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Does the age of metal-dissolved organic carbon complexes influence binding of metals to fish gills?
ELSEVIER
Aquatic
Toxicology
35 (1996) 253-264
Does the age of metal-dissolved organic carbon complexes influence binding of metals to fish gills? Lydia Hollis”, Kent Burnisonb, Richard
C. Playlea>*
“Department of Biology, Wiljiid Laurier University, Waterloo, Ont., N2L 3C5, Canada “National Water Research Institute, Environment Canada, Burlington, Ont., L7R 4A6, Canada Accepted
20 March
1996
Abstract We investigated the effect of aging on the strength of dissolved organic carbon (DOC) complexation of metals, as indicated by the ability of DOC to keep copper and cadmium off trout gills. The addition of S-20 mg C 1-l DOC reduced the toxicity of Cu and Cd to small rainbow trout (Oncorhynchus mykiss, 1 g). Exposures were to 0.2 PM Cu and 0.03 PM Cd for 2 weeks, in synthetic soft water. The DOC was isolated from surface waters of a marsh through reverse osmosis. Copper was kept off the gills by DOC but Cd was not, in agreement with the relative binding strengths of the metals to DOC and to the gills themselves. Aging 0.5 pM Cu, 0.1 pM Cd, and 5 mg C 1-l DOC solutions for 3 weeks did not change the ability of DOC to keep Cu or Cd off trout gills: 3 week old and fresh metal-DOC solutions kept Cu off the gills equally well, whereas Cd was always only partially kept off the gills by DOC. Our results indicate that efforts to model metal-gill interactions in fish from waters of differing chemistries and DOC content do not need to consider the age of metalDOC complexes, because metal-DOC complexes do not become appreciably stronger with time. Keywords:
Dissolved
organic
carbon;
Copper;
Cadmium;
Fish;
Gills
1. Introduction Fish gills are the first organ to be affected by many waterborne metals (e.g. Wood, 1992). We have been determining metal-gill binding constants for modelling metal-gill interactions and prediction of toxic effects of waterborne metals to fish (Cu and Cd, Playle et al., 1993a; Playle et al., 1993b; Ag, Janes and Playle, 1995; Co, Pb, Hg, R.C. Playle, unpublished data, 1995 and 1996). Dissolved organic carbon (DOC) is an important modifying agent of metal toxicity to fish (e.g. *Corresponding
author.
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Spry and Wiener, 1991; Welsh et al., 1993) and acts by complexing metals so that less free metal is available to bind to the gills. It is important to investigate whether the age of metal-DOC complexes influences the protective effect of DOC against metal binding to fish gills. From our previous work, DOC and fish gills both bind metals relatively strongly, with conditional equilibrium binding constants of log Kou_noo = 9.1, log &-uoo = 7.4, and log K~~-noo = 9.0, compared with gill binding constants of log Kou-sill= 7.4, log Kod-ai]t = 8.6, and log KAs_sill= 10.0 (Playle et al., 1993b; Janes and Playle, 1995). From these values, DOC binds Cu about 50 times better than Cu binds to gills, but the gills bind Cd and Ag about 16 times and about 10 times better, respectively, than does DOC. For Cd, greater than expected binding to the gills is likely related to active uptake of Cd through Ca channels (Verbost et al., 1989). Dissolved organic carbon is a general term for poorly defined breakdown products of organic material. In freshwater the source of DOC is mainly plant material. Freshwater DOC is composed of humic and fulvic acids, with carboxyl and phenol groups mainly being responsible for metal chelation (Morel and Hering, 1993). In previous work, we showed that to a first approximation the source and size fraction of DOC does not influence the protective effect of DOC against Cu accumulation on or in fish gills (Playle et al., 1993a). Aging of metal-DOC solutions, however, could increase the apparent metal binding constant of DOC for a metal if metalDOC bonds become stronger with time (e.g. over 2 days, Clark and Choppin, 1990). Similarly, DOC could better prevent metal accumulation on or in gills if the slower, double exchange reaction of metal deposition onto the gills increases in importance with aging. The double exchange reaction would develop at the expense of metal-ligand dissociation which first releases free metal which then can bind to the gills (e.g. adjunctive versus disjunctive mechanisms; Morel and Hering, 1993, pp. 395-405). In the present work, we investigated changes in the protective effects of DOC against Cu and Cd binding to gills of rainbow trout, as an indication of changes in the strength of DOC complexation of metals. Both Cu and Cd are of environmental concern, are gill toxicants, and affect ionoregulation in fish by interfering with Na uptake (Cu; e.g. Lauren and McDonald, 1986) and with Ca uptake (Cd; e.g. Verbost et al., 1989; Wicklund Glynn et al., 1994). We used natural DOC isolated from a marsh, as opposed to terrestrial humic acid, to make our experiments as environmentally relevant as possible. We aged our metal-DOC solutions for 3 weeks, much longer than the 2 days which gave increased strength of binding (from 4 to 38% strong bonds; Clark and Choppin, 1990). Copper binds well to DOC but relatively weakly to gills, whereas Cd binds less well to DOC than to fish gills (see above); use of these two metals in our trout and softwater system was expected to demonstrate biologically relevant, time-related changes in metal binding strength of DOC.
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2. Materials and methods Rainbow trout (Oncorhynchus mykiss) were obtained from Rainbow Springs Hatchery in Thamesford, Ontario. Fish were held in low Ca water (see below) produced using a reverse osmosis system (Culligan of Canada, Ltd., Mississauga, Ontario). Fish were fed Martin’s Starter Food (Martin Feed Mills, Elmira, Ontario), but were not fed during experiments. A 15 day experiment was run in five randomly arranged polyethylene buckets, each containing 10 1 of aerated soft water (Ca, 240 + 30 PM (n = 20); Na, 2620 k 170 PM (n = 20); 11°C pH 7.64kO.02 (n = 25); mean? 1 SEM). Thirty or 31 trout (1.45 + 0.05 g, n = 137) were placed randomly into each of the buckets. Six fish were sampled from each bucket at 5 h, 2 days, and 8 days, and the remaining fish (n = 3-12) at 15 days, and their gills analyzed for Cd and Cu (see below). Each bucket corresponded to one of five treatments: 1. 2. 3. 4. 5.
no added DOC, no added metals; no added DOC, plus metals (Cu and Cd); 5 mg C 1-l DOC, plus metals; 10 mg C 1-l DOC, plus metals; 20 mg C 1-r DOC, plus metals.
