DDE in oyster tissue were taken on days 3, 7, 14, 21, and 28
of depuration. The test was conducted 13 December 1979-8 February
1980.
PCP Test
Oysters were exposed to a nominal concentration of 10 ppb PCP
for 144 hours. Samples for chemical analysis of PCP in water and
oyster tissue were taken at 3, 6, 12, 24, 48, 96 and 144 hours of
exposure. Following exposure, samples for chemical analysis of
PCP in oyster tissue were taken at 3, 6, and 9 hours of depuration.
The test was-conducted 20-26 February 1980.
TCB Test
Oysters were exposed to a nominal concentration of 25 ppb TCB
for 128 hours. Samples for chemical analysis of TCB in water and
oyster tissue were taken at 9, 18, 36, and 84 hours of exposure.
Oysters were sampled at 128 hours of exposure, but no water samples
were taken because an electrical power failure occurred and the
syringe pump did not function for an hour immediately before the
sampling. Following exposure, samples for chemical analysis of TCB
in oyster tissue were taken at 4.5, 6, 9, 12, 24, and 48 hours of
depuration. The test was conducted 13-19 December 1979.
CHEMICAL ANALYSES
DDE Analysis
Water—Each 100-mJl water sample was measured volumetrically
and placed in an amber glass bottle with a Teflon-lined screw
103
-------
cap. The sample was quantitatively transferred to a 250-m£ separatory
funnel and was extracted twice with 20-mJl portions of methylene chloride.
Each time the solvent layer was allowed to separate and was passed
through a bed of anhydrous sodium sulfate (approximately 20 g) into a
round-bottom flask. The sodium sulfate bed was rinsed with an additional
100 ml of the solvent after the last extraction. The combined extract
was concentrated to approximately 5 ml on a rotary evaporator at
ambient temperature. The remaining methylene chloride was quantitative-
ly transferred to a 15-mJZ. centrifuge, tube with two 5-ml washes of hexane-.
The tube was placed in a Kontes® tube heater (temperature 35°C) and the
solvents evaporated to 5 ml under a stream, of purified nitrogen. The
remaining sample was diluted to 10 ml with hexane and analyzed by
electron capture gas chromatography.
Recoveries of spiked samples showed that the method was 96±6%
efficient. The limit of detection was 0.02 yg/lfbased on. a. l-£ sample.
Tissue—Each sample of oyster tissue was weighed (wet weight)
and then homogenized with 10 mJl of acetonitrile by using a Polytron©
tissue grinder. Each sample was then centrifuged and the supernatant
was passed through filter paper into a 250-mJZ. separatory funnel.. The
process was repeated and the filter paper was rinsed with 10 ml of
acetonitrile. The extract was combined with 150 mJl of 2% sodium
sulfate-in-water (w/w). The sample was then back-extracted with one
20-mZ portion of methylene chloride. After phase separation, the
solvent was passed through a bed of sodium sulfate (approximately 20 g),
into a round-bottom flask. The sodium sulfate bed was rinsed with
two 20-mJZ, portions of methylene chloride. The sample was concentrated
on a rotary evaporator at ambient temperature to a volume of
approximately 5 ml. The remaining solvent was quantitatively transferred
to a 15-m£ centrifuge tube by using two 5-mJl hexane. rinses. The volume
was further reduced to 1 ml by using a Kontes tube heater at 35°C and
a stream of purified nitrogen. The 1 ml of concentrate was transferred
to a 9-millimeter (mm) chromaflex florisil column topped with
approximately 5 g.of sodium sulfate. The transfer was made quan-
titatively by using two 5-ml hexane rinses. The florisil column
had been previously rinsed with 20 ml of hexane. After the extract
had., filtered ."into the column, the column was eluted with one 20-mA
portion of 6% ethyl ether-in-petroleum ether (v/v). The eluate
was collected in a 25-m£ concentrator tube, the volume adjusted as
necessary with hexane, and the sample was analyzed by electron capture
gas chromatography.
As the concentration of DDE in the tissue samples increased,
the florisil cleanup step became unnecessary and was discontinued.
The samples analyzed after 1.7 days were diluted after back-
extraction rather than being concentrated.
Recoveries of spiked samples showed that the method was 85±5%
efficient. The limit of detection was 0.02 ug/g, based on a 5-g sample.
All chemicals used in the water and tissue analyses were
reagent grade or better.
104
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The extracts were analyzed by gas-liquid chromatography by
using the following chromatographic conditions:
Instrument: Perkin-Elmer Sigma 2
Detector: 63Ni electron capture
Column: Glass, 1.3-m x 2-mm ID
Packing: 3% 0V 101 on 80/100 mesh Gaschrom Q
Oven temperature (isothermal): 200°C
Injector temperature: 250°C
Detector temperature: 250°C
Carrier gas: Argon/methane (10%) at 20 cubic cm (cc)/minute (min)
Attenuation:. 16
Amp range: 4
PCP Analysis
Water—Each 100-mA water sample was measured volumetrically
and placed in an amber glass bottle with a Teflon-lined screw cap-
The sample was quantitatively transferred to a 250-raJl
separatory funnel, acidified to a pH of 1-2 with concentrated
sulfuric acid, and extracted with one 20-mA portion of hexane.
Following phase separation, the hexane fraction was collected
in a 25-m£ concentrator tube and 2 mi of that fraction removed
for derivatization. Approximately 0.1 mi of ethereal diazomethane
was added, the sample tube was capped, the mixture was shaken
and incubated for 15 min. Purified nitrogen was then bubbled
through the sample until the yellow color was removed (approximately
5-10 min). The volume was then increased to 5 mi by the addition
of hexane and the sample.was analyzed by using gas-liquid
chromatography.
Recoveries of spiked samples showed that the method was
102±8% efficient. The limit of detection was 1.0 vg/i,
based on a 100-ra£ sample.
Tissue—Each sample of oyster tissue was weighed (wet weight)
and homogenized with 20 mi of acetonitrile by using a Polytron®
tissue grinder. Each sample was centrifuged and the supernatent
decanted into a 250-m£ separatory funnel. The process was then
repeated. Following the second extraction, 100 mi of 2% sodium
sulfate in water (w/w) was added to each sample. The sample was
acidified with sulfuric acid to a pH of 1-2 and back-extracted
with one 2Q-mi portion of hexane. After phase separation, the
hexane fraction was collected in a 25-m.i concentrator tube. An
aliquot of this fraction (2 m£ at the beginning of uptake and the
end of depuration; 1 m& for all other samples) was then taken and
105
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PCP was derivatized as described for the seawater samples. Each
sample was diluted as necessary and analyzed by using gas-liquid
chromatography.
Recoveries of spiked samples showed that the method was 94±11%
efficient. The limit of detection was 0.01 vg/g, based on a
5-g sample.
The extracts were analyzed by gas-liquid chromatography by
using the following chromatographic conditions:
Instrument: Perkin Elmer Sigma 2
Detector: 63Ni electron capture
Column: Glass,.. 1.8-m. x 2-ram ID
Packing: 3% OV-101 on 80/100 mesh Gaschrora Q
Oven temperature (isothermal): 140°C
Injector temperature: 250°C
Detector temperature: 250°C
Carrier gas: Argon/methane (5%) at 30 cc/min
Attenutation: 8
Amp range: 3
All solvents used in the extraction procedures and to prepare
standard solutions were pesticide, grade. All chemicals used were
reagent grade or better.
TCB Analysis
Water—Each 100-mZ water sample was measured volumetrically
and placed in an amber glass bottle with a Teflon-lined screw cap.
The sample was quantitatively transferred to a 250-m£ separatory
funnel, partitioned with one 20-m& portion of hexane, and analyzed
by electron capture gas chromatography.
Recoveries of spiked samples showed that the method was 96±6%
efficient. The limit, of detection was 0.05 vg/l, based on a
1-^ sample.
Tissue—Each sample of oyster tissue was weighed (wet
weight) and then homogenized with 10 m£ of acetonitrile by using
a Polytron tissue grinder. Each sample was then centrifuged
and the supernatent was passed through filter paper into a 250-mi
106
-------
separatory funnel. The process was repeated and the filter paper
was rinsed with 10 mJl of acetonitrile. The extract was combined.
with 150 m£ of 2% sodium sulfate-in-water (w/w). The sample was
then back-extracted with one 20-m£ portion of petroleum ether.
After phase separation, the ether was passed through a bed of
sodium sulfate (approximately 20 g) into a round-bottom flask.
The sodium sulfate bed was rinsed with two 20-mi portions of
petroleum ether. The sample was concentrated on a rotary
evaporator at ambient temperature to a volume of approximately
10 mi, quantitatively transferred to a 25-mJl concentrator tube,
and the volume was further evaporated to 1 mi by using a Kontes
tube heater at 30°C and a stream of purified nitrogen. The 1 ml
of concentrate was transferred to a 9-mm chromaflex florisil
column topped with approximately 5 g of sodium sulfate. The
transfer was made quantitatively by using two 5-mi hexane
rinses. The florisil column had been previously rinsed with 20 mi
of hexane. After the extract had filtered into the column, the
column was eluted with one 2Q-mi portion of 15% ethyl ether-in-
petroleum ether (v/v) . The eluate was collected in a 25-mJZ,
concentrator tube, the volume adjusted to 20 mi with petroleum
ether, and the sample was analyzed by electron capture gas
chromatography.
The high concentrations of TCB in the tissue samples made the
florisil cleanup step unnecessary. The samples analyzed after
18 hours were diluted after back-extraction rather than being
concentrated.
Recoveries of spiked samples showed that the method was
83±6% efficient. The limit of detection was 0.1 vg/g, based on a
5-g sample.
All chemicals used in water and tissue analyses were reagent
vgrade or better.
The extracts were analyzed by gas-liquid chromatography by
using the following chromatographic conditions:
Instrument: Perkin-Elmer Sigma- 2
Detector: 63Ni electron capture
Column: Glass, 1.8-m x 2-mm ID
Packing: 3% OV-101 on 80/100 mesh Gaschrora Q
Oven temperature (isothermal): 100°C
Injector temperature: 250°C
Detector temperature: 250°C
Carrier gas: Argon/methane (10%) at 20 cc/min
Attenuation: 4
Amp range: 3 107
-------
SECTION 4
RESULTS AND DISCUSSION
DDE TEST
The mean measured concentration of DDE in seawater in
the 1.0 yg/& nominal concentration during the uptake phase of
the test was 0.3 yg/&- No DDE was detected in the control
(Table 1). " . ' .
Oysters continuously bioaccumulated DDE throughout the
28-day uptake phase. The mean measured concentration of DDE
in the whole-body tissue of four separate oysters sampled from
the exposure aquarium on day 28 was 21.42 yg/g (Table 2 and
Figure 1).
A bioaccumulation factor of 71,00OX was calculated by
dividing the mean measured concentration of DDE in the tissue
of oysters sampled on day 28 of update (21.42 yg/g) by the
average concentration of DDE measured in the exposure water
during the uptake phase (0.3 yg/JZ.) .
Oysters were sampled after 3 days of depuration, although
not required by the method. The mean measured concentration of
DDE in. whole-body tissue of 4 separate oysters was 31.47 yg/g,
significantly higher than that measured in oysters sampled on
day 28 of the uptake phase (21.42 yg/g). The concentration in
individual oysters ranged from 4.35-56.41 yg/g. We have no
rational explanation for this aberrant sample.
Oysters depurated DDE after being placed in flowing seawater.
After 7 days of depuration, the mean measured concentration of
DDE in tissue was 18.18 yg/g. After 28 days, oysters had depurated
93% of the DDE which they had accumulated. The mean measured
concentration of DDE in the whole-body tissue of 4 oysters sampled
after 28 days of depuration was 1.41 yg/g (Table 2 and Figure 2).
No significant mortality was observed during the test. Three
dead oysters were found in the control aquarium and one dead ,
oyster in the exposure aquarium during the transfer of .oysters to
clean depuration tanks.
During the study (uptake and depuration), the salinity and
temperature of the seawater ranged from 20-34 °/0o and 11-18°C,
respectively; measured concentrations of DO remained >_90% of
saturation and pH was 7.8-8.3.
108
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PGP TEST
The mean measured concentration of PCP in seawater in the
10 yg/fc nominal concentration during the uptake phase of the
test was 10 ug/i (Table 3) . Most chroma tographs of water sam-
ples taken from the control aquarium had a small peak which
suggested a trace amount of PCP in the seawater. Previous
studies with PCP have indicated that this peak is not PCP, but
an interference inherent to the method of analysis.
The bioaccumulation of PCP by oysters had apparently reached
steady-state after 12 hours of exposure. The mean measured
concentration of PCP in whole-body tissue of four oysters sampled
at 12 hours was 0.39 wg/g (Table 4)- The exposure was continued,
to 144 hours, during which time the mean measured concentration
of PCP in whole-body tissue of oysters sampled at 5 different
intervals was 0.37 ug/g. Analysis of variance demonstrated
that there was no statistically significant difference in the
concentrations of PCP in oyster tissue sampled at the 5 intervals.
Graphic interpolation of the data as a line of best fit
constructed from a 3rd degree polynominal regression equation
on a Hewlett-Packard 9 8 ISA computer indicated that steady-state
was reached at approximately 36 hours of exposure (Figure 3} .
A. BCF of 37X was calculated by dividing the mean measured
concentration of PCP in the tissue of oysters sampled during
steady-state (0.37 ug/g) by the mean measured concentration of
PCP measured in the exposure water during the same period
(10
Oysters rapidly depurated PCP after being placed in flowing
seawater. After 9 hours of depuration, oysters had depurated
92% of the PCP which they had accumulated; the mean measured
concentration in tissue was 0.03 ug/g (Table 4 and Figure 4).
No mortality was observed during the test.
During the study (uptake and depuration) , the salinity and
temperature of the seawater ranged from 25-30 °/oo and 13-16°C,
respectively; measured concentrations of DO remained >100% of
saturation and pH was 8.0.
TCB TEST
The mean measured concentration of TCB in seawater in the
25 ug/& nominal concentration during the uptake phase of the
test was 8.8 ug/Z. No TCB was detected in the control (Table 5).
