- Email: [email protected]
Depuration kinetics of hexachlorobenzene in the clam, Macoma nasuta
0306492/90
Camp. Biochem. Physiol.Vol. 96C, No. 2, pp. 327-331, 1990
$3.00 + 0.00
PergamonPressplc
Printedin Great Britain
DEPURATION KINETICS OF HEXACHLOROBENZENE IN THE CLAM, MACOMA NASUTA B. L.
BOESE, M. WINSOR,*
H.
LEE
II, D. T. SPECHT and K. C. RUKAVINA
Pacific Ecosystems Branch, Environmental Research Laboratory-Narragansett, U.S. Environmental Protection Agency, and lAScI Inc., Hatfield Marine Science Center, Newport, Oregon 97365, U.S.A. Telephone (503) 867-4037 (Received 22 February 1990)
(HCB) in the clam, Mucoma seawater. 2. Depuration was not correlated with ventilation volume, nor did the amount of sediment ingested during depuration have a significant effect. 3. The half-life for HCB in M. nusufa was estimated to he 16 days with a bioconcentration factor of 3490 (wet weight basis).
Abstract-l.
The depuration
rate constant for [‘4C]hexachlorohenzene
nusuta, was determined following a short-term exposure to HCB contaminated
CPW = Concentration of pollutant is assumed to be constant. KU= Uptake rate constant. Kd = Depuration rate constant. r = Time.
INTRODUCTION Hexachlorobenzene (HCB) is a persistent environmental pollutant that bioaccumulates in tissues of marine and fresh water fish (Young and Heesen, 1978; Niimi, 1979; Tsui and McCart, 1981). Once taken up by these tissues, biodegradation is very slow with pentachlorophenol (Sanborn et al., 1977) and polar conjugates (Metcalf et al., 1973) as possible metabolites in aquatic organisms. Numerous studies have been done on HCB uptake and depuration in
fish (Sanborn et al., 1977; Veith et al., 1979; Kosian et al., 1981; Niimi and Cho, 1981; Oliver and Niimi, 1983). In these studies the HCB bioconcentration factors (BCFs) varied from 5000 to 40,000 (wet weight basis) with half-lives (t,,,) from a few days to several years. Although several studies have examined HCB uptake in invertebrates (Boese et al., 1988; Ekelund et al., 1987; Oliver, 1987; Knezovich and Harrison, 1988), only Oliver’s (1987) study estimate a BCF. Oliver (1987) found that the BCF of HCB in oligochaete worms was 3120 when calculated on a wet weight basis, with a t,,Z of 27 days. However, in Oliver’s study the worms were buried in the contaminated sediment and the BCF calculated using the interstitial water concentration. As a portion of the HCB body burden was likely due to the ingestion of particulates, the reported BCF value may be in error. Bioconcentration factors are commonly estimated from non-equilibrium exposures using a kinetic uptake model (Branson et al., 1975; Davies and Dobbs, 1984), where the change in tissue concentration with time is calculated by: dTR/dt = K,CPW
- K,,TR
TR = Pollutant tissue residue concentration organism.
in
Mention of trade names or commercial products does not constitute endorsement or recommendation of use by the Environmental Protection Agency. CSPC %,2--H
When steady-state pollutant tissue concentrations are attained (i.e. dTR/dt = 0), the BCF is estimated by: BCF = KU/K,.
(2)
In previous work with HCB in the deposit-feeding clam, Mucomu nusutu, we have shown that KU is highly dependent upon the flow of water ventilated across the gills (FW) (Boese et al., 1988). K,, may likewise be dependant on the amount of water ventilated or the amount of sediment ingested during depuration. To provide additional HCB depuration data from an invertebrate, we determined Kd in M. nusutu using water-borne [14C]HCB as the uptake route, followed by depuration in uncontaminated water and sediment. The uptake phase was conducted in clam ventilation chambers (clamboxes) (Specht and Lee, 1989) that allow for the direct measurement of ventilation rate and a more accurate prediction of the tissue residue in each individual at the beginning of the depuration phase (see Boese et al., 1988). The depuration phase was also conducted in the clamboxes to test whether there was a relationship between depuration and ventilation rate or to the rate at which ingested sediment flows through the gut (sediment processing rate or FS). MATERIALS AND METHODS
(1)
where:
in water which
One to two weeks prior to the experiment, M. nasuta (25-32 mm length) were collected from the Idaho Point mud flat of Yaquina Bay, Newport, Oregon, and maintained in sediment from the collection site in flow-through seawater aquaria. Forty-eight hours before experiment initiation, 15 clams were placed into individual clamboxes (Fig. 1) according to the method of Specht and Lee (1989). Clams were exposed for three days in flow-through seawater (42 ml/hr, 1.0 pm filtered, salinity = 25%) which 327
328
B. L. BOESEet al.
3
v
7
Y-
I I I I
Jac7
2 /_
1
d’
-IL Fw
Overflow
I I I
-l-
Fig. I. Macoma nasuta clambox (uptake phase configuration). (a) HCB dosing solution inflow, (b) inhalant siphon, (c) exhalant siphon, (d) dental dam membrane. Ventilation volume (FW) measurements taken from exhalant chamber overflow standpipe. Arrows indicate direction of water ventilated by clam. During the depuration phase, 5Og of sediment was added to the inhalant chamber.