Nominal metal additions were 0.2 pM Cu and 0.03 pM Cd. Measured DOC and total metal concentrations taken during the experiment are given in Section 3. Measurement of DOC and metal concentrations throughout the experiment would account for any losses of DOC or metals from the solutions. Cadmium was added as CdC12*HZ0 (Baker Chemical Co., Phillipsburg, NJ) and Cu was added as CuS04*5H20 (AnalaR, BDH Toronto, Ontario). Depending on the number of fish left in a bucket, 2 or 3 1 of treatment water were replaced daily with water that was aging as the experiment progressed. This experiment was not replicated because of our limited supply of DOC concentrate. Added DOC was concentrated from surface waters of Luther Marsh, near Grand Valley, Ontario (43”54’N, 80”24’W) through reverse osmosis (Filmtec NF40 membrane, Minneapolis, MN; molecular weight cutoff 400 Da) after glass fibre filtration (1 pm). This process concentrates molecules of over 400 Da, so the isolated Luther Marsh DOC was a mixture of both fulvic and humic acids. Concentration of DOC by physical means such as reverse osmosis and ultrafiltration is considered better than by chemical means because large volumes of water can be processed with little modification of the DOC (Clair et al., 1991). Our concentrated DOC contained Cu, either because of contact with Cu in the reverse osmosis unit or because Cu was preferentially retained during the concentration process. To remove Cu, the DOC concentrate was passed through a cation exchange resin (Dowex 5OW-X8, 20-50 mesh, H-form, Baker Chemical Co., Phillipsburg, NJ). This treatment made the DOC concentrate more stable (e.g. no precipitation of material during storage), likely because metal removal eliminated the development of metal-DOC crossbridges leading to precipitate formation.
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A second experiment with fresh or aged (3-week-old) metal or metal-DOC solutions was run in six 2-l polyethylene containers, each containing five randomly placed trout (1.20 + 0.08 g, n = 56) in 1 1 of aerated synthetic soft water (Ca, 9 f 1 uM (n = 12); Na, 1260 + 20 uM (n = 12); 18°C pH 6.91 ? 0.03 (n = 24)). The same batch of synthetic soft water was used for all treatments (e.g. aerated for 3 weeks, then DOC or metals added if necessary), to assure similar water chemistry in all treatments. Fish gills were sampled after 4 h and were analyzed for Cu and Cd concentrations; sampling time meant that fish were exposed to the solutions between 4 and 5 h. This experiment was run in duplicate on 2 different days. Each container in the experiment corresponded to one of six treatments: 1. 2. 3. 4. 5. 6.
no added DOC, no added metals, aged 3 weeks (SW); no added DOC, plus metals (Cu and Cd), aged 3 weeks (Aged Metals); 3-week-old SW with metals added at the start of the 4 h exposure (Fresh Metals) ; added DOC, added metals, aged 3 weeks (Aged DOC, Aged Metals); 3-week-old SW with DOC and metals added at the start of the 4 h exposure (Fresh DOC, Fresh Metals); added DOC, no added metals, aged 3 weeks, with metals added at the start of the 4 h exposure (Aged DOC, Fresh Metals).
Nominal DOC additions were 5 mg C l-l, and nominal Cu plus Cd additions were 0.5 uM and 0.1 PM, respectively; measured values taken during the experiments are given in Section 3. After exposure to the various treatments in both experiments, fish were killed with a blow to the head, both sets of gills were excised, and gills were rinsed for 10 s in 100 ml of synthetic soft water. Extracted gills were weighed and then digested in 10 times their weight of 1 N HNOs (Ultrex II Ultrapure HNOs, Baker Analyzed reagent) for 3 h at about 80°C. Gill digests were shaken, left to settle for 10 min, then the supernatant was diluted 10 times with E-pure deionized water (Fisher Scientific Ltd., Unionville, Ont.). Gill metal concentrations were measured on a graphite furnace atomic absorption spectrophotometer (Varian AA-1275 with GTA-95 atomizer) using 10 ~1 injection volumes and N2 gas. Operating conditions were 30 s drying time at 90°C 12 s at 120°C and 4 s at 1800 or 2300°C during which Cd or Cu, respectively, were read. Samples were read against standards prepared from Fisher certified standards. Water samples were taken during the experiments for analysis of Na, Ca, Cu, Cd, and DOC. Water Na and Ca concentrations were measured using a Perkin Elmer Model 3100 atomic absorption spectrophotometer. Total water Cu and Cd concentrations were measured using the methods described for gill metal analysis (above). Water DOC concentrations were analyzed using a Beckman model 915B total carbon analyzer, after acidification and sparging to remove inorganic carbon (error less than 5% at 5 mg C 1-l). Water pH was measured using a Radiometer PHM82 meter with GK2401C combination electrode. Error bars on line graphs represent the 95% confidence interval (CZ) about the
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mean of fish gill samples (usually six). Bar graphs show 95% CI about the mean for eight to ten fish gill samples; clear bars represent control treatments with no metals or DOC added. For all graphs, an ANOVA followed by a Student-Newman-Keuls procedure was used for multiple comparison of mean values. P < 0.05, P < 0.01, and P < 0.001 significance levels are represented by *, **, and ***, respectively.
3. Results Small rainbow trout (Oncorhynchus mykiss, about 1 g) were exposed to 0.28-0.51 uM total Cu and 0.052-0.057 uM total Cd in synthetic soft water for 15 days with the addition of 5-20 mg C 1-l dissolved organic carbon (DOC) which had been isolated from a marsh. Thirty percent of the fish died when exposed to 0.28 uM Cu and 0.05 uM Cd in the absence of added DOC (background DOC 3.0 mg C 1-l; Fig. 1). The addition of 5 mg C 1-l DOC partially protected the fish against the toxic effects of Cu and Cd (13% died), and the addition of 10 or 20 mg C 1-l DOC fully protected the fish against Cu and Cd toxicity (Fig. 1). Measured DOC concentrations (in mg C l-l, mean + 1 SEM, n = 4 for each) in the five treatments were: 1. 2. 3. 4. 5.
no added DOC (2.5 + 0.4) no added metals; no added DOC (3.0+0.4), plus metals; 7.6kO.2 DOC, plus metals; 14.5 ?I 0.2 DOC, plus metals; 26.4 + 0.4 DOC, plus metals.
