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Relationship between chronic toxicity and bioaccumulation of copper, cadmium and zinc as affected by water hardness and humic acid
Aquatic Toxicology, 8 (1986) 149-161
149
Elsevier
AQT 00195
RELATIONSHIP BETWEEN CHRONIC TOXICITY AND BIOACCUMULATION OF C O P P E R , C A D M I U M A N D ZINC AS A F F E C T E D BY W A T E R HARDNESS AND HUMIC ACID
ROBERT W. WINNER and JOSEPH D. GAUSS
Department of Zoology, Miami University, Oxford, OH 45056, U.S.A. (Received 19 November 1985; accepted 28 February 1986)
Using daphnids, we have evaluated the effects of water hardness and humic acid (HA) on the chronic toxicity and bioaccumulation of Cu, Cd and Zn. Although changes in water hardness and HA concentration changed bioaccumulation or chronic toxicity, or both, of each of the three metals, there was no consistent relationship between changes in toxicity and changes in bioaccumulation. Only for Cu in hard water was there a positive correlation between toxicity and bioaccumulation. The uptake of 65Zn by molted exoskeletons suggests that changes in water chemistry do indeed modify the bioavailability of metals as would be expected. That is, an increase in the concentration of Ca 2 ÷ or Mg 2 + or the increased chelation of metals by HA should decrease bioavailability. The complex storage, transformation and excretion processes in multiceUular animals, however, result in there being a poor correlation among bioavailability, bioaccumulation and toxicity. Key words: water hardness; humic acid; bioaccumulation; chronic toxicity; Cu; Cd; Zn; Daphnia
i NTRODUCTION
Although it is generally assumed that the chemical matrix of exposure water will affect the bioaccumulation and toxicity of heavy metals, there are relatively few studies in which either chronic toxicity or bioaccumulation of metals has been evaluated under conditions where the important components of water chemistry are known and have been systematically manipulated to determine their effect. We can find only two references which provide data on the effects of water chemistry on both chronic toxicity and bioaccumulation o f one or more metals. H u m i c acid (HA) was found to reduce both the toxicity to and bioaccumulation of Cd by the green alga, Selenastrum capricornutum (Sedlacek et al., 1983) and to increase the Cd-induced mortality, reduce the Cu-induced mortality, but to have no effect on the bioaccumulation o f either metal by daphnids (Winner, 1984). I f one looks for data dealing with the effects of water chemistry on either toxicity or bioaccumulation o f metals, the data base is somewhat larger but still rather limited. Equally important, the conclusions as to the effects of water chemistry on toxicity or bioaccumulation are frequently not in agreement a m o n g the published 0166-445X/86/$03.30 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
150 studies. The two most frequently studied aspects of water chemistry are the effects o f water hardness and humic substances on toxicity or bioaccumulation. The greatest number o f such papers deal with Cu or Cd. There is general agreement that H A reduces the acute and chronic toxicity of Cu (Zitko et al., 1973; Brown et al., 1974; Winner 1984, 1985). However, there are only two studies which evaluate the effects of H A on bioaccumulation of Cu (Winner, 1984, 1985). The direct effects o f water hardness on Cu toxicity are in dispute. The most recent U.S. E P A Water Quality Criteria for Copper (U.S. E P A , 1980) concludes that hardness reduces the acute, but has little effect on the chronic, toxicity o f Cu. Most of the cited literature, however, deals with acute toxicity and is research in which the effects of hardness and alkalinity have not been separated and in which the organic content of the test water was unknown. In those few cases where hardness has been evaluated at constant alkalinities, both effect ( C h a k o u m a k o s et al., 1979; Miller et al., 1980) and no effect (Zitko et al., 1976) on Cu toxicity have been reported. No references were found on the effects of water hardness on the bioaccumulation of Cu. There is contradictory information on the effect of H A on bioaccumulation of Cd by freshwater and marine organsisms. Some investigators (George and C o o m b s , 1977; R a m a m o o r t h y and Blumhagen, 1984) have reported that H A increased the bioaccumulation of Cd; others (Poldoski, 1979; Hung, 1982) report that H A reduced Cd bioaccumulation and one (Winner, 1984) that H A had no effect on bioaccumulation. Only one of these studies (Winner, 1984) related bioaccumulation to toxicity. The literature dealing with the effects of water hardness on Cd toxicity is also replete with contradictions. One study (Carroll et al., 1979) found that Ca 2 ÷ reduced the acute toxicity of Cd to b r o o k trout (Salvelinus fontinalis) while another (Zitko and Carson, 1976) reported that neither Ca 2 + nor Mg 2+ had any effect on the acute toxicity of Cd to juvenile Atlantic salmon (Salmo salar). One paper (Poldoski, 1979) has evaluated the short-term (2-day) effect of Ca 2 ÷ on Cd bioaccumulation by Daphnia magna and concluded that an increase in Ca caused a reduction in bioaccumulation. Several papers (Phillips, 1976; George et al., 1978; Wright, 1978) have reported that marine crustaceans and molluscs accumulate Cd faster in seawater of reduced salinity. Since the dilution technique reduces the concentration of all inorganic and organic constituents, this, however, does not necessarily indicate that the increase in bioaccumulation is due to a decrease in hardness. None of these studies has evaluated interactions a m o n g water chemistry and the bioaccumulation and toxicity of Cd. In those few cases where the effects of hardness on Zn toxicity have been evaluated in the absence of concurrent changes in alkalinity, an increase in test water hardness has resulted in a decrease in acute Zn toxicity (Carter, 1976; Zitko and Carson, 1976; H o l c o m b e and Andrew, 1978). The independent effect of water hardness on chronic Zn toxicity does not seem to have been evaluated. There are even fewer
151
data to document the effect of hardness on bioaccumulation of Zn, and these involve such short-term exposures (i.e., 24 h, Carter and Nicholas, 1978) or comparison of bioaccumulation at a single high Ca concentration (2000 mg Ca/l) with bioaccumulation in distilled water (Matthiessen and Brafield, 1977) that they are of little value in predicting what would occur under longer-term exposures in more realistic levels of water hardness. We could find only one paper (Zitko et al., 1973) which evaluated the effect of H A on acute Zn toxicity. The authors concluded that H A had no effect on the acute toxicity of Zn to juvenile Atlantic salmon. We could find no published data on the effects of H A on chronic toxicity or bioaccumulation of Zn. In view of the paucity of data on interactions a m o n g water chemistry and the toxicity and bioaccumulation of heavy metals, the objective of the present study was to evaluate the effects of water hardness and H A on the toxicity and bioaccumulation of Cu, Cd and Zn.
