The Precision of the ASTM Bioconcentration Test
(U.S.)  Environmental Research  Lab.
Duluth,  MN
Oct 80
                   U.S. DEPARTMENT OF COMMERCE
                 National Technical Information Service
                                KTIS

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                                             EPA-600/3-81-022
                United States                       FEBRUARY 1981
                Environmental Protection
                Agency                 .            D09l-!6!'«
&EPA        Research  and
                Development
                THE PRECISION OF THE ASTM
                BIOCONCENTRATION TEST
                Prepared for

                OFFICE OF TOXIC SUBSTANCES
                Prepared  by


                Environmental Research
                Laboratory
                Duluth MN 55804

                AND

                Center for Lake Superior
                Environmental Studies
                Superior, WI  54880

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                                    TECHNICAL REPORT DATA
                             (Please read Instructions on the reverse before completing)
  DEPORT NO.
 'PA-600/3-81-022-
                                                            3. RECIPIENT'S ACCESSION NO.
 -AUTHORS Patricia Kosian, Armond Lemke, Karen  Studders,
id Gilman Veith
 fTTITLE AND SUBTITLE

 The Precision  of the ASTM  Bioconcentration Test
             5. REPORT DATE
                February 1981 Issuing  Date,
                                                            8. PERFORMING ORGANIZATION CODE
                                                            8. PERFORMING ORGANIZATION REPORT NO.
S. PERFORMING ORGANIZATION NAME ANO ADDRESS
                                                             10. PROGRAM ELEMENT NO.
 Environmental  Research Laboratory-Duluth, U.S.  EPA
 620] Congdon Boulevard, Duluth,  Minnesota  55804
             11. CONTRACT/GRANT NO.
•«. SPONSORING AGENCY NAME ANO ADDRESS
                                                             13. TYPE OF REPORT ANO PERIOD COVERED
 Environmental  Research Laboratory-Duluth, U.S. EPA,
 6201  Congdon Boulevard, Duluth,  Minnesota  55804
              f. SPONSORING AGENCY CODE
IS. SUPPLEMENTARY NOTES
JATA8STRACT

      The ASTM method  for  measuring  the  bioconcentration  factor  (BCF)  of chemicals
 was evaluated using 1,2,4-trichlorobenzene (TCB),  hexachlorobenzene (HCB), and
  ,p'-DDE (DDE).  Four  replicate, 28-day exposures  of the chemicals  to fathead
  'innows were used to determine the  precision of the test method.  Using the 28-day
 values, the  mean ( + S.D.)  BCF for TCB, HCB,  and DDE were  1,700 (_+70) ,  35,000
 (_*3,300),  and 50,000  (^4,800), respectively.   The  results showed  that steady-state,
 residues are not attained for highly  bioaccumulative chemicals  in the 28-day
 exposure,  and the calculation of the  BCF by dividing the 28 day residues by the mean
 water concentration is  inadequate.  Two alternate  methods of calculating the BCF are
 d iscussed.
17.
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EPA Form 2220-1 ("•»• 4-77)   pnevious eoi TPON is OBSOLETE

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                                                              EPA 600/3-81-022
                                                              February 1981
              The Precision of  Che ASTM  Bioconcencracion  Tesc
                                    by

Patricia Kosian^, Arroond Lerake  , Karen Scudders^, and Oilman Veith*
               -Environmental Research Laboracory-Duluch

                   U.S. Environmental Protection Agency

                          6201 Congdon  Boulevard

                         Duluth, Minnesota   55804  '  '  . '
                                    and
           ^Center for Lake Superior Environmental Studies

                     University of Wisconsin-Superior

                        Superior, Wisconsin   54880
                               October,  1980

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                                 INTRODUCTION.