No water sample was taken at the last sampling period (128 hours)
because of an electrical power failure.
The bioaccumulation of TCB by oysters had apparently reached
steady-state after 9 hours of exposure. The mean concentration of
109
-------
VTCB in whole-body tissue of four oysters sampled at that time
was 1.12 yg/g (Table 6). The exposure was continued to 128
hours, during which time the mean concentration of TCB in whole-
body tissue of oysters sampled at 5 different intervals was
1.24 yg/g. Analysis of variance demonstrated that there was no
statistically significant difference in the concentrations of
TCB. in oyster tissue sampled at the 5 intervals.
Graphic interpolation of the data as a line of best fit
constructed from a 3rd degree polynominal regression equation
on a Hewlett-Packard 9815A computer indicated that steady-state
was reached at approximately 24 hours of exposure (Figure 5).
A bioaccumulation factor of 141X was calculated by dividing
the average concentration of TCB in whole-body tissue of oysters
during steady-state (1.24 yg/g) by the average concentration of
TCB in.the exposure water during the same period (8.8 yg/£).
t
After 24 hours in TCB-free, flowing seawater, oysters had
depurated virtually all the TCB which they'had accumulated. The
mean measured concentration of TCB in the whole-body tissue
of 4 oysters sampled after 24 hours of depuration was 0.03 ug/g;
TCB was not detected in 2 of those oysters sampled (Table 6 and
Figure 6).
No significant mortality was observed during the test. ' Two
dead oysters were found in the exposure aquarium during the trans-
fer of oysters to a clean depuration tank. One oyster appeared
to have been dead before the test began because the shell was
filled with mud. There was no mortality in the control.
During the study, the salinity and temperature of the
seawater ranged from 26-31 °/oo and 14-18°C, respectively;
measured concentrations of DO remained >j92% of saturation and
pH was 8.2.
SUMMARY
Oysters bioaccumulated the three test materials, but the
extent and rate of bioaccumulation varied widely among the
three tests. The highest BCF, 71,-OOOX, was calculated for DDE;
the BCF for TCB was 14IX and that for PCP was 37X. Oysters
quickly bioaccumulated both TCB and PCP and appeared to have
reached a steady-state condition by 9 and 12 hours of exposure,
respectively. Bioaccumulation of DDE increased gradually
throughout the 28-day exposure period.
The test materials" were depurated by oysters in a manner
similar to that by which they were bioaccumulated. Oysters
rapidly depurated TCB and PCP «90% after 24 hours), while
DDE was gradually depurated over the 28-day period (93% on
day 23) .
110
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REFERENCE
American Society for Testing and Materials. 1979. Committee
E35.21; Standard practice for conducting bioaccumulation
tests with fishes and saltwater bivalve molluscs, draft
no. 9.
Ill
-------
TABLE 1. Measured concentrations of p,p'-DDE in seawater during the uptake phase
of a bioaccumulation study with eastern oysters (Crassostrea virginica). The
test was conducted in flowing, unfiltered natural seawater of 26-34 °/0o
salinity and 12-18°C temperature. The nominal exposure concentration was
1.0
Sampling period
(day)
1.8
3.5
7
14
21
28
Concentration of DDE (ug/A;ppb)
in water samples
Control
N.D.b
N.D.
N.D.
N.D.
N.D.
N.D.
#1
0.2
0.1
0.3
0.4
0.3
0,4
#2
0.1
0.2
0.3
0.2
0.3
0.4
xa
0.2
0.2
0.3
0.3
0.3
0.4
aOverall mean measured concentration =0.3 pg/fc.
bNot detected; <0.02 pg/£.
-------
TABLE 2. Measured concentrations of p,p'-DDE in whole-body tissue of eastern
oysters (Crassostrea virginica) during a bioaccumulation study conducted
in flowing, unfiltered natural seawater. Concentrations in p
are for dry weight; all others are for wet weight. The mean
exposure concentration during the uptake phase was 0.3 pg/fc.
Sampling period
(day)
UPTAKE PHASE 1.8
3.5
7
14
21
28
DEPURATION 7
PHASE
14
21
28
Oyster tissue concentrations (pg
Control
Tracea
N.D.
N.D.
N.D.
N.D.
Tracea
N.D.
N.D.
N.D.
N.D.
#1
0.81
2.50
(41)
10.54
9.74
14.94
10.93
(225)
25.62
8.50
7.27
0.61
(13)
#2
N.D.b
3.12
(64)
11.06
14.13
8.86
16.34
(297)
15.27
1.36
3.59
0.52
(10)
#3
0.68
1.34
(49)
8.78
5.12
26.32
24.51
(359)
19.21
2.16
8.62
1.77
(28)
#4
N.D.
4.41
(71)
10.36
16.89
11.43
33.90
(422)
12.60
3.84
1.05
2.73
(44)
DDE/g)
X
0.74
2.84
(56)
10.19
11.47
15.39
21.42
(323)
18.18
3.97
5.13
1.41
(24)
larentheses
measured
S.D.
1.28
(14)
0.98
5.16
7.70
10.02
(86)
5.66
3.20
3.45
1.05
(16)
^Visible peak, but below measurable detection limit.
detected; <0.02
-------
mean measured concentration of DDE in seawater during the exposure was 0.3
The line of best fit was constructed from a 3rd degree polynomial regression
equation on a Hewlett-Packard 9815A computer.
23.03 .
en
w
D
H
25
•Z
o
H
W
U
2
O
U
1S.B3 -
IU.W
5.K0
7.QQ
TB;
DAYS OF EXPOSURE
-------
following 28^3ays of exposure i^ a mean Pleasured Concentration of O.r3 pg/fc.
The line of best fit was constructed from a 3rd degree polynomial regression
equation on a Hewlett-Packard 9815A computer.
2S1.E3 -
tr»
w
CO
H
EH
2
O
H
s
W
U
2
O
-------
TABLE 3. Measured concentrations of pentachlorophenol (PGP) in seawater during the
uptake phase of a bioaccumulation study with eastern oysters (Crassostrea
CTi
virginica) . The test was conducted in flowing, unfiltered natural sea-
water of 26-30 °/oo salinity and 13-16°C temperature. The nominal exposure
concentration was 10 pg/fc.
Concentration of PCP (pg/fc;ppb)
Sampling period
(hr)
3
6
12
24
48
96
144
Control
N.D.b
Trace0-
Trace
Trace
Trace
Trace
N.D.
in water
#1
10
10
9
9
10
9
8
samples
#2
9
8
13
8
10
10
10
xa
10
9
11
8
10
10
9
aOverall mean measured concentration = 10
bNot detected; <1 yg/fc.
°Visible peak, but below measurable detection limit.
-------
TABLE 4. Measured concentrations of pentachlorophenol (PGP) in whole-body tissue of
eastern oysters (Crassostrea virginica) during a bioaccumulation study conducted
in flowing, unfiltered natural seawater. Concentrations in parentheses are
dry weight; all others are for wet weight. The mean measured exposure conce
tration during the uptake phase was 10 yg/H.
Oyster tissue concentration (pg PCP/g of tissue)
Sampling period (hr)
UPTAKE PHASE 3
6
12
24
48
96
144
DEPURATION PHASE 3
6
9
Control
0.01
0,02
0.01
0.02
0.02
0.01
Tracea
0.02
Trace
0.01
(0.01)
#1
0.03
0.28
(3.43)
0.27
0.36
0.31
(5.86)
0.41
0.32
0.40
0.06
0.03
(0.25)
n
0.26
0.03
(0.33)
0.35
\
0.38
0.34
(6.09)
0.35
0.43
0.13
0.05
0.03
(0.38)
#3
0.04
0.29
(3.71)
0.46
0.42
0.32
(3.57)
0.38
0.35
0.26
0.07
0.03
(0.39)
#4
0.01
0.23
(2.99)
0.49
0.42
0.31
(4.61)'
0.36
0.35
0.17
0.06
0.03
(0.31)
X
0.08
0.21
(2.62)
0.39
0.40
0.32
(5.03)
0.38
0.36
0.24
0.06
0.03
(0.33)
S.D.
0.12
0.12
(1.55)
0.10
0.03
0.01
(1.17)
0.03
0.05
0.12
0.01
(0.07)
visible peak, but below measurable detection limit.
-------
i.iecia measured concentration ot pep in seawater during the exposure was
10 \ig/i. The line of best fit was constructed from a 3rd degree polynomial
regression equation on a Hewlett-Packard 9815A computer.
H.HK
W
D
CO
CO
H
O
H
EH
W
CJ
2
O
u
0.33
1.00
ha.
HOURS OF EXPOSURE
-------
pentacnj^ropnenoi^Dy eastern oyster* (Crasso^rea virg^nica)
folrowing 14hours or exposure to a mean measured concentration of 10 pg/fc.
The line of best fit was constructed from a 3rd degree polynomial regression.
equation on a Hewlett-Packard 9815A computer.
cn
in
H
EH
2
'-'H
W
U
2
O
O
H.JS
HOURS OF DEPURATION
-------
TABLE 5. Measured concentrations of 1,2,4-trichlorobenzene (TCB) in seawater during
the uptake phase of a bioaccumulation study with eastern oysters (Crassostrea
virginica). The test was conducted in flowing, unfiltered natural seawater
of 26-31 °/oo salinity and 14-18°C temperature. The nominal exposure concen-
tration was 25 vg/i.
ro
o
Sampling period
(hr)
a
b
c
9
18
36
84
128
Overall mean measured
Not detectable; <0.05
Concentration of TCB (yg/fc;ppb)
in water samples
Control #1 #2 xa
N.D.b 9.4 12.0 10.7
N.D. 7.8 7.6 7.7
N.D. 6.8 6.3 6.6
N.D. 10.1 10.5 10.3
_c _ _ _
concentration = 8.8 pg/A.
vg/SL.
electrical power failure.
-------
TABLE 6. Measured concentrations of 1,2,4-trichlorobenzene (TCB) in whole-body tissue
of eastern oysters (Crassostrea virginica) during a bioaccumulation study con-
ducted in flowing, unfiltered natural seawater. Concentrations in parenthese
are for dry weight; all others are for wet weight. The mean measured- expos,ur<
concentration during the uptake phase was 8.8 yg/Jl.
Sampling period (hr)
UPTAKE PHASE 9
18
36
84
128
DEPURATION PHASE 4.5
6
9
12
24
Control
N.D.a
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Oyster
#1
1.31
(20.57)
3.43
0.92
1.35
1.45
(26.48)
2.99
1.00
0.46
1.16
0.02
tissue concentrations
#2
0.08
(1.40)
1.16
0.49
1.44
1.75
(35.20)
0.40
1.49
0.11
0.33
0.04
#3
0.99
(14.28)
1.72
0.69
0.82
1.15
(27.68)
0.43
0.37
0.70
0.93
N.D.
(ug TCB/g)
#4
2.08
(21.00)
0.91
1.17
0.50
1.37
(27.16)
1.54
0.52
0.14
0.82
N.D.
X
1.12
(14.31)
1.81
0.82
1.03
1.43
(29.13)
1.34
0.85
0.35
0.81
0.03
S.D.
0.83
(9.14)
1.14
0.29
0.45
0.25
(4.08)
1.22
0.51
0.28
0.35
*Not detected; <0.1 yg/g.
-------
virginica. vne mean measured concentration ot TCb in seawater auring trie
exposure was 8.8 pg/8,. The line of best fit was constructed from a 3rd degree
polynomial regression equation on a.Hewlett-Packard 9815A computer.
I.SB ..
tr>
w
D
CO
CO
H
O
H
H
W
CJ
2
O
U
I.E0
B.EH -.
0.0H
120.00
HOURS OF EXPOSURE
-------
following 128 liburs of dfcposure tifc a mean Measured fbncentraflLon of 8!% pg/£
The line of best fit was constructed from a 3rd degree polynomial regression
equation on a Hewlett-Packard 9815A computer.
7..K3 ,.
en
W
D
CO
01
o
H
H
W
U
2
O
U
n.nn
HOURS OF DEPURATION
-------
APPENDIX A
Results of Chemical Analyses for Routine Characterization of Selected Chemical
Constituents in Bionomics Marine Research Laboratory Seawater
Chemical Constituent
Concentration (mg/&;ppm)
Arsenic
Cadmium
Chromium
Copper
Mercury
Nickel
Zinc
Lead
Total Phosphate as P
Ammonia Nitrogen as N
Nitrate Nitrogen as N
Nitrite Nitrogen as N
Total Petroleum Hydrocarbons
Sulfides
Pesticides
Polychlorinated Biphenyls
1979 Rangea
<0.001-0.006
<0.01-0.002
<0.01-0.008
<0.01-0.02
<0.0005-0.0007
<0.01-0.02
<0.02-0.05
<0.02
<0.02
0.14-0.42
<0.01
<0.01
<5.0
<1.0
None detected*5
None detected0
April 1980
0.013
<0.01
<0.01
<0.001
0.002
0.02
0.02
0.05
0.02
<0.01
0.10
<0.01
<5.0
<0.01
None detected
None detected
Water samples were collected from Bionomics Marine Research Laboratory seawater
system after the mixing station in the wet lab.
aRange of concentrations are based on two sampling periods.
bPesticides: BHC, lindane, heptachlor, heptachlor epoxide, aldrin, dieldrin, endrin,
perthane, DDE, TDE (DDD), DDT, methoxychlor, endosulfan, strobane, toxaphene, kelthane,
and chlordane all <0.005 yg/£;ppb.
GPolychlorinated Biphenyls: Aroclor® 1016, 1232, 1248, 1260, 1221, 1242, and 1254
all <0.05 yg/£;ppb.