contained dissolved [i4C]HCB (nominal dose = 2.5 ng/ml, 59.3 nCi/mg HCB). This dosing solution was prepared by dissolving [14C]HCB in acetonitrile solvent (0.4 pCi/ml), diluting this stock solution with flow-through seawater which was delivered to each clambox (1 l/d) via ceramic piston pump (Lab Pump Jr, Fluid Metering Inc.). Details of this procedure have been previously published (Boese et al., 1988). To avoid sorption of the dissolved HCB to particulates, no sediment was placed in the clamboxes during the uptake phase of the experiment. Ventilation volume was determined daily by measuring exhalant chamber overflow volume (Fig. 1). Water samples (2 ml) for radioactivity analysis were taken from the inhalant and exhalant chambers daily. Radioactivity of water samples were determined by liquid scintillation counter (LSC) (Packard Instrument Co., Model 2000CA) in a biodegradable LSC cocktail (Optifluor, Packard Instrument Co.). To determine whether the uptake followed a previously described relationship (Boese et al., 1988), five clams were sampled after three days of exposure. These clams were removed from their clamboxes, frozen for two hours (- lO”C), weighed and shucked. While frozen, the soft-tissue was dissected into four tissue portions, (I) foot; (2) mantlesiphon-gill-adductor muscles (MSGA); (3) viscera or gut; and (4) pallial fluid (water contained between the valves). These portions were separately weighed and analyzed for radioactivity. Radioactivity of tissue samples were determined by LSC after combustion with a sample oxidizer (Packard Instrument Co., Model 306A). _ For the 10 clams remaining on Day 3, the [i4C]HCB dosed seawater was replaced with clean water and 50 g of uncontaminated sediment (1 mm sieved) from the collection site was placed in the inhalant side of each clambox. Each clambox was supplied with flow-through uncontaminated seawater at the same temperature, salinity and flow rate as in the uptake phase. During this depuration phase, frozen sediment wafers (2g) were added to each clambox three times weekly to maintain an adequate food supply (Specht and Lee, 1989). Ventilation and radioactivity of water samples were measured by the same procedures used in the uptake phase of the experiment. Individual clams were removed from clamboxes for radioactivity analysis after 2, 4, 8, 12, 16, 20, 34, 48, 60, and 74 days of depuration to provide a time series for estimation of the depuration rate. Sampling clams individually, as opposed to using fewer time intervals with replication, maximized the spread of the data in the time series which provided the greatest statistical power in defining the regression curve. Dissections and
analytical procedures were identical to those used during the uptake phase of the experiment. Fecal pellets were collected from the exhalant chamber during the depuration phase of the experiment. Collected fecal pellets from each clam were stored in minimal seawater at 4°C until the completion of the experiment. At the end of the experiment, fecal pellets were rinsed in distilled water to remove salt from the samples, placed on tared filter paper, air-dried for 24 hr at room temperature, and weighed ( f 0.1 mg). The radioactivity of these fecal pellet samples was not determined due to loss of HCB from -pellets duhng the drying process (see Boese et al., 1990). Data analysis
HCB tissue residues at the start of the depuration phase (i.e., t = 0) were calculated in each of the ten depurated clams using a previously established linear relationship between total HCB gill exposure and HCB tissue residue in M. nasuta (Boese ef al., 1988). This relationship was modified by the addition of data from the clams sampled at the end of the uptake phase. Total gill exposure to HCB was estimated by summing the product of daily ventilation volume times the mean daily HCB water concentration in each clambox. Depuration was modeled by: TR, = TRO(emM’)
(3)
where TR, = Measured HCB tissue residue at time f TR, = Calculated tissue residue at the start of the depur-
ation phase (time = 0). In a previous study using a similar dosing system, HCB exposure concentrations, and hence tissue residues, tended to vary significantly among clamboxes (Boese et al., 1988). Because of this tissue residues are reported as the per cent of the calculated initial HCB residue (at t = 0) remaining at depuration time t (XTR,): %TR, = (TR,/T&)(lOO).
(4)
A linear regression of the natural log of this value (% TR,) on the number of days that the clam depurated was performed, and the regression coefficient (slope) used as an estimate of the depuration rate constant. The effects of ventilation volume and sediment processing rate on this relationship were assessed using multiple linear regression (Sokal and Rohlf, 1981). Ventilation volume and sediment processing effects on depuration phase tissue residues were tested using correlation analysis (Sokal and Rohlf, 1981). Using the depuration and uptake rate constants, tissue concentrations at any given time may be determined by: TR, = (CPW*K,)/K,,(l
- eeK“‘)
(5)
Assuming that depuration during the three day uptake phase was minimal, Ku was estimated by the mean daily uptake of the clams sampled at the end of the uptake phase. As exposure time approaches infinity, the emKdrterm in Equation 5 approaches zero. Thus the equilibrium tissue residue (TR,) for a given HCB water concentration may be calculated by:
= (cPw*K”)/K, %mx
(6)
The time (t,) required to reach a given fraction (F) of T&,, is then: $= -ln(l
- F)/K,
where: F = decimal fraction of Co,,,.
(7)
Kinetics of hexachlorobenzene
329
Table I. Uptake phase results and exposure conditions. CPW = HCB concentration in water, FWup = total uptake phase ventilation volume, HCB Exposure = total weight-specific exposure to HCB from ventilated water. HCB Tissue Residues are for each of the dissected tissue fraction and the combined amount. CPW values are means of 6 measurements (standard error). Clam 5 was considered an outlier and was not included in mean values with the exception of CPW values Clam no. I
2 3 4 5 Mean SE N
CPW Mean (SE) nalml
FWup ml
0.87 (0.09) 0.86 (0.51) 0.87 (0.37) 0.79 (0.28) 1.13 (0.50) 0.90 0.058 5
630 520 190 530 I260 470 96 4
HCB Tissue Residues Gut Pal.
HCB Exposure
MSGA
nala
w/8
nnln
w/g
w/g
w/g
338 368 139 374 1253 300 56 4
319 297 112 286 968 250 48 4
110 203 lost 213 503 180 33 3
335 484 114 395 927 330 79 4
27 58 43 56 137 469 7.1 4
214 284 NA 261 669 253 20.6 4
Foot
Combined
*Significantly different from means of other tissue fractions, ANOVA, p 9 0.05. RESULTS
Table 1 summarizes the results from the five uptake phase clams and Table 2 summarizes the results from the 10 clams sampled during the depuration phase. Only three of the five uptake phase clams were used to revise the previously published relationship between soft-tissue residues and gill exposures to HCB (Boese et al., 1988). An oxidizer malfunction invalidated a portion of the data from clam three. Clam five data was also not used as it ventilated water at a rate that was far in excess of any previously measured h4. nasuta ventilation values (Boese et al., 1988; Specht and Lee, 1989), possibly due to an undetected leak. When data from the three remaining clams were combined with data from a previously published determination (Boese et al., 1988), the resulting relationship between weight-specific HCB gill exposure (rig/g wet tissue weight) and tissue residue (rig/g wet tissue weight) was:
J 0
10
a0
so
Depuration
40
IS
Time
es
7s
a0
(Days)
Fig. 2. Relationship between depuration time and amount of HCB remaining in Mucoma nasutasoft-tissue. First order
regression equation for this data is: TR = 7.02 + 0.73 (HCB Exposure)
r* = 0.9.
(8)
HCB exposure is the product of the concentration of HCB in the water in an individual clambox times the amount of water ventilated per gram wet tissue weight by the clam. When the depurated combined tissue residues were normalized as the natural log of the percent of HCB remaining (Equation 4), the depuration rate was constant over time (Fig. 2): ln(%TR,) = 4.7 - (0.043t)
r* = 0.90.