     The  purpose of  chis  study was  to evaluate the bioconcentrat ion factor

(BCF) X test method  suggested  by ASTM (1)  through the participation in an

interlaboratory round-robin  testing program.   Although an evaluation of the

round-robin  tests  will  be  published elsewhere, this laboratory examined the

precision of the method  in  four replicate  tests  using three chemicals  as well

as several different methods of estimating the bioconcentration factor from

the exposure data.   Hexachlorobenzene CHCB),  p,p'-DDE (DDE) and

1,2,4-trichlorobenzene  (TCB) were  selected for the round-robin evaluation

because they were  anticipated  to exhibit varying depuration rate constants

and bioconcentrat ion factors while  minimizing complications of metabolism and

of the need  for using dissimilar analytical methods.

     Discussions of  the  bioconcent rat ion process hav-? been published (2, 3,

A, 5).  Bioconcentrat ion  is defined as  the direct uptake of a chemical into

aquatic organisms  through  the  gill  or other membranes.  The bioconcentrat ion

factor is the rat-io of  the chemical residue  in the fish  tissue and the

concentration of the chemical  in the water after a steady-state is observed.

  Branson  et al. C2)  proposed that  the uptake  process  can be modeled by the

first order  relationship:

                      d£E. s KICW -  K2cF
                       dt

where Cw  and Cp are  the  chemical concentrations  in the water and fish,

respectively, and  Kj and  K2 are che uptake and depuration rate constants,

respectively.  Since steady-state  is defined 'as  the point where dC/dt  = 0,   it

is clear  that chemicals  which  have  small depuration rate constants, i.e.

K« -> 0, will require  long  exposure  t irnes in order co observe steady-scac-e .

                                       1

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Consequently, one  method  to estimate the BCF using the ratio of CF/CW ac




the end of an arbitrary exposure  period may only be an accurate measure of




the bioconcentration  factor for chemicals with appreciable depuration rates




where steady-state  is  reached quickly.   For chemicals which are not  depurated




rapidly the  ratio  may  underestimate the steady-state bioconcentration factor




because the  residues continue to  increase throughout the  exposure.




     Branson et  al. (2) proposed  that  this problem can be alleviated by




defining the bioconcentration factor as the ratio of the  rate  constants,




Kj/K2«  This eliminates the need  to expose fish until steady-state  is




achieved, but it  introduces the uncertainity of extrapolating  beyond the




exposure data and  the  tendency to amplify variability in  the analytical




measurements by  dividing  by a small number.  The computer program for this




model provided by  Dow  Chemical called  BIOFAC was used as  a second method to




estimate the bioconcentration factor in this study.




     A third method used  to estimate the bioconcentracion factor was similar




to the BIOFAC in  that  it  assumed  the uptake was a first  order  rate  process.




Integrating  the  uptake equation gives  CF = (K^/K2)CWCl-e~k2c).




If BCF 2. Ki/K2,  then CF/CW = BCF(l-e~k2c).  This is similar to




the equation proposed  by  Ernst (3).  Therefore, if the values  of Cp/Cw




are measured for varying  time periods,  t, a non-linear least squares analysis




can be used  to fit  the data and estimate the steady-state BCF.   This least




squares analysis of the data was  compared to the other methods  of estimating




the bioconcentration  factor.

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                            EXPERIMENTAL PROCEDURES




Exposure System




     The case  syscem  consisted of a syringe injeccor exposure system




described by DeFoe  (6).   The  three  chemicals were tested simultaneously in




quadruplicate  by  preparing acetone  stock solutions containing appropriate




amounts of HCB, DDE,  and  TCB  to produce exposure concentrations  of approxi-




mately 0.2, 0.15,  and 20  ug/1, respectively, when 8 ul of acetone was




injected into  each  liter  of dilution  water*  The control tanks received an




equal quantity of  acetone.




          The  test  water  was  unfiltered Lake Superior water heated to 21°C_+




0.5°C and contained greater than 90%  DO saturation.  Hardness and pH measured




at the initiation  of  the  test  were  45-47 mg/1  (as CaCOj) and 7.8, respec-




tively.  Other chemical characteristics of Lake Superior water may be found




in Biesinger and  Christensen  (7).