MATERIALS AND METHODS
All toxicity and bioaccumulation experiments were initiated with neonate daphnids ( < 24 h old) maintained individually in 40 ml of test water in 50-ml beakers covered with watch glasses. All tests were run at 20 +_ l ° C on a 16-h photoperiod in environmental chambers. Each animal was fed a daily ration o f Chlamydomonas reinhardtii (0.3 or 0.1 mg ash-free dry wt. for Daphnia magna and Daphnia pulex, respectively) from a vitamin-enriched culture (Winner and Farrell, 1976). At this time, any mortalities were recorded and offspring removed from the beakers. In the Cu and Cd experiments, animals were maintained in soft (58 m g / l hardness as CaCO3), medium-hard (115 m g / l hardness) or hard (230 mg/l) waters at a constant alkalinity of 100 mg/1. These waters were produced by adding reagent-grade salts to an ultrapure water (Winner, 1985). In the Zn experiments, a pond water was diluted with ultrapure water to a hardness of 50 mg/1 and an alkalinity of 49-50 mg/1. Test waters of 100 and 200 mg/1 hardness were prepared by adding appropriate weights of CaSO4 2 H 2 0 and MgSO4 while maintaining the 2:1 Ca:Mg ratio of the original pond water. H u m i c acid was added to some test waters as the technical grade sodium salt (Aldrich Chemical Co.). Humic acid concentrations in test water are expressed as ash-free dry weight. Metals were added as CuSO4 5H20, CdSO4 8H20 and ZnSO4 7H20. Animals were transferred into freshly prepared test solutions on M - W - F for the duration of experiments. Cu and Cd concentrations were measured by flameless, atomic absorption spectroscopy in each batch of freshly prepared test water and in the aged test waters at the time animals were transferred. Measured concentrations were within 10070 of nominal concentrations and did not decrease significantly over the 2- or 3-day exposure periods. Zn levels are given as nominal concentrations. In the Cu experiments, mean p H varied from
152
8.4 to 8.7 and did not differ significantly ( P < 0 . 0 5 ) a m o n g treatments (i.e., hardnesses, metal or H A concentrations). The higher p H values developed during the periods between transfer of animals to fresh media and were due to photosynthetic activity of the algal food (Winner, 1985). In the Cd experiments, mean p H varied f r o m 8.3 to 8.8 and also did not differ significantly a m o n g treatments. As for Cu, the lower p H values were for test waters prior to biological conditioning by algae. Treatment also had no significant effect on p H in the Zn experiments where mean p H varied f r o m 8.2 to 8.4. D. pulex was used in chronic Cu and Cd bioassays and D. magna in chronic Zn bioassays. Chronic toxicity was evaluated by comparing control and treatment survivorship curves by the Gehan-Wilcoxon Xz analysis using the SAS Survtest (Reinhardt, 1980). D. magna was used in all bioaccumulation experiments because of its larger mass which permitted fewer animals to be used in the analysis. It has previously been shown (Winner and Farrell, 1976) that D. magna and D. pulex do not differ in their sensitivity to a chronic Cu stress and that D. magna is even more sensitive to a chronic Cd stress than is D. pulex (Winner, unpubl, data). In any event, if bioaccumulation and toxicity of the metals are related to water chemistry of the exposure water, changes in water chemistry should have a consistent effect on bioaccumulation and toxicity. Copper and Cd bioaccumulations were determined by exposing animals to selected metal concentrations in the appropriate h a r d n e s s - H A matrix for 7 days (Cu) or 20-28 days (Cd). Animals were killed, dried to a constant weight, and individually weighed to the nearest 0.01 mg on a Cahn 25 electrobalance. Accumulated metal concentrations were determined by flameless, atomic-absorption spectroscopy after digesting animals in Baker Instra-analyzed HNO3. Reagent blanks and metal standards were processed along with each batch of animals. Zinc bioaccumulation was determined by individually exposing animals to selected Zn concentrations in appropriate water h a r d n e s s - H A combinations with the addition of 0.01/~Ci 65Zn/ml. Freshly molted exoskeletons from adult animals in Zn-free water were also exposed to 65Zn to determine Zn ' u p t a k e ' by the skeletal surface. In some experiments, an attempt was made to differentiate between physical adsorption o f Zn to exoskeletal surfaces and uptake of Zn by microorganisms associated with the exoskeleton. This was done by measuring Zn accumulation on exoskeletons exposed to Zn with or without the addition of two antibiotics (penicillin and streptomycin). Zinc accumulation was estimated by periodically transferring exposed animals or exoskeleton through two rinses of Znfree water and then transferring them, with a minimum of water, into 4-ml vials for counting in a Beckman B i o g a m m a spectrophotometer. After counting, and prior to being returned to their culture beakers, animals were measured from anterior edge of head to base of caudal spine (Winner, 1981). Dry weight was then calculated f r o m a body length-dry weight regression equation (Gauss, 1985). Copper and Cd data
153 are given as #g metal/g Daphn& and Zn data as/~g Zn accumulated/g Daphn&. Differences in accumulated Cu and Cd by animals, and Zn by exuviae, were detected by one-factor A N O V A and Duncan's new multiple range test (Duncan, 1955); and for accumulated Zn by animals by one-factor A N O V A , corrected for repeated measures on a single animal, and the Bonferroni multiple range test (Miller, 1981). All statements of significant differences are based on accepting P < 0 . 0 5 . RESULTS
Copper Interactive effects of water hardness and H A on the toxicity and bioaccumulation o f Cu in daphnids have been described (Winner, 1985). Results pertinent to the present discussion are summarized in Fig. 1. An increase in water hardness f r o m 58 to 115 m g / l resulted in a significant decrease in whole-body bioaccumulation in either the presence or absence o f 0.75 p p m H A (Fig. 1, left). Under neither o f these conditions, however, was there much, if any, change in the chronic Cu no-observableeffect concentration (NOEC) (Fig. 1, right). A further increase in hardness from 115 to 230 mg/1 had no additional effect on the bioaccumulation or toxicity of Cu in the absence of HA. However, in the presence of H A , changing exposure condition f r o m medium-hard to hard water resulted in a significant increase in whole-body bioaccumulation and a decrease in the N O E C o f Cu. Dialysis experiments (Table I) have shown that the inorganic Cu concentration is actually higher in hard water than in medium water when both contain the same H A and total Cu concentrations.