About half the background DOC was from the fish themselves (e.g. shed mucus); DOC in the water without fish was about 1.5 mg C 1-l. Total Cd concentrations (in PM, n=4 for each) were 0.002t0.001, 0.052+0.001, 0.054?0.003, 0.057+0.004,
0 Fig. 1. Survival of 1 g rainbow trout in synthetic pM Cd (Metals) and 7.626.4 mg C 1-l dissolved DOC (to reach 7.6 mg C 1-t) reduced the toxicity 20 mg C 1-l DOC (to reach 14.5 and 26.4 mg C toxicity. Background DOC was between 2.5 and clarity.
soft water plus 0.28-0.51 pM Cu and 0.052-0.057 organic carbon (DOC). The addition of 5 mg C 1-l of the metal mixture, and the addition of 10 and l-t, respectively) fully protected against Cu and Cd 3.0 mg C 1-t. Some symbols have been offset for
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and 0.056 2 0.003, respectively. Total Cu concentrations (in uM, n = 4 for each) were 0.09t0.01, 0.28kO.02, 0.33+0.01, 0.42f0.02, and 0.51 40.01, respectively. Elevated concentrations of Cu in treatments containing DOC reflect residual Cu in the Luther Marsh DOC concentrate. Cadmium and Cu accumulation by gills of trout was measured after 5 h, 2 days, and at 1 and 2 week exposures to the Cu, Cd, and DOC solutions. Gill Cd concentrations increased from initial values of about 3 nmol Cd g-l wet tissue to lo-14 nmol Cd 8-l wet tissue after 8 days in all treatments containing Cd, regardless of DOC concentration (Fig. 2). Gill Cu concentrations were more variable, and by the end of the 2 week exposure period, the only significant increase in gill Cu compared with the controls was from fish from the metal mixture with no added DOC (Fig. 3), the most toxic solution (Fig. 1). We investigated whether the age of the metal-DOC mixture affected the protective effect of DOC against Cu and Cd accumulation by trout gills by exposing trout for 4 h to freshly made metal or metal-DOC mixtures or to mixtures that had been aged for 3 weeks. DOC additions of 5 mg C llr were chosen on the basis of the previous experiment (e.g. partial but not complete protection against Cu and Cd toxicity, Fig. 1). We originally tried this experiment with the lower Cu and Cd concentrations used in the first experiment (above), but did not get significant Cu and Cd accumulations by trout gills in short exposures (data not presented), so we increased the nominal Cu and Cd additions by about two fold. Addition of 5 mg C 1-l DOC kept 0.52-0.61 nM Cu off trout gills equally well whether the metal-DOC mixtures were freshly made or were 3 weeks old (e.g. gill Cu concentrations were not above background in the three DOC treatments, Fig. 4). If aging produced stronger metal-DOC bonds then the Aged DOC, Aged Metals mixture would have kept Cu off the gills best, but there were no significant differ-
Fig. 2. Accumulation of Cd by gills of rainbow trout held in synthetic soft water plus 0.28-0.51 pM Cu and 0.052-0.057 nM Cd (Metals) and 2.5-26.4 mg C 1-l DOC. Between 2 and 8 days exposure, highly significant accumulations of Cd developed on or in trout gills in all treatments containing Cd f 95% CZ about the mean of gills from six (‘P< 0.05, “P< 0.01, ***P < 0.001). Error bars represent fish at each point, except for day 15, where rz= 12, 12, 9, 3, and 11 from top to bottom of legend. Symbols are offset for clarity.
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26.4 DDC plus Metals 14.5 DDC plus Metals 7 6 DDC pl”E Metals 3.0 DOC plus Metals
.
2 5 DOC, no Metals
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Fig. 3. Accumulation of Cu by gills of rainbow trout held in synthetic soft water plus 0.28-0.51 PM Cu and 0.052-0.057 PM Cd (Metals) and 2.5-26.4 mg C 1-l DOC. The only significant increase in gill Cu above background was at 15 days in the Cu and Cd exposure with no added DOC. Symbols are offset for clarity. See caption of Figure 2 for more details.
ences between the three DOC treatments. About half as much Cu accumulated on fish exposed to the Aged Metals treatment (metal solution aged for 3 weeks) compared with Fresh Metals, because Cu was lost from solution during aging: measured Cu was 0.32 yM in Aged Metals compared with 0.42 nM in Fresh Metals (below). This was the only instance of either Cu or Cd binding to the polyethylene
Fig. 4. Accumulation of Cu by gills of rainbow trout exposed for 4 h to fresh or 3-week-old Cu (about 0.5 PM) and Cd (about 0.14 PM) mixtures with and without the addition of 5 mg C 1-r DOC. Addition of 5 mg C I-’ DOC kept Cu off the gills (e.g. less Cu accumulation than in Fresh Metals (crosses) and not significantly greater than SW controls) whether the fish were exposed to fresh or aged metal-DOC solutions. Low Cu accumulation in the Aged Metals treatment was because Cu was lost from solution while that treatment was aging (see text). Eight to ten fish per bar (from left to right, n= 10, 10, 9, 9, 8, and lo), +95% CI. Statistical comparisons indicated were made against background gill Cu (SW controls; asterisks) and between Fresh Metals and other treatments in which metals were added (crosses).
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*** T
B
m
Metals DOC plusMetals
Fig. 5. Accumulation of Cd by gills of rainbow trout exposed for 4 h to fresh or 3-week-old Cu (about 0.5 PM) and Cd (about 0.14 FM) mixtures with and without the addition of 5 mg C 1-l DOC. Addition of 5 mg C 1-l DOC reduced slightly the amount of Cd accumulating on or in the gills (less Cd accumulation than in Fresh Metals, but still significantly above SW controls). There was no influence of metal-DOC aging on the reduction of Cd accumulation by trout gills. f 95% Cl, eight to ten fish per bar (see caption of Figure 4). Statistical comparisons indicated were made against background gill Cd (open bar; asterisks) and between Fresh Metals and other treatments to which metals were added (crosses).
