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Factors affecting the evaluation of chemicals in laboratory experiments using marine organisms
ECOTOXICOLOGY
AND
ENVIRONMENTAL
SAFETY
3, 90-98 (1979)
Factors Affecting the Evaluation of Chemicals in Laboratory Experiments Using Marine Organism9 W. ERNST Institut
fiir
Meeresforschung,
2850 Bremerhaven, Received
August
Federal
Republic
of Germany
16, 1978
Two marine invertebrates, the common mussel, Mytilus edulis, and the polychaete, Lanice have been selected to demonstrate effects influencing the determination of concentration factors using o-HCH, y-HCH, and pentachlorophenol as test substances. Bioconcentration factors were measured at steady-state concentrations in a static system. Species difference and lipid content of the animals appeared to seriously affect bioconcentration factors. Variation of temperature, and metabolic activity, as conjugation of PCP in the mussels, showed no remarkable effect. A tentative translation of laboratory data to the environment has been made with acceptable results.
conchilega,
One of the criteria for the ecotoxicological evaluation of organic chemicals is their bioconcentration in aquatic organisms. Bioconcentration potentials can be measured in laboratory experiments but it is an important requirement that these results should be of a predictive value so that a reliable extrapolation to natural ecosystems can be achieved. There are numerous factors which contribute to the complexity of natural environments and may have an influence on bioaccumulation so that their elucidation in every respect seems to be impracticable. Furthermore, an extensive simulation of nature on a laboratory scale is not feasible. For that reason, rather simple test procedures and the knowledge of the key factors governing the processes to be investigated are assumed to be a reasonable approach. Marine organisms are representatives of an environment that is the ultimate sink for numerous chemicals, and animals from coastal areas can especially act as monitor organisms. From an experimental point of view they are preferably suitable as test organisms because they can display their full physiological activity at low ambient temperatures; because of the lower volatility of the test substances at this temperature static bioaccumulation experiments are favored. From our previous experiments, it seemed likely that species differences, lipid content, temperature, and metabolism of the compounds may have an effect on the establishment of bioconcentration factors. This paper deals with the elucidation of these parameters using the common mussel, Mytifus edulis, and the polychaete worm, Lanice conchilega, as test organisms, and a-HCH, y-HCH, and pentachlorophenol (PCP) as test substances. These substances were chosen, because they are outstanding representatives among the organohalogen comr Paper presented at the meeting on “Scientific Basis for the Ecotoxicological Environmental Chemicals” August 16- 18, 1978, Vienna. 0147-6513/79/010090-09$02.00/O Copyright 8 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
90
Assessment of
FACTORS
AFFECTING
EVALUATION
OF CHEMICALS
91
pounds in our coastal region and have been analyzed in our studies over long periods. A tentative prediction of environmental levels of these compounds in animals is made on the basis of the laboratory data. TEST ANIMALS The common mussel was chosen as a reference organism in the bioaccumulation studies for the following reasons: (1) it is a sedentary animal and can be maintained without food through the experiment; (2) the partition of the substances is achieved in rather short periods due to the high filtration rate of more than 2 liters. hr’ *g dry wt-l (Widdows, 1978) for the size class used; (3) the wet weight of the animals is 2-3 g, thus providing sufficient material for analysis when using whole individuals or dissected tissues. Although not as suitable as Mytilus in laboratory experiments, the polychaete L. conchilega has been selected in order to demonstrate species differences in accumulation tests. Lanice is a sedentary animal, highly abundant in our coastal area and behaves extraordinarily in bioconcentration (Ernst and Weber, 1978), thus contributing to the efforts of translating experimental results to natural conditions. DETERMINATION
OF THE STEADY
STATE
Bioconcentration factors at steady state conditions are increasingly acknowledged because they are more predictive and, for the purpose of comparison, better reproducible than those obtained within fixed periods of time before the steady state is reached. Usually test animals are exposed to a constant concentration of the substance in the water and are analyzed after different times until a plateau level is reached. In most bioaccumulation experiments fish is used, which requires considerable periods for equilibration. A valuable method for an acceleration of this procedure has been described by Branson (1975) for muscle tissue of the rainbow trout including a computerized calculation of bioconcentration factors. However, as a rule, relatively high numbers of animals are necessary to accomplish reasonable average values, and working at constant concentrations is more laborious. On the basis of the advantageous properties of mussels, mentioned above, it seemed justified to employ a static method, which has been described in more detail (Ernst, 1977) and is very briefly demonstrated by means of Fig. 1. The animals are exposed in an aquarium to a given initial concentration of the substance; according to the uptake of the substance into the animals the concentration in the water decreases along the curve. On the other hand, animals eliminate increasingly with increasing substance levels in their tissues. Both processes finally lead to a “steady state,” which is characterized by a concentration of the substance in the water, C w,t, that is practically constant with time. The concentration factor is then the quotient of the concentration in the animals, CA,,, and the water, CWst.The uptake proceeds very rapidly during the initial 50 hr and is terminated in mussels after about 8 days. Similar patterns of accumulation were observed by Mason and Rowe (1976) for dieldrin and endrin in the eastern oyster, Crassostrea virginica.