-------
APPENDIX D-2
BIOCONCENTRATION TEST: INTER-LABORATORY COMPARSION
USING THE EASTERN OYSTER
by
William W. Walker
Gulf Coast Research Laboratory
Ocean Springs, Mississippi 39564
Contract No. 68-03-2860
Project Officer
Steven C. Schimmel
U.S. Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, Florida 32561
August 29, 1980
-------
ABSTRACT
Simultaneously with other selected laboratories, flow-through bioconcen-
tration evaluations were conducted using p_,p_'-DDE, 1,2,4-trichlorobenzene, and
pentachlorophenol as toxicants and the Eastern oyster, Crassostrea virginica,
as the test animal. Testing consisted of an uptake phase in which one set of
oysters was exposed to a single concentration of each toxicant in dilution
water until steady state was reached, at which time toxicant delivery was
terminated and the exposed oysters allowed to depurate.
For DDE, steady state was reached in 22-24 days with a maximum DDE tissue
concentration of 87.94 jJg/g- The mean DDE water concentration during 28 days
of uptake was 0.66 M8/l> resulting in a bioconcentration factor of 133,242.
During a 28-day depuration phase, the DDE tissue concentration decreased to
30.76 (Jg/gf approximately 35 percent of the maximum detected. In the case of
trichlorobenzene, steady state was reached after only five days with a TCB
tissue concentration of 24.7 (Jg/g. The mean TCB water concentration through
five days was 52.0 |JgA> leading to a bioconcentration factor of 475. Steady
state was reached most rapidly with PCP, requiring only 19 hours and producing
a maximum PCP tissue concentration of 1.50 |Jg/g- The mean PCP water concen-
tration through 19 hours was 10.4 pg/1, resulting in a bioconcentration factor
of 144.2.
125
-------
CONTENTS
Abstract ............................... 126
List of Tables ............................ 128
1. Introduction ......................... 129
2. Recommendations ........................ 130
3. Materials and Methods . . . . ................. 131
Diluent Water ...................... I31
Test Animals ....................... I31
Test Chemicals ...................... 131
Analytical Procedures .................. ^3^
Tissue ....................... 1
Water ........................ l33
4. Results and Discussion ....................
TCB ...........................
PCP ........................... 135
127
-------
TABLES
Number Page
1 DDE Concentration in Test Tissue and Water During 28 Days
Exposure Followed by 28 Days Depuration 137
2 TCB Concentration in Test Tissue and Water During 168 Hours
Exposure Followed by 31.5 Hours Depuration 138
3 PCP Concentration in Test Tissue and Water During 115 Hours
Exposure Followed by 19 Hours Depuration . . 139
A-l Temperature, pH, Salinity, and Dissolved Oxygen Parameters
During Uptake and Depuration Phases, DDE/Oyster
Bioaccumulation Test 140
A-2 Temperature, pH, Salinity and Dissolved Oxygen
Parameters During Uptake and Depuration Phases,
DDE/Oyster Bioaccumulation Test 142
A-3 Temperature, pH, Salinity, and Dissolved Oxygen
Parameters During Uptake and Depuration Phases,
PGP/Oyster Bioaccumulation Test 142
128
-------
SECTION 1
INTRODUCTION
Flow-through bioconcentration evaluations using p_,p_'-DDE, 1,2,4-
trichlorobenzene, and pentachlorophenol as toxicants and the Eastern oyster,
Crassostrea virginica, as test animal were conducted at the Gulf Coast
Research Laboratory in accordance with EPA/ASTM "Proposed Standard Practice
for Conducting Bioconcentration Tests with Fishes and Saltwater Bivalve
Molluscs." Testing consisted of an uptake phase in which one set of oysters
was exposed to a single concentration of each toxicant in dilution water until
steady state was reached, at which time toxicant delivery was terminated and
the exposed oysters allowed to depurate until 90 percent of the accumulated
test material had dissipated from the tissue or 28 days of depuration had
elapsed. A control group of oysters exposed to dilution water to which no
toxicant was added was included for each test material.
These tests were conducted simultaneously with other selected labora-
tories, and the resultant data should, in part, satisfy the need for a series
of comparison tests to be utilized by EPA enforcement personnel in making
performance judgements in connection with the Toxic Substance Control Act
Program.
129
-------
SECTION 2
RECOMMENDATIONS
While a project of this nature cannot be expected to generate recommenda-
tions of the same magnitude as purely research-oriented investigations, certain
observations do come to light and merit consideration. In this particular
case, attention should be given to the suitability of the Eastern oyster as a
test animal for bioconcentration evaluations. The oyster is, of course, of
commercial value in many regions of the United States and at the same time
represents a food product that is consumed directly by man. In addition to
these favorable criteria, the oyster is quite easily collected in a variety of
size ranges, it can be transported relatively large distances with minimum
attention and precautions, and it is easily maintained under laboratory
conditions. And, the oyster's proficiency of accumulating in its tissues
materials from the water column is somewhat unique with respect to other
commonly-used test animals. All things considered, it appears that the
Eastern oyster represents an animal that is ideally suited for use as a test
animal for bioaccumulation investigations.
130
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SECTION 3
MATERIALS AND METHODS
DILUENT WATER
All water utilized in the tests described herein was natural water pumped
from Davis Bayou, a tributary to the Mississippi Sound estuary system. Water
was pumped into three 500-gallon and two 250-gallon settling tanks and then
into three 500-gallon tanks located within the Toxicology testing laboratory.
From these final holding-tanks, bay water was pumped into a constant-level
head box and delivered via siphons to test and control aquaria. Diluent water
was intensely aerated in the head box using Silent Giant air pumps. Tempera-
ture was constantly recorded and maintained at 22 ± 2 C during all test pro-
cedures, and an attempt was made to keep the salinity at or above 12 g/1
(ppt) either by waiting for the natural salinity to reach this level or by the
addition of artificial salts. Hardness, alkalinity, specific conductance,
TOG, and particulate matter of the dilution water were determined periodically
throughout the contract period.
TEST ANIMALS
The Eastern oyster (Crassostrea virginica) was utilized as the test
animal for all tests described herein. Oysters ranging in size from four to
six centimeters valve height were collected by dredging from Biloxi Bay,
Mississippi and held at the Oyster Hatchery of the Gulf Coast Research
Laboratory. As needed, test animals were transported to the Toxicology
Laboratory, cleaned of barnacles and muscles, and acclimated for a minimum of
four days in a water bath receiving diluent water from the overflow standpipe
in the aforementioned head box. During acclimation, all oysters were care-
fully scrutinized for signs of disease, stress, and physical damage. Injured,
dead, or abnormal individuals were discarded, as were those that did not
deposit feces or pseudofeces.
TEST CHEMICALS
DDE, trichlorobenzene, and pentachlorophenol were supplied by the
Environmental Protection Agency. Also furnished by EPA were water spikes and
oyster tissue blanks and extracts for the three test materials for use in an
analytical chemistry performance evaluation. These quality assurance tests
were completed and the resultant data sent to the project officer on December
7, 1979.
In the actual test procedures, each test material was delivered to its
respective test aquarium at a constant rate using a Harvard infusion pump.
131
-------
Toxicant was pumped through Teflon microtubing to a mixing tube attached to
the siphon supplying diluent water to the test chamber. Siphons supplying the
control aquaria were also equipped with mixing tubes but received no toxicant.
To minimize splashing, mixing tubes extended nearly to the bottom of both test
and control chambers, and all chambers were covered. The entire testing area
was enclosed by curtains to reduce the effect of external movement on the test
animals. Delivery of toxicant to the test chamber was initiated four to six
days prior to the addition of animals, and toxicant-amended water was analyzed
for accuracy as well as consistency with respect to the concentration of test
material. To initiate a test (i.e., zero hour), tissue, test water, and
control water samples were collected, and appropriate oysters were placed in
test and control aquaria.
ANALYTICAL PROCEDURES
Tissue
For tissue collection, oysters were measured and opened at the hinge.
Tissue was removed, rinsed lightly with diluent water, and placed in chem-
ically clean beakers for extraction. Each sample of tissue consisted of four
replicates of test tissue, duplicate fortified controls, duplicate reagent
blanks, and at periodic intervals, duplicate control tissue. With respect to
extraction, procedures were essentially identical for DDT and TCB, and these
will be discussed together. Approximately five grams of tissue was homogen-
ized with 20 ml pesticidequality acetonitrile using a Tekmar STD tissue
homogenizer, the spike (1.2 MS DDE or 47.7 [Jg TCB) added, and a second
homogenization with an additional 20 ml acetonitrile carried out. Each sample
was" then centrifuged for four minutes at 10,000 rpm and 10 C using an IEC
B-20A refrigerated centrifuge. The supernatant liquid was then added to 75 ml
of two percent aqueous sodium sulfate and the resultant mixture extracted
three times with 40 ml pesticidequality hexane. The three hexane extracts
were combined, dried by passage through anhydrous sodium sulfate, and evapor-
ated to near-dryness using a Brinkman-Buchi Rotovapor-R equipped with a Haake
FK refrigeration system. Each sample was then quantitatively transferred to a
pre-washed Florisil column (three gm Florisil topped with Na2S04 and pre-
washed with 10 ml five percent diethyl ether in hexane) and eluted with 40 ml
five percent diethyl ether in hexane. Each cleaned extract was then evaporated
to near-dryness, brought up to 10 ml with hexane, and analyzed for residual
test material. Two Tracor MT-222 gas chromatographs equipped with Ni63
electron-capture detectors, Varian series 8000 autosamplers and CSI Super-
grator 3A programmable integrators were used for all DDE and TCB analyses.
Six-foot glass columns packed with either 1.5% OV-17 + 1.95% QF-1 or 6% QF-1 +
4% SE-30 on Chromosorb W, HP, 100/120 mesh were employed, with detector and
inlet temperatures of 285 and 235 C, respectively. Column temperature was
185 C for DDE and 165 C for TCB. Nitrogen served as carrier and purge gas for
all test materials, with flow rates of 100 and 50 ml/min for DDE and TCB,
respectively.
For PCP analysis, tissue samples were extracted with a 1:1 v:v mixture of
dichloromethane and hexane, both pesticidequality solvents. During homogen-
ization, 6N HC1 was utilized to adjust the pH to 2-3, and a 9.07 pg PCP spike
was added. Each extract was centrifuged as previously described and dried
132
-------
through sodium sulfate without back-extraction. Each dried extract was then
evaporated to near-dryness to assure complete removal of dichloromethane and
methylated with two ml etheral diazomethane. Following methylation, each
extract was cleaned through Florisil, evaporated, brought to 10 ml with
hexane, and analyzed for residual PCP as described for DDE.
Water
Test and control water was sampled by siphoning from mid-depth in the
test and control aquaria. As was the case with tissue, extraction procedures
were similar for DDE and TCB but modified for PCP. For DDE and TCB, one liter
of test or control water (spiked with 1.2 |Jg DDE or 47.7 [Jg TCB) was extracted
with 400 ml.hexane:acetone (1:1 v/v) mixture. Following separation, the
hexane (with some acetone) layer was removed and the aqueous fraction extracted
a second time with 400 ml of the hexane:acetone mixture and a third and fourth
time with 100 ml hexane only. The combined extracts were then washed with
distilled water to remove the acetone and dried through sodium sulfate. Each
extract was evaporated, made to 10 ml, and analyzed by electron-capture gas
chromatographic methods previously described for DDE and TCB.
For PCP, each water sample was acidified and extracted twice with 200 ml
dichloromethane and twice with 100 ml dichloromethane. The combined extracts
were dried through sodium sulfate, evaporated to near-dryness, methylated, and
brought to 10 ml with hexane. Gas chromatographic analyses were identical to
those described for the PCP tissue samples.
133
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SECTION 4
RESULTS AND DISCUSSION
DDE
The data presented in Tables 1 and A-l are concerned with DDE portion of
these bioaccumulation tests. Delivery of DDE to the system was initiated on
February 17, with the desired DDE test water concentration being 1.20 [Jg/1.
Test water samples collected on February 19, 20, 21, 22, 23, and 24 revealed
respective DDE concentrations of 1.08, 1.14, 1.17, 0.95, and 0.56, and
0.42 |Jg/l. At zero hour (February 25) of the actual test, DDE concentration
in the test water was 1.11 pg/1. The low levels experienced on February 23
and 24 are thought not to be representative of the actual situation but rather
a consequence of freezing and holding these particular samples. With the
exception of those collected on February 23 and 24, all samples were extracted
immediately following collection. No DDE was detected in any control samples.
Test oysters were added to exposure and control chambers on February 25,
and tissue and water samples collected as indicated in Table 1. We feel that
we reached steady state at 22 or 24 days with a maximum DDE tissue concentra-
tion of 87.94 |Jg/g. The somewhat reduced DDE tissue concentrations observed
at 26 and 28 days appear resultant of correction for recovery from fortified
control samples on these days. This information is listed on pages 19 and 20
of the carbon notebook pages and indicates essentially that while one replicate
fortified control is quite close to the desired concentration of 1.2 |Jg/g, the
other is considerably higher, resulting in fortified control recoveries in
excess of 100% and, hence, reduced corrected test tissue concentrations. DDE
concentration in the test water ranged from 0.26 to 1.14 |Jg/l during the
28-day uptake phase for an average concentration of 0.66 (Jg/1. Using a tissue
concentration of 87.94 |Jg/g and the mean water concentration of 0.66 |Jg/l, a
bioconcentration factor of 133,242 can be calculated.
Delivery of DDE to the test chamber was discontinued on March 24, and
depuration was allowed to continue through April 21 (28 days). Upon termina-
tion of depuration, DDE tissue concentration had dropped to 30.76 (Jg/g- Heavy
rains and reduced salinities were experienced during the latter part of the
depuration phase. Temperature, pH, salinity, and dissolved oxygen parameters
throughout the 56-day test period are listed in Table A-l.
Tissue and water fortified controls were prepared and extracted simulta-
neously with each test tissue and water sample. Control tissue was spiked
with 1.2 MS DDE, and recoveries (during uptake) ranged from 106 to 132 percent.
Similarly, control water was spiked to contain 1.2 (Jg/1 DDE, and recoveries
ranged from 98 to 112 percent. Control tissue samples were collected on
134
-------
February 25, March 10, March 24, and April 21. Respective DDE residues were
0.06, 0.01, 0.05, and 0.03 (Jg/g. Control water samples were collected on
February 25, March 10, and April 15, and respective DDE concentrations were
zero, 0.06, and 0.5 M8/1- Reagent blanks were analyzed at each sampling time,
and in no case was there any indication of solvent contamination.