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Fig. !. Effect of water hardness (S = 58, M = 115, H = 230 mg/l) and humic acid (HA) on bioaccumulation (left) by and the chronic NOEC of copper (right) to daphnids.
154 TABLE 1 Effect of water hardness and humic acid (HA) on the concentration of inorganic Cu as measured by dialysis
Water hardness (mg CaCO~/I)
Inorganic Cu (#g/l) Exp. 1 ....... Bag 1 Bag 2
115 230
25.2 41.2
26.0 42.9
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Bag 2
x Inorganic Cu (SE)
28.9 36.8
28.6 42.5
27.2 (0.93) 41.1 (1.51)
Forty ml of Cu-free H20 was placed in dialysis bags (Spectrapore 6; M W cutoff 1,000) and immersed in one liter of water of the same hardness containing 40/~g C u / l and 1.5 mg HA/I. After 72 h, inorganic Cu was estimated by analyzing the copper concentration within the dialysis bags by flameless, atomicabsorption spectroscopy. Means are significantly different (Student's t-test, P < 0 . 0 5 ) .
Presumably, this is due to Cu 2 + being displaced from binding sites on H A by Ca 2 +, Mg 2+ , or both, in the hard water. However, even though H A caused an increase in toxicity and a decrease in b i o a c c u m u l a t i o n of Cu in hard water compared to
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Fig. 2. Effect of water hardness (top) and humic acid (bottom) on the survival of D. pulex exposed to 15/~g Cd/l. Each curve is for an initial cohort of l0 neonates. Increasing water hardness from soft (58 m g / l ) to hard (230 mg/l) significantly (P_<0.05) increased survival, whereas the addition of H A to m e d i u m - h a r d water significantly decreased survival.
155
medium-hard water, the pattern was not consistent when the effect o f H A on bioaccumulation and toxicity o f Cu are compared for hard and soft water. Animals accumulated more Cu in soft water but Cu was more toxic in hard water under those circumstances. Finally, although the effect o f H A on toxicity and bioaccumulation varied a m o n g the water hardnesses, H A consistently reduced chronic toxicity o f Cu in each of the three waters (Fig. 1, right). Readers are referred to Winner (1985) for more detailed information and statistical treatment o f the Cu experiments. Cadmium An increase in water hardness from 57 mg/1 to either 115 (not shown) or 230 mg/1 (Fig. 2, top) resulted in a significant decrease in chronic Cd toxicity as estimated from survivorship curves over a 40-day exposure period to 15 #g Cd/1. Animals exposed to Cd in soft and hard water over a 28-day exposure period and analyzed for whole-body Cd concentration on days 5, 10, 20 and 28, however, exhibited no significant differences in bioaccumulation o f Cd in the two waters (Fig. 3). Wholebody Cd concentrations were still increasing after 28 days o f exposure. As previously reported (Winner, 1984), H A had an unexpected effect on Cda
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Fig. 3. Bioaccumulation of Cd by D. magna exposed to 7.5/zg C d / l in soft (left) and hard (right) water. Each mean is based o0 a sample size of 10. Means (+ SE) not sharing the same letter are significantly (P_< 0.05) different.
156 induced mortality under conditions of chronic (40-day) exposure. In medium-hard water, animals died off more rapidly when exposed to 15 #g Cd/1 in the presence o f 0.75 mg H A / I than when exposed in the absence of H A (Fig. 2, bottom). This H A effect also occurs in soft and hard water (Winner, unpubl, data). When animals were exposed to 7.5/~g Cd/1 over a 20-day exposure period, and body loads of Cd measured on days 5, 10 and 20, however, animals exposed in the presence of 0.75 mg H A / I did not have significantly different body loads than did animals exposed in the absence of H A (Fig. 4). Again, animals had not reached a steady state in respect to Cd concentration after 20 days of exposure. Zinc An increase in the hardness of exposure water resulted in a significant reduction in chronic Zn toxicity as estimated from survivorship curves over a 50-day exposure to 125/zg Zn/1 (Fig. 5, top). However, the effect of water hardness on Zn toxicity was not accompanied by a reduction in Zn bioaccumulation. As measured by Zn accumulation over a 24-day exposure in soft and medium-hard water, animals had a significantly higher body load of Zn in soft water on only one of the 8 days of measurement (Fig. 6, top). The chronic toxicity o f Zn was also significantly reduced by H A over a 50-day exposure to 125/xg Zn/l in soft water (Fig. 5, bottom). As was the case for water hardness, however, H A did not significantly reduce the accumulab
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157
tion of Zn by daphnids over a 24-day exposure period to 50 #g Z n / l . Body loads of Zn were lower in animals exposed in presence of H A on only one o f the 8 days o f measurement (Fig. 6, bottom) but also were significantly higher at one time in the exposure period. Although neither water hardness nor H A had any effect on bioaccumulation of Zn, both consistently reduced the accumulation of Zn by molted exoskeletons (Fig. 7). This association of Zn with exoskeletal surfaces may not be entirely passive since the presence of antibiotics significantly reduced the accumulation of Zn on the exoskeletons (Fig. 7). This could be due to either an inhibition of uptake by microorganisms associated with the exoskeletal surfaces or to a competition between Zn binding sites on these surfaces and binding sites on the antibiotic molecules. DISCUSSION
It is clear, in the case o f all three metals, that a change in water chemistry does not consistently have the same effect on metal bioaccumulation and toxicity.
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Fig. 5. Effect of water hardness (top) and humic acid (bottom) on the survival o f D. magna exposed to 125 #g Zn/1. Each curve is for an initial cohort o f 10 neonates. Increasing water hardness from soft (52 mg/l) to m e d i u m - h a r d (102 mg/I), or the addition of humic acid to soft water significantly ( P ~ 0 . 0 5 ) increased survival.