92
W. ERNST UPTAKE
CA, 1.k, cw ,,= cF. ‘w,,
d 0 -t(h)
50
100
150
FIG. 1. Typical equilibration curve for mussels during uptake of a substance from water. C,,: Initial concentration in sea water aquaria; CW.: steady-state concentration in the seawater; CA,,: concentration of the substance in mussels at steady state; K,: rate constant for uptake (hr-I); K,: rate constant for loss (hr-I). Concentration factor CF = CAs~Cws,.
ELIMINATION -- % dt T
=
‘4
k2.CA
=ti k,
FIG. 2. Elimination in mussels. C,: Concentration of the substance in animals; C,,: concentration of the substance in animals at f = 0 (usually identical with CAStin Fig. 1); K,: rate constant for elimination.
The quantitative description of the elimination via the elimination constant k, gives a useful indication of the retention of a substance in the animal. The elimination can be measured, when exposed animals are transferred to clean water and analyzed at different times during elimination, applying the following equations (Fig. 2): CA = CA,,-e-k’t, In CA = In CA0 - k,t,
FACTORS
AFFECTING
EVALUATION TABLE
OF CHEMICALS
93
1
CONCENTFUTIONFACTORSFOR a-HCH, y-HCH, AND PCP IN Mytilus edulis AND Lunice conchilega FOR WET TISSUES AT 10°C” Substance a-HCH ;FpC”
Mytilus
Lanice
105 390 139
2750 3830 1240
N Sea water: 33 %Osalinity for Mytilus and 27 %a salinity for Lanice. Initial substance concentrations in sea water were 2-5 pg 1-l.
where CA = concentration of the substance in animals at time t and CA0 = concentration of the substance in animals at the beginning of elimination (to). It should be noted that these constants should be used cautiously because some simplifications have been made regarding the compartments of the animal. Very often the initial elimination of the substance proceeds faster resulting in higher k, values and lower half-lives respectively and for this, the calculation of k, values depends substantially on the period chosen in the course of elimination. Nevertheless, standardized experiments will provide a reasonable chance to make these values comparable. SPECIES DIFFERENCE Concentration factors for three test substances in the animal are shown in Table 1. It can readily be seen that Lanice exhibits a considerably higher bioaccumulation by a factor of more than 10 compared with Myths. This different behavior is confirmed when comparing elimination rates (Table 2). As with PCP, no measurable elimination could be detected in Lanice during the experimental period. Results from the literature, although not directly comparable since different TABLE
2
HALF-LIVES IN HOURSFOR a-HCH, yHCH, AND PCP IN Mytilus edulis AND Lanice conchilega” Substance a-HCH ;FpC”
Mytilus 19 22 78
Lmice n.m.* 113 500’
” For salinity of sea water see Table 1. * Not measured. c Experimental period with no measurable elimination.