     The test  organism used  in the  bioconcentration cest was the fathead




minnow (Pimephales  promelas).obtained from the Environmental Research




Laboratory-Duluth  culture.  Juvenile  fish weighing 0.1-0.15 g were fed a




daily diet of  frozen  brine  shrimp CSan Francisco Bay Brand) supplimented- wic.h




dry trout chow pellets (Glenoce Mills).  The trout chow was purchased for




experimental work  and was  previously  shown to  be free of. significant




quantities of  pesticides.   Handling,  holding and acclimation procedures for




the fish were  followed according to the ASTM guidelines for conducting




bioconcentration  tests (1).




     After chemical analyses  verified stable exposure concentrations in the




test chambers, 40  fish were transferred to each tank containing 27 liters of




water.  The flow  rate of  incoming water was 250 ml per minute.  Samples of 4




fish from each tank were  randomly removed on day 0, 2, 4. 8. 16, and 28 of

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che uptake phase  and  on  day  35, 49,  and  56  of the depuration phase.  The fish




were removed from che  canks  and placed  in a beaker of ice wacer.  Afcer all




movement ceased,  chey  were blotted  dry,  weighed,  and frozen in solvenc-rinsed




glass vials.




     The water  samples were  siphoned  direccly from che  tank into a 500 ml




volumetric flask  (250  ml  for  control  canks)  co  which 25  ml  of  hexane had




previously been added.   A 1.5  inch  teflon coated  stirring bar  was placed in




che flask and the sample  was  vorcex mixed for 1 hr.   The cwo phases were




allowed to separate  for  0.5  hr, and  a 1.5 ml  aliquot of  hexane was




transferred Co a gas  chromotography  injection vial for  analysis.




     The accuracy of  che  analytical mechods was checked  by determining che




percent recovery of known amounts of  I,2,4-crichlorobenzene,




hexachlorobenzene, and p,p'-DDE  in  che  wacer.  The percent recovery for che




water analysis was 95.4%, 98.6%,  and  102.8%,  for  TCB, .HCB,  and DDE,




respectively (N=6).   The. water concentrations were corrected for percent




recovery.




Analytical Methods




     Composite whole  fish  samples were  homogenized with  40 gm  of granular




anhydrous Na2SO^  (Mailinckrodt Inc.).   The  powdered  homogenate was




transferred co a  300' ml  chromacographic  column  and eluted with 250 ml  of




redistilled hexane into  a 250  ml  volumetric flask.  Because of the high




concentration, no cleanup procedure was  performed.  After the  necessary




dilutions were made,  quantitation of  TCB, HCB,  and DDE  was performed by gas




chromatography.




     The lipid content of each tissue sample  was  determined gravimetrically




using a drying period  of 15  minutes  ac  110°C.  The lipid content ranged from

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6.1% co 9.8% with  a mean  value  of  8.44  +_ 0.8 with N=23.


     The gas chromacographic  analyses were  performed on  a 5730A


Hewlett-Packard gas chromatograph.  with  an auto sampler and a Hewlett-Packard
                e
3354B lab automation data  system.   The  gas  chromatograph was equipped with a


^Ni electron capture detector  held at  300°C.   The injection port


temperature was 2500C.  The 6 ft 2 mr x  3 mm (OD)  glass column was packed wich

1.5% SP-2250/1.95% SP-2401 on 100/120 mesh  supelcoport (Supelco Inc.).  The


carrier gas was 5% methane  in argon wich a  flow rate of  40 ml per minute.


     The water samples  were analyzed at a programmed column temperature of

140°C co 190°C ac 4"C per  rainuce.   The  cissue  samples were, anlayzed at a

programmed column  temperature of 100° to 220°C at 8°C per minute for TCB and


HCB and at 200°C isothermal for DDE.  The percenc recovery for spiked tissue


samples were 102% wich  N=3 for  TCB 99%  with N=3 for HCB  and 111% with N=3 for


DDE.  The tissue concencracions were correcced for recovery.