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Although it has not been demonstrated previously that changes in water chemistry may differentially modify metal toxicity and bioaccumulation, there are several studies (Brown, 1977; Dixon and Sprague, 1981) which show an inverse correlation between whole-body accumulation and toxicity. There are several possible explanations for the fact that whole-body metal accumulation and toxicity may be poorly, or even negatively, correlated. The most probable is that much o f the accumulated metal is in some nontoxic form (Dixon and Sprague, 1981) such as being complexed with a metabolically inactive protein, for example, a metallothionein. It is also possible that the only important fraction o f the total metal accumulated is concentrated in some relatively small target organ or organelles and that significant changes in metal concentrations o f that target, or targets, are too small a fraction o f the changes in whole-body metal concentration to be detected. Finally, in some
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E X P O S U R E TIME ( H R S ) Fig. 7. Effect of water hardness (left), humic acid in soft water (middle), and antibiotics (right) on Zn accumulation by cast exoskeletons of D. magna exposed to 50/zg Zn/l. Means (+_ SE) joined by solid lines for a treatment or sharing the same letter at a sampling time are not significantly (P_<0.05) different. Each mean is based on a sample size of 5.
cases, the disparity between bioaccumulation and toxicity may be due to the toxic action occurring at an organism-water interface such as the gill surfaces. Some of the differences in published results on the effect o f water chemistry on metal bioaccumulation may reflect differences in the time interval over which bioaccumulation was measured. For example, on the basis o f a 2-day exposure (Poldoski, 1979), it was concluded that H A significantly reduced whole-body Cd accumulation by D. magna. However, it is obvious from the present study that, although D. magna had still not reached a Cd equilibrium after a 28-day exposure, there was no change in whole-body Cd loads between days 10 and 20 in the experiment dealing with the effect o f water hardness on Cd bioaccumulation (Fig. 3). If additional data had not been collected we might have concluded that Cd body loads had reached a steady state after 10 days o f exposure. In another study (Rainbow et al., 1980), it was reported that H A reduced bioaccumulation o f Cd by the barnacle, Semibalanus balanoides, over a 7-day exposure but had no effect over a 15- or 30-day exposure. Of the three heavy metals studied, only for Zn did body burdens decrease under conditions o f continuous exposure. We are aware o f only two other studies that show similar reductions. Sticklebacks, Gaterosteus aculeatus, were able to reduce accumulated Zn levels to near control values despite continued exposure to 1 or 4 mg Zn/l (Matthiessen and Brafield, 1977). The gut and gills were hypothesized as probable routes o f Zn excretion. The shrimp, Crangon crangon, after a rapid initial uptake o f Cd, lost metal for several days before again increasing body levels (Dethlefsen, 1978). A n incipient oversaturation o f available binding sites was hypothesized as the reason for the unexpected loss o f Cd.
160 The u p t a k e o f Z n by molted exoskeletons in the present s t u d y suggests that the effects o f water hardness a n d H A o n Z n b i o a v a i l a b i l i t y are what w o u l d be predicted f r o m our k n o w l e d g e o f a q u a t i c chemistry, i.e., that b i o a v a i l a b i l i t y w o u l d be reduced by c o m p e t i t i o n with other divalent cations or by chelation o f Z n with H A . If the effect o f antibiotics o n a c c u m u l a t i o n o f Z n b y the exoskeletons is due to bacterial i n h i b i t i o n , it w o u l d suggest that b i o a c c u m u l a t i o n o f metals is m o r e closely related to their b i o a v a i l a b i l i t y in m i c r o o r g a n i s m s t h a n it is in multicellular a n i m a l s which have longer life spans a n d m o r e c o m p l e x systems for storing, t r a n s f o r m i n g a n d excreting metals. I n a n y event, it seems clear that, for a q u a t i c a n i m a l s , b i o a v a i l a b i l i t y c a n n o t be t r a n s l a t e d directly into b i o a c c u m u l a t i o n , n o r b i o a c c u m u l a t i o n into toxicity. U n t i l these relationships are better u n d e r s t o o d , it is p r o b a b l y m i s l e a d i n g to infer the m e t a l - e x p o s u r e history or p o t e n t i a l toxicity o f a b o d y o f water f r o m metal conc e n t r a t i o n s of o r g a n i s m s collected f r o m that b o d y . It w o u l d also seem that o u r u n d e r s t a n d i n g o f the effects o f water chemistry o n metal toxicity is i n a d e q u a t e to allow use o f water chemistry as a tool for predicting levels o f a metal which w o u l d protect the biota, or fractions thereof, in a p a r t i c u l a r b o d y o f water.
ACKNOWLEDGEMENT This research was s u p p o r t e d by cooperative a g r e e m e n t n o . 809224010 with the E n v i r o n m e n t a l Research L a b o r a t o r y , U.S. E P A , Corvallis, O R .
REFERENCES Brown, V.M., T.L. Shaw and D.G. Shurber, 1974. Aspects of water quality and the toxicity of copper to rainbow trout. Water Res. 8, 797-803. Brown, B.E., 1977. Uptake of copper and lead by a metal-tolerant isopod Asellus meridianus Rac. Freshw. Biol. 7, 235-244. Carroll, J.J., S.J. Ellis and W.S. Oliver, 1979. Influence of hardness constituents on the acute toxicity of cadmium to brook trout (Salvelinusfontinalis). Bull. Environ. Contam. Toxicol. 22, 575-581. Carter, J.G.T., 1976. The effect of dissolved zinc on the larvae of Simulium orantipes Skuse (Diptera: Nematocera). Ph.D. Dissertation, Australian Nat. Univ. Carter, J.G.T. and W.L. Nicholas, 1978. Uptake of zinc by the aquatic larvae of Simulium ornatipes (Diptera: Nematocera). Aust. J. Mar. Freshw. Res. 29, 299-309. Chakoumakos, C., R.C. Russo and R.V. Thurston, 1979. Toxicity of copper to cutthroat trout (Salmo clarki) under different conditions of alkalinity, pH and hardness. Environ. Sci. Technol. 13,213-219. Dethlefsen, V., 1978. Uptake, retention and loss of cadmium by brown shrimp (Crangon crangon). Meeresforsch 26, 137-152. Dixon, D.G. and J.B. Sprague, 1981. Copper bioaccumulation and hepatoprotein synthesis during acclimation to copper by juvenile rainbow trout. Aquat. Toxicol. 1, 69-81. Duncan, D.B., 1955. Multiple range and multiple F tests. Biometrics 11, 1-42. Gauss, J.D., 1985. Influence of water hardness and humic acid on zinc toxicity and accumulation in Daphnia magna Strauss. Ph.D. Dissert., Miami Univ., Oxford, OH, U.S.A.