94
W. ERNST
methods were used, contribute likewise to the fact that species differences have to be considered. In a terrestrial-aquatic ecosystem bioaccumulation factors for PCP were 205 for daphnia, 21 for snail, and 132 for fish (Lu et al., 1977). In the bluegill, Lepomis macrochirus, concentration factors from 10 to 350 were measured for PCP in various tissues after 8 days of exposure (Pruitt et al., 1977), whereas Kobayashi and Akitake (1975) reported lOOO-fold concentration of PCP in the goldfish, Carassius auratus.
Half-lives of the three substances appear to be rather high inA4ytilus; this is due to the inclusion of elimination data of a 400-hr elimination period. From reasons mentioned above, half-lives are increasing at longer elimination periods. In fact these half lives are reduced by a factor of 4 and more, when the initial period of 20-50 hr is chosen for the calculation. In the rainbow trout similar half lives for PCP were observed in various tissues over a short elimination period of 24 hr (Glickman et al., 1977).
LIPID
CONTENT
OF TEST ANIMALS
In analytical work whole animals were used and the results were usually expressed on a wet weight basis. Average values from 5- 10 animals from batches sampled at the same time and location were comparable, but variation among individuals was considerably high with standard deviations of up to +30% (n = 10). Furthermore, great differences in bioconcentration factors were observed in the polychaete collected at different times of the year. Determination of the lipid contents of mussels exhibited that a correlation exists between lipid contents and concentration factors on a wet basis (Fig. 3). Correspondingly, standard deviations on a fat weight basis were better than 216%. As with polychaetes, a similar, but less distinct, correlation could be shown so far; obviously other factors besides
.
. l.. 1.0
. .
.
.
.
Date of collection . = Nov /Dec.1975 o=
April
. = Jun
I 100 +
FIG. weight
Concentration
3. Plot of lipid contents of mussels basis. Lipids = extractables without
1976 1976
, 200 factor
vs concentration factors further specification.
for
y-HCH,
calculated
on a wet
FACTORS AFFECTING
EVALUATION TABLE
95
OF CHEMICALS
3
CONCENTRATIONFACTORSFOR PCP AND y-HCH IN Myths edulis AT DIFFERENT TEMPERATURESOFTHE SEAWATER(33 %O SALINITY) ON WET WEIGHT BASIS” Concentration Temperature (“C)
PCP 326 304 324
5 10 15
factor y-HCH
177 (12 421) 142 (12 222) 151 (12 223)
n Values in brackets: related to lipid.
lipids may be additionally this species.
responsible
EFFECT
for variation
of concentration
factors in
OF TEMPERATURE
In most experiments a constant temperature of 10°C for the sea water was chosen, but in order to extrapolate the results to the environment it seems necessary to cover a temperature range from 5 to 15°C. From preliminary results with mussels no remarkable effect on the concentration factor for cr-HCH, y-HCH, and PCP could be stated (Table 3). The only discernible effect was a faster rate of uptake of the substances at 15°C (Fig. 4); this has been found also with other compounds.
. .
I 1
FIG.
15°C.
4. Equilibration
Time
50 (h)
I
1
I
100
150
200
curve for y-HCH in mussels at different temperatures. Circles: 5°C; squares:
96
W. ERNST
METABOLISM Among marine invertebrates mussels do not substantially degrade environmental chemicals as far as the majority of organohalogen compounds is concerned. However, conjugation with sulfate is well known, e.g., for naphthol in the crab, Maia squinado (Corner et al., 1973) and for PCP in the short-necked clam, Tapes philippinurum (Kobayashi et al., 1970). In our experiments mussels likewise conjugated PCP with sulfate very readily. The formation of the conjugate during equilibration is exhibited in Fig. 5. In repeated runs with varying PCP concentrations a reproducible pattern of conjugation could be achieved, showing that the formation of the sulfated PCP did not affect the concentration factor. TRANSLATION
OF LABORATORY
RESULTS
TO THE ENVIRONMENT
Experimentally derived concentration factors can serve for a prediction of environmental levels of the compounds in animals, if the concentration of these substances in the surrounding water is known over periods long enough to attain steady-state conditions. Although these investigations are expensive and not easy to perform they are extraordinarily important, at least with selected compounds, to prove the usefulness of laboratory data from an ecotoxicological point of view. From our analytical results in a coastal area in combination with experimental concentration factors tentative predictions of the environmental contamination of mussels and Lunice with a-HCH, y-HCH, and PCP have been made. The results in Table 4 give an indication how the predicted values match the actual field observations. CONCLUSION The evaluation of numerous chemicals in view of their ecotoxicology is based on a number of criteria one of which is bioconcentration. Bioconcentration in aquatic ecosystems proceeds primarily via uptake from water. Biomagnification along food webs occurs most likely with various compounds and should be carefully considered but the processes are rather complex, difficult to survey, and should be excluded from an accelerated testing for the time being. Their contribution to the complex of accumulation may either be estimated from more sophisticated tests or from appropriate calculations.