(9)
ln(%TR,) = 4.6 - (0.043t)
r2 = 0.91
slope of this equation, O.O43/day, is the depuration rate constant (I&), which yields a t,,, of 16 days. No significant correlation (P > 0.05) was found between total or weight-specific ventilation volumes and tissue residues during depuration. A similar lack of correlation (P > 0.05) was observed between total or weight normalized sediment processing rates and tissue residues. The relationship between tissue residues and time was not improved by addition of either The
Table 2. Depuration phase results. CPW = mean HCB concentration in water during uptake phase, FWup = total uptake phase ventilation volume phase, Total Up = Estimate HCB uptake from Equation 8, Dep. = length of depuration, FWdep = total depuration phase ventilation volume, FS = total wt. of depuration phase fecal pellets. HCB Tissue Residues are for each of the dissected tissue fraction and the combined amount Clam no. 6 7 8 9 10 11 12 13 14 15 Mean SE N
CPW n8lmt 0.70 0.99 0.73 0.94 0.85 0.98 1.10 0.90 I .08 0.73 0.90 0.05 IO
FWup ml 960 855 520 310 550 880 425 750 410 860 600 84 10
Total Up n8l8 305 478 428 215 229 370 229 359 299 245 399 48 10
DeP. days 2 4 8 12 16 20 34 48 60 74
HCB Tissue Residues FWdep FS MSGA ml m8 ngl8 375 0 252 485 17 374 900 41 122 770 4 158 1475 22 206 3955 44 140 3240 65 117 4380 42 73 6160 310 16 I 1,270 128 19
Foot n8lg
Gut n8/8
Pal. ng/8
Combined rip/g
268 288 128 90 177 159 49 78 10 15
481 542 lost 159 207 564 115 71 13 20
51 51 lost 20 34 30 26 13 5 3
260 322 NA 118 144 222 85 50 11 14
B. L. BOESEet
330
ventilation volume or sediment processing using a multiple linear regression (P > 0.05). All dissected tissue portions, with the exception of pallial fluid, accumulated the same HCB concentration (Table I), and depurated HCB at the same rate (Table 2). Pallial fluid bioconcentrated HCB to a level about 20% that of the other tissue portions (Table 1). Using the mean uptake rate from the uptake phase clams (135 rig/g wet weight/day) and the derived depuration rate constant, the equilibrium tissue residue for HCB at the mean exposure concentration (0.9 ng/ml) was calculated to be 3140 rig/g wet flesh weight (Equation 6). Thus, the estimated BCF of HCB in M. nasuta is 3490 and the time required to reach 95% of the equilibrium tissue residue was estimated at 70 days (Equation 7). These results change slightly when the uptake rate is adjusted for depuration occurring during the uptake phase. Using our calculated depuration rate constant (O.O43/day) and Equation 5, an uptake rate constant was back-calculated to be 7% greater (144 rig/g--day), with a resulting 7% increase in the equilibrium body burden and BCF (3350 rig/g and 3720 respectively). As clams did not grow appreciably during the depuration phase, normalizing tissue residues for growth dilution was not necessary. Water samples taken from the exhalant chamber during both the uptake and depuration phases contained detectable amounts of [i4C]HCB (mean = 5 DPM/ml) over background levels. Mean exhalant chamber DPM did not vary significantly during uptake and depuration phases. The exception to this was clam five, where net DPM in the exhalant chamber ranged from 50 to 130 DPM/ml. Although exhalant water samples were above background levels, further analysis was not done as these data were considered to be unreliable due to the high fugacity of HCB dissolved in water, and the variable residence times within the exhalant chamber of clamboxes (Boese et al., 1988). DISCUSSION
One of the assumptions of this kinetic approach to determining BCF is that depuration is a constant (Branson et al., 1975; Davies and Dobbs, 1984). However, in a previous study (Boese et al., 1988) individual variation in uptake of HCB in M. nasuta could be largely explained by differences in the amount of HCB exposure at the gill surface, which is a function of the individuals ventilation rate. It seemed possible that individual variations in depuration rates could also be related to the bioenergetics of the individual, as measured by ventilation or sediment processing rate. For example, large ventilation or sediment processing rates might indicate a higher metabolic rate, possibly resulting in a faster depuration of pollutants and their metabolites. Another mechanism could be that the greater the flow of water across gills or sediment across the gut lining would increase the exchange (diffusion rate) and enhance depuration. In the present experiment this was not found to be the case, as ventilation rates and fecal pellet production rates did not correlate with HCB loss. Our results, however, encompassed a narrow range of metabolic rates. Perhaps a wider range of ventilation and sediment processing rates (e.g. those
al.
induced by seasonal temperature and food supply differences) would elicit an effect. Additionally, more complex depuration models may be necessary for compounds that are more readily metabolized than HCB (Spacie and Hamelink, 1982). HCB uptake and depuration kinetics in fish (mainly rainbow trout, Salmo gairdneri) have been measured in several studies. Rainbow trout tended to depurate HCB at a slower rate than we observed in M. nasuta, with T,,* values that ranged from 65 to 770 days (Niimi and Cho, 1981; Niimi and Plazzo, 1985; Norheim and Roald, 1985). However, all of these values were derived from HCB that was taken up via diet or intraperitoneal injection. The estimated BCF value for M. nasuta can be compared to three fish studies which used waterborne HCB as the route of exposure (Veith et al., 1979; Kosian et al., 1981; Oliver and Niimi, 1983). The BCF values in these studies ranged from 5500 to 39,000 which are in excess of the BCF for HCB in M. nasuta by as much as 10 fold. The apparent difference between fish and M. nasutu values is likely due to the greater lipid content of fish. Lipophilic compounds such as HCB will tend to preferentially accumulate in tissue lipids, as evidenced by the reduction in fish BCF variability when normalized on a lipid weight basis (Veith, 1975; Lieb et al., 1974). Fish tend to have considerably greater lipid content than mollusks, which use glycogen rather than fats for energy storage. M. nasuta is especially low in lipid content, with immature clams of the size range we used having a mean of 0.4% lipid content on a wet weight basis (R. C. Randall, unpublished data) as determined by a chloroform-methanol extraction (Bligh and Dyer, 1959) that was modified for small (1 g) tissue sample. In rainbow trout, lipid content may be as much as 20 times greater than this (Wheaton and Lawson, 1985), which may explain the differences in BCFs and retention times. In the only comparable invertebrate study (Oliver, 1987), oligochaete worms (Tubifex tubifex and Limnodrilus hofmeisteri) were exposed to HCB in contaminated sediments. In that study, the BCF value from interstitial water was 3 120 (wet weight basis), which is very similar to the value obtained in the present study. The lipid content of these oligochaetes (about 1% of wet body weight) was also similar to M. nasuta. Within a given fish species, organs may take up and retain HCB with varying affinities (Sanbom et al., 1977; Norheim and Roald, 1985). This was not the case in M. nasuta, where, with the exception of pallial fluid, all of the dissected tissue portions took up and depurated HCB at approximately the same rate. It is not surprising that pallial fluid was the exception to this, as the majority of the pallial fluid in M. nusuta is composed of water that has been pulled into the mantle cavity from the external environment (Specht and Lee, 1989). The lipid content of the immature N. nasuta used in the present study is lower than that of gravid clams. M. nasutu and other bivalve species vary seasonally in lipid content with substantial decreases associated with spawning (Gabbot, 1976; Davis and Wilson, 1983). As a result the BCF for HCB is also likely to vary seasonally. The higher lipid content of gonads may make these tissue the most important
Kinetics of hexarhlorobenzene uptake site for lipophilic pollutants such as HCB, and as a result may be the most fertile site for research on chronic toxicity of these compounds in clams. Acknowledgements-Thanks go to Judy Pelletier of AScI Corporation, who assisted in data gathering and reduction. Thanks also to Gary Chapman, Lawrence Curtis, and Wayne Munns Jr for their review of the manuscript. Environmental Research Laboratory-Narragansett contribution number N081. REFERENCES
Boese B. L., Lee H. II and Specht D. T. (1988) Efficiency of uptake of hexachlorobenzene from water by the tellinid clam, Macoma nasuta. Aquatic Tox. 12, 345-356. Boese B. L., Lee H. II, Specht D. T., Randall R. C. and Winsor M. (1990) Comparison of aqueous and solidphase uptake for hexachlorobenzene in the tellinid clam, Macoma nasuta (Conrad): a mass balance approach. Environ. Toxicol. Chem. 9, 221-232. Bligh E. G. and Dyer W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917.