161 George, S.G. and T.L. Coombs, 1977. The effects of chelating agents on the uptake and accumulation of cadmium by Mytilus edulis. Mar. Biol. 39, 261-268. George, S.G., E. Carpene and T.L. Coombs, 1978, The effect of salinity on the uptake of cadmium by the common mussel, Mytilus edulis (L.). In: Physiology and behavior of marine organisms, edited by D.S. McLusky and A.J. Berry, Proc. 12th Eur. Mar. Biol. Symp., Pergamon Press, pp. 189-193. Holcombe, G.W. and R.W. Andrew, 1978. The acute toxicity of zinc to rainbow and brook trout: comparisons in hard and soft water. U.S. EPA, EPA-600/3-78-094, Duluth, MN, U.S.A. Hung, Y.W., 1982. Effects of temperature and chelating agents on cadmium uptake in the American oyster. Bull. Environ. Contam. Toxicol. 28, 546-551. Matthiessen, P. and A.E. Brafield, 1977. Uptake and loss of dissolved zinc by the stickleback Gasterosteus aculeatus (L.). J. Fish Biol. 10, 399-410. Miller, R.G., Jr., 1981. Simultaneous statistical inference. Springer-Verlag Co., New York. Miller, T.G. and W.C. Mackay, 1980. The effects of hardness, alkalinity and pH of test water on the toxicity of copper to rainbow trout (Salmo gairdneri). Water Res. 14, 129-133. Phillips, D.J.H., 1976. The common mussel Mytilus edulis as an indicator of pollution by zinc, cadmium, lead and copper. 1. Effects of environmental variables on uptake by metals. Mar. Biol. 38, 56-69. Poldoski, J.E., 1979. Cadmium bioaccumulation assays. Their relationship to various ionic equilibria in Lake Superior water. Environ. Sci. Tech. 13, 701-706. Rainbow, P.S., A.G. Scott. E.A. Wiggins and R.W. Jackson, 1980. Effect of chelating agents on the accumulation of cadmium by the barnacle, Semibalanus balanoides, and complexation of soluble Cd, Zn, and Cu. Mar. Ecol. Prog. Ser. 2, 143-152. Ramamoorthy, S. and K. Blumhagen, 1984. Uptake by Zn, Cd, and Hg by fish in the presence of competing compartments. Can. J. Fish. Aquat. Sci. 41, 750-756. Sedlacek, J., T. Kallqvist and E. Gjessing, 1983. Effect of aquatic humus on uptake and toxicity of cadmium to Selenastrurn capricornutum Printz. In: Aquatic and terrestrial humic materials, edited by R.F. Christman and E.T. Gjessing, Ann Arbor Sci., Ann Arbor, MI, pp. 495-516. U.S. Environmental Protection Agency, 1980. Ambient water quality criteria for copper. EPA 440/5-80-036. U.S. Govt. Print. Office, Washington, DC. Winner, R.W., 1981. A comparison of body length, brood size and longevity as indices of chronic copper and zinc stresses in Daphnia magna. Environ. Pollut. (Ser. A). 26, 33-37. Winner, R.W., 1984. The toxicity and bioaccumulation of cadmium and copper as affected by humic acid. Aquat. Toxicol. 5, 267-274. Winner, R.W., 1985. Bioaccumulation and toxicity of copper as affected by interactions between humic acid and water hardness. Water Res. 19, 449-455. Winner, R.W. and M.P. Farrell, 1976. Acute and chronic toxicity of copper to four species of Daphnia. J. Fish. Res. Board Can. 33, 1685-1691. Wright, D.A., 1977. The effect of salinity on cadmium uptake by the tissues of the shore crab, Caroinus maenas (L.). J. Exp. Biol. 63, 137-146. Zitko, P. and W.G. Carson, 1976. A mechanism of the effects of water hardness on the lethality of heavy metals to fish. Chemosphere 5, 299-303. Zitko, P., W.V. Carson and W.G. Carson, 1973. Prediction of incipient lethal levels of copper to juvenile Atlantic salmon in the presence of humid acid by cupric electrode. Bull. Environ. Contam. Toxicol. 10, 265-271.
149
Elsevier
AQT 00195
RELATIONSHIP BETWEEN CHRONIC TOXICITY AND BIOACCUMULATION OF C O P P E R , C A D M I U M A N D ZINC AS A F F E C T E D BY W A T E R HARDNESS AND HUMIC ACID
ROBERT W. WINNER and JOSEPH D. GAUSS
Department of Zoology, Miami University, Oxford, OH 45056, U.S.A. (Received 19 November 1985; accepted 28 February 1986)
Using daphnids, we have evaluated the effects of water hardness and humic acid (HA) on the chronic toxicity and bioaccumulation of Cu, Cd and Zn. Although changes in water hardness and HA concentration changed bioaccumulation or chronic toxicity, or both, of each of the three metals, there was no consistent relationship between changes in toxicity and changes in bioaccumulation. Only for Cu in hard water was there a positive correlation between toxicity and bioaccumulation. The uptake of 65Zn by molted exoskeletons suggests that changes in water chemistry do indeed modify the bioavailability of metals as would be expected. That is, an increase in the concentration of Ca 2 ÷ or Mg 2 + or the increased chelation of metals by HA should decrease bioavailability. The complex storage, transformation and excretion processes in multiceUular animals, however, result in there being a poor correlation among bioavailability, bioaccumulation and toxicity. Key words: water hardness; humic acid; bioaccumulation; chronic toxicity; Cu; Cd; Zn; Daphnia
i NTRODUCTION
Although it is generally assumed that the chemical matrix of exposure water will affect the bioaccumulation and toxicity of heavy metals, there are relatively few studies in which either chronic toxicity or bioaccumulation of metals has been evaluated under conditions where the important components of water chemistry are known and have been systematically manipulated to determine their effect. We can find only two references which provide data on the effects of water chemistry on both chronic toxicity and bioaccumulation o f one or more metals. H u m i c acid (HA) was found to reduce both the toxicity to and bioaccumulation of Cd by the green alga, Selenastrum capricornutum (Sedlacek et al., 1983) and to increase the Cd-induced mortality, reduce the Cu-induced mortality, but to have no effect on the bioaccumulation o f either metal by daphnids (Winner, 1984). I f one looks for data dealing with the effects of water chemistry on either toxicity or bioaccumulation o f metals, the data base is somewhat larger but still rather limited. Equally important, the conclusions as to the effects of water chemistry on toxicity or bioaccumulation are frequently not in agreement a m o n g the published 0166-445X/86/$03.30 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
150 studies. The two most frequently studied aspects of water chemistry are the effects o f water hardness and humic substances on toxicity or bioaccumulation. The greatest number o f such papers deal with Cu or Cd. There is general agreement that H A reduces the acute and chronic toxicity of Cu (Zitko et al., 1973; Brown et al., 1974; Winner 1984, 1985). However, there are only two studies which evaluate the effects of H A on bioaccumulation of Cu (Winner, 1984, 1985). The direct effects o f water hardness on Cu toxicity are in dispute. The most recent U.S. E P A Water Quality Criteria for Copper (U.S. E P A , 1980) concludes that hardness reduces the acute, but has little effect on the chronic, toxicity o f Cu. Most of the cited literature, however, deals with acute toxicity and is research in which the effects of hardness and alkalinity have not been separated and in which the organic content of the test water was unknown. In those few cases where hardness has been evaluated at constant alkalinities, both effect ( C h a k o u m a k o s et al., 1979; Miller et al., 1980) and no effect (Zitko et al., 1976) on Cu toxicity have been reported. No references were found on the effects of water hardness on the bioaccumulation of Cu. There is contradictory information on the effect