TABLE TENTATIVE y-HCH,
4
PREDICTION OF ENVIRONMENTAL CONCENTRATION OF a-HCH, AND PCP IN Lunice conchilega AND Mytilus edulis ON THE BASIS OF EXPERIMENTALLY DERIVED CONCENTRATION FACTORS a-HCH
y-HCH Animal
P”
m
Lanice Mytilus
7 1
4-14 -1
20 1
in field samples.
Values
n p: Predicted;
m: measured
P
PCP m 23-57 l-2
are in nanograms
P
m
120 10
160 3-10
per gram
wet weight.
2.5
.
5.0
5OC
. '.
2.5
20
.
10%
i
1.0
1OT
10%
.
1 0 --,
l .
. exposureterminated
I 50 Tfme
100
150
200
( h)
FIG. 5. Equilibration curve for [‘“C]PCP and formation of conjugated [‘“C]PCP concentrations in seawater, and different temperatures. Open conjugated); full circles: free [‘“C]PCP.
97
[‘“C]PCP in mussels at different circles: total [*T]PCP (free and
W. ERNST
98
Bioconcentration factors obtained via uptake from water should be established at steady-state conditions as far as possible. Reproducible data can be achieved with the common mussel, M. edulis, within relatively short periods using a static test. Different lipid contents of the animals have been found to be the cause for rather differing concentration factors in individuals, if the results are expressed on a wet weight basis. In order to standardize test methods, the lipid contents of the animals should be regarded. Bioconcentration factors considerably depend on the species chosen, even in the same test procedure. If threshold concentrations of organic pollutants in water should be established, these species differences have to be considered at least by an appropriate margin of safety. Temperature of the seawater in the range of 5- 15°C and sulfate conjugation with PCP does not seem very likely to interfere with the determination of concentration factors. ACKNOWLEDGMENTS I thank Frau R. Ernst and Frau I. Johannsen for helpful cooperation supported by the Deutsche Forschungsgemeinschaft.
and assistance. This work was
REFERENCES BRANSON, D. R., BLAU,G. E.,ALEXANDER,H. C.,ANDNEELY, W. B. (1975). Bioconcentrationof2,2’, 4,4’-tetrachlorobiphenyl in rainbow trout as measured by an accelerated test. Trans. Amer. Fish. Sot. 104, 785-792.
CORNER,E. D., KILVINGTON, C. C., AND O’HARA, S. C. M. (1973). Qualitative studies on the metabolism of naphthalene in Maia Squinado (Herbst). J. Mar. Biol. Ass. U.K. 53, 819-832. ERNST,~. (1977). Determination of the bioconcentration potential of marine organisms. A steady state approach. I. Bioconcentration data for seven chlorinated pesticides in mussels (Mytilus edulis) and their relation to solubility data. Chemosphere 6, 731-740. ERNST, W., AND WEBER, K. (1978). Chlorinated phenols in estuarine bottom fauna. Chemosphere 7, 867-872.
GLICKMAN, A. H., STATHAM, C. N., Wu, A., AND LECH, J. L. (1977). Studies on the uptake, metabolism, and disposition of pentachlorophenol and pentachloroanisole in rainbow trout. Toxicof. Appl.
Pharmacol.
41, 649-658.