containers (see Section 4). DOC concentrations in the soft water exposures (mean, in mg C 1-l ; followed by the two DOC measurements taken, one per replicate) from left to right in Fig. 4 were: SW (4.8; 4.3, 5.4), Aged Metals (5.0; 5.3, 4.7) Fresh Metals (4.6; 4.7, 4.6) Aged DOC, Aged Metals (7.7; 8.2, 7.2) Fresh DOC, Fresh Metals (9.7; 9.2, 10.2), and Aged DOC, Fresh Metals (8.6; 8.9, 8.2). Total Cu concentrations (in PM, mean f 1 SEM, II = 4 for each) were 0.11 f 0.05, 0.32 ? 0.02 (Cu lost from solution), 0.42 + 0.05, 0.52 ? 0.01, 0.60 f 0.02, and 0.61 2 0.03, respectively. The DOC contained some Cu, so Cu concentrations in the DOC treatments were 0.1-0.2 l.tM higher than in the Fresh Metals exposure. In this experiment, DOC concentrations in treatments without added DOC were higher (4.3-5.3 mg C 1-l) than in the previous experiment (2.5-3.0 mg C l-l), likely because mucus shed by the fish accumulated in a smaller volume of water (1 vs. 10 1). For Cd there was also no difference between Cd accumulation by gills of trout held in 3-week-old or fresh metal-DOC mixtures. Gill Cd accumulations were reduced somewhat by the addition of 5 mg C 1-l DOC compared with the Cd and Cu exposures in the absence of DOC (significantly less than Fresh Metals, but still significant Cd accumulation compared with SW), but there was no effect of aging (e.g. no significant differences between the three DOC treatments; Fig. 5). In contrast with Cu, there was no loss of Cd from solution in the Aged Metals treatment, thus there was no difference in Cd accumulation by the gills of fish exposed to aged or fresh metal solution in the absence of DOC. Total Cd (in uM, mean + 1 SEM, n = 4 for each) and DOC concentrations (in mg C 1-l) mean, n = 2 for each) in the
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soft water exposures, from left to right in Fig. 5, were: SW (Cd=0.013 2 0.004, DOC = 4.8), Aged Metals (Cd = 0.134 +.0.002, DOC=5.0), Fresh Metals (Cd = 0.139 + 0.005, DOC = 4.6), Aged DOC, Aged Metals (Cd = 0.138 & 0.003, DOC = 7.7) Fresh DOC, Fresh Metals (Cd = 0.155 + 0.002, DOC = 9.7) and Aged DOC, Fresh Metals (Cd = 0.151 + 0.003, DOC = 8.6).
4. Discussion The primary objective of our Cu, Cd, and dissolved organic carbon (DOC) experiments with trout was to determine whether the age of metal-DOC complexes affects how well DOC keeps a metal off gills of a fish. Three-week-old metal-DOC solutions did not keep Cu or Cd off trout gills better than fresh metal-DOC solutions: Cu was kept off the gills equally well in all metal exposures with the addition of 5 mg C ll’ DOC, while Cd was only partially (but equally) kept off the gills by the addition of 5 mg C ll’ DOC (Figs. 4 and 5). These results will simplify modelling of metal-gill interactions for predictive purposes (Playle et al., 1993b; Janes and Playle, 1995) because they indicate that experimentally determined metal-DOC binding constants are robust and do not need to be altered to reflect time from formation of the metal-DOC complexes. The binding constants are also robust with respect to source or size fraction of DOC (Playle et al., 1993a), which further simplifies modelling of metal-gill interactions. In other words, DOC concentration is a ‘master variable’ which needs to be considered in predictive modelling of metal binding to fish gills, but the history of the DOC and its source can be ignored. We recognize that future work using natural DOC isolated from different sources will be needed to prove the generality of this statement, but our conclusions agree with those of Cabaniss and Shuman (1988) who concluded that pH and ionic strength were more important than DOC source when considering variations in Cu-DOC binding. We thought initially that aged metal-DOC solutions might better protect fish gills against metal accumulation, on the basis of time allowing the formation of stronger metal-DOC bonds (Clark and Choppin, 1990) and on the basis of a switch to adjunctive from disjunctive metal-gill interactions (Morel and Hering, 1993). With concentrations of humic acid and Cu similar to ours, Hering and Morel (1990) found that disjunctive and adjunctive reactions were of about equal importance (terrestrial humic acid 5.4 and 10.8 mg C 1-l; Cu, about 0.05 uM). Clark and Choppin (1990) found that trivalent europium (Eu) was 100% bound by humic acid after just 15 min contact time, but only 4% of the bonds were high affinity. After 2 days contact time, 38% of the Eu-humic acid bonds were high affinity. The humic acid used by Clark and Choppin (1990) was chemically extracted from sediments of a Florida lake, and had an average molecular weight of 8000 Da. Development of higher affinity Eu-humic acid bonds over 2 days was thought to result from cation movement to sites inside the humic acid molecules and to folding of humic acid, trapping Eu inside (Clark and Choppin, 1990). Chemical shifts such as these are not discounted by our results obtained using a physically isolated DOC
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containing a mixture of fulvic and humic acids of over 400 Da. Rather, our results indicate that even if metal-DOC bonds do become stronger with time, the changes in metal-DOC binding strength are not biologically important. That is, breaking of stronger metal-DOC bonds (if they develop) is still fast relative to the time scale of even our short, 4 h metal exposures. In our experiments DOC kept Cu off gills better than it kept Cd off trout gills (Figs. 2-5). This result is a function of relatively strong binding of Cu to DOC (log &u_noC = 9.1) and relatively weak binding of Cu to gills (log Kou_gill= 7.4), and the weaker binding of Cd to DOC relative to Cd binding at gills (log &d-noo = 7.4, log KC&gill= 8.6; Playle et al., 1993b). In our experiments we did not differentiate between Cu and Cd bound to the gill surface and Cu and Cd in the interior of the gills. Gill toxicants such as Cu and Cd must first adsorb to the gills before subsequent toxic action either at the gill surface or gill interior (Simkiss and Taylor, 1989). Strong enough complexation of metals by complexing ligands such as DOC will reduce or eliminate metal adsorption on the gill surface and therefore subsequent internalization of a metal. Thus, for the purposes of this study of changes in metal-DOC binding relative to metal-gill binding, it was not necessary to treat extracted gills with a complexing agent to separate surface bound from interior metal (e.g. with EDTA; Verbost et al., 1989). Dissolved organic carbon reduced the toxicity of Cu and Cd to trout over 2 weeks (Fig. l), but it is not clear through what mechanism the protection arose. Cadmium accumulated on or in trout gills even in the presence of DOC (Fig. 2), in accord with the relatively strong binding of Cd to gills and its weak binding to DOC. This Cd accumulation by trout gills does not correlate with toxicity of the metal mixture (Fig. 1). Although the Cu accumulation results are not definitive, by the end of the experiment the only significant accumulation of Cu by the gills was from trout exposed to the metal mixture in the absence of added DOC, and the amount of Cu on or in the gills (although not significant) decreased as DOC concentration increased (Fig. 3, 15 days). The relative lack of Cu accumulation on or in the gills in the more toxic solutions might be related to mucus sloughing of Cu; histological examination of the gills via light or electron microscopy might have indicated damage to the gills in the absence of significant Cu accumulation. However, in the absence of histological work it appears, for these concentrations of Cu, Cd, and DOC in our synthetic soft water system, that Cu was more likely the toxic agent to the trout. About one-third of the added Cu in the Aged Metals mixture was lost from solution during aging, presumably through reactions of Cu with the sides of the polyethylene containers. Thus, little Cu bound to trout gills exposed 4 h to the Aged Metals solution (Fig. 4). In contrast, Cu was not lost from solution during aging with DOC. This property of DOC is well known: DOC keeps metals in solution by binding them, preventing them from precipitating from solution (Urban et al., 1990; Livens, 1991; Watras et al., 1995). In this manner, DOC tends to elevate total metal concentrations in water at the same time as it tends to detoxify metals in solution. In conclusion, we showed that the protective effect of DOC against Cu and Cd
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accumulation by trout gills is not affected by age of the metal-DOC complexes. Earlier results of ours indicated that source and size fraction of DOC do not influence the protective effects of DOC. Together, these results make modelling of metal-gill interactions for predictive purposes simpler because experimentally determined metal-DOC equilibrium constants are robust values which do not need to be altered to reflect DOC history or source.