of H A on bioaccumulation of Cd by freshwater and marine organsisms. Some investigators (George and C o o m b s , 1977; R a m a m o o r t h y and Blumhagen, 1984) have reported that H A increased the bioaccumulation of Cd; others (Poldoski, 1979; Hung, 1982) report that H A reduced Cd bioaccumulation and one (Winner, 1984) that H A had no effect on bioaccumulation. Only one of these studies (Winner, 1984) related bioaccumulation to toxicity. The literature dealing with the effects of water hardness on Cd toxicity is also replete with contradictions. One study (Carroll et al., 1979) found that Ca 2 ÷ reduced the acute toxicity of Cd to b r o o k trout (Salvelinus fontinalis) while another (Zitko and Carson, 1976) reported that neither Ca 2 + nor Mg 2+ had any effect on the acute toxicity of Cd to juvenile Atlantic salmon (Salmo salar). One paper (Poldoski, 1979) has evaluated the short-term (2-day) effect of Ca 2 ÷ on Cd bioaccumulation by Daphnia magna and concluded that an increase in Ca caused a reduction in bioaccumulation. Several papers (Phillips, 1976; George et al., 1978; Wright, 1978) have reported that marine crustaceans and molluscs accumulate Cd faster in seawater of reduced salinity. Since the dilution technique reduces the concentration of all inorganic and organic constituents, this, however, does not necessarily indicate that the increase in bioaccumulation is due to a decrease in hardness. None of these studies has evaluated interactions a m o n g water chemistry and the bioaccumulation and toxicity of Cd. In those few cases where the effects of hardness on Zn toxicity have been evaluated in the absence of concurrent changes in alkalinity, an increase in test water hardness has resulted in a decrease in acute Zn toxicity (Carter, 1976; Zitko and Carson, 1976; H o l c o m b e and Andrew, 1978). The independent effect of water hardness on chronic Zn toxicity does not seem to have been evaluated. There are even fewer
151
data to document the effect of hardness on bioaccumulation of Zn, and these involve such short-term exposures (i.e., 24 h, Carter and Nicholas, 1978) or comparison of bioaccumulation at a single high Ca concentration (2000 mg Ca/l) with bioaccumulation in distilled water (Matthiessen and Brafield, 1977) that they are of little value in predicting what would occur under longer-term exposures in more realistic levels of water hardness. We could find only one paper (Zitko et al., 1973) which evaluated the effect of H A on acute Zn toxicity. The authors concluded that H A had no effect on the acute toxicity of Zn to juvenile Atlantic salmon. We could find no published data on the effects of H A on chronic toxicity or bioaccumulation of Zn. In view of the paucity of data on interactions a m o n g water chemistry and the toxicity and bioaccumulation of heavy metals, the objective of the present study was to evaluate the effects of water hardness and H A on the toxicity and bioaccumulation of Cu, Cd and Zn.
MATERIALS AND METHODS
All toxicity and bioaccumulation experiments were initiated with neonate daphnids ( < 24 h old) maintained individually in 40 ml of test water in 50-ml beakers covered with watch glasses. All tests were run at 20 +_ l ° C on a 16-h photoperiod in environmental chambers. Each animal was fed a daily ration o f Chlamydomonas reinhardtii (0.3 or 0.1 mg ash-free dry wt. for Daphnia magna and Daphnia pulex, respectively) from a vitamin-enriched culture (Winner and Farrell, 1976). At this time, any mortalities were recorded and offspring removed from the beakers. In the Cu and Cd experiments, animals were maintained in soft (58 m g / l hardness as CaCO3), medium-hard (115 m g / l hardness) or hard (230 mg/l) waters at a constant alkalinity of 100 mg/1. These waters were produced by adding reagent-grade salts to an ultrapure water (Winner, 1985). In the Zn experiments, a pond water was diluted with ultrapure water to a hardness of 50 mg/1 and an alkalinity of 49-50 mg/1. Test waters of 100 and 200 mg/1 hardness were prepared by adding appropriate weights of CaSO4 2 H 2 0 and MgSO4 while maintaining the 2:1 Ca:Mg ratio of the original pond water. H u m i c acid was added to some test waters as the technical grade sodium salt (Aldrich Chemical Co.). Humic acid concentrations in test water are expressed as ash-free dry weight. Metals were added as CuSO4 5H20, CdSO4 8H20 and ZnSO4 7H20. Animals were transferred into freshly prepared test solutions on M - W - F for the duration of experiments. Cu and Cd concentrations were measured by flameless, atomic absorption spectroscopy in each batch of freshly prepared test water and in the aged test waters at the time animals were transferred. Measured concentrations were within 10070 of nominal concentrations and did not decrease significantly over the 2- or 3-day exposure periods. Zn levels are given as nominal concentrations. In the Cu experiments, mean p H varied from
152
8.4 to 8.7 and did not differ significantly ( P < 0 . 0 5 ) a m o n g treatments (i.e., hardnesses, metal or H A concentrations). The higher p H values developed during the periods between transfer of animals to fresh media and were due to photosynthetic activity of the algal food (Winner, 1985). In the Cd experiments, mean p H varied f r o m 8.3 to 8.8 and also did not differ significantly a m o n g treatments. As for Cu, the lower p H values were for test waters prior to biological conditioning by algae. Treatment also had no significant effect on p H in the Zn experiments where mean p H varied f r o m 8.2 to 8.4. D. pulex was used in chronic Cu and Cd bioassays and D. magna in chronic Zn bioassays. Chronic toxicity was evaluated by comparing control and treatment survivorship curves by the Gehan-Wilcoxon Xz analysis using the SAS Survtest (Reinhardt, 1980). D. magna was used in all bioaccumulation experiments because of its larger mass which permitted fewer animals to be used in the analysis. It has previously been shown (Winner and Farrell, 1976) that D. magna and D. pulex do not differ in their sensitivity to a chronic Cu stress and that D. magna is even more sensitive to a chronic Cd stress than is D. pulex (Winner, unpubl, data). In any event, if bioaccumulation and toxicity of the metals are related to water chemistry of the exposure water, changes in water chemistry should have a consistent effect on bioaccumulation and toxicity. Copper and Cd bioaccumulations were determined by exposing animals to selected metal concentrations in the appropriate h a r d n e s s - H A matrix for 7 days (Cu) or 20-28 days (Cd). Animals were killed, dried to a constant weight, and individually weighed to the nearest 0.01 mg on a Cahn 25 electrobalance. Accumulated metal concentrations were determined by flameless, atomic-absorption spectroscopy after digesting animals in Baker Instra-analyzed HNO3. Reagent blanks and metal standards were processed along with each batch of animals. Zinc bioaccumulation was determined by individually exposing animals to selected Zn concentrations in appropriate water h a r d n e s s - H A combinations with the addition of 0.01/~Ci 65Zn/ml. Freshly molted exoskeletons from adult animals in Zn-free water were also exposed to 65Zn to determine Zn ' u p t a k e ' by the skeletal surface. In some experiments, an attempt was made to differentiate between physical adsorption o f Zn to exoskeletal surfaces and uptake of Zn by microorganisms associated with the exoskeleton. This was done by measuring Zn accumulation on exoskeletons exposed to Zn with or without the addition of two antibiotics (penicillin and streptomycin). Zinc accumulation was estimated by periodically transferring exposed animals or exoskeleton through two rinses of Znfree water and then transferring them, with a minimum of water, into 4-ml vials for counting in a Beckman B i o g a m m a spectrophotometer. After counting, and prior to being returned to their culture beakers, animals were measured from anterior edge of head to base of caudal spine (Winner, 1981). Dry weight was then calculated f r o m a body length-dry weight regression equation (Gauss, 1985). Copper and Cd data