KOBAYASHI, K., AKITAKE, H., AND TOMIYAMA, T. (1970). Studies on the metabolism of pentachlorophenate, a herbicide, in aquatic organisms. III. Isolation and identification of aconjugated PCP yielded by a shell-fish, Tapes philippinarum. Bull. Jap. Sot. Sci. Fish. 36, 103-108. KOBAYASHI, K., AND AKITAKE, H. (1975). Studies on the metabolism of chlorophenols in fish. I. Absorption and excretion of PCP in goldfish. Bul/. Jap. Sot. Sci. Fish. 41, 87-92. Lu, P. -Y., METCALF, R. L., AND COLE, L. K. (1977). The environmental fate ofi4C-pentachlorophenol in laboratory model ecosystems. Abstract In Symposium on Pentachlorophenol (K. Rango Rao, and N. L. Richards, ed.) p. 6. U.S. Environmental Protection Agency and the University of West Florida, Pensacola, Fla. MASON, J. W., AND ROWE, D. R. (1976). The accumulation and loss of dieldrin and endrin in the eastern oyster. Arch. Environ. Contam. Toxicol. 4, 349-360. PRUITT, G. W., GRANTHAM, B. J., AND PIERCE, H. R. (1977). Accumulation and elimination of pentachlorophenol by the bluegill, Lepomis macrochirus. Trans. Amer. Fish. Sot. 106, 462-465. WIDDOWS, J. (1978). Combined effects ofbody size, food concentration and season on the physiology of Mytilus edulis. .I. Mar. Biol. Ass. U.K. 58, 109-124.
AND
ENVIRONMENTAL
SAFETY
3, 90-98 (1979)
Factors Affecting the Evaluation of Chemicals in Laboratory Experiments Using Marine Organism9 W. ERNST Institut
fiir
Meeresforschung,
2850 Bremerhaven, Received
August
Federal
Republic
of Germany
16, 1978
Two marine invertebrates, the common mussel, Mytilus edulis, and the polychaete, Lanice have been selected to demonstrate effects influencing the determination of concentration factors using o-HCH, y-HCH, and pentachlorophenol as test substances. Bioconcentration factors were measured at steady-state concentrations in a static system. Species difference and lipid content of the animals appeared to seriously affect bioconcentration factors. Variation of temperature, and metabolic activity, as conjugation of PCP in the mussels, showed no remarkable effect. A tentative translation of laboratory data to the environment has been made with acceptable results.
conchilega,
One of the criteria for the ecotoxicological evaluation of organic chemicals is their bioconcentration in aquatic organisms. Bioconcentration potentials can be measured in laboratory experiments but it is an important requirement that these results should be of a predictive value so that a reliable extrapolation to natural ecosystems can be achieved. There are numerous factors which contribute to the complexity of natural environments and may have an influence on bioaccumulation so that their elucidation in every respect seems to be impracticable. Furthermore, an extensive simulation of nature on a laboratory scale is not feasible. For that reason, rather simple test procedures and the knowledge of the key factors governing the processes to be investigated are assumed to be a reasonable approach. Marine organisms are representatives of an environment that is the ultimate sink for numerous chemicals, and animals from coastal areas can especially act as monitor organisms. From an experimental point of view they are preferably suitable as test organisms because they can display their full physiological activity at low ambient temperatures; because of the lower volatility of the test substances at this temperature static bioaccumulation experiments are favored. From our previous experiments, it seemed likely that species differences, lipid content, temperature, and metabolism of the compounds may have an effect on the establishment of bioconcentration factors. This paper deals with the elucidation of these parameters using the common mussel, Mytifus edulis, and the polychaete worm, Lanice conchilega, as test organisms, and a-HCH, y-HCH, and pentachlorophenol (PCP) as test substances. These substances were chosen, because they are outstanding representatives among the organohalogen comr Paper presented at the meeting on “Scientific Basis for the Ecotoxicological Environmental Chemicals” August 16- 18, 1978, Vienna. 0147-6513/79/010090-09$02.00/O Copyright 8 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
90
Assessment of
FACTORS
AFFECTING
EVALUATION
OF CHEMICALS
91