Acknowledgements
We thank Dr. Chris Wood and Dr. Gord McDonald, McMaster University, Hamilton, Ontario, Canada, for the use of their graphite furnace for metal analyses, and Donna Nuttley for technical assistance and DOC analyses. This research was supported by grants from the Canadian Network of Toxicology Centers and from the Natural Sciences and Engineering Research Council of Canada. References Cabaniss, S.E. and Shuman, MS., 1988. Copper binding by dissolved organic matter: II. Variation in type and source of organic matter. Geochim. Cosmochim. Acta, 52: 195-200. Clair, T.A., Kramer, J.R., Sydor, M. and Eaton, D., 1991. Concentration of aquatic dissolved organic matter by reverse osmosis. Water Res., 25: 1033-1037. Clark, S.B. and Choppin, G.R., 1990. Kinetics of rare earth metal binding to aquatic humic acids. In: D.C. Melchior and R.L. Bassett (Editors), Chemical Modeling of Aqueous Systems II. American Chemical Society, Washington, DC. Hering, J.G. and Morel, F.M.M., 1990. Kinetics of trace metal complexation: ligand-exchange reactions. Environ. Sci. Technol., 24: 242-252. Janes, N. and Playle, R.C., 1995. Modeling silver binding to gills of rainbow trout (Oncorhynchus mykiss). Environ. Toxicol. Chem., 14: 1847-1858. Laurkn, D.J. and McDonald, D.G., 1986. Influence of water hardness, pH, and alkalinity on the mechanisms of copper toxicity in juvenile rainbow trout, Salmo gairdneri. Can. J. Fish. Aquat. Sci., 43: 1488-1496. Livens, F.R., 1991. Chemical reactions of metals with humic material. Environ. Pollut., 70: 183-208. Morel, F.M.M. and Hering, J.G., 1993. Principles and Applications of Aquatic Chemistry. John Wiley and Sons, New York, 588 pp. Playle, R.C., Dixon, D.G. and Burnison, K., 1993a. Copper and cadmium binding to fish gills: modification by dissolved organic carbon and synthetic ligands. Can. J. Fish. Aquat. Sci., 50: 2667-2677. Playle, R.C., Dixon, D.G. and Burnison, K., 1993b. Copper and cadmium binding to fish gills: estimates of metal-gill stability constants and modelling of metal accumulation. Can. J. Fish. Aquat. Sci., 50: 2678-2681. Simkiss, K. and Taylor, M.G., 1989. Metal fluxes across the membranes of aquatic organisms. Rev. Aquat. Sci., 1: 173-188. Spry, D.J. and Wiener, J.G., 1991. Metal bioavailability and toxicity to fish in low-alkalinity lakes: a critical review. Environ. Pollut., 71: 243-304. Urban, N.R., Gorham, E., Underwood, J.K., Martin, F.B. and Ogden, J.G. III, 1990. Geochemical processes controlling concentrations of Al, Fe, and Mn in Nova Scotia Lakes. Limnol. Oceanogr., 35: 1516-1534. Verbost, P.M., VanRooij, J., Flik, G., Lock, R.A.C. and Wendelaar Bonga, SE., 1989. The movement of cadmium through freshwater trout branchial epithelium and its interference with calcium transport. J. Exp. Biol., 145: 185-197.
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Watras, C.J., Morrison, K.A. and Host, J.S., 1995. Concentration of mercury species in relationship to other site-specific factors in the surface waters of northern Wisconsin lakes. Limnol. Oceanogr., 40: 556-565. Welsh, P.G., Skidmore, J.F., Spry, D.J., Dixon, D.G., Hodson, P.V., Hutchinson, N.J. and Hickie, B.E., 1993. Effect of pH and dissolved organic carbon on the toxicity of copper to larval fathead minnow (Pifflephalespromelas) in natural lake waters of low alkalinity. Can. J. Fish. Aquat. Sci., 50: 1356-1362. Wicklund Glynn, A., Norrgren, L. and Mfissener, A., 1994. Differences in uptake of inorganic mercury and cadmium in the gills of the zebrafish, Brachydanio rerio. Aquat. Toxicol., 30: 13-26. Wood, C.M., 1992. Flux measurements as indices of Ht and metal effects on freshwater fish. Aquat. Toxicol., 22: 239-264.