153 are given as #g metal/g Daphn& and Zn data as/~g Zn accumulated/g Daphn&. Differences in accumulated Cu and Cd by animals, and Zn by exuviae, were detected by one-factor A N O V A and Duncan's new multiple range test (Duncan, 1955); and for accumulated Zn by animals by one-factor A N O V A , corrected for repeated measures on a single animal, and the Bonferroni multiple range test (Miller, 1981). All statements of significant differences are based on accepting P < 0 . 0 5 . RESULTS
Copper Interactive effects of water hardness and H A on the toxicity and bioaccumulation o f Cu in daphnids have been described (Winner, 1985). Results pertinent to the present discussion are summarized in Fig. 1. An increase in water hardness f r o m 58 to 115 m g / l resulted in a significant decrease in whole-body bioaccumulation in either the presence or absence o f 0.75 p p m H A (Fig. 1, left). Under neither o f these conditions, however, was there much, if any, change in the chronic Cu no-observableeffect concentration (NOEC) (Fig. 1, right). A further increase in hardness from 115 to 230 mg/1 had no additional effect on the bioaccumulation or toxicity of Cu in the absence of HA. However, in the presence of H A , changing exposure condition f r o m medium-hard to hard water resulted in a significant increase in whole-body bioaccumulation and a decrease in the N O E C o f Cu. Dialysis experiments (Table I) have shown that the inorganic Cu concentration is actually higher in hard water than in medium water when both contain the same H A and total Cu concentrations.
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154 TABLE 1 Effect of water hardness and humic acid (HA) on the concentration of inorganic Cu as measured by dialysis
Water hardness (mg CaCO~/I)
Inorganic Cu (#g/l) Exp. 1 ....... Bag 1 Bag 2
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Forty ml of Cu-free H20 was placed in dialysis bags (Spectrapore 6; M W cutoff 1,000) and immersed in one liter of water of the same hardness containing 40/~g C u / l and 1.5 mg HA/I. After 72 h, inorganic Cu was estimated by analyzing the copper concentration within the dialysis bags by flameless, atomicabsorption spectroscopy. Means are significantly different (Student's t-test, P < 0 . 0 5 ) .
Presumably, this is due to Cu 2 + being displaced from binding sites on H A by Ca 2 +, Mg 2+ , or both, in the hard water. However, even though H A caused an increase in toxicity and a decrease in b i o a c c u m u l a t i o n of Cu in hard water compared to
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Fig. 2. Effect of water hardness (top) and humic acid (bottom) on the survival of D. pulex exposed to 15/~g Cd/l. Each curve is for an initial cohort of l0 neonates. Increasing water hardness from soft (58 m g / l ) to hard (230 mg/l) significantly (P_<0.05) increased survival, whereas the addition of H A to m e d i u m - h a r d water significantly decreased survival.
155
medium-hard water, the pattern was not consistent when the effect o f H A on bioaccumulation and toxicity o f Cu are compared for hard and soft water. Animals accumulated more Cu in soft water but Cu was more toxic in hard water under those circumstances. Finally, although the effect o f H A on toxicity and bioaccumulation varied a m o n g the water hardnesses, H A consistently reduced chronic toxicity o f Cu in each of the three waters (Fig. 1, right). Readers are referred to Winner (1985) for more detailed information and statistical treatment o f the Cu experiments. Cadmium An increase in water hardness from 57 mg/1 to either 115 (not shown) or 230 mg/1 (Fig. 2, top) resulted in a significant decrease in chronic Cd toxicity as estimated from survivorship curves over a 40-day exposure period to 15 #g Cd/1. Animals exposed to Cd in soft and hard water over a 28-day exposure period and analyzed for whole-body Cd concentration on days 5, 10, 20 and 28, however, exhibited no significant differences in bioaccumulation o f Cd in the two waters (Fig. 3). Wholebody Cd concentrations were still increasing after 28 days o f exposure. As previously reported (Winner, 1984), H A had an unexpected effect on Cda
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156 induced mortality under conditions of chronic (40-day) exposure. In medium-hard water, animals died off more rapidly when exposed to 15 #g Cd/1 in the presence o f 0.75 mg H A / I than when exposed in the absence of H A (Fig. 2, bottom). This H A effect also occurs in soft and hard water (Winner, unpubl, data). When animals were exposed to 7.5/~g Cd/1 over a 20-day exposure period, and body loads of Cd measured on days 5, 10 and 20, however, animals exposed in the presence of 0.75 mg H A / I did not have significantly different body loads than did animals exposed in the absence of H A (Fig. 4). Again, animals had not reached a steady state in respect to Cd concentration after 20 days of exposure. Zinc An increase in the hardness of exposure water resulted in a significant reduction in chronic Zn toxicity as estimated from survivorship curves over a 50-day exposure to 125/zg Zn/1 (Fig. 5, top). However, the effect of water hardness on Zn toxicity was not accompanied by a reduction in Zn bioaccumulation. As measured by Zn accumulation over a 24-day exposure in soft and medium-hard water, animals had a significantly higher body load of Zn in soft water on only one of the 8 days of measurement (Fig. 6, top). The chronic toxicity o f Zn was also significantly reduced by H A over a 50-day exposure to 125/xg Zn/l in soft water (Fig. 5, bottom). As was the case for water hardness, however, H A did not significantly reduce the accumulab
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157
tion of Zn by daphnids over a 24-day exposure period to 50 #g Z n / l . Body loads of Zn were lower in animals exposed in presence of H A on only one o f the 8 days o f measurement (Fig. 6, bottom) but also were significantly higher at one time in the exposure period. Although neither water hardness nor H A had any effect on bioaccumulation of Zn, both consistently reduced the accumulation of Zn by molted exoskeletons (Fig. 7). This association of Zn with exoskeletal surfaces may not be entirely passive since the presence of antibiotics significantly reduced the accumulation of Zn on the exoskeletons (Fig. 7). This could be due to either an inhibition of uptake by microorganisms associated with the exoskeletal surfaces or to a competition between Zn binding sites on these surfaces and binding sites on the antibiotic molecules. DISCUSSION
It is clear, in the case o f all three metals, that a change in water chemistry does not consistently have the same effect on metal bioaccumulation and toxicity.