Branson D. R., Blau G. E., Alexander H. C. and Neely W. B. (1975) Bioconcentration of 2,2’,4,4’-tetrachlorobiphenyl in rainbow trout as measured by an accelerated test. Trans. Am. Fish. Sot. 104, 785-792. Davies R. P. and Dobbs A. J. (1984) The prediction of bioconcentration of fish. Water Res. 18, 1253-1262. Davis J. P. and Wilson J. G. (1983) Seasonal changes in tissue weight and biochemical composition of the bivalve Nucula turgida in Dublin Bay with reference to gametogenesis. Neth. J. Sea Res. 17, 84-95. Ekelund R., Granmo A., Berggren, Renberg M. L. and Wahlberg C. (1987) Influence of suspended solids on bioavailability of hexachlorobenzene and lindane to the deposit-feeding marine bivalve, Abra nitida (Miiller). Bull. environ. Contam. Toxicol. 38, 500-508.
Gabbot P. A. (1976) Energy metabolism. In Marine Mussels, their Ecology and Physiology (Edited by Bayne B. L.), pp. 294-355. Cambridge University Press; Cambridge. Knezovich J. P. and Harrison F. L. (1988) The bioavailability of sediment-sorbed chlorobenzenes to larvae of the midge Chironomus decorus. Ecotox. Environ. Safety 15, 226-241. Kosian P., Lemke A., Studders K. and Veith G. (1981) The precision of the ASTM bioconcentration test. U.S. Environmental Protection Agency, ERL-Duluth MN. EPA-600/S3-81-022. Lieb A. J., Bills D. D. and Sinnhuber R. 0. (1974) Accumulation of dietary PCB (Aroclor 1254) by rainbow trout. J. Agric. Food Chem. 22, 638-642.
Metcalf R. L., Kapoor I. P., Lu P. Y., Schuth C. K. and Sherman P. (1973) Model ecosystem studies of the environ-
331
mental fate of 6 organochlorine pesticides. Environ. hlth Persp. 4, 35-44. Niimi A. J. (1979) Hexachlorobenzene (HCB) levels in Lake Ontario salmonids. Bull. environ. Contam. Toxicol. 23, 20-24. Niimi A. J. and Cho C. Y. (1981) Elimination of hexachlorobenzene (HCB) by rainbow trout (Salmo gairdneri), and an examination of its kinetics in Lake Ontario salmonids. Can J. Fish Aquat. Sci. 38, 1350-1356. Niimi A. J. and Palazzo V. (1985) Temperature effect on the elimination of pentachlorophenol, hexachlorob-enzene and miirex bv rainbow trout (Salmo aairdneri). Water Res. 19, 205-207. Norheim G. and Roald S. 0. (1985) Distribution and elimination of hexachlorobenzene, octachlorostyrene and decachlorobiphenyl in rainbow trout, Salmo gairdneri. Aquaf. Tox. 6, 13-24.
Oliver B. G. (1987) Biouptake of chlorinated hydrocarbons from laboratory-spiked and field sediments by oligochaete worms. Environ. Sci. Technol. 21, 785-790..
_
Oliver B. G. and Niimi A. J. (1983) Bioconcentration of chlorobenzenes from water by‘raindow trout: correlations with partition coefficients and environmental residues. Environ. Sci. Technol. 17, 287-291.
Sanborn J. R., Childers W. F. and Hansen L. G. (1977) Uptake and elimination of [i4Clhexachlorobenzene (HCB) by the green sunfish, Lepomis cyanellus Raf., after feeding contaminated food. J. Agric. Food Chem. 25, 551-553. Sokal R. R. and Rohlf F. J. (1981) Biometry. 2nd edition, p. 776. W. H. Freeman, San Francisco. Spacie A. and Hamelink J. L. (1982) Alternative models for describing the bioconcentration of organics in fish. Enuiron. Toxicol. Chem. 1, 309-320.
Specht D. T. and Lee H. II (1989) Direct measurement technique for determining ventilation rate in the depositfeeding clam, Macoma nasuta (Bivalvia, Tellinaceae). Mar. Biol. 101, 211-218. Tsui P. T. P. and McCart P. J. (1981) Chlorinated hydrocarbon residues and heavy metals in several fish species from the Cold Lake area in Alberta, Canada. Intern. J. Environ. anal. Chem. 10, 277-285. Veith B. D. (1975) Baseline concentration of PCBs and DDT in Lake Michigan fish. Pets. Monk J. 9, 21-29. Veith G. D., DeFoe D. L. and Bergstedt B. V. (1979) Measuring and estimating the bioconcentration factor of chemicals in fish. J. Fish. Res. Board Can. 36, 1040-1048. Wheaton F. W. and Lawson T. B. (1985) Processing Aquatic Food Products. Wiley, New York. Young D. R. and Heesen T. C. (1978) DDT, PCB, and chlorinated benzenes in the marine ecosystem off Southern California. In Water Chlorination: Enuironmenfal Impact and Health Effects (Edited by Jolley R., Gorchev H. and Hamilton D. H. Jr), Vol. 2, pp. 267-290. Ann Arbor, Michigan.