TCB
The data in Tables 2 and A-2 are concerned with the trichlorobenzene
portion of these tests. Delivery of TCB to the system was begun on May 7, and
the desired TCB test water concentration was 50 (Jg/1. Test water samples
collected on May 8, 9, 10, and 11 revealed actual TCB levels of 54.8, 53.1,
60.3, and 52.3 (Jg/1- Oysters were added on May 12, at which point the concen-
tration of TCB in the test water was 52.2 (Jg/1. Tissue and water samples were
collected as indicated in Table 2, and TCB concentration determined. Steady
state was reached after only five days with a TCB tissue concentration of
24.7 |Jg/g. The mean TCB water concentration through five days was 52.0 |Jg/l>
leading to a bioconcentration factor of 475.
Delivery of TCB to the test system was terminated at 0830 on May 19, and
depuration was allowed to continue until 1600 on May 20. At the end of depu-
ration, TCB tissue concentration was found to be only 2.5 (Jg/g- Temperature,
salinity, pH, and dissolved oxygen parameters for the 200-hour test period are
listed in Table A-l.
As was the case for DOE, tissue and water fortified controls were pre-
pared and extracted along with test tissue and water samples. Control tissue
was spiked with 47.7 Mg TCB, and recoveries (during uptake) ranged from 61 to
96 percent. Control water was similarly spiked, and TCB recovery ranged from
91 to 104 percent. Control tissue samples were analyzed for TCB residue on
May 12, May 15, May 19, and May 20, and respective TCB concentrations were
zero, 0.003, 0.003, and 0.001 (Jg/g. Control water samples were analyzed on
the above dates and failed to indicate the presence of TCB in the diluent
water. As was the case with DDE, TCB reagent blanks did not indicate solvent
contamination.
PCP
Data regarding the pentachlorophenol portion of these evaluations is
presented in Tables 3 and A-3. Delivery of PCP to the system was initiated on
July 7, and the desired PCP test water concentration was approximately 9.5-
10 [Jg/1- Test water samples were collected on July 10, 11, 14, and 15, and
revealed respective PCP concentrations of 9.93, 12.04, 11.04, and 11.58 (JgA-
Oysters were added on August 16, at which time the concentration of PCP in the
test water was determined to be 10.78 (Jg/1. Tissue and water samples were
collected as indicated in Table 3, and PCP concentration determined. Steady
state was reached after only 19 hours incubation with a tissue concentration
of 1.50 (Jg/g (mean of F, G, H, and I samples). The mean water concentration
through 19 hours was 10.4 |Jg/l, leading to a bioconcentration factor of 144.2.
Delivery of PCP to the test aquarium was terminated at Q815 on July 21,
and depuration was allowed to continue until 1100 on July 22. Upon termination
135
-------
of depuration, PGP tissue concentration was found to be only 0.07 (Jg/g.
Temperature, salinity, pH, and dissolved oxygen parameters for the 134-hour
PCP test period are listed in Table A-3.
As was the case with DDE and TCB, fortified (spiked) tissue and control
samples were analyzed at each sampling period, and these recovery data are
listed in Table 3. Control tissue and water samples (no spike) were analyzed
on July 16, 19, 21, and 22. Respective PCP residues were 0.018, 0.014, 0.015,
and 0.021 |Jg/g in control tissue and 0.021, 0.013, 0.015, and 0.06 |jg/l in
control water. These figures indicate that PCP was contained in our diluent
water in detectable amounts, not surprising for a compound as ubiquitous as
pentachlorophenol. Further, as indicated on pages 18-47 of the attached
yellow sheets, our reagent blanks (indicated by prefix code RB) tested positive
for PCP and ranged from 0.0029 to 0.0420 (Jg/40 ml solvent in tissues and from
0.0013 to 0.0635 pg/1 in water. The data in Table 3 has not been corrected
for reagent blank PCP.
136
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TABLE 1. DDE CONCENTRATION3 IN TEST TISSUE AND WATER DURING 28 DAYS EXPOSURE FOLLOWED BY 28 DAYS DEPURATION
!-*
00
Code
A
B
C
D
E
F
G
H
I
J
K
L
M
N
0
P
Q
R
S
T
U
V
Date
2/25
2/26
2/27
2/28
2/29
3/2
3/5
3/7
3/10
3/12
3/15
3/18
3/20
3/22
3/24
3/25
3/26
3/28
4/2
4/8
4/15
4/21
Time
of
Day
0830
0830
0830
0830
0830
0830
0830
0830
0830
0830
0830
0830
0830
0830
0830
0830
0830
0830
0830
0830
0830
0830
Days
of
Incubation
0
1
2
3
4
6
9b
11
14
16
19
22
24
26
28
1
2
4
9
15
22
28
% Recovery from
Test Tissue Fortified
Concentration Control Tissue
11 *• 1 T3l-»
0.06
1.58
7.10
16.06
15.51
26.47
34.63
35.76
49.80
c
64.72
89.66
86.21
68.44
76.59
~ ~ "•— Depuration rn
88.25
69.55
68.39
55.73
42.54
29.84
30.76
117
106
113
118
124
108
107
117
112
c
132
111
112
128
114
ase~~ — — — — — — —
108
123
112
108
101
101
100
Test Water
Concentration
1.11
0.51
0.54
0.49
0.50
0.32
0.40
0.74
1.14
0.26
0.27
1.11
0.98
0.52
1.01
0.21
0.16
0.05
0.15
0.10
0.04
0.06
% Recovery from
Fortified
Control Water
100
102
103
104
108
98
100
105
108
98
112
113
106
105
102
102
104
108
113
105
100
107
DOE concentration is expressed as micrograms per gram (ug/g) tissue on a wet-weight basis and as micrograms
per liter (ug/1) water. All values are corrected for recoveries from fortified controls.
On these dates, power failures occurred at our laboratory. Safeguards in our toxicant delivery system are
such that an interruption of power terminates the delivery of both toxicant and dilution water. The resultant
"static periods" were 10 hours on February 26 and nine hours on March 5.
°Tissue samples were not collected this date.
-------
TABLE 2. TCB CONCENTRATION IN TEST TISSUE AND WATER DURING 168 HOURS EXPOSURE
FOLLOWED BY 31.5 HOURS DEPURATION
OJ
00
Code
A
B
C
D
E
F
n
\j
H
1
J
K
L
M
Date
5/12
5/12
5/12
5/13
5/13
5/15
5/17
5/19
5/19
5/19
5/19
5/20
5/20
Time
of
Day
0830
1300
1700
0230
2030
0830
0900
0830
1300
1700
2300
0700
1600
Hours
of
Incubation
0
4.5
8.5
18
36
72
120.5
168
4.5
8.5
14.5
22.5
31.5
Test Tissue
Concentration
— — — — Up L
-------
TABLE 3. PCP CONCENTRATION IN TEST.TISSUE AND WATER DURING 115 HOURS EXPOSURE
FOLLOWED BY 19 HOURS DEPURATION
oo
Code
A
B
C
D
E
F
G
11
I
J
K
L
M
N
0
Date
7/16
7/16
7/16
7/16
7/16
7/17
7/18
7/19
7/20
7/21
7/21
7/21
7/21
7/21
7/22
Hours
of
Incubation
0
1
2.5
4.8
9.5
19
43
67
91
115
2
4.8
9.5
14.3
19
Test Tissue
Concentration
0.02
0.14
0.56
0.65
0.86
1.46
1.58
1.50
1.47
1.91
0.93
0.44
0.14
0.08
0.07
% Recovery From
Fortified
Control Tissue
83
90
106
95
105
97
90
99
144
104
• nv.
Depuration Phase
100
106
100
96
96
Test Water
Concentration
10.8
10.4
11.4
10.4
9.2
10.2
11.3
11.5
15.9
11.5
0.36
0.08
0.01
0.01
0.01
% Recovery From
Fortified
Control Water
102
99
100
101
110
95
104
105
98
103
104
104
100
105
104
PCP concentration is expressed as micrograms per gram (M8/g) tissue on a wet-weight basis and as micro-
grams per liter ((Jg/1) water. All values are corrected for recoveries from fortified controls.
-------
TABLE A-l. TEMPERATURE, pH, SALINITY, AND DISSOLVED OXYGEN
PARAMETERS DURING UPTAKE AND DEPURATION PHASES,
DDE/OYSTER BIOACCUMULATION TEST
Date
2/25
2/26
2/26
2/27
2/27
2/28
2/28
2/29
3/1
3/2
3/3
3/4
3/5
3/5
3/6
3/7
3/8
3/9
3/10
3/11
3/12
3/13
3/14
3/15
3/16
3/17
3/18
3/19
3/20
3/21
3/22
3/24
3/25
3/26
3/27
3/28
3/29
3/30
3/31
3/31
4/1
4/2
4/3
4/4
4/5
Temp. (°C)
22.5
21.5
22
22
22
22
22.5
22.5
22
20
20.5
22
23
24
23
22.5
23
22
23
22
21.5
22.5
21
21
21
21
21
21
22
22
22
22
22
22.5
22.5
22.5
22.5
22.5
22.5
—
22.5
22.5
22.5
22.5
22.5
pH Salinity (ppt)
7.2
6.9
7.1
7.5
7.4
7.5
7.2
7.5
7.6
7.6
7.1
7.4
7.4
7.5
7.4
7.4
7.3
7.35
7.2
7.1
7.2
7.4
7.2
7.2
7.2
7.3
7.1
7.3
7.1
7.0
7.4
7.2
7.1
7.1
7.1
7.1
7.1
7.2
7.2
—
7.1
7.0
7.2
7.2
7.1
11
10
11
14
14
12
13
14
16
13
14
18
18
18
18
18
16
15
13
11
12
11
10
11
17
10
10
7
5
4
9.5
12.5
8
7
15
12
5
2
4
6
12
12
8
6
4
D.O. (ppm)
8.4
8.4
8.3
8.6
8.4
8.4
8.2
8.4
8.6
7.7
7.8
7.7
6.9
7.1
7.1
7.2
6.9
7.1
—
7.1
7.6
6.2
6.9
7.5
6.6
6.9
6.6
6.4
7.7
7.4
7.8
6.8
7.3
6.6
6.0
6.8
6.6
7.2
7.2
—
6.7
7.3
6.7
6.8
—
140
-------
TABLE A-l. (CONTINUED)
Date
4/6
4/7
4/8
4/9
4/10
4/11
4/13
4/14
4/15
4/16
4/17
4/18
4/19
4/20
4/21
Temp. (°C)
22.5
22.5
22.5
22.5
22.5
22.5
22.5
23
23
22.5
23
23
23
22.5
23
pH
7.1
7.0
7.1
7.1
7.16
7.1
7.2
7.1
7.0
7.1
6.9
6.85
6.9
6.9
6.9
Salinity (ppt)
7
6
5
5
4
4
3
3
3
4
3
4
3.5
5
4
D.O. (ppm)
6.3
5.3
7.0
6.8
6.4
6.4
6.8
6.8
7.1
6.8
7.1
6.5
6.4
6.4
6.3
141
-------
TABLE A-2. TEMPERATURE, pH, SALINITY, AND DISSOLVED OXYGEN PARAMETERS
DURING UPTAKE AND DEPURATION PHASES, TCB/OYSTER BIOACCUMULATION TEST
Date
5/12
5/13
5/14
5/15
5/16
5/17
5/18
5/19
5/19
5/19
5/19
5/20
Time of Day
0900
0300
0800
0800
0830
0930
1830
0700
0900
1300
1700
0730
Temp. (8C)
24
22
22
22
22
22
—
—
21
—
—
21.5
pH Salinity (ppt)
7.0
7.0
7.0
7.0
7.0
7.0
—
—
7.0
—
—
7.0
16
12
8.5
11
14 '
14
10
8
10
10
10
8
D.O. (ppm)
7.5
6.0
5.7
6.0
6.1
6.1
—
—
6.0
—
—
5.8
TABLE A-3. TEMPERATURE, pH, SALINITY, AND DISSOLVED-OXYGEN PARAMETERS
DURING UPTAKE AND DEPURATION PHASES, PCP/OYSTER BIOACCUMULATION TEST
Date Sampling Code Temp, (°C)
7/16
7/16
7/17
7/18
7/19
7/20
7/21
7/22
A
C
F
G
H
I
J
0
21
21.5
- 21
21
21
21
21
20.5
pH Salinity (ppt)
7.4
7.4
7.5
7.5
7.5
7.5
7.5
7.5
18
18
20
21
21
20
20
22
D.O. (ppm)
6.3
7.0
7.0
7.0
6.5
6.8
6.3*
6.5
142
-------
September 1980
TOXICITY TESTING INTER-LABORATORY COMPARISON
Eastern Oyster Bioconcentration Testing using
p,p'-DDE, 1,2,4-Trichlorobenzene and Pentachlorophenol
APPENDIX D-3
by
MARINE BIOASSAY LABORATORIES
Watsonville, California 95076
Contract No. 68-03-2858
Final Report
for
Steven C. Schinunel, Project Officer
U.S. ENVIRONMENTAL PROTECTION AGENCY
Environmental Research Laboratory
Sabine Island
Gulf Breeze, Florida 32561
143
-------
ABSTRACT
This research program was designed as an interlaboratory comparison
of the ASTM Proposed Standard Practice for Conducting Bioconcentration
Tests with Fishes and Saltwater Bivalve Molluscs. Four laboratories were
selected by USEPA to perform biocencentration tests with identical test
species and chemicals. This report presents results obtained by Marine
Bioassay Laboratories, one of the four contractors,
A system for conducting flow-through bioconcentration tests was
developed. In an environmentally controlled laboratory, test chemicals
and high-quality dilution water were metered into mixing tubes and thence
into non-absorbing aquaria containing experimental animals. The animal
population was as uniform as possible with regard to size and environmental/
genetic history. Effluents from the aquaria were scrubbed in an activated
carbon column prior to discharge from the laboratory. The test animals used
were eastern oysters (Crassostrea virginica) . The three tests chemicals
were p,p'-DDE, 1,2,4-Trichlorobenzene and Pentachlorophenol.