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                                     RESULTS




      The  results  of Che biocoricentration tests using TCB are presented in




 Table  I.   The  data show that the water concentrations decreased) approximately




 20%  from  the  initial  concentrations during the 28 day exposures.   TCB was not




 detectable in  the initial test fish but had accumulated to 15.8-18.4 ug/g




 after  two  days  of exposure.  After the initial rapid uptake, the




 concentrations  increased slowly to maximum concentrations of 1-9.0-26.3 ug/g




 in  the  four  exposures.  The ratio of .Cp/Cw for TCB  in Exposure 1  ranged




 from 1,080 after  two  days to 1,640 after 28 days.  The Cp/Cw in Exposure




 2 ranged  from  1,170 after two days to 1,770 after 28 days.  The Cp/Cw in




 Exposure  3 ranged from 1,070 after two days to 1,800 after 28 days.  The  •




 Cp/Cw  in  Exposure 4 ranged  from 1,070 after 2 days  to 1,730 after 28




 days.   TCB was  eliminated rapidly from fish during  the depuration phase of




 the  study.   All fish  contained 0.60 ug/g .or less seven days after the




 chemical  ceased to be added.  After 21 days in clean water the fish contained




 approximately  0.1 ug/g.




      The  results  of the bioconcentration tests using HCB are presented in




 Table  2.   The  water concentration decreased approximatley 15% during the




 exposure  and  the  mean'concentrations were all approximately 0.15  ug/1.  HCB




 concentrations  in the test  fish were initially about 0.09 ug/g.  After two




 days of exposure, the HCB concentration increased ten-fold to approximately  1




 Mg/g.   .In  contrast to the TCB exposures, HCB residues continually increased




. during  the 28-day tests to  maximum concentrations ranging from 4.55 to 6.22




 ug/g in the  four  tests.  The Cp/Cw in Exposure 1 ranged from 5,700 after




 cwo  days  to  33,000 after 28 days.  The Cp/Cw in Exposure 2 ranged from




 7,400  after  two days  to 32,000 after 28 days.  The  ranges of two  day and  28




 day  CF/CW in Exposures 3 and 4 were 5,700 to 39,000 and 5,400 to 37.000.

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                      TABLE  I.   Summary  of  Analytical  Results (ran 1,2,4-Tr ichlorobeniene Bloconcentratlon Tests
               Exposure  1
Exposure 2
Exposure 3
Exposure 4
Test
Day
0
2
4
8
16
28
35
49
56
Water
14. 6a
14.6
13.9
13.3
12.3
11.3
0.1
<0.l
<0.1
Fish

<0.0lb
I5.ed
15.4
16.1
19.0
18.5
0.25
MA
0.03
Llpld Cr
<*) cw
6.06C
7.99 .1,080
7.55 1,100
MA 1,200
9.28 1.540
9.22 1.640
8.77
6.63
5.82
Water
13.8
13.8
13.7
13.5
12.9
12.4
O.I
<0.01

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                          TABLE  2.   Summary of Analytical Results  trom Hexachlorobenzene Bio'concentratIon Tests
Exposure 1
Test
Day
0
2
4
8
16
28
35
49
56
Water

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respectively.   HCB concentrations decreased from approximately 5-6 ug/g co




approximately  1-2  ug/g  during the 28 days depuration study.




     The  results  of the bioconcentration tests using DDE are presented in




Table 3.  The  water concentrations in the DDE exposure also decreased about




15% during  the exposures and the mean concentrations were all approximately




0.13 ug/1.   DDE concentrations  in the test  fish were initially 0.10 ug/g  and




these residues increased about  9-fold to approximately 0.9 ug/g after two




days of exposure.   Like HCB  exposures,  the  fish in the DDE tests continually




accumulated  DDE throughout  the  test  and a steady-state condition was not




observed.  The Cp/Cw in Exposure 1 ranged from 6,200 after two days to




48,00 after  28 days.   In Exposure -2,  the CF/CW ranged from 6,600 after




two days  to  46,000 after .28  days.  The  range of two day and 28 day Cp/Cw




in Exposures 3 and 4 were 7,700 to 57,000 and 5,500 to 50,000,  respectively.




DDE concentrations declined  approximately 2 ug/g in che four tests during the




depuration study.




     The  results  of .these tests are  presented graphically in Figure 1.