Camp. Biochem. Physiol.Vol. 96C, No. 2, pp. 327-331, 1990
$3.00 + 0.00
PergamonPressplc
Printedin Great Britain
DEPURATION KINETICS OF HEXACHLOROBENZENE IN THE CLAM, MACOMA NASUTA B. L.
BOESE, M. WINSOR,*
H.
LEE
II, D. T. SPECHT and K. C. RUKAVINA
Pacific Ecosystems Branch, Environmental Research Laboratory-Narragansett, U.S. Environmental Protection Agency, and lAScI Inc., Hatfield Marine Science Center, Newport, Oregon 97365, U.S.A. Telephone (503) 867-4037 (Received 22 February 1990)
(HCB) in the clam, Mucoma seawater. 2. Depuration was not correlated with ventilation volume, nor did the amount of sediment ingested during depuration have a significant effect. 3. The half-life for HCB in M. nusufa was estimated to he 16 days with a bioconcentration factor of 3490 (wet weight basis).
Abstract-l.
The depuration
rate constant for [‘4C]hexachlorohenzene
nusuta, was determined following a short-term exposure to HCB contaminated
CPW = Concentration of pollutant is assumed to be constant. KU= Uptake rate constant. Kd = Depuration rate constant. r = Time.
INTRODUCTION Hexachlorobenzene (HCB) is a persistent environmental pollutant that bioaccumulates in tissues of marine and fresh water fish (Young and Heesen, 1978; Niimi, 1979; Tsui and McCart, 1981). Once taken up by these tissues, biodegradation is very slow with pentachlorophenol (Sanborn et al., 1977) and polar conjugates (Metcalf et al., 1973) as possible metabolites in aquatic organisms. Numerous studies have been done on HCB uptake and depuration in
fish (Sanborn et al., 1977; Veith et al., 1979; Kosian et al., 1981; Niimi and Cho, 1981; Oliver and Niimi, 1983). In these studies the HCB bioconcentration factors (BCFs) varied from 5000 to 40,000 (wet weight basis) with half-lives (t,,,) from a few days to several years. Although several studies have examined HCB uptake in invertebrates (Boese et al., 1988; Ekelund et al., 1987; Oliver, 1987; Knezovich and Harrison, 1988), only Oliver’s (1987) study estimate a BCF. Oliver (1987) found that the BCF of HCB in oligochaete worms was 3120 when calculated on a wet weight basis, with a t,,Z of 27 days. However, in Oliver’s study the worms were buried in the contaminated sediment and the BCF calculated using the interstitial water concentration. As a portion of the HCB body burden was likely due to the ingestion of particulates, the reported BCF value may be in error. Bioconcentration factors are commonly estimated from non-equilibrium exposures using a kinetic uptake model (Branson et al., 1975; Davies and Dobbs, 1984), where the change in tissue concentration with time is calculated by: dTR/dt = K,CPW
- K,,TR
TR = Pollutant tissue residue concentration organism.
in
Mention of trade names or commercial products does not constitute endorsement or recommendation of use by the Environmental Protection Agency. CSPC %,2--H
When steady-state pollutant tissue concentrations are attained (i.e. dTR/dt = 0), the BCF is estimated by: BCF = KU/K,.
(2)
In previous work with HCB in the deposit-feeding clam, Mucomu nusutu, we have shown that KU is highly dependent upon the flow of water ventilated across the gills (FW) (Boese et al., 1988). K,, may likewise be dependant on the amount of water ventilated or the amount of sediment ingested during depuration. To provide additional HCB depuration data from an invertebrate, we determined Kd in M. nusutu using water-borne [14C]HCB as the uptake route, followed by depuration in uncontaminated water and sediment. The uptake phase was conducted in clam ventilation chambers (clamboxes) (Specht and Lee, 1989) that allow for the direct measurement of ventilation rate and a more accurate prediction of the tissue residue in each individual at the beginning of the depuration phase (see Boese et al., 1988). The depuration phase was also conducted in the clamboxes to test whether there was a relationship between depuration and ventilation rate or to the rate at which ingested sediment flows through the gut (sediment processing rate or FS). MATERIALS AND METHODS
(1)
where:
in water which
One to two weeks prior to the experiment, M. nasuta (25-32 mm length) were collected from the Idaho Point mud flat of Yaquina Bay, Newport, Oregon, and maintained in sediment from the collection site in flow-through seawater aquaria. Forty-eight hours before experiment initiation, 15 clams were placed into individual clamboxes (Fig. 1) according to the method of Specht and Lee (1989). Clams were exposed for three days in flow-through seawater (42 ml/hr, 1.0 pm filtered, salinity = 25%) which 327
328
B. L. BOESEet al.
3
v
7
Y-
I I I I
Jac7
2 /_
1
d’
-IL Fw
Overflow
I I I
-l-
Fig. I. Macoma nasuta clambox (uptake phase configuration). (a) HCB dosing solution inflow, (b) inhalant siphon, (c) exhalant siphon, (d) dental dam membrane. Ventilation volume (FW) measurements taken from exhalant chamber overflow standpipe. Arrows indicate direction of water ventilated by clam. During the depuration phase, 5Og of sediment was added to the inhalant chamber.