Animal and water samples were collected on a prescribed schedule. Con-
centration of the test chemical in tissue and water were analyzed by gas
liquid chromatography/electron capture detection, and these data formed the
basis for calculation of uptake rate constants,.depuration rate constants
and bioconcentration factors.
The data resulting from bioconcentration studies on all three test
chemicals were extremely variable, and possible reasons for this variability
were discussed. The experience gained by this laboratory in utilizing the
Standard Practice was reviewed and evaluated, and suggestions were advanced
for possible modification.
This report was submitted in fulfillment of Contract No. 68-03-2858 by
Marine Bioassay Laboratories under the sponsorship of the U.S. Environmental
Protection Agency. This report covers the period 1 November, 1979 to
30 September, 1980, and work was completed as of 20 September, 1980.
144
-------
CONTENTS
Abstract [[[ 144
List of Figures ..... . ................................ . . ____ . ....... 146
List of Tables [[[ 147
1 . Introduction ............................................... 148
2 . Discussion and Recommendations ....... . ..................... 149
3 . Materials and Methods ...................................... 151
4 . Experimental Procedures . . ........................ . .........
Bioconcentration Tests - p,p ' -DDE ..................... 155
Biocon centra tion Tests - 1,2,4-Trichlorobenzene ....... 156
Bioconcentration Tests - Pentachlorophenol ............ 157
5 . Experimental Results
Data Analysis - p ,p ' -DDE . . ........... . ....... . ........ 158
Data Analysis - 1,2 , 4-Trichlorobenzene ...... .......... 165
Data Analysis - Pentachlorophenol ..................... 172
References [[[ 179
BdJbliography [[[ 18°
Appendix ......... . ................................................. 181
-------
LIST OF FIGURES
DDE
FIGURE 1: Bioconcentration Curves: Tank 2 - Uptake and Depuration 160
FIGURE 2: Water Concentrations: Tank 2 161
FIGURE 3: Bioconcentration Curves: Tank 3 - Uptake and Depuration 162
FIGURE 4: Water Concentrations: Tank 3 163
1,2,4-Trichlorobenzene
FIGURE 5: Bioconcentration Curves: Tank 4 - Uptake and Depuration 167
FIGURE 6: Water Concentrations: Tank 4 168
FIGURE 7: Bioconcentration Curves: Tank S - Uptake and Depuration
FIGURE 8: Water Concentrations: Tank 5
Pentachloropheno1
FIGURE 9: Bioconcentration Curves: Tank 4 - Uptake and Depuration 174
FIGURE 10: Water Concentrations: Tank 4 175
FIGUS2 11: Bioconcentration Curves: Tank 5 - Uptake and Depuration 176
FIGURE'12: Water Concentrations i Tank 5 177
146
-------
LIST OF TABLES
DDE
TABLE 1: Summary of Regression Equations and Parameters 159
TABLE 2: Summary of Calculations - Rate Constants, BCF's etc
1,2,4-Trichlorobenzene
TABLE 3: Summary of Regression Equations and Parameters
TABLE 4: Summary of Calculations - Rate Constants, BCF's etc 171
Pentachlorophenol
TABLE 5: Summary of Regression Equations and Parameters 173
TABLE 6: Summary of Calculations - Rate Constants, BCF's etc 178
147
-------
SECTION 1
INTRODUCTION
The United States Environmental Protection Agency (USEPA) has con-
tracted with a number of laboratories to perform bioconcentration tests
with eastern oysters (Grassestrea virginica), utilizing procedures described
in the ASTM Proposed Standard Practice for Conducting Bioconcentration Tests
with Fishes and Saltwater Bivalve Molluscs. Three compounds were specified
by USEPA for testing. Results were requested in terms of steady-state
and asymptotic bioconcentration factors (BCF) and uptake and depuration
rate constants for each specified compound. Test compounds p,p'-DDE [DDE],
1,2,4-Trichlorobenzene [TCG], and Pentachlorophenol [PCP] were provided
by USEPA.
Marine Bioassay Laboratories (MBL), as one of the contractors, performed
these tests at Davenport, California during the period 15 April, 1980
through 26 June, 1980.
USEPA will compare results received from all contractors and issue
an inter-laboratory comparison report which will serve as a basis for
evaluation of current methodologies for bioconcentration studies.
148
-------
SECTION 2
DISCUSSION AND RECOMMENDATIONS
MBL has long recognized the limitations of generally utilized methods
for bioconcentration (bioaccumulation studies). We feel that incorporation
of kinetic information would be useful both for better understanding of the
mechanisms involved and in terms of the potential practical applications.
We therefore welcomed the opportunity to participate in this interlaboratory
comparison of the ASTM Proposed Standard Practice and feel we have thereby
gained much valuable insight and experience. This portion of the report
attempts to summarize that experience and, in particular, to elucidate
the problems inherent in properly performing these bioconcentration tests.
We chose to meter the test compounds into the tanks via Mariotte
bottles because we felt that the alternative method of utilizing metering
pumps would be too vulnerable to electrical power failure. While the
probability of a power failure on any given day at our location is quite
small, that probability projected over a 28-day period becomes much more
significant. We did experience difficulty in maintaining toxicants at
constant levels because of clogged nozzles, brief failure of our temperature
control system, and slight variations in rate of dilution water flow due
to growth of fouling organisms in the system.
We enumerate these problems to illustrate the extremely demanding
requirements for quality control and surveillance of this complex and
interdependent system over such long time periods. There are many critical
components and failure or malfunction of any one of them produced adverse
effects. In simpler bioassay procedures we have been accustomed to thinking
in terms of 95% levels of confidence in our control systems. In these tests,
however the usually acceptable 5% level of possible error becomes amplified
both because of the longer times involved and because of the complexity of
the system. The level of effort required to perform these tests properly,
then, is increased by at least an order of magnitude. We found that we
were better able to anticipate and plan for problems as our experience
increased, and we feel that our overall level of performance in the oyster
project was higher than in the fathead minnow bioconcentration study which
we recently completed.
We found consistently wide variations in uptake and depuration rates
among individual organisms, even when our control systems were functioning
well. One obvious source of such variability in bivalve studies is the
tendency -for such animals to "clam up", i.e., close their valves tightly
together and simply stop pumping. In this case they are effectively isola-
ted from their aquatic environment and are not exposed to the test compound.
The effects of such behavior would be particularly noticeable during short-
term bioconcentration tests. There are undoubtedly other inherent physio- .
logical differences among individuals, and these together were a source of
149
-------
variability in our data. The effects of our somewhat variable toxicant
concentrations were compounded by the biological differences among test
animals, and produced data which were fairly widely scattered.
Extreme data variability results in lower confidence limits in the
reduced data and difficulties in fitting models to them. Because of the
lack of documentation of appropriate models in the Standard Practice and
the general unavailability of the suggested BIOFAC and NONLIN programs,
we had to search the literature and use the best model we could find for
calculating rate constants and BCF's. -This simple two-compartment model
was probably too simple to fi't our data well. Further, the use of ANOVA
to compare S, S+2 and S+4 data proved to be inadequate, in some cases, to
verify attainment of steady state. We feel a better estimate of steady
state would be obtained by calculating percent of steady state reached
at the end of the uptake phase.
Based on our experience, then, we offer the following suggestions
for modification of the Standard Practice as written.
(a) The necessity for extremely high levels of reliability in environ-
mental control and toxicant delivery systems should be emphasized.
(b) We feel it possible that some of the variability of our data might
be statistically based as suggested by J.L. Hamelink (1). He suggests that
there is a general tendency for the variability about the mean (i.e., the
standard deviation for n = 4} to increase with time. If we assume that we
have a. normally distributed population of test organisms in the tank with
a given mean and standard deviation, then as we reduce the population
(or sample the oysters), the probability of sampling an individual whose
response characteristics lie at the extremes of the distribution is
markedly increased. We suggest that either the initial experimental popula-
tion size be increased to offset these probability effects or that a
"probability factor" be generated to normalize the data (i.e. correct for
predictable variability).
(c) As data reduction techniques, formulae and programs become
standardized and available, much more direction and documentation must be
provided in the Standard Practice. The absence of such guidelines can
only make comparisons among experiments difficult and confusing.
(d) Finally, and most importantly, we consider ourselves a reasonably
capable and competent lab which has ^a considerable background of experience
in conventional bioassay projects. We, however, had problems with this
technique. It was just not feasible for us to appreciate the stringent
requirements for precision, surveillance and quality assurance feedback
and fine-tuning until we had gone through it a few times. We therefore
conclude that a necessary pre-requisite for attempting this procedure is
experience.
150
-------
SECTION 3
MATERIALS AND METHODS
Up to 5000 gallons per minute of uncontaminated open-coast seawater
was continuously delivered to the salmon release station adjacent to the
MBL Davenport bioassay facility by one or both of two parallel cast-iron
pump systems. Prom the intake pump, MBL's 50 GPM plastic pump delivered
water to a 500 gallon reservoir from which it flowed by gravity (20 foot
head) into the bioassay laboratory. The float switch controlling the
MBL pump was set to maintain the reservoir at least 60% full at all times.
From laboratory distribution lines the water was passed through a temperature
regulation tank equipped with a 1000-watt Vycor immersion heater, a thermo-
regulator and a submersible pump which insured aeration and thorough mixing.
Temperature-controlled water (15°C ± 1°C) flowed into a constant-head
device, and thence into a main aquarium distribution line. Final flow
rates were precisely controlled by varying the length and diameter of
flexible feeder tubes which discharged into 25 liter glass aquaria via
glass mixing tubes. The entire water system was of glass or plastic
construction and was non-toxic to aquatic organisms.
The laboratory was uniformly illuminated at an intensity of
l.SxlO1^ quanta/cm2/sec by cool-white fluorescent lamps. Photoperiod
was., maintained at 16-hour light/8-hour dark cycle with a 15-minute
transitional period.
Laboratory air temperature was maintained at 17°C t 2°C by an air
conditioner and electric space heaters working in tandem and controlled
by a common thermostat and relay system.
Concentrations of test compounds were maintained by metering stock
solutions at constant rates into glass mixing tubes which were receiving
constant flows of dilution water. Metering devices were slightly different
for each test compound, but were all variations of the Marietta bottle
principle. Concentrations were controlled either by varying the strength
of stock solutions or by adjusting the size of the orifice in the glass
delivery tubes (thus adjusting the flow rate).
Effluent from test tanks was passed through an activated carbon column
(75 cm x 125 cm diameter) prior to discharge from the laboratory. Cleaned
effluent water samples were analyzed at weekly intervals to monitor clean
up effectiveness.
Oysters were purchased from Cotuit Oyster Company, Cotuit Massachusetts,
and were selected from a single year class and population. All oysters
were acclimated to MBL water for at least 3 weeks prior to initiation of
testing.
151
-------
Octanol-water partition coefficients were used to calculate expected
time to steady-state for each test compound, after Leo, Hansch and Elkins
(2). . These estimates were further revised by preliminary bioconcentration
experiments.
Water samples were collected by glass siphon tube from the approximate
center of test aquaria, and prepared for analysis by hexane extraction.
In preparation for analysis of DDE and TCG, tissue samples were homogenized
(Tissuemizer, Model SDK) three times in a total of 9 ml of acetonitrile.
The acetonitrile extract was washed in 25 ml of 2% aqueous sodium sulfate,
and the test compound recovered by extracting three times with a total of
10 ml of hexane. Final clean-up was achieved by passing the hexane extract
through a florosil column.
For analysis of PCP, tissues were prepared differently. The acetoni-
trile homogenate was prepared and added to 2% sodium sulfate as usual. This
mixture was made alkaline with sodium hydroxide and extracted three times
with five ml portions of hexane. The hexane phase was discarded. The
washed mixture was then acidified with sulfuric acid and extracted with
three more five ml portions of hexane. This acid hexane phase was retained
and one ml was methylated by addition of one ml of diazomethane methylating
reagent, prepared according to USEPA specifications. After 20 minutes the
methylation reaction was quenched by addition of one ml of methanol contain-
ing 20% water. The hexane phase was withdrawn and analyzed for PCP content.
All test compounds were analyzed by gas-liquid chromatography/electron
capture detection on a Perkin-Elmer Sigma II system equipped with a Sigma
10 printer/plotter data processor. The method of internal standardization
was used to quantify the amounts of test compound present in water and
tissue samples. This method has operational advantages in analytical
situations where only one compound is to be measured. The internal standard
must be structurally similar to the test compound and be resolved from other
peaks; it must also elute from the column close to the test compound peak.
For calibration of the internal standard, a mixture of known weights of
test compound and internal standard is prepared and chromatographed. Peak
areas are measured and a relationship between area ratio and weight ratio
is thereby established. During analysis of unknowns, an accurately known
amount .of internal standard is added to each sample and the mixture is
chromatographed. Area ratios are measured and from these, by use of the
area/weight relationship established during calibration, weight of the test
compound in the sample is calculated. Internal standard compounds used
with each test compound are identified in the appropriate following section
of the report.
Data were processed on a Tektronix 4051 graphic computer with 32K RAM,
and the plotting was done by Tektronix 4662 interactive digital plotter.
Since we were unable to gain access to an appropriate non-linear regression
program (e.g. BIOFAC or NONLIN), we followed the method described by
Blanchard, et al (3) to estimate constants and bioconcentration factors.
The basis of this method is outlined below.
152
-------
Data Analysis - General
The reversible reaction used to describe the movement of test compounds
into and out of fish is as follows.