Figure l(a)  illustrates that  the TCB  exposure produced the only resemblence




of steady-state for the three chemicals.  Figures l(b) and l(c) clearly show




that the  28  day bioconcentration factor underestimates the BCF for the




bioaccumulative chemicals.   In  comparing the uptake curves in Figure 1, it




must be recognized  that the  mean water  concentrations in the TCB exposures




were approximately 100  times  those in the HCB and DDE exposures and chat  che




absolute  residue concentrations are  not a measure of the bioaccumulation




potential of the  chemicals.   Moreover,, the  apparent variability in




concentrations  in  Figure 1  refleccs  variations in che water concentrations in




addition  to  the analytical  and  biological variations.

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                             TABLE 3.  Summary of Analytical Results from p.p'-DDE Bioconcentration Tests
               Exposure I
Exposure 2
Exposure 3
Exposure 4
Test
Gay
0
2
4
8
16
28
,35
49
56
Water
(ug/l)
0.15
0.15
0.14
0.13
0.11
0. 12
0.02
<0.01
<0.01
Fish
(ug/q)
0.10
0.94
1.19
2.39
3.81
5.74
5.25
NA
3.48
Llpld Cc
(?) *£
6.06
7.99 6,200
7.55 8,500
NA 18.000
9.28 35,000
9.22 48,000
8.77
6.63
5.82
Water
(ug/i)
0.13
0. 13
0.18
0.18
0.16
0. 14
<0.01

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     The data are  presented  on  a  more  comparative basis in Figure 2 in which




Che ratio Cp/Cw  is  plotted versus time of exposure.   This plot corrects




for the different  exposure concentrations within the four tests with each




chemical.  Moreover,  the  relative accumulation between chemicals is




apparent.  This  figure gives a  visual  summary  of the precision of the




exposures on a scale  which should encompass  most chemicals tested by this




method.




     Figure 3 presents the data from  the  depuration  phase of- this study.   The




data show that the  DDE concentration  in  fish remained'essentially constant




during the 54 days  in clean  water.  HCB  was  eliminated more rapidly than. DDE




while the residues  of TCB declined  rapidly to  near the detection limit within




one week.  These data on  elimination  rates are inversely related to the BCF




which is expected  from the bioconcentration  kinetics.   If BCF at steady-state




is the uptake rate  divided by the depuration rate, chemicals with very small




depuration rates should have large  BCF if the  uptake rate is comparable to




other lipophilic chemicals.

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                                  DISCUSSION




     This scudy demonstrates  thac  che proposed method provides a reproducible




test for measuring  the  bioconcentrat ion factor.  Using the 28 day BCF values




for the four tests,  Che  mean  (+_ S.D.) BCF for TCB was 1,700 (_+70) and the




range was 1,600 to  1,800 in  the four tests.  The mean (^ S.D.) BCF for HCB




was 35,000 (+3,300)  and  the  range  was 32,000 to 39,000.   The mean (+_ S.D.)




BCF for DDE was 50,000  (+4,800) and che range was 46,000 to 56,000.




     The greatest concern  in  estimating the BCF in the bioconcentration rest




is not the method of testing,  but  rather the method of calculating the BCF.




As stated previously, the  use  of the 28 days BCF can only be an adequate




measure of the bioaccumulation potential when the 28 day BCF is




representative of steady-state residues.  The 28-day BCF for HCB and DDE were




clearly not at steady-state.




     To .compare different  methods  of estimating BCF from a given set of




uptake and depuration data,  the data were also analyzed  using a modified




BIOFAC computer program  and  a non-linear cjirve-f itt ing program, CANDLES,




developed at ERL-D.   Table 4 presents t'fie results of the analyses.  These




results demonstrate  that all  three methods of estimating the BCF give




essentially the same  values  for TCB, which can be expected since this




chemical was tested  to  near  steady-state.  However, both che BIOFAC and




CANDLES program estimated  that che sceady-scace BCF for HCB and DDE are




substantially higher than  is  estimated  from the 28 day value.  In the case of




HCB, the BCF estimated  from BIOFAC was 52,000 and that from CANDLES was




48,000.  Compared co che 35,000 escimated  from the 28 day ratio of Cp/Cw,




che latter method is  clearly  inappropriate.  For DDE, che BIOFAC method




established a steady-state BCF of  180,000 while CANDLES estimated 110,00




compared co che 28 day  value  of 50,000.