pounds in our coastal region and have been analyzed in our studies over long periods. A tentative prediction of environmental levels of these compounds in animals is made on the basis of the laboratory data. TEST ANIMALS The common mussel was chosen as a reference organism in the bioaccumulation studies for the following reasons: (1) it is a sedentary animal and can be maintained without food through the experiment; (2) the partition of the substances is achieved in rather short periods due to the high filtration rate of more than 2 liters. hr’ *g dry wt-l (Widdows, 1978) for the size class used; (3) the wet weight of the animals is 2-3 g, thus providing sufficient material for analysis when using whole individuals or dissected tissues. Although not as suitable as Mytilus in laboratory experiments, the polychaete L. conchilega has been selected in order to demonstrate species differences in accumulation tests. Lanice is a sedentary animal, highly abundant in our coastal area and behaves extraordinarily in bioconcentration (Ernst and Weber, 1978), thus contributing to the efforts of translating experimental results to natural conditions. DETERMINATION
OF THE STEADY
STATE
Bioconcentration factors at steady state conditions are increasingly acknowledged because they are more predictive and, for the purpose of comparison, better reproducible than those obtained within fixed periods of time before the steady state is reached. Usually test animals are exposed to a constant concentration of the substance in the water and are analyzed after different times until a plateau level is reached. In most bioaccumulation experiments fish is used, which requires considerable periods for equilibration. A valuable method for an acceleration of this procedure has been described by Branson (1975) for muscle tissue of the rainbow trout including a computerized calculation of bioconcentration factors. However, as a rule, relatively high numbers of animals are necessary to accomplish reasonable average values, and working at constant concentrations is more laborious. On the basis of the advantageous properties of mussels, mentioned above, it seemed justified to employ a static method, which has been described in more detail (Ernst, 1977) and is very briefly demonstrated by means of Fig. 1. The animals are exposed in an aquarium to a given initial concentration of the substance; according to the uptake of the substance into the animals the concentration in the water decreases along the curve. On the other hand, animals eliminate increasingly with increasing substance levels in their tissues. Both processes finally lead to a “steady state,” which is characterized by a concentration of the substance in the water, C w,t, that is practically constant with time. The concentration factor is then the quotient of the concentration in the animals, CA,,, and the water, CWst.The uptake proceeds very rapidly during the initial 50 hr and is terminated in mussels after about 8 days. Similar patterns of accumulation were observed by Mason and Rowe (1976) for dieldrin and endrin in the eastern oyster, Crassostrea virginica.
92
W. ERNST UPTAKE
CA, 1.k, cw ,,= cF. ‘w,,
d 0 -t(h)
50
100
150
FIG. 1. Typical equilibration curve for mussels during uptake of a substance from water. C,,: Initial concentration in sea water aquaria; CW.: steady-state concentration in the seawater; CA,,: concentration of the substance in mussels at steady state; K,: rate constant for uptake (hr-I); K,: rate constant for loss (hr-I). Concentration factor CF = CAs~Cws,.
ELIMINATION -- % dt T
=
‘4
k2.CA
=ti k,
FIG. 2. Elimination in mussels. C,: Concentration of the substance in animals; C,,: concentration of the substance in animals at f = 0 (usually identical with CAStin Fig. 1); K,: rate constant for elimination.
The quantitative description of the elimination via the elimination constant k, gives a useful indication of the retention of a substance in the animal. The elimination can be measured, when exposed animals are transferred to clean water and analyzed at different times during elimination, applying the following equations (Fig. 2): CA = CA,,-e-k’t, In CA = In CA0 - k,t,
FACTORS
AFFECTING
EVALUATION TABLE
OF CHEMICALS
93
1
CONCENTFUTIONFACTORSFOR a-HCH, y-HCH, AND PCP IN Mytilus edulis AND Lunice conchilega FOR WET TISSUES AT 10°C” Substance a-HCH ;FpC”
Mytilus
Lanice
105 390 139
2750 3830 1240
N Sea water: 33 %Osalinity for Mytilus and 27 %a salinity for Lanice. Initial substance concentrations in sea water were 2-5 pg 1-l.