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Does the age of metal-dissolved organic carbon complexes influence binding of metals to fish gills? Lydia Hollis”, Kent Burnisonb, Richard
C. Playlea>*
“Department of Biology, Wiljiid Laurier University, Waterloo, Ont., N2L 3C5, Canada “National Water Research Institute, Environment Canada, Burlington, Ont., L7R 4A6, Canada Accepted
20 March
1996
Abstract We investigated the effect of aging on the strength of dissolved organic carbon (DOC) complexation of metals, as indicated by the ability of DOC to keep copper and cadmium off trout gills. The addition of S-20 mg C 1-l DOC reduced the toxicity of Cu and Cd to small rainbow trout (Oncorhynchus mykiss, 1 g). Exposures were to 0.2 PM Cu and 0.03 PM Cd for 2 weeks, in synthetic soft water. The DOC was isolated from surface waters of a marsh through reverse osmosis. Copper was kept off the gills by DOC but Cd was not, in agreement with the relative binding strengths of the metals to DOC and to the gills themselves. Aging 0.5 pM Cu, 0.1 pM Cd, and 5 mg C 1-l DOC solutions for 3 weeks did not change the ability of DOC to keep Cu or Cd off trout gills: 3 week old and fresh metal-DOC solutions kept Cu off the gills equally well, whereas Cd was always only partially kept off the gills by DOC. Our results indicate that efforts to model metal-gill interactions in fish from waters of differing chemistries and DOC content do not need to consider the age of metalDOC complexes, because metal-DOC complexes do not become appreciably stronger with time. Keywords:
Dissolved
organic
carbon;
Copper;
Cadmium;
Fish;
Gills
1. Introduction Fish gills are the first organ to be affected by many waterborne metals (e.g. Wood, 1992). We have been determining metal-gill binding constants for modelling metal-gill interactions and prediction of toxic effects of waterborne metals to fish (Cu and Cd, Playle et al., 1993a; Playle et al., 1993b; Ag, Janes and Playle, 1995; Co, Pb, Hg, R.C. Playle, unpublished data, 1995 and 1996). Dissolved organic carbon (DOC) is an important modifying agent of metal toxicity to fish (e.g. *Corresponding
author.
Tel.: (519) 884 1970, ext. 3407; fax: (519) 746 0677.
0166-445X/96/$15.00 Copyright PIIS 66-445X(96)00793-X
0 1996 Elsevier
Science B.V. All rights
reserved
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Spry and Wiener, 1991; Welsh et al., 1993) and acts by complexing metals so that less free metal is available to bind to the gills. It is important to investigate whether the age of metal-DOC complexes influences the protective effect of DOC against metal binding to fish gills. From our previous work, DOC and fish gills both bind metals relatively strongly, with conditional equilibrium binding constants of log Kou_noo = 9.1, log &-uoo = 7.4, and log K~~-noo = 9.0, compared with gill binding constants of log Kou-sill= 7.4, log Kod-ai]t = 8.6, and log KAs_sill= 10.0 (Playle et al., 1993b; Janes and Playle, 1995). From these values, DOC binds Cu about 50 times better than Cu binds to gills, but the gills bind Cd and Ag about 16 times and about 10 times better, respectively, than does DOC. For Cd, greater than expected binding to the gills is likely related to active uptake of Cd through Ca channels (Verbost et al., 1989). Dissolved organic carbon is a general term for poorly defined breakdown products of organic material. In freshwater the source of DOC is mainly plant material. Freshwater DOC is composed of humic and fulvic acids, with carboxyl and phenol groups mainly being responsible for metal chelation (Morel and Hering, 1993). In previous work, we showed that to a first approximation the source and size fraction of DOC does not influence the protective effect of DOC against Cu accumulation on or in fish gills (Playle et al., 1993a). Aging of metal-DOC solutions, however, could increase the apparent metal binding constant of DOC for a metal if metalDOC bonds become stronger with time (e.g. over 2 days, Clark and Choppin, 1990). Similarly, DOC could better prevent metal accumulation on or in gills if the slower, double exchange reaction of metal deposition onto the gills increases in importance with aging. The double exchange reaction would develop at the expense of metal-ligand dissociation which first releases free metal which then can bind to the gills (e.g. adjunctive versus disjunctive mechanisms; Morel and Hering, 1993, pp. 395-405). In the present work, we investigated changes in the protective effects of DOC against Cu and Cd binding to gills of rainbow trout, as an indication of changes in the strength of DOC complexation of metals. Both Cu and Cd are of environmental concern, are gill toxicants, and affect ionoregulation in fish by interfering with Na uptake (Cu; e.g. Lauren and McDonald, 1986) and with Ca uptake (Cd; e.g. Verbost et al., 1989; Wicklund Glynn et al., 1994). We used natural DOC isolated from a marsh, as opposed to terrestrial humic acid, to make our experiments as environmentally relevant as possible. We aged our metal-DOC solutions for 3 weeks, much longer than the 2 days which gave increased strength of binding (from 4 to 38% strong bonds; Clark and Choppin, 1990). Copper binds well to DOC but relatively weakly to gills, whereas Cd binds less well to DOC than to fish gills (see above); use of these two metals in our trout and softwater system was expected to demonstrate biologically relevant, time-related changes in metal binding strength of DOC.
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2. Materials and methods Rainbow trout (Oncorhynchus mykiss) were obtained from Rainbow Springs Hatchery in Thamesford, Ontario. Fish were held in low Ca water (see below) produced using a reverse osmosis system (Culligan of Canada, Ltd., Mississauga, Ontario). Fish were fed Martin’s Starter Food (Martin Feed Mills, Elmira, Ontario), but were not fed during experiments. A 15 day experiment was run in five randomly arranged polyethylene buckets, each containing 10 1 of aerated soft water (Ca, 240 + 30 PM (n = 20); Na, 2620 k 170 PM (n = 20); 11°C pH 7.64kO.02 (n = 25); mean? 1 SEM). Thirty or 31 trout (1.45 + 0.05 g, n = 137) were placed randomly into each of the buckets. Six fish were sampled from each bucket at 5 h, 2 days, and 8 days, and the remaining fish (n = 3-12) at 15 days, and their gills analyzed for Cd and Cu (see below). Each bucket corresponded to one of five treatments: 1. 2. 3. 4. 5.
no added DOC, no added metals; no added DOC, plus metals (Cu and Cd); 5 mg C 1-l DOC, plus metals; 10 mg C 1-l DOC, plus metals; 20 mg C 1-r DOC, plus metals.