Cf
K2
where:
C = concentration of the compound in water (ppb)
C = concentration of the compound in fish (ppb)
1C ^ rate constant of uptake (ml/g/hr)
K_ = rate constant of depuration (hr~ )
For this two-compartment model, changes in C are described by the
differential equation
dCf/dt = K1Cw - K2Cf
With initial conditions of t=0, C = 0 and C = a constant, this equation
may be solved as follows w
Cf" (KlCw)/K2) x (1 " ^^
In our experimental data, C was not constant both because of uptake by
the fish and because of imperfect diluter performance, and the simple
analytical solution of the second equation was not strictly true. The rate
constants were, therefore, estimated by the following graphic/computer
method:
First, C_ was plotted vs. time for the uptake period, and a curve was
fitted to these points by linear regression. In most cases this regression
curve turned out to be a simple straight line. The slope of this line was
used to calculate the uptake rate constants at each sampling poing. From
these values the average uptake rate was calculated. In the cases where
regression analysis yielded a curve rather than a straight line, the slope
was calculated from tangents to the curve at time points corresponding
to the sampling times in the experimental design. Note that the slope
is the term dC_/dt in the first equation. The C_ value of the regression
curve at each of the sampling times was recorded.
Secondly, a plot of InC vs time for the depuration period was drawn
and yielded a straight line of slope K-.
153
-------
Third, averaged (time weighted) Cw values were plotted vs time and regression
analyses were used to fit the best line to these points. C^ values at desig-
nated sampling times were interpolated from this curve and recorded.
Values for K^ at each sampling time were then calculated from the first
equation using the values of slope, K2, Cw and Cf derived in steps 1-3 above,
and from these an average K-j_ was calculated.
Asymptotic bioconcentration factor (BCF = K^/) was calculated.
Expected tissue concentration at steady state was calculated by the for-
mula (BCF)
Percent steady state achieved was calculated by the formula
Actual C
x 100.
_^ . _
Expected C
Steady state BCF was calculated by Cf/Cw at steady state or end of
uptake phase.
Single Factor Analysis of Variance (ANOVA) was applied to the data
where the end of the uptake was not clearly predictable.
154
-------
SECTION 4
EXPERIMENTAL PROCEDURES
BIOCONCENTRATION TESTS - DDE
A. Organisms
Two days prior to initiation of DDE exposure, 60 eastern oysters
were stocked into experimental tanks 2 and 3 and control tank 1. Oyster
weights were estimated by eye and weighed 3.72 grams ±0.98 grams (Tank 2)
and 3.54 ± 0.91 grams (Tank 3. During the course of DDE uptake and depura-
tion (56 days) one oyster died in Tank 2.
B. Experimental methods
A primary stock solution of DDE in dimethyl formamide (DMF) was
prepared. Aliquots of the primary stock were diluted daily with distilled
water to form a working stock, which was metered into the glass mixing
tubes from a Mariotte bottle whose rate of flow was set by adjusting the
orifice size of its glass outlet tube. Final DMF concentration in the
tanks was 0.001 ml/1. Dilution water flow rate through the mixing tubes
was 100 ml/minute throughout uptake and depuration. DDE concentrations
in the experimental tanks were measured at frequent intervals over the
uptake phase, and ranged from 0.00024 to 0.00057 ug/ml (Tank 2) and
0.00033 to 0.00060 ug/ml (Tank 3).
Since it was not expected that steady state would be reached for DDE
uptake by 28 days, the sampling schedule was planned using 28 days as the
nominal "S" point. Four oysters from each tank were sampled at times
corresponding to S/16 (1.75 days)", S/8 (3.5 days), S/4 (7 days), S/2 (14
days) and S (28 days). Since steady state was not expected at 28 days, "S"
was considered equal to D and the S+2 and S+4 samples were eliminated.
Depuration was begun immediately after the 28-day oysters were collected,
and depuration tissue samples were taken at U+D/4 (35 days), U+D/2 (42 days),
W-3D/4 (49 days) and U+D (56 days) . Duplicate 250 ml water samples were
collected at the time of oyster sampling.
Water and tissue samples were extracted and analyzed as detailed above.
The internal standard employed for DDE analysis was Aldrin. Spiked blank
samples were run and recovery averaged 100% from water and 101% from tissue.
Control oysters were sampled and analyzed at the beginning and end of the
uptake phase and at the end of the depuration phase.
155
-------
BIOCONCENTRATION TESTS - 1,2,4 Trichlorobenzene (TCG)
A. Organisms
On 13 May, 1980, 60 eastern oysters were stocked into each of tanks
4 and 5 and allowed to acclimate for two days prior to initiation of TCG
flow. Oyster weights were estimated by eye and averaged 4.10 ± 1.08 (Tank 4)
and 4.18 ± 1.09 (Tank 5). There were no mortalities in either experimental
tank during the 12-day uptake and depuration period.
B. Experimental Methods
Acetone was used as the carrier solvent for TCG and the test compound
was metered into the mixing tubes from Mariotte bottles. Final acetone
concentration in the tanks was 0.2 ml/1. Flow rate of dilution water was
300 ml/min throughout uptake and depuration. TCG concentrations were
measured during uptake at the times of tissue sampling, and ranged from
0.00446 to 0.004535 ug/ml (Tank 4) and from 0.00393 to 0.003995 ug/ml
(Tank 5).
The oyster sampling schedule was planned with an anticipated steady
state at 96 hours. Four oysters from each tank were sampled at times
corresponding to S/16 (6 hours), S/8 (12 hours), S/4 (24 hours), 3S/4
(72 hours), S (96 hours), S + 1 day (120 hours), S + 2 days (144 hours),
and S +• 7 days = U (264 hours) . Depuration was begun immediately following
the S + 7 sample, and samples were removed at U+D/4 (312 hours), U+D/2
(336 hours), U+3D/4 (360 hours) and U+D (384 hours).
Water and tissue samples were extracted and analyzed as detailed
above. The internal standard employed for TCG analysis was 1,2,3
trichlorobenzene (TCD). Spiked blanks were analyzed periodically and
recovery averaged 95% from water and 97% from tissue. Control fish
were sampled and analyzed at the beginning and end of uptake and at the
end of depuration. A sample from each batch of frozen brine shrimp was
analyzed to ensure no uncontrolled input of chlorinated hydrocarbons.
156
-------
BIOCONCENTRATION TESTS - Pentachlorophenol (PCP)
A. Organisms
On 17 June, 1980, 60 eastern oysters were stocked into each of the
tanks 4 and 5 and allowed to acclimate for two days prior to initiation
of HXB flow. Oyster weights were estimated by eye and averaged 0.402 ±1.29
grams (Tank 4) and 4,51 ± 1.36 (Tank 5) . There were no mortalities recorded
in either experimental tank during the 6-day uptake and depuration period.
B. Experimental Methods
PCP was dissolved in 0.02M sodium carbonate and the test compound was
metered into the mixing tubes from Mariotte bottles. Flow rate of dilution
water was 1000ml/minute throughout uptake and depuration. PCP concentration
in the experimental tanks was measured at intervals during the uptake phase
and ranged from 0.00042 to 0.00392 ug/ml (Tank 4) and 0.00063 to 0.00886
ug/ml (Tank 5) .
The sampling schedule was planned anticipating steady state at 16 hours.
Four oysters were sampled from each tank at S/16 (1 hour), S/8 (2 hours),
S/4 (4 hours), S/2 (8 hours), 3S/4 (12 hours) and S (16 hours). Steady
state.was established by sampling at 5+2 days (64 hours) and S-f-4 days (112
hours). Depuration was begun immediately after the 112 hour samples were
taken and samples were removed at U+D/4 (116 hours) U+D/2 (120 hours) ,
U+3d/4 (124 hours) and 0+D (128 hours). Duplicate 250 ml water samples
were collected at time of tissue sampling throughout uptake and depuration.
Water and tissue samples were extracted and analyzed as detailed above.
The internal standard employed for PCP analysis was Aldrin. Spiked blank
samples were analyzed, and recovery averaged 93% from water and 80% from
tissue. Control oysters were sampled and analyzed at the beginning of
the uptake phase and at the end of the depuration phase.
157
-------
SECTION 5
EXPERIMENTAL RESULTS
DATA ANALYSIS: p,p'-DDE
Results of tissue and water analyses (experimental data) are tabulated
in Appendix Tables A-l and A-2. Uptake and depuration curves are illustrated
in Figure 1 (Tank 2) and Figure 3 (Tank 3). Water concentrations during the
uptake phase are illustrated in Figure 2 (Tank 2) and Figure 4 (Tank 3). The
regression equations and their parameters used to best fit the curves to
these rather variable data are summarized in Table 1.
Rate Constants and BCF's (see Table 2) were derived from the interpolated
values of each regression line (see Appendix Table A-3).
It should be noted that our treatment of the data showed the depuration
curve in Tank 3 to have a positive slope i.e., that the tissue concentration
continued to increase after DDE input was discontinued. Water analyses
confirmed an insignificant concentration of DDE in the water during depuration
(Appendix Table A-2) . This anomalous result can only be due to the extreme
variability of the data.
158
-------
TABLE 1
Summary of Regression Equations and Parameters
Constituent/
Phase
Regression Equation
Standard Error Coefficient of
of Estimate Determination
DDE Uptake
Tank 2
f(x) = 0.1177X + 0.1527
1.2096
0.4864
DDE Depuration
Tank 2
£(») = 3.4893 *
0.4302
0.0065
DDE Uptake
Tank 3
f(x) = 0.19724x + 0.2946
1.5895
0.6079
DDE Depuration
Tank 3
f(x) = 4.0412 * e(°-°1435x)
0.6694
0.0454
-------
(a)
(b)
: 6
3 4
o
a
ia
20
2E
i.
o
! <
.«»
o
I
Is
3
I
G 10 1C 20
Tin* Elop**^ Tin*
i
FIGURE 1 Bioconcentration Curves: DDE Tank 2 (a) Uptake (b) Depuration
25
30
-------
cr>
6.BE-4
G.EEH
2 6.8E-H
•
4.6E--4
o
L
c
3.EE-H
3.BE--4
2.BE-I
•f mean value
|0 1C 28 26
FIGURE 2 Water Concentrations: DDE Tank 2
-------
(a)
(b)
rv;
12
~ 18
e
e
o
o
0
IB
IE
Tim
20
30
IB
H
•
"
3 .
1 *
0
10 IE 20
El <*>••
38
FIGURE 3 Bioconcentration Curves: DDE Tank 3 (a) Uptake (b) Depuration
-------
cr>
oo
a.QE-4
6.CE-4
6.0E-4
4.EE-4
0
L
3.BE--I
+ mean value
10 IS 20 26 30
•d Tim*
FIGURE 4 Water Concentrations: DDE Tank 3
-------
TABLE 2
Summary of Calculations
DDE
Tank 2
Tank 3
Units
RATE CONSTANTS
Uptake : K.
Depuration : K_
BIOCONCENTRATION FACTORS
28-day BCP (Cf/cw)
Asymptotic BCF (K./K,)
303 . 303 362 . 351 nl/g/day
-0.00343 0.01435 day"1
9194.1 12,673.4
88,426.5 25,250.9
STEADY STATE
Percent of SS at 28 day
ANOVA P-value
10.4
50.2
164
-------
DATA ANALYSIS: 1,2,4-Trichlorobenzene
Results of tissue and water analyses (experimental data) are tabulated
in Appendix Tables A-4 and A-5. Uptake and depuration curves are illustrated
in Figure 5 (Tank 4) and Figure 7( Tank 5) . Water concentrations during the
uptake phase are illustrated in Figure 6 (Tank 4) and Figure S (Tank 5) . The
regression equations and their parameters used to best fit the curves to
these rather variable data are summarized in Table 3.
Rate Constants and BCF's (see Table 4) were derived from the interpolated
values of each regression line (see Appendix Table A-6).
165
-------
TABLE 3
Summary of Regression Equations and Parameters
Constituent/
Phase
Regression Equation
Standard Error Coefficient of
of Estimate Determination
en
cr>
1,2,4-Trichlorobenzene
Uptake Tank 4 f(x) = 0.0639x + 0.0653
1,2,4-Trichlorobenzene 03036x1
Depuration Tank 4 f(x) = 1.0237 * el U-UJUJOX'
0.2478
0.8632
0.4406
0.6130
1 , 2 , 4-Trichlorobenzene
Uptake Tank 5 f(x) = 0.00424x + 0.1266
1,2, 4-Trichlorobenzene
Depuration Tank 5 f(x) - 0.87490 * e* u-
0.2161
0.9808
0.3201
0.5885
-------
(a)
(b)
1.2
1 '
8
I...
c
"»
i: 0.4
0
20
40 00
*4 Tin*
60
1-
1.0
4
1.2
I
0
8.2
0
100
40 ea
Tin*
ea
I0a
FIGURE:5 Bioconcentration Curves: 1,2,4-Trichlorobenzene Tank 4 (a) Uptake x(b) Depuration
-------
t—•
en
00
0.08454
0.004S3
^ 0.00452
~i
g 0.00461
* 0.0046
g
0
^ 0.00448
L
§ 0.00448
0.00447
0.00440
(
+ mean value
•
i
t
+ 4-
. . . .
•
• Illllllll
i 20 40 ao eo 10
Elop««d Tl*« Chour«>
FIGURE 6 Water Concentrations! 1,2,4-Trichlorobenzene Tank 4
-------
(a)
(b)
en
10
£
3
I
0.8
e.e
a.7
8.6
e.c
0.4
0.3
0.2
3.t
a
o
20
40 60
»••<* Tim*
tao
FIGURE 7 Bioconcentration Curves: 1,2,4-Trichlorobenzene Tank 5 (a) Uptake (b) Depuration
-------
0.00388
J 0.00396
3 0.00387
j
"« 0.00380
£ 0.00386
0.00384
0.00383
C
+ mean value
i •
4-
*
P •
1 * • *
•V
• ••••••ill
1 20 40 00 60 10
E|op«*d
FIGURE 8 Water Concentrations: 1,2,4-Trichlorobenzene Tank 5
-------
TABLE 4
Summary of Calculations
1,2,4-Trichlorobenzene
Tank 4
Tank 5
Units
RATE CONSTANTS
Uptake: K.
Depuration: K_
BJOCONCENTRATION FACTORS
96-hour BCP (C_/C )
Asymptotic BCF (K-/K-)
STEADY STATE
Percent of SS at 96 hours
* ANOVA F-value
3.444
-0.0304
149.39
113.44
132.1
1.785 n.s.