where CA = concentration of the substance in animals at time t and CA0 = concentration of the substance in animals at the beginning of elimination (to). It should be noted that these constants should be used cautiously because some simplifications have been made regarding the compartments of the animal. Very often the initial elimination of the substance proceeds faster resulting in higher k, values and lower half-lives respectively and for this, the calculation of k, values depends substantially on the period chosen in the course of elimination. Nevertheless, standardized experiments will provide a reasonable chance to make these values comparable. SPECIES DIFFERENCE Concentration factors for three test substances in the animal are shown in Table 1. It can readily be seen that Lanice exhibits a considerably higher bioaccumulation by a factor of more than 10 compared with Myths. This different behavior is confirmed when comparing elimination rates (Table 2). As with PCP, no measurable elimination could be detected in Lanice during the experimental period. Results from the literature, although not directly comparable since different TABLE
2
HALF-LIVES IN HOURSFOR a-HCH, yHCH, AND PCP IN Mytilus edulis AND Lanice conchilega” Substance a-HCH ;FpC”
Mytilus 19 22 78
Lmice n.m.* 113 500’
” For salinity of sea water see Table 1. * Not measured. c Experimental period with no measurable elimination.
94
W. ERNST
methods were used, contribute likewise to the fact that species differences have to be considered. In a terrestrial-aquatic ecosystem bioaccumulation factors for PCP were 205 for daphnia, 21 for snail, and 132 for fish (Lu et al., 1977). In the bluegill, Lepomis macrochirus, concentration factors from 10 to 350 were measured for PCP in various tissues after 8 days of exposure (Pruitt et al., 1977), whereas Kobayashi and Akitake (1975) reported lOOO-fold concentration of PCP in the goldfish, Carassius auratus.
Half-lives of the three substances appear to be rather high inA4ytilus; this is due to the inclusion of elimination data of a 400-hr elimination period. From reasons mentioned above, half-lives are increasing at longer elimination periods. In fact these half lives are reduced by a factor of 4 and more, when the initial period of 20-50 hr is chosen for the calculation. In the rainbow trout similar half lives for PCP were observed in various tissues over a short elimination period of 24 hr (Glickman et al., 1977).
LIPID
CONTENT
OF TEST ANIMALS
In analytical work whole animals were used and the results were usually expressed on a wet weight basis. Average values from 5- 10 animals from batches sampled at the same time and location were comparable, but variation among individuals was considerably high with standard deviations of up to +30% (n = 10). Furthermore, great differences in bioconcentration factors were observed in the polychaete collected at different times of the year. Determination of the lipid contents of mussels exhibited that a correlation exists between lipid contents and concentration factors on a wet basis (Fig. 3). Correspondingly, standard deviations on a fat weight basis were better than 216%. As with polychaetes, a similar, but less distinct, correlation could be shown so far; obviously other factors besides
.
. l.. 1.0
. .
.
.
.
Date of collection . = Nov /Dec.1975 o=
April
. = Jun
I 100 +
FIG. weight
Concentration
3. Plot of lipid contents of mussels basis. Lipids = extractables without
1976 1976
, 200 factor
vs concentration factors further specification.
for
y-HCH,
calculated
on a wet
FACTORS AFFECTING
EVALUATION TABLE
95
OF CHEMICALS
3
CONCENTRATIONFACTORSFOR PCP AND y-HCH IN Myths edulis AT DIFFERENT TEMPERATURESOFTHE SEAWATER(33 %O SALINITY) ON WET WEIGHT BASIS” Concentration Temperature (“C)
PCP 326 304 324
5 10 15
factor y-HCH
177 (12 421) 142 (12 222) 151 (12 223)
n Values in brackets: related to lipid.
lipids may be additionally this species.
responsible
EFFECT
for variation
of concentration
factors in
OF TEMPERATURE
In most experiments a constant temperature of 10°C for the sea water was chosen, but in order to extrapolate the results to the environment it seems necessary to cover a temperature range from 5 to 15°C. From preliminary results with mussels no remarkable effect on the concentration factor for cr-HCH, y-HCH, and PCP could be stated (Table 3). The only discernible effect was a faster rate of uptake of the substances at 15°C (Fig. 4); this has been found also with other compounds.