contained dissolved [i4C]HCB (nominal dose = 2.5 ng/ml, 59.3 nCi/mg HCB). This dosing solution was prepared by dissolving [14C]HCB in acetonitrile solvent (0.4 pCi/ml), diluting this stock solution with flow-through seawater which was delivered to each clambox (1 l/d) via ceramic piston pump (Lab Pump Jr, Fluid Metering Inc.). Details of this procedure have been previously published (Boese et al., 1988). To avoid sorption of the dissolved HCB to particulates, no sediment was placed in the clamboxes during the uptake phase of the experiment. Ventilation volume was determined daily by measuring exhalant chamber overflow volume (Fig. 1). Water samples (2 ml) for radioactivity analysis were taken from the inhalant and exhalant chambers daily. Radioactivity of water samples were determined by liquid scintillation counter (LSC) (Packard Instrument Co., Model 2000CA) in a biodegradable LSC cocktail (Optifluor, Packard Instrument Co.). To determine whether the uptake followed a previously described relationship (Boese et al., 1988), five clams were sampled after three days of exposure. These clams were removed from their clamboxes, frozen for two hours (- lO”C), weighed and shucked. While frozen, the soft-tissue was dissected into four tissue portions, (I) foot; (2) mantlesiphon-gill-adductor muscles (MSGA); (3) viscera or gut; and (4) pallial fluid (water contained between the valves). These portions were separately weighed and analyzed for radioactivity. Radioactivity of tissue samples were determined by LSC after combustion with a sample oxidizer (Packard Instrument Co., Model 306A). _ For the 10 clams remaining on Day 3, the [i4C]HCB dosed seawater was replaced with clean water and 50 g of uncontaminated sediment (1 mm sieved) from the collection site was placed in the inhalant side of each clambox. Each clambox was supplied with flow-through uncontaminated seawater at the same temperature, salinity and flow rate as in the uptake phase. During this depuration phase, frozen sediment wafers (2g) were added to each clambox three times weekly to maintain an adequate food supply (Specht and Lee, 1989). Ventilation and radioactivity of water samples were measured by the same procedures used in the uptake phase of the experiment. Individual clams were removed from clamboxes for radioactivity analysis after 2, 4, 8, 12, 16, 20, 34, 48, 60, and 74 days of depuration to provide a time series for estimation of the depuration rate. Sampling clams individually, as opposed to using fewer time intervals with replication, maximized the spread of the data in the time series which provided the greatest statistical power in defining the regression curve. Dissections and
analytical procedures were identical to those used during the uptake phase of the experiment. Fecal pellets were collected from the exhalant chamber during the depuration phase of the experiment. Collected fecal pellets from each clam were stored in minimal seawater at 4°C until the completion of the experiment. At the end of the experiment, fecal pellets were rinsed in distilled water to remove salt from the samples, placed on tared filter paper, air-dried for 24 hr at room temperature, and weighed ( f 0.1 mg). The radioactivity of these fecal pellet samples was not determined due to loss of HCB from -pellets duhng the drying process (see Boese et al., 1990). Data analysis
HCB tissue residues at the start of the depuration phase (i.e., t = 0) were calculated in each of the ten depurated clams using a previously established linear relationship between total HCB gill exposure and HCB tissue residue in M. nasuta (Boese ef al., 1988). This relationship was modified by the addition of data from the clams sampled at the end of the uptake phase. Total gill exposure to HCB was estimated by summing the product of daily ventilation volume times the mean daily HCB water concentration in each clambox. Depuration was modeled by: TR, = TRO(emM’)
(3)
where TR, = Measured HCB tissue residue at time f TR, = Calculated tissue residue at the start of the depur-
ation phase (time = 0). In a previous study using a similar dosing system, HCB exposure concentrations, and hence tissue residues, tended to vary significantly among clamboxes (Boese et al., 1988). Because of this tissue residues are reported as the per cent of the calculated initial HCB residue (at t = 0) remaining at depuration time t (XTR,): %TR, = (TR,/T&)(lOO).
(4)
A linear regression of the natural log of this value (% TR,) on the number of days that the clam depurated was performed, and the regression coefficient (slope) used as an estimate of the depuration rate constant. The effects of ventilation volume and sediment processing rate on this relationship were assessed using multiple linear regression (Sokal and Rohlf, 1981). Ventilation volume and sediment processing effects on depuration phase tissue residues were tested using correlation analysis (Sokal and Rohlf, 1981). Using the depuration and uptake rate constants, tissue concentrations at any given time may be determined by: TR, = (CPW*K,)/K,,(l
- eeK“‘)
(5)
Assuming that depuration during the three day uptake phase was minimal, Ku was estimated by the mean daily uptake of the clams sampled at the end of the uptake phase. As exposure time approaches infinity, the emKdrterm in Equation 5 approaches zero. Thus the equilibrium tissue residue (TR,) for a given HCB water concentration may be calculated by:
= (cPw*K”)/K, %mx
(6)
The time (t,) required to reach a given fraction (F) of T&,, is then: $= -ln(l
- F)/K,
where: F = decimal fraction of Co,,,.
(7)
Kinetics of hexachlorobenzene
329
Table I. Uptake phase results and exposure conditions. CPW = HCB concentration in water, FWup = total uptake phase ventilation volume, HCB Exposure = total weight-specific exposure to HCB from ventilated water. HCB Tissue Residues are for each of the dissected tissue fraction and the combined amount. CPW values are means of 6 measurements (standard error). Clam 5 was considered an outlier and was not included in mean values with the exception of CPW values Clam no. I
2 3 4 5 Mean SE N
CPW Mean (SE) nalml
FWup ml
0.87 (0.09) 0.86 (0.51) 0.87 (0.37) 0.79 (0.28) 1.13 (0.50) 0.90 0.058 5
630 520 190 530 I260 470 96 4
HCB Tissue Residues Gut Pal.
HCB Exposure
MSGA
nala
w/8
nnln
w/g
w/g
w/g
338 368 139 374 1253 300 56 4
319 297 112 286 968 250 48 4
110 203 lost 213 503 180 33 3
335 484 114 395 927 330 79 4
27 58 43 56 137 469 7.1 4
214 284 NA 261 669 253 20.6 4
Foot
Combined
*Significantly different from means of other tissue fractions, ANOVA, p 9 0.05. RESULTS
Table 1 summarizes the results from the five uptake phase clams and Table 2 summarizes the results from the 10 clams sampled during the depuration phase. Only three of the five uptake phase clams were used to revise the previously published relationship between soft-tissue residues and gill exposures to HCB (Boese et al., 1988). An oxidizer malfunction invalidated a portion of the data from clam three. Clam five data was also not used as it ventilated water at a rate that was far in excess of any previously measured h4. nasuta ventilation values (Boese et al., 1988; Specht and Lee, 1989), possibly due to an undetected leak. When data from the three remaining clams were combined with data from a previously published determination (Boese et al., 1988), the resulting relationship between weight-specific HCB gill exposure (rig/g wet tissue weight) and tissue residue (rig/g wet tissue weight) was:
J 0
10
a0
so
Depuration
40
IS
Time
es
7s
a0
(Days)
Fig. 2. Relationship between depuration time and amount of HCB remaining in Mucoma nasutasoft-tissue. First order
regression equation for this data is: TR = 7.02 + 0.73 (HCB Exposure)
r* = 0.9.
(8)
HCB exposure is the product of the concentration of HCB in the water in an individual clambox times the amount of water ventilated per gram wet tissue weight by the clam. When the depurated combined tissue residues were normalized as the natural log of the percent of HCB remaining (Equation 4), the depuration rate was constant over time (Fig. 2): ln(%TR,) = 4.7 - (0.043t)
r* = 0.90.