3.407
-0.0328
133.76
103.93
128.7
0.386 n.s.
ml/g/hr
hr'1
Bartlett's Test
0.125 n.s. 1.914 n.s.
n.s. indicates not significant at alpha level 0.05
±
4- ANOVA calculated for values at S, S+lday, S+2days, S+7days
171
-------
DATA ANALYSIS: Pentachlorophenol
Results of tissue and water analyses (experimental data) are tabulated
in Appendix Tables A-8 and A-9. Uptake and depuration carves are illustrated
in Figure 9 (Tank 4) and Figure 11 (Tank 5). Water concentrations during the
uptake phase are illustrated in Figure 10 (Tank 4) and Figure 12 (Tank 5).
The regression equations and their parameters used to best fit the curves to
these rather variable data are summarized in Table 5.
Kate Constants and BCF's (see Table 6) were derived from the interpo-
lated values of each regression line (see Appendix Table A-10).
In our treatment of. the data we have used two approaches to estimate
whether steady-state was reached at the end of the uptake phase:
a) ANOVA was used to determine whether tissue concentrations at time
St S+2 days and S+4 days were different from one another, and
b)percent of steady-state reached'at the nominal S point was calculated.
For data from both Tanks 4 and 5 the two methods gave conflicting answers.
ANOVA suggested that steady-state had not been reached at 96 hours.
However, Bartlett's test indicated that data from both tanks to be non-
homogeneous during times S through S+4 days which questions the validity
of ANOVA for these data. The alternative procedure (b) indicated that
100% of the steady state concentration had been achieved at 96 hours.
172
-------
TABLE 5
Summary of Regression Equations and Parameters
Constituent/
Phase
Regression Equation
Standard Error Coefficient of
of Estimate Determination
OJ
Pentachlorophenol
Uptake Tank 4
Pentachlorophenol
Depuration Tank 4
Pentachlorophenol
Uptake Tank 5
Pentachlorophenol
Depuration Tank 5
f(x) = 0.02054x + 0.26023
f(x) = 0.88602 * e(-°-01802x)
f(x) = O.Q3581X -I- 0.22466
f(x) = 2.37023 * e(~°*
0.5321
0.8892
0.5483
1.1902
0.04668
0.01440
0.12299
0.10155
-------
(a)
(b)
1.8
1.6
ft
I '•<•
0*
9 1.2
fe
v»
§ 8.8
.**
0
j; 8.0
0
c 8 4
o "'^
u
8.2
0
3.B
3
Z 2.S
1 1.6
«•
0
<•
I !
5
0.E
0
0 2 4 6 6 10 12 14 10
»d Tl»«
0
0
10 12 14 10
FIGURE 9 Bioconcentration Curvest Pentachlorophenol Tank 4 (a) Uptake (b) Depuration
-------
en
r\
~i
X
&
$
|
M*
«
t.
1
j
0CIA4
• w^
0.0036
0.003
0.0026
0.002
0.0016
I.0E-3
6.0E-4
a
r
•
•
t
•
•
, *
•»•
•»•
1-
•
• •
. •«•
•
||||iti>
0
4 9 8 10 12 M 10
FIGURE 10 Water Concentrations: Pentachlorophenol Tank 4
-------
(a)
(b)
1.6
l.e
1.2
0
J;
0
8.2
0
a
«
B
fc
r
s -»
02-406
Elop««
-------
f\
I
3
|
L
i
0.0024
0.0022
0.002
0.00|«
0.00IB
0.00H
0.0012
I00B09
8.0E-4
R Off -A
\ mean value
•
•*•
'
t
f
. ' t +
i 1 1, i 1 i 1 1
0
0
10 12 |4 18
FIGURE 12 Water Concentrations: Pentachlorophenol Tank 5
-------
TABLE 6
Summary of Calculations
Pentachlorophenol
Tank 4
Tank 5
Units
RATE CONSTANTS
Uptake: KL 22.576
Depuration: K_ -0.01802
BIOCONCENTRATTCN FACTORS
16-hour BCP (Cf/Cw)
Asymptotic BCF
STEADY STATE
Percent of SS at 16 hours 100
"ANOVA P-value 4.203*
429.78
1252.83
65.130
-0.06711
1009.61
970.50
100
6.724*
ml/g/hr
hr
-1
Bartlett's Test
2.568*
2.515*
* indicates significant result at alpha level 0.05
j. —
4> ANOVA performed on values at S, S+4hr, S+8hr, S+2days, S+4days
178
-------
REFERENCES
1. BamelinJc, J.L. Current Bioconcentration Test Methods and Theory.
In: Aquatic Toxicology and Hazard Evaluation Proceedings of the
First Annual Symposium on Aquatic Toxicology. Am. Soc for Testing
and Materials, Philadelphia, Pennsylvania, pp. 149-161
2. Leo, A., C. Hansch and D. EUcins. Partition Coefficients and Their
Uses. Chemical Reviews, 71(6): pp. 525-616, December 1971.
3. Blanchard, F.A., I.T. Takahashi, H.C. Alexander, and E.A. Bartlett.
Uptake, Clearance, and Bioconcentration of 14C-Sec-Butyl-4-Chlorodiphenyl
Oxide in Rainbow Trout. In: Aquatic Toxicology and Hazard Evaluation
Proceedings of the First Annual Symposium on Aquatic Toxicology.
Am. Soc. for Testing and Materials, Philadelphia, Pennsylvania.
pp. 162-177
179
-------
BIBLIOGRAPHY
Marking, L.L. and R.A. Kimerle, eds. 1979. Aqua-tic Toxicology, Proceedings
of the Second Annual Symposium on Aquatic- Toxicology, Am. Soc. for
Testing and Materials, Philadelphia, Pennsylvania. 392 pp.
Mayer, F.L. and J.L. Hamelink. 1977. Aquatic Toxicology and Hazard Evalua-
tion, Proceedings fo the First Annual Symposium on Aquatic Toxicology.
Am. Soc. for Testing and Materials, Philadelphia, Pennsylvania. 307 pp.
Sokal, R.R. and F.J. Rohlf. 1969. Biometry. W.H. Freeman and Co.,
San Francisco, California. 776 pp.
Zar, J.H. 1974. Biostatistical Analysis, Prentice-Hall, Inc. Engelewood
Cliffs, N.J. 620 pp.
180
-------
APPENDIX D-3
181
-------
CONTENTS OF APPENDIX
DDE
A-l: Bioconcentration Data Results , 183
A-2: Water Concentrations 185
A-3: Interpolated Values from Regression Analyses ... 186
1,2,4-Trichloroben2ene
A-4: Bioconcentration Data Results . 187
A-5: Water Concentrations .. 188
A-6: Interpolated Values from Regression Analyses 189
A-7: Analysis of Variance at Steady State 19°
Pentachlorophenol
A-8: Bioconcentration Data Results 191
A-9: Water Concentrations 193
A-10: Interpolated Values from Regression Analyses 1^4
A-11: Analysis of Variance at Steady State 195
182
-------
TABLE A-l
Bioconcentration Results Oyster - DDE
CO
co
Tank #2
Code
S/16
1.75 days
(42 hours)
S/8
3.5 days
Sample
Size (g)
2.28
3.51
2.64
3.62
4.22
3.08
3.84
ugDDE/
Sample
0.82
0.22
3.25
5.46
0.72
0.26
0.03
(84 hours) 4.11 1.4
S/4
7 days
5.58
3.80
1.90
(168 hours) 4.97
S/2
14 days
5.30
3.48
3.50
(336 hours) 2.93
S(U)
28 days
2.92
4.47
2.36
(672 hours) 4.95
U+D/4
7 days
1
2.19
3.60
3.28
(168 hours) 4.64
U+D/2
14 days
3.58
4.64
3.47
(336 hours) 2.68
14
2.1
4.1
0.59
7.5
4.5
4.3
6.5
4.7
7.8
18
16
9.3
8.1
14
28
20
17
13
15
ug DDE/g
0.36
0.06
1.2
1.5
0.17
0.08
0.01
0.34
2.5
0.56
2.1
0.12
1.4
1.3
1.2
2.2
1.6
1.7
7.6
3.2
3.2
2.2
4.3
6.0
5.5
3.6
3.9
5.5
Tank #3
Code
S/16
1.75 days
Sample
Size (g)
4.16
4.22
3.71
(42 hours) 3.77
S/8
3.5 days
2.32
3.40
2.25
(84 hours) 2.68
S/4
7 days
2.77
4.25
2.92
(168 hours) 3.13
S/2
14 days
4.14
3.52
2.21
(336 hours) 2.92
S(U)
28 days
4.13
5.09
2.64
(672 hours) 4.04
U+D/4
7 days
5.21
3.38
3.37
(168 hours) 5.81
U+D/2
14 days
249
2.42
3.43
(336 hours) 3.88
ug DDE/
Sample
2.72
4.40
5.25
0.97
1.0
0.46
2.1
6.2
4.8
11
2.2
3.3
8.5
14
12 .
8.1
10
22
28
18
8.1
7.8
11
26
36
32
13
18
ug DDE/g
0.65
1.0
1.4
0.26
0.34
0.13
0.94
2.3
1.7
2.6
0.75
1.1
2.1
4.1
5.4
2.8
2.5
4.3
11
4.5
1.6
2.3
3.1
4.5
16
13
3.9
4.7
-------
TABLE A-l
Bioconcentration Results Oyster - DDE;
Tank 0 2 Sample ugDDE/
Code Size (g) Sample ugDDE/g
5.18 19 3.6
IH3D/4 3.86 13 3.5
19 days 4.48 9.0 2.0
(504 hours) 4.59 , 18 4.0
3.00 6.4 2.1
U+D 3.62 10 3.0
28 days 5.13 14 2.8
(672 hours) 2.44 7.5 3.1
Tank *» 3 Sample ugDDE/
Code Size (g) Sample ugDDE/g
2.67 30 11
U+3D/4 4.05 41 10
19 days 3.07 5.0 1.6
(504 hours) 3.99 14 3.5
5.35 13 6.0
U+D 3.80 18 4.6
28 days 3.22 14 4.5
(672 hours) 3.47 2.9 8.4
-------
TABLE A-2
Code
Water Concentration
ODE
S/16
1.75 days
S/8
3.5 days
S/4
7 days
10 days
S/2
14 days
17 days
3S/4
21 days
24 days
S(U)
28 days
O+D/4
35 days
W-D/2
42 days
U+3D/4
49 days
Elapsed Time
(hours)
0
42
84
168
240
336
408
504
576
672
Concentration Tank 2
(ug/ml)
0.00047
0.00042
0.00049
0.00044
0.00036
0.00024
0.00044
0.00036
0.00057
0.00048
0.00050
0.00045
0.00036
0.00030
0.00039
0.00039
0.00042
0.00038
0.00037
0.00032
0.00003
0.00010
0.00002
0.00001
0.00006
0.00008
Concentration Tank 3
(ug/ml)
0.00050
0.00046
0.00051
0.00046
0.00047
0.00042
0.00060
0.00054
0.00038
0.00035
0.00036
0.00033
0.00045
0.00042
0.00045
0.00060
0.00052
0.00052
0.00046
0.00042
0.00007
0.00001
0.00002
0.00005
0.00005
0.00003
185
-------
TABLE A-3 Summary of Interpolated Values from Regression Analyses
00
Sampling Point
T
(days)
DDE Uptake Tank 2
0
1.75
3.5
7
14
28
DDE Uptake Tank 3
0
1.75
3.5
7
14
28
Tissue Concentration Hater Concentration
C.
t.
(ug/g)
[f(x) - 0.1177x + 0.1527]
0.1527
0.3587
0.5646
0.9765
1.8002
3.4478
[f (xj = 0.1972x + 0.29457]
0.2947
0.6400
0.9850
1.6753
3.0559
5.8171
C
w
(ug/ml)
0.000445
0.000465
0.000331
0.000363
0.000480
0.000375
0.000480
0.000485
0.000452
0.000544
0.000371
0.000459
Rate Constants
*i
1
(ml/g/day)
265.671
255.764
361.440
333.469
258.072
345.403
•
402.023
387.662
405.012
318.308
413.336
247.766
-------
TABLE A-4
Bioconcentration Results Oyster - 1,2,4-Trichlorobenzene
00
•--J
Tank ft 4
Code
S+2
144 hours
S+7(U)
U+D/4
24 hours
U+D/2
48 hours
U+3D/4
72 hours
U+D
96 hours
Sample
Size (g)
5.10
3.24
3.27
5.84
2.38
3.60
3.22
3.53
5.12
6.25
3.73
4.32
3.59
3.98
1.61
6.88
5.21
3.41
2.53
5.34
4.24
3.76
5.16
6.39
U9TCG/
Sample
5.2
2.4
1.0
3.7
2.9
6.5
3.5
3.0
0.70
3.8
1.7
2.6
0.47
0.84
0.43
3.1
1.7
1.5
0.11
0.24
0.08
0,10
2.07
0.27
ugTCG/g
1.0
0.75
0.30
0.63
1.2
1.8
1.1
0.85
0.14
0.61
0.45
0.61
0.13
0.21
0.27
0.46
0.33
0.43
0.04
0.04
0.02
0.03
0.40
0.04
Tank #5
Code
S+2
144 hours
S+7(U)
IHD/4
24 hours
U+D/2
48 hours
U+3D/4
72 hours
IttD
96 hours
Sample
Size (g)
6.17
2.31
4.06
3.34
2.50
2.15
3.17
3.69
4.46
3.32
3.46
3.54
4.93
3.63
3.70
1.97
4.36
4.80
5.52
6.63
6.18
2.97
2.81
4.63
ugTCG/
Sample
7.9
2.2
0.27
2.8
2.0
1.7
2.2
3.3
6.4
1.3
3.0
1.8
0.74
0.09
2.69
0.20
0.04
0.46
0.38
0.22
0.63
0.05
0.11
0.97
ugTCG/g
1.3
0.96
0.07
0.83
0.81
0.77
0.68
0.90
1.4
0.4
0.9
0.5
0.15
0.03
0.73
0.10
0.01
0.10
0.07
0.04
0.10
0.02
0.04
0.21
-------
TABLE A-5
Code
Water Concentrations
1,2,4-Trichlorobenzene
Elapsed Time
(hours)
Concentration
(ug/ml)
Tank 4
Concentration
(ug/ml)
Tank 5
0
S/16 6
s/a 12
S/4 24
S/2 48
3S/4 72
S 96
0.00447
0.00445
0.00448
0.00449
0.00448
0.00449
0.00450
0.00450
0.00448
0.00450
0.00454
0.00453
0.00452
0.00453
0.00393
0.00395
0.00395
0.00395
0.00394
0.00395
0.00396
0.00397
0.00395
0.00395
0.00399
0.00400
0.00398
0.00398
188
-------
TABLE A-6 Summary of Interpolated Values from Regression Analyses
eo
Sampling Point Tissue Concentration Water Concentration Bate Constants
T
(hours)
1 , 2 , 4-Trichlorobenzene
0
6
12
24
48
72
96
1,2, 4-Trichlorobenzene
0
6
12
24
48
72
96
c.
t
(ug/g)
Tank 4 f (x)
0.06529
0.10365
0.14200
0.21871
0.3721
0.5255
0.6790
Tank 5 f(x)
0.1266
0.1521
0.1775
0.2284
0.3302
0.4319
0.5337
C
(ug/ml)
= 0.00639x + 0.0653
0.00446
0.00448
0.00449
0.00450
0.00450
0.00453
0.00453
- 0.00424x + 0.1266
0.00394
0.00395
0.00395
0.00396
0.00396
o!fl0398
0.00399
k,
1
(ml/g/hour)
1.8772
2.1288
2.3833
2.8955
3.9304
4.9325
5.9612
2.1294
2.3357
2.5464
2.9614
3.8040
4.6225
5.4473
-------
TABLE A-7 Analysis of Variance at Steady State
1,2,4-Trichlorobenzene
Groups S, S+l, S+2, S+7
Source
Degrees of
Freedom •
Sum of Mean Square F-value
Squares
TANK 4
Total
Groups
Error
15
3
12
2.4599
0.7591
1.7008
0.2530
0.1417
Bartlett's Test - 0.1255 n.s.