Nominal metal additions were 0.2 pM Cu and 0.03 pM Cd. Measured DOC and total metal concentrations taken during the experiment are given in Section 3. Measurement of DOC and metal concentrations throughout the experiment would account for any losses of DOC or metals from the solutions. Cadmium was added as CdC12*HZ0 (Baker Chemical Co., Phillipsburg, NJ) and Cu was added as CuS04*5H20 (AnalaR, BDH Toronto, Ontario). Depending on the number of fish left in a bucket, 2 or 3 1 of treatment water were replaced daily with water that was aging as the experiment progressed. This experiment was not replicated because of our limited supply of DOC concentrate. Added DOC was concentrated from surface waters of Luther Marsh, near Grand Valley, Ontario (43”54’N, 80”24’W) through reverse osmosis (Filmtec NF40 membrane, Minneapolis, MN; molecular weight cutoff 400 Da) after glass fibre filtration (1 pm). This process concentrates molecules of over 400 Da, so the isolated Luther Marsh DOC was a mixture of both fulvic and humic acids. Concentration of DOC by physical means such as reverse osmosis and ultrafiltration is considered better than by chemical means because large volumes of water can be processed with little modification of the DOC (Clair et al., 1991). Our concentrated DOC contained Cu, either because of contact with Cu in the reverse osmosis unit or because Cu was preferentially retained during the concentration process. To remove Cu, the DOC concentrate was passed through a cation exchange resin (Dowex 5OW-X8, 20-50 mesh, H-form, Baker Chemical Co., Phillipsburg, NJ). This treatment made the DOC concentrate more stable (e.g. no precipitation of material during storage), likely because metal removal eliminated the development of metal-DOC crossbridges leading to precipitate formation.
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A second experiment with fresh or aged (3-week-old) metal or metal-DOC solutions was run in six 2-l polyethylene containers, each containing five randomly placed trout (1.20 + 0.08 g, n = 56) in 1 1 of aerated synthetic soft water (Ca, 9 f 1 uM (n = 12); Na, 1260 + 20 uM (n = 12); 18°C pH 6.91 ? 0.03 (n = 24)). The same batch of synthetic soft water was used for all treatments (e.g. aerated for 3 weeks, then DOC or metals added if necessary), to assure similar water chemistry in all treatments. Fish gills were sampled after 4 h and were analyzed for Cu and Cd concentrations; sampling time meant that fish were exposed to the solutions between 4 and 5 h. This experiment was run in duplicate on 2 different days. Each container in the experiment corresponded to one of six treatments: 1. 2. 3. 4. 5. 6.
no added DOC, no added metals, aged 3 weeks (SW); no added DOC, plus metals (Cu and Cd), aged 3 weeks (Aged Metals); 3-week-old SW with metals added at the start of the 4 h exposure (Fresh Metals) ; added DOC, added metals, aged 3 weeks (Aged DOC, Aged Metals); 3-week-old SW with DOC and metals added at the start of the 4 h exposure (Fresh DOC, Fresh Metals); added DOC, no added metals, aged 3 weeks, with metals added at the start of the 4 h exposure (Aged DOC, Fresh Metals).
Nominal DOC additions were 5 mg C l-l, and nominal Cu plus Cd additions were 0.5 uM and 0.1 PM, respectively; measured values taken during the experiments are given in Section 3. After exposure to the various treatments in both experiments, fish were killed with a blow to the head, both sets of gills were excised, and gills were rinsed for 10 s in 100 ml of synthetic soft water. Extracted gills were weighed and then digested in 10 times their weight of 1 N HNOs (Ultrex II Ultrapure HNOs, Baker Analyzed reagent) for 3 h at about 80°C. Gill digests were shaken, left to settle for 10 min, then the supernatant was diluted 10 times with E-pure deionized water (Fisher Scientific Ltd., Unionville, Ont.). Gill metal concentrations were measured on a graphite furnace atomic absorption spectrophotometer (Varian AA-1275 with GTA-95 atomizer) using 10 ~1 injection volumes and N2 gas. Operating conditions were 30 s drying time at 90°C 12 s at 120°C and 4 s at 1800 or 2300°C during which Cd or Cu, respectively, were read. Samples were read against standards prepared from Fisher certified standards. Water samples were taken during the experiments for analysis of Na, Ca, Cu, Cd, and DOC. Water Na and Ca concentrations were measured using a Perkin Elmer Model 3100 atomic absorption spectrophotometer. Total water Cu and Cd concentrations were measured using the methods described for gill metal analysis (above). Water DOC concentrations were analyzed using a Beckman model 915B total carbon analyzer, after acidification and sparging to remove inorganic carbon (error less than 5% at 5 mg C 1-l). Water pH was measured using a Radiometer PHM82 meter with GK2401C combination electrode. Error bars on line graphs represent the 95% confidence interval (CZ) about the
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mean of fish gill samples (usually six). Bar graphs show 95% CI about the mean for eight to ten fish gill samples; clear bars represent control treatments with no metals or DOC added. For all graphs, an ANOVA followed by a Student-Newman-Keuls procedure was used for multiple comparison of mean values. P < 0.05, P < 0.01, and P < 0.001 significance levels are represented by *, **, and ***, respectively.
3. Results Small rainbow trout (Oncorhynchus mykiss, about 1 g) were exposed to 0.28-0.51 uM total Cu and 0.052-0.057 uM total Cd in synthetic soft water for 15 days with the addition of 5-20 mg C 1-l dissolved organic carbon (DOC) which had been isolated from a marsh. Thirty percent of the fish died when exposed to 0.28 uM Cu and 0.05 uM Cd in the absence of added DOC (background DOC 3.0 mg C 1-l; Fig. 1). The addition of 5 mg C 1-l DOC partially protected the fish against the toxic effects of Cu and Cd (13% died), and the addition of 10 or 20 mg C 1-l DOC fully protected the fish against Cu and Cd toxicity (Fig. 1). Measured DOC concentrations (in mg C l-l, mean + 1 SEM, n = 4 for each) in the five treatments were: 1. 2. 3. 4. 5.
no added DOC (2.5 + 0.4) no added metals; no added DOC (3.0+0.4), plus metals; 7.6kO.2 DOC, plus metals; 14.5 ?I 0.2 DOC, plus metals; 26.4 + 0.4 DOC, plus metals.