(9)
ln(%TR,) = 4.6 - (0.043t)
r2 = 0.91
slope of this equation, O.O43/day, is the depuration rate constant (I&), which yields a t,,, of 16 days. No significant correlation (P > 0.05) was found between total or weight-specific ventilation volumes and tissue residues during depuration. A similar lack of correlation (P > 0.05) was observed between total or weight normalized sediment processing rates and tissue residues. The relationship between tissue residues and time was not improved by addition of either The
Table 2. Depuration phase results. CPW = mean HCB concentration in water during uptake phase, FWup = total uptake phase ventilation volume phase, Total Up = Estimate HCB uptake from Equation 8, Dep. = length of depuration, FWdep = total depuration phase ventilation volume, FS = total wt. of depuration phase fecal pellets. HCB Tissue Residues are for each of the dissected tissue fraction and the combined amount Clam no. 6 7 8 9 10 11 12 13 14 15 Mean SE N
CPW n8lmt 0.70 0.99 0.73 0.94 0.85 0.98 1.10 0.90 I .08 0.73 0.90 0.05 IO
FWup ml 960 855 520 310 550 880 425 750 410 860 600 84 10
Total Up n8l8 305 478 428 215 229 370 229 359 299 245 399 48 10
DeP. days 2 4 8 12 16 20 34 48 60 74
HCB Tissue Residues FWdep FS MSGA ml m8 ngl8 375 0 252 485 17 374 900 41 122 770 4 158 1475 22 206 3955 44 140 3240 65 117 4380 42 73 6160 310 16 I 1,270 128 19
Foot n8lg
Gut n8/8
Pal. ng/8
Combined rip/g
268 288 128 90 177 159 49 78 10 15
481 542 lost 159 207 564 115 71 13 20
51 51 lost 20 34 30 26 13 5 3
260 322 NA 118 144 222 85 50 11 14
B. L. BOESEet
330
ventilation volume or sediment processing using a multiple linear regression (P > 0.05). All dissected tissue portions, with the exception of pallial fluid, accumulated the same HCB concentration (Table I), and depurated HCB at the same rate (Table 2). Pallial fluid bioconcentrated HCB to a level about 20% that of the other tissue portions (Table 1). Using the mean uptake rate from the uptake phase clams (135 rig/g wet weight/day) and the derived depuration rate constant, the equilibrium tissue residue for HCB at the mean exposure concentration (0.9 ng/ml) was calculated to be 3140 rig/g wet flesh weight (Equation 6). Thus, the estimated BCF of HCB in M. nasuta is 3490 and the time required to reach 95% of the equilibrium tissue residue was estimated at 70 days (Equation 7). These results change slightly when the uptake rate is adjusted for depuration occurring during the uptake phase. Using our calculated depuration rate constant (O.O43/day) and Equation 5, an uptake rate constant was back-calculated to be 7% greater (144 rig/g--day), with a resulting 7% increase in the equilibrium body burden and BCF (3350 rig/g and 3720 respectively). As clams did not grow appreciably during the depuration phase, normalizing tissue residues for growth dilution was not necessary. Water samples taken from the exhalant chamber during both the uptake and depuration phases contained detectable amounts of [i4C]HCB (mean = 5 DPM/ml) over background levels. Mean exhalant chamber DPM did not vary significantly during uptake and depuration phases. The exception to this was clam five, where net DPM in the exhalant chamber ranged from 50 to 130 DPM/ml. Although exhalant water samples were above background levels, further analysis was not done as these data were considered to be unreliable due to the high fugacity of HCB dissolved in water, and the variable residence times within the exhalant chamber of clamboxes (Boese et al., 1988). DISCUSSION
One of the assumptions of this kinetic approach to determining BCF is that depuration is a constant (Branson et al., 1975; Davies and Dobbs, 1984). However, in a previous study (Boese et al., 1988) individual variation in uptake of HCB in M. nasuta could be largely explained by differences in the amount of HCB exposure at the gill surface, which is a function of the individuals ventilation rate. It seemed possible that individual variations in depuration rates could also be related to the bioenergetics of the individual, as measured by ventilation or sediment processing rate. For example, large ventilation or sediment processing rates might indicate a higher metabolic rate, possibly resulting in a faster depuration of pollutants and their metabolites. Another mechanism could be that the greater the flow of water across gills or sediment across the gut lining would increase the exchange (diffusion rate) and enhance depuration. In the present experiment this was not found to be the case, as ventilation rates and fecal pellet production rates did not correlate with HCB loss. Our results, however, encompassed a narrow range of metabolic rates. Perhaps a wider range of ventilation and sediment processing rates (e.g. those
al.
induced by seasonal temperature and food supply differences) would elicit an effect. Additionally, more complex depuration models may be necessary for compounds that are more readily metabolized than HCB (Spacie and Hamelink, 1982). HCB uptake and depuration kinetics in fish (mainly rainbow trout, Salmo gairdneri) have been measured in several studies. Rainbow trout tended to depurate HCB at a slower rate than we observed in M. nasuta, with T,,* values that ranged from 65 to 770 days (Niimi and Cho, 1981; Niimi and Plazzo, 1985; Norheim and Roald, 1985). However, all of these values were derived from HCB that was taken up via diet or intraperitoneal injection. The estimated BCF value for M. nasuta can be compared to three fish studies which used waterborne HCB as the route of exposure (Veith et al., 1979; Kosian et al., 1981; Oliver and Niimi, 1983). The BCF values in these studies ranged from 5500 to 39,000 which are in excess of the BCF for HCB in M. nasuta by as much as 10 fold. The apparent difference between fish and M. nasutu values is likely due to the greater lipid content of fish. Lipophilic compounds such as HCB will tend to preferentially accumulate in tissue lipids, as evidenced by the reduction in fish BCF variability when normalized on a lipid weight basis (Veith, 1975; Lieb et al., 1974). Fish tend to have considerably greater lipid content than mollusks, which use glycogen rather than fats for energy storage. M. nasuta is especially low in lipid content, with immature clams of the size range we used having a mean of 0.4% lipid content on a wet weight basis (R. C. Randall, unpublished data) as determined by a chloroform-methanol extraction (Bligh and Dyer, 1959) that was modified for small (1 g) tissue sample. In rainbow trout, lipid content may be as much as 20 times greater than this (Wheaton and Lawson, 1985), which may explain the differences in BCFs and retention times. In the only comparable invertebrate study (Oliver, 1987), oligochaete worms (Tubifex tubifex and Limnodrilus hofmeisteri) were exposed to HCB in contaminated sediments. In that study, the BCF value from interstitial water was 3 120 (wet weight basis), which is very similar to the value obtained in the present study. The lipid content of these oligochaetes (about 1% of wet body weight) was also similar to M. nasuta. Within a given fish species, organs may take up and retain HCB with varying affinities (Sanbom et al., 1977; Norheim and Roald, 1985). This was not the case in M. nasuta, where, with the exception of pallial fluid, all of the dissected tissue portions took up and depurated HCB at approximately the same rate. It is not surprising that pallial fluid was the exception to this, as the majority of the pallial fluid in M. nusuta is composed of water that has been pulled into the mantle cavity from the external environment (Specht and Lee, 1989). The lipid content of the immature N. nasuta used in the present study is lower than that of gravid clams. M. nasutu and other bivalve species vary seasonally in lipid content with substantial decreases associated with spawning (Gabbot, 1976; Davis and Wilson, 1983). As a result the BCF for HCB is also likely to vary seasonally. The higher lipid content of gonads may make these tissue the most important
Kinetics of hexarhlorobenzene uptake site for lipophilic pollutants such as HCB, and as a result may be the most fertile site for research on chronic toxicity of these compounds in clams. Acknowledgements-Thanks go to Judy Pelletier of AScI Corporation, who assisted in data gathering and reduction. Thanks also to Gary Chapman, Lawrence Curtis, and Wayne Munns Jr for their review of the manuscript. Environmental Research Laboratory-Narragansett contribution number N081. REFERENCES
Boese B. L., Lee H. II and Specht D. T. (1988) Efficiency of uptake of hexachlorobenzene from water by the tellinid clam, Macoma nasuta. Aquatic Tox. 12, 345-356. Boese B. L., Lee H. II, Specht D. T., Randall R. C. and Winsor M. (1990) Comparison of aqueous and solidphase uptake for hexachlorobenzene in the tellinid clam, Macoma nasuta (Conrad): a mass balance approach. Environ. Toxicol. Chem. 9, 221-232. Bligh E. G. and Dyer W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917.