1.7852 n.s.
TANK 5
Total
Groups
Error
15
3
12
1.7508
0.1541
1.5968
0.0514
0.1331
Bartlett's Test » 1.9144 n.s.
0.3859 n.s.
n.s. indicates not significant at 0.05 alpha level.
190
-------
TABLE A-8
Bioconcentration Results Oyster - Pentachlorophenol
Tank # 4
Code
S/16
(1 hour)
S/8
(2 hours)
(2 hours)
S/4
(4 hours)
S/2
(8 hours)
S
Sample
Size (g)
1
4.42
4.43
3.28
5.63
3.31
4.00
5.31
4.89
3.50
3.51
6.27
1.65
3.81
5.63
2.05
4.57
4.61
4.45
5.92
(16 hours) 3.49
S+4 hr
2.89
3.95
3.86
(20 hours) 3.82
S+8 hr
2.92
3.23
3.92
(24 hours) 5.70
UgPCP/
sample
0.44
1.0
0.70
2.9
0.58
0.30
0.28
0.31
6.3
3.5
0.65
0.69
7.1
0.65
0.73
0.69
5.5
0.90
0.35
0.54
0.27
5.6
0.74
0.22
0.90
7.4
5.7
1.2
ugPCP/g
0.10
0.24
0.21
0.51
0.18
0.08
0.05
0.06
1.8
1.0
0.10
0.42
1.8
0.12
0.36
0.15
1.2
0.20
0.06
0.15
0.09
1.4
0.19
0.06
0.31
2.3
1.5
0.21
Tank « 5
Code
S/16
(1 hour)
S/8
(2 hours)
S/4
(4 hours)
S/2
(8 hours)
S
Sample
Size (g)
2.00
5.10
3.89
3.05
3.75
3.89
4.80
3.89
7.29
4.06
5.13
3.13
4.63
4.95
7.12
5.60
4.85
5.84
3.98
(16 hours) 4.05
S+4 hr
•
6.44
3.00
4.47
(20 hours) 6.43
S+8 hr
5.40
4.33
4.05
(24 hours) 5.46
ugPCP/
Sample
0.21
4.0
0.32
5.1
0.95
0.45
0.36
0.27
0.40
0.81
0.36
1.1
0.40
7.6
9.6 ..
1.4
0.28
0.36
4.4
6.5
0.16
2.7
0.36
0.32
21
22
12
20
ugPCP/g
0.10
0,78
0.08
1.7
0.25
0.12
0.08
0.07
0.06
0.20
0.07
0,36
0.09
1.5
1.3
0.25
0.06
0.06
1.1
1.6
0.03
0.91
0.06
0.05
3.9
5.1
3.0
3.7
-------
TABLE A-8
Bioconcentration Restuls Oyster - Pentachlorophenol
ro
Tank ft 4
Code
S+2 days
Sample
Size (g)
7.06
2.68
3.52
(72 hours) 6.68
S+4 days
4.01
6.23
5.51
(120 hours) 4.44
D/4
3.62
6.91
2.69
(124 hours) 3.37
D/2
2.78
2.88
2.89
(128 hours) 2.73
3D/4
2.26
3.64
2.82
(132 hours) 3.93
D
3.82
5.42
3.21
(136 hours) 4.78
D+4 hours
2.79
4.34
2.06
(140 hours) 2.75
ugPCB'
sample
21
16
21
2.1
28
17
8.2
18
4.4
4.8
2.7
2.1
0.65
0.88
0.83
9.6
5.3
12
3.7
2.5
1.6
5.5
3.5
4.8
3.1
1.1
2.4
0.38
ug PCP/g
3.0
6.0
6.1
0.31
6.9
2.7
1.5
4.0
1.2
0.69
1.00
0.62
0.23
0.31
0.29
3.5
2.3
3.2
1.3
0.63
0.42
1.0
1.1
1.0
1.1
0.26
1.2
0.14
Tank ft 5
Code
S+2 days
Sample
Size (g)
4.28
3.53
5.94
(72 hours) 5.64
S+4 days
1.23
6.93
4.77
(120 hours) 7.09
D/4
3.71
5.37
2.71
(124 hours) 5.74
D/2
4.17
3.29
5.56
(128 hours) 3.79
3D/4
4.81
5.62
3.27
(132 hours) 4.87
D
2.96
5.45
3.03
(136 hours) 3.29
P+4 hours
2.97
2.22
6.33
(140 hours) 3.62
ugPCP/
Sample
21
21
17
14
9.9
16
21
17
2.8
5.6
4.3
18
11
14
30
32
13
12
2.4
0.62
0.40
19
3.2
19
5.0
2.7
0.93
3.0
-
ug PCP/g
4.9
6.0
2.9
2.5
8.1
2.2
4.5
2.4
0.75
1.0
1.6
3.2
2.6
4.2
5.4
8.4
2.8
2.1
0.74
0.13
0.13
3.4
1.0
5.7
1.7
1.2
0.15
0.82
-------
TABLE A-9
Water Concentrations
Pentachlorophenol
Code
Elapsed Time
(hours)
Concentration Tank 4 Concentration Tank 5
(tag/ml)• (ug/ml) .
S/16
S/8
S/4
S/2
S
S+4 hrs
S+8 hrs
S+2 days
S+4 days
D/4
D/2
3D/4
D
D4-4 hr
1
2
4
8
16
20
24
72
120
124
128
132
136
140
0.00073
0.00042
0.00152
0.00125
0.00392
0.00157
0.00120
0.00090
0.00181
0.00073
0.00151
0.00152
0.00112
0.00161
0.00083
0.00083
0.00133
0.00175
0.00105
0.00064
0.00246
0.00038
0.00055
0.00033
0.00168
0.00008
0.00043
0.00027
0.0226
0.00087
0.00099
0.00136
0.00090
0.00077
0.00107
o.oo in
0.00074
0.00093
0.00247
0.00886
0.00063
0.00120
0.00433
0.00090
0.00181
0.00162
0.00031
0.00047
0.00107
0.00022
0.00050
0.00067
0.00042
0.00028
0.00034
0.00041
193
-------
TABLE A-10 Summary of Interpolated Values from Regression Analyses
Sampling Point
T
(hours)
Pentachlorophenol
1
2
4
8
16
Pentachlorophenol
1
2
4
a
16
Tissue Concentration
C.
t
(ug/g)
Uptake Tank 4 f(x) =
0.2808
0.3013
0.3424
0.4245
0.5888
Uptake Tank 5 f(x) -
0.26047
0.29627
0.36789
0.51112
0.79759
Hater Concentration
C
w
(ug/ml)
0.02054x + 0.26022
0.00058
0.00138
0.00276
0.00161
0.00137
0.03581x + 0.22466
0.00157
0.00196
0.00080
0.00094
0.00079
Rate Constants
It
(ml/g/hour)
44.138
18.818
9.678
17.509
22.737
33.943
28.415
75.624
74.586
113.084
-------
TABLE A-11 Analysis of Variance at Steady State
Pentachlorophenol'
Groups S, S+4hr, S+8hr, S+2day, S+4day
Source
TANK 4
Total
Groups
Error
Bartletts
TANK 5
Total
Groups
Error
Bartlett's
Degrees of
Freedom
19
4
15
Test = 2.568*
19
4
15
Test m 2.515*
Sum of Mean Square
Squares
93.76
49.55 12.39
44.21 2.95
98.83
63.45 15.86
35.38 2.36
F-value
4.203*
•
6.724*
* indicates significant result at alpha level 0.05
195
-------
GLOSSARY
asymptotic bioconcentration factor: The BCF calculated for infinite
from uptake and depuration rate constants derived, for an appropriate
compartmental model.
bioconcentration: The net accumulation of a material directly from water
into and onto aquatic organisms resulting from uptake and depuration.
bioconcentration factor (BCF): The quotient of the concentration of a
material in one or more tissues of aquatic organisms at a particular
tiTne during exposure divided by the concentration at the same time
of that same material in the solution which contains the organisms.
depuration: The elimination of a material from organisms.
depuration carver A line obtained by plotting the measured concentration
of test material in aquatic organisms vs time during the depuration
phase of a bioconcentration test.
depuration phase: The portion of a bioconcentration test after the uptake
phase and in which the organisms are in water to which no test material
has been added.
depuration rate constant(s): The mathematically derived value(s) which
express how fast a test material is eliminated from previously exposed
test organisms when placed in water to which no test material has
been added; usually reported in units of hour"1.
steady-state bioconcentration factor: A BCF that.is unchanged for at least
> four days at a constant concentration of the material in the solution
containing the organisms, i.e., the BCF that exists when uptake and
depuration are equal for at least several days.
uptake: The sorption of a material into and- onto aquatic organisms.
uptake curve: A line obtained by plotting the measured concentration of
test material in aquatic organisms vs time during the uptake phase
of a bioconcentration test.
uptake phase: The portion of a bioconcentration test during which the
organisms are exposed to a solution of the test material.
uptake rate constant(s): The mathematically derived value(s) which express
how fast a test material is accumulated by an aquatic species during
a bioconcentration test; usually reported in units of litres/grain/hour.
196
-------
APPENDIX E
Method for Sampling Oysters
The oyster should be opened with an oyster knife by separating the
valves at the hinge. Once separated, the knife should then sever the
adductor muscle at the point of attachment to the left (upper) valve.
Care should be taken not to penetrate the mantle. Discard the
upper valve and shake the right valve (with attached living tissue)
three times to remove excess water. Sever the adductor muscle at the
attachment to the right valve. The living oyster tissue should then be
intact, free of the valves, and ready for analysis.
197
-------
APPENDIX F
Methods for Lipid Analyses
All tissues were lyophilized to dryness prior to extraction.
Neutral Lipids - similar to the tissue method of Schimmel et al. (1979)
for the analysis of EPN and leptophos, except that the combined hexane
extract was not cleaned by florisil, but was dried to constant weight
as percentage lipid by dry weight. Marine Sci. 22 193-203 (1979).
Polar lipids. Total lipids - homogenized by the method of Schimmel et
al. (1979) with a mixture of chloroform/methanol/water, as suggested by
Bligh, E.G. and W.J. Dyer (1959) Can. J. Biochem. Physiol. 37, 911.
For polar lipid determination, the tissue was first extracted for
neutral lipids, then for polar lipids.
A general reference:
Laboratory techniques in biochemistry and molecular biology
General editors: T.S. Work and E. Work. Techniques of
Lipidology: Isolation, analysis, and identification of lipids
Morris Kates, North-Holland/America, Elsevier, 1972
198
-------
ERL,GB POR 133
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
5. REPORT DATE
Results: Interlaboratory Comparison-Bioconcentration
Tests Using The Eastern Oyster
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Steven C. Schimmel and Richard L. Garnas
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
10. PROGRAM ELEMENT NO.
A87E1A
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency
Office of Research and Development
Environmental Research Laboratory
Gulf Breeze. Florida 32561
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA/600/04
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Four laboratories (one EPA and three contract labs) participated in a
bioconcentration interlaboratory comparison study using the eastern oyster
(Crassostrea virginica). Three chemicals were selected for study, including
p,p'-DDE, pentachlorophenol (PCP), and 1,2,4-trichlorobenzene (KB). The test
method used was that of the American Society for Testing and Materials (ASTM)
"Standard Practice for Conducting Bioconcentration tests with Fishes and Saltwater
Bivalve Molluscs" (Draft 9). Calculated steady-state bioconcentration factors
(BCFs) for p,p'-DDE ranged from 27,000 to 92,000; that for PCP was 34 to 82;
and that for TCB was 115 to 264. The .high-to-low ratio (an estimated of the
"worse case" variability, derived by dividing the highest BCF by the lowest
BCF for each chemical) was 3.4 for p,p'-DDE, 2.4 for PCP and 2.3 for TCB.
These results indicate that the ASTM Method can generate reproducible results
even though the participating laboratories varied in their experience and in
their water quality characteristics and were widely separated geographically.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Pollution
Mollusca
Bioconcentration tests
ASTM Method
Crassostrea virginica
06/F
18. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (This Report)
unclassified
21. NO. OF PAGES
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
------- |