. .
I 1
FIG.
15°C.
4. Equilibration
Time
50 (h)
I
1
I
100
150
200
curve for y-HCH in mussels at different temperatures. Circles: 5°C; squares:
96
W. ERNST
METABOLISM Among marine invertebrates mussels do not substantially degrade environmental chemicals as far as the majority of organohalogen compounds is concerned. However, conjugation with sulfate is well known, e.g., for naphthol in the crab, Maia squinado (Corner et al., 1973) and for PCP in the short-necked clam, Tapes philippinurum (Kobayashi et al., 1970). In our experiments mussels likewise conjugated PCP with sulfate very readily. The formation of the conjugate during equilibration is exhibited in Fig. 5. In repeated runs with varying PCP concentrations a reproducible pattern of conjugation could be achieved, showing that the formation of the sulfated PCP did not affect the concentration factor. TRANSLATION
OF LABORATORY
RESULTS
TO THE ENVIRONMENT
Experimentally derived concentration factors can serve for a prediction of environmental levels of the compounds in animals, if the concentration of these substances in the surrounding water is known over periods long enough to attain steady-state conditions. Although these investigations are expensive and not easy to perform they are extraordinarily important, at least with selected compounds, to prove the usefulness of laboratory data from an ecotoxicological point of view. From our analytical results in a coastal area in combination with experimental concentration factors tentative predictions of the environmental contamination of mussels and Lunice with a-HCH, y-HCH, and PCP have been made. The results in Table 4 give an indication how the predicted values match the actual field observations. CONCLUSION The evaluation of numerous chemicals in view of their ecotoxicology is based on a number of criteria one of which is bioconcentration. Bioconcentration in aquatic ecosystems proceeds primarily via uptake from water. Biomagnification along food webs occurs most likely with various compounds and should be carefully considered but the processes are rather complex, difficult to survey, and should be excluded from an accelerated testing for the time being. Their contribution to the complex of accumulation may either be estimated from more sophisticated tests or from appropriate calculations.
TABLE TENTATIVE y-HCH,
4
PREDICTION OF ENVIRONMENTAL CONCENTRATION OF a-HCH, AND PCP IN Lunice conchilega AND Mytilus edulis ON THE BASIS OF EXPERIMENTALLY DERIVED CONCENTRATION FACTORS a-HCH
y-HCH Animal
P”
m
Lanice Mytilus
7 1
4-14 -1
20 1
in field samples.
Values
n p: Predicted;
m: measured
P
PCP m 23-57 l-2
are in nanograms
P
m
120 10
160 3-10
per gram
wet weight.
2.5
.
5.0
5OC
. '.
2.5
20
.
10%
i
1.0
1OT
10%
.
1 0 --,
l .
. exposureterminated
I 50 Tfme
100
150
200
( h)
FIG. 5. Equilibration curve for [‘“C]PCP and formation of conjugated [‘“C]PCP concentrations in seawater, and different temperatures. Open conjugated); full circles: free [‘“C]PCP.
97
[‘“C]PCP in mussels at different circles: total [*T]PCP (free and
W. ERNST
98
Bioconcentration factors obtained via uptake from water should be established at steady-state conditions as far as possible. Reproducible data can be achieved with the common mussel, M. edulis, within relatively short periods using a static test. Different lipid contents of the animals have been found to be the cause for rather differing concentration factors in individuals, if the results are expressed on a wet weight basis. In order to standardize test methods, the lipid contents of the animals should be regarded. Bioconcentration factors considerably depend on the species chosen, even in the same test procedure. If threshold concentrations of organic pollutants in water should be established, these species differences have to be considered at least by an appropriate margin of safety. Temperature of the seawater in the range of 5- 15°C and sulfate conjugation with PCP does not seem very likely to interfere with the determination of concentration factors. ACKNOWLEDGMENTS I thank Frau R. Ernst and Frau I. Johannsen for helpful cooperation supported by the Deutsche Forschungsgemeinschaft.
and assistance. This work was
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