Branson D. R., Blau G. E., Alexander H. C. and Neely W. B. (1975) Bioconcentration of 2,2’,4,4’-tetrachlorobiphenyl in rainbow trout as measured by an accelerated test. Trans. Am. Fish. Sot. 104, 785-792. Davies R. P. and Dobbs A. J. (1984) The prediction of bioconcentration of fish. Water Res. 18, 1253-1262. Davis J. P. and Wilson J. G. (1983) Seasonal changes in tissue weight and biochemical composition of the bivalve Nucula turgida in Dublin Bay with reference to gametogenesis. Neth. J. Sea Res. 17, 84-95. Ekelund R., Granmo A., Berggren, Renberg M. L. and Wahlberg C. (1987) Influence of suspended solids on bioavailability of hexachlorobenzene and lindane to the deposit-feeding marine bivalve, Abra nitida (Miiller). Bull. environ. Contam. Toxicol. 38, 500-508.
Gabbot P. A. (1976) Energy metabolism. In Marine Mussels, their Ecology and Physiology (Edited by Bayne B. L.), pp. 294-355. Cambridge University Press; Cambridge. Knezovich J. P. and Harrison F. L. (1988) The bioavailability of sediment-sorbed chlorobenzenes to larvae of the midge Chironomus decorus. Ecotox. Environ. Safety 15, 226-241. Kosian P., Lemke A., Studders K. and Veith G. (1981) The precision of the ASTM bioconcentration test. U.S. Environmental Protection Agency, ERL-Duluth MN. EPA-600/S3-81-022. Lieb A. J., Bills D. D. and Sinnhuber R. 0. (1974) Accumulation of dietary PCB (Aroclor 1254) by rainbow trout. J. Agric. Food Chem. 22, 638-642.
Metcalf R. L., Kapoor I. P., Lu P. Y., Schuth C. K. and Sherman P. (1973) Model ecosystem studies of the environ-
331
mental fate of 6 organochlorine pesticides. Environ. hlth Persp. 4, 35-44. Niimi A. J. (1979) Hexachlorobenzene (HCB) levels in Lake Ontario salmonids. Bull. environ. Contam. Toxicol. 23, 20-24. Niimi A. J. and Cho C. Y. (1981) Elimination of hexachlorobenzene (HCB) by rainbow trout (Salmo gairdneri), and an examination of its kinetics in Lake Ontario salmonids. Can J. Fish Aquat. Sci. 38, 1350-1356. Niimi A. J. and Palazzo V. (1985) Temperature effect on the elimination of pentachlorophenol, hexachlorob-enzene and miirex bv rainbow trout (Salmo aairdneri). Water Res. 19, 205-207. Norheim G. and Roald S. 0. (1985) Distribution and elimination of hexachlorobenzene, octachlorostyrene and decachlorobiphenyl in rainbow trout, Salmo gairdneri. Aquaf. Tox. 6, 13-24.
Oliver B. G. (1987) Biouptake of chlorinated hydrocarbons from laboratory-spiked and field sediments by oligochaete worms. Environ. Sci. Technol. 21, 785-790..
_
Oliver B. G. and Niimi A. J. (1983) Bioconcentration of chlorobenzenes from water by‘raindow trout: correlations with partition coefficients and environmental residues. Environ. Sci. Technol. 17, 287-291.
Sanborn J. R., Childers W. F. and Hansen L. G. (1977) Uptake and elimination of [i4Clhexachlorobenzene (HCB) by the green sunfish, Lepomis cyanellus Raf., after feeding contaminated food. J. Agric. Food Chem. 25, 551-553. Sokal R. R. and Rohlf F. J. (1981) Biometry. 2nd edition, p. 776. W. H. Freeman, San Francisco. Spacie A. and Hamelink J. L. (1982) Alternative models for describing the bioconcentration of organics in fish. Enuiron. Toxicol. Chem. 1, 309-320.
Specht D. T. and Lee H. II (1989) Direct measurement technique for determining ventilation rate in the depositfeeding clam, Macoma nasuta (Bivalvia, Tellinaceae). Mar. Biol. 101, 211-218. Tsui P. T. P. and McCart P. J. (1981) Chlorinated hydrocarbon residues and heavy metals in several fish species from the Cold Lake area in Alberta, Canada. Intern. J. Environ. anal. Chem. 10, 277-285. Veith B. D. (1975) Baseline concentration of PCBs and DDT in Lake Michigan fish. Pets. Monk J. 9, 21-29. Veith G. D., DeFoe D. L. and Bergstedt B. V. (1979) Measuring and estimating the bioconcentration factor of chemicals in fish. J. Fish. Res. Board Can. 36, 1040-1048. Wheaton F. W. and Lawson T. B. (1985) Processing Aquatic Food Products. Wiley, New York. Young D. R. and Heesen T. C. (1978) DDT, PCB, and chlorinated benzenes in the marine ecosystem off Southern California. In Water Chlorination: Enuironmenfal Impact and Health Effects (Edited by Jolley R., Gorchev H. and Hamilton D. H. Jr), Vol. 2, pp. 267-290. Ann Arbor, Michigan.