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Although it has not been demonstrated previously that changes in water chemistry may differentially modify metal toxicity and bioaccumulation, there are several studies (Brown, 1977; Dixon and Sprague, 1981) which show an inverse correlation between whole-body accumulation and toxicity. There are several possible explanations for the fact that whole-body metal accumulation and toxicity may be poorly, or even negatively, correlated. The most probable is that much o f the accumulated metal is in some nontoxic form (Dixon and Sprague, 1981) such as being complexed with a metabolically inactive protein, for example, a metallothionein. It is also possible that the only important fraction o f the total metal accumulated is concentrated in some relatively small target organ or organelles and that significant changes in metal concentrations o f that target, or targets, are too small a fraction o f the changes in whole-body metal concentration to be detected. Finally, in some
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E X P O S U R E TIME ( H R S ) Fig. 7. Effect of water hardness (left), humic acid in soft water (middle), and antibiotics (right) on Zn accumulation by cast exoskeletons of D. magna exposed to 50/zg Zn/l. Means (+_ SE) joined by solid lines for a treatment or sharing the same letter at a sampling time are not significantly (P_<0.05) different. Each mean is based on a sample size of 5.
cases, the disparity between bioaccumulation and toxicity may be due to the toxic action occurring at an organism-water interface such as the gill surfaces. Some of the differences in published results on the effect o f water chemistry on metal bioaccumulation may reflect differences in the time interval over which bioaccumulation was measured. For example, on the basis o f a 2-day exposure (Poldoski, 1979), it was concluded that H A significantly reduced whole-body Cd accumulation by D. magna. However, it is obvious from the present study that, although D. magna had still not reached a Cd equilibrium after a 28-day exposure, there was no change in whole-body Cd loads between days 10 and 20 in the experiment dealing with the effect o f water hardness on Cd bioaccumulation (Fig. 3). If additional data had not been collected we might have concluded that Cd body loads had reached a steady state after 10 days o f exposure. In another study (Rainbow et al., 1980), it was reported that H A reduced bioaccumulation o f Cd by the barnacle, Semibalanus balanoides, over a 7-day exposure but had no effect over a 15- or 30-day exposure. Of the three heavy metals studied, only for Zn did body burdens decrease under conditions o f continuous exposure. We are aware o f only two other studies that show similar reductions. Sticklebacks, Gaterosteus aculeatus, were able to reduce accumulated Zn levels to near control values despite continued exposure to 1 or 4 mg Zn/l (Matthiessen and Brafield, 1977). The gut and gills were hypothesized as probable routes o f Zn excretion. The shrimp, Crangon crangon, after a rapid initial uptake o f Cd, lost metal for several days before again increasing body levels (Dethlefsen, 1978). A n incipient oversaturation o f available binding sites was hypothesized as the reason for the unexpected loss o f Cd.
160 The u p t a k e o f Z n by molted exoskeletons in the present s t u d y suggests that the effects o f water hardness a n d H A o n Z n b i o a v a i l a b i l i t y are what w o u l d be predicted f r o m our k n o w l e d g e o f a q u a t i c chemistry, i.e., that b i o a v a i l a b i l i t y w o u l d be reduced by c o m p e t i t i o n with other divalent cations or by chelation o f Z n with H A . If the effect o f antibiotics o n a c c u m u l a t i o n o f Z n b y the exoskeletons is due to bacterial i n h i b i t i o n , it w o u l d suggest that b i o a c c u m u l a t i o n o f metals is m o r e closely related to their b i o a v a i l a b i l i t y in m i c r o o r g a n i s m s t h a n it is in multicellular a n i m a l s which have longer life spans a n d m o r e c o m p l e x systems for storing, t r a n s f o r m i n g a n d excreting metals. I n a n y event, it seems clear that, for a q u a t i c a n i m a l s , b i o a v a i l a b i l i t y c a n n o t be t r a n s l a t e d directly into b i o a c c u m u l a t i o n , n o r b i o a c c u m u l a t i o n into toxicity. U n t i l these relationships are better u n d e r s t o o d , it is p r o b a b l y m i s l e a d i n g to infer the m e t a l - e x p o s u r e history or p o t e n t i a l toxicity o f a b o d y o f water f r o m metal conc e n t r a t i o n s of o r g a n i s m s collected f r o m that b o d y . It w o u l d also seem that o u r u n d e r s t a n d i n g o f the effects o f water chemistry o n metal toxicity is i n a d e q u a t e to allow use o f water chemistry as a tool for predicting levels o f a metal which w o u l d protect the biota, or fractions thereof, in a p a r t i c u l a r b o d y o f water.
ACKNOWLEDGEMENT This research was s u p p o r t e d by cooperative a g r e e m e n t n o . 809224010 with the E n v i r o n m e n t a l Research L a b o r a t o r y , U.S. E P A , Corvallis, O R .
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