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   TABLE 4.  Comparison of  BCF  Values  Computed  by These  Mechods
   Method




ASTM, CF/CW at 28 days




BIOFAC




CANDLES
                                Estimated  Bioconcentracion  Factor
TCB




1,700




1,600




1,500
 HCB




35,000




52,000




48,000
  DDE




 50,000




180,000




110,000
                                      13

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                             REFERENCES




American  Society for Testing and Materials.  1979.  Proposed standard




      practice for conducting bioconcentration tests with fishes and




     •saltwater  bivalve molluscs.  Draft No. 10.  Philadelphia, PA.




Branson,  D.  R.,  G. E. Blau, H.  C.  Alexander, and W. B. Neely.  1975.




      Bioconcentration of 2,2',4,4'-tetrachiorobiphenyl in rainbow trout




      as  measured by an accelerated test.  Trans. Am. Fish.  Soc.  4:




      785-792.




Ernst, W.   1977.  Determination of the bioconcentration potential of




      marine  organisms - A steady state approach.  Chemosphere 11:




      731-740.




Hamelink,  J.  L., R. C. Waybrant, and R. C. Ball.  1971.  A proposal:




      Exchange equilibria control the- degree chlorinated hydrocarbons are




      biologically magnified in  lentic environments.  Trans. Am. Fish.




      Soc.  -100(2): 207-214.




Veith, G.  D., D. L. DeFoe, and B.  V. Bergstedt.   1979.  Measuring and




      estimating  the bioconcentration factor of chemicals in fish.  J.




     'Fish.  Res.  Board Can. 36:   1040-1048.




.DeFoe, D.  L.   1975.  Multichannel  toxicant injection system for




      flow-through bioassays.  J. Fish. Res. Board Can. 32(4): 544-546.




Biesinger,  K. E., and G. M. Christensen.  1972.   Effects of various




      metals  on survival, growth, reproduction and metabolism of Daphnia




      magna.   J.  Fish. Res. Board Can. 29(12): 1691-1700.

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                                LIST OF FIGURES




Figure l(a)   .  Uptake  and  depuration  of 1,2,4-trichlorobenzene in fathead




              .  minnows  (Pimephales promelas).




Figure Kb)     Uptake  and  depuration  of hexachlorobenzene  in fathead minnows




                (Pimephales promelas).




Figure l(c)     Uptake  and  depuration  of p.p'DDE  in  fathead  minnows




                (Pimephales promelas).




Figure 2        Uptake  of TCB,  HCB,  and DDE  in fathead  minnows  (Pimephales




                promelas).




Figure 3        Depuration  of  TCB,  HCB, and  DDE in fathead  minnows




                (Pimephales promelas).
                                      15

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                                          Depuration
26
                                                  Exposure I
                                          •———- Exposure 2
                                          •-	• Exposures
                                                  Exposure 4
                        24      32      4O
                       TIME (.days)
48

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   IO
    8
o
o:
O

o
O
LL
    O
      0
                  Uptake
      j	1
                             Depuration
                                     • Exposure I
                                    ^ Exposure 2
                                     • Exposures
                                     x Exposure 4
8
16      24      32
     TIME (days)
40
48
56

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   10
8
O
or
O

O
O 2
   0
      o
                 Uptake
                                       Depuration
                                 .X.
                                                   o Exposure I
                                                  _ Exposure 2
                                                   • Exposures
                                                  .* Exposure4
      I	I
                                     I    I
          8
16     24      32     4O
     TIME (days)
48
56

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CD
Q
                                                  *. TC8
     0
68      10     12

    TIME (days)
14
                                                  16

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   5r
   4-
                                                    HC8
'
o
9
                                                  -. TCS
   0-
        3O     34     38     42     46

                       TIME (days)
50
54

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