About half the background DOC was from the fish themselves (e.g. shed mucus); DOC in the water without fish was about 1.5 mg C 1-l. Total Cd concentrations (in PM, n=4 for each) were 0.002t0.001, 0.052+0.001, 0.054?0.003, 0.057+0.004,
0 Fig. 1. Survival of 1 g rainbow trout in synthetic pM Cd (Metals) and 7.626.4 mg C 1-l dissolved DOC (to reach 7.6 mg C 1-t) reduced the toxicity 20 mg C 1-l DOC (to reach 14.5 and 26.4 mg C toxicity. Background DOC was between 2.5 and clarity.
soft water plus 0.28-0.51 pM Cu and 0.052-0.057 organic carbon (DOC). The addition of 5 mg C 1-l of the metal mixture, and the addition of 10 and l-t, respectively) fully protected against Cu and Cd 3.0 mg C 1-t. Some symbols have been offset for
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and 0.056 2 0.003, respectively. Total Cu concentrations (in uM, n = 4 for each) were 0.09t0.01, 0.28kO.02, 0.33+0.01, 0.42f0.02, and 0.51 40.01, respectively. Elevated concentrations of Cu in treatments containing DOC reflect residual Cu in the Luther Marsh DOC concentrate. Cadmium and Cu accumulation by gills of trout was measured after 5 h, 2 days, and at 1 and 2 week exposures to the Cu, Cd, and DOC solutions. Gill Cd concentrations increased from initial values of about 3 nmol Cd g-l wet tissue to lo-14 nmol Cd 8-l wet tissue after 8 days in all treatments containing Cd, regardless of DOC concentration (Fig. 2). Gill Cu concentrations were more variable, and by the end of the 2 week exposure period, the only significant increase in gill Cu compared with the controls was from fish from the metal mixture with no added DOC (Fig. 3), the most toxic solution (Fig. 1). We investigated whether the age of the metal-DOC mixture affected the protective effect of DOC against Cu and Cd accumulation by trout gills by exposing trout for 4 h to freshly made metal or metal-DOC mixtures or to mixtures that had been aged for 3 weeks. DOC additions of 5 mg C llr were chosen on the basis of the previous experiment (e.g. partial but not complete protection against Cu and Cd toxicity, Fig. 1). We originally tried this experiment with the lower Cu and Cd concentrations used in the first experiment (above), but did not get significant Cu and Cd accumulations by trout gills in short exposures (data not presented), so we increased the nominal Cu and Cd additions by about two fold. Addition of 5 mg C 1-l DOC kept 0.52-0.61 nM Cu off trout gills equally well whether the metal-DOC mixtures were freshly made or were 3 weeks old (e.g. gill Cu concentrations were not above background in the three DOC treatments, Fig. 4). If aging produced stronger metal-DOC bonds then the Aged DOC, Aged Metals mixture would have kept Cu off the gills best, but there were no significant differ-
Fig. 2. Accumulation of Cd by gills of rainbow trout held in synthetic soft water plus 0.28-0.51 pM Cu and 0.052-0.057 nM Cd (Metals) and 2.5-26.4 mg C 1-l DOC. Between 2 and 8 days exposure, highly significant accumulations of Cd developed on or in trout gills in all treatments containing Cd f 95% CZ about the mean of gills from six (‘P< 0.05, “P< 0.01, ***P < 0.001). Error bars represent fish at each point, except for day 15, where rz= 12, 12, 9, 3, and 11 from top to bottom of legend. Symbols are offset for clarity.
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26.4 DDC plus Metals 14.5 DDC plus Metals 7 6 DDC pl”E Metals 3.0 DOC plus Metals
.
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Fig. 3. Accumulation of Cu by gills of rainbow trout held in synthetic soft water plus 0.28-0.51 PM Cu and 0.052-0.057 PM Cd (Metals) and 2.5-26.4 mg C 1-l DOC. The only significant increase in gill Cu above background was at 15 days in the Cu and Cd exposure with no added DOC. Symbols are offset for clarity. See caption of Figure 2 for more details.
ences between the three DOC treatments. About half as much Cu accumulated on fish exposed to the Aged Metals treatment (metal solution aged for 3 weeks) compared with Fresh Metals, because Cu was lost from solution during aging: measured Cu was 0.32 yM in Aged Metals compared with 0.42 nM in Fresh Metals (below). This was the only instance of either Cu or Cd binding to the polyethylene
Fig. 4. Accumulation of Cu by gills of rainbow trout exposed for 4 h to fresh or 3-week-old Cu (about 0.5 PM) and Cd (about 0.14 PM) mixtures with and without the addition of 5 mg C 1-r DOC. Addition of 5 mg C I-’ DOC kept Cu off the gills (e.g. less Cu accumulation than in Fresh Metals (crosses) and not significantly greater than SW controls) whether the fish were exposed to fresh or aged metal-DOC solutions. Low Cu accumulation in the Aged Metals treatment was because Cu was lost from solution while that treatment was aging (see text). Eight to ten fish per bar (from left to right, n= 10, 10, 9, 9, 8, and lo), +95% CI. Statistical comparisons indicated were made against background gill Cu (SW controls; asterisks) and between Fresh Metals and other treatments in which metals were added (crosses).
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*** T
B
m
Metals DOC plusMetals
Fig. 5. Accumulation of Cd by gills of rainbow trout exposed for 4 h to fresh or 3-week-old Cu (about 0.5 PM) and Cd (about 0.14 FM) mixtures with and without the addition of 5 mg C 1-l DOC. Addition of 5 mg C 1-l DOC reduced slightly the amount of Cd accumulating on or in the gills (less Cd accumulation than in Fresh Metals, but still significantly above SW controls). There was no influence of metal-DOC aging on the reduction of Cd accumulation by trout gills. f 95% Cl, eight to ten fish per bar (see caption of Figure 4). Statistical comparisons indicated were made against background gill Cd (open bar; asterisks) and between Fresh Metals and other treatments to which metals were added (crosses).
containers (see Section 4). DOC concentrations in the soft water exposures (mean, in mg C 1-l ; followed by the two DOC measurements taken, one per replicate) from left to right in Fig. 4 were: SW (4.8; 4.3, 5.4), Aged Metals (5.0; 5.3, 4.7) Fresh Metals (4.6; 4.7, 4.6) Aged DOC, Aged Metals (7.7; 8.2, 7.2) Fresh DOC, Fresh Metals (9.7; 9.2, 10.2), and Aged DOC, Fresh Metals (8.6; 8.9, 8.2). Total Cu concentrations (in PM, mean f 1 SEM, II = 4 for each) were 0.11 f 0.05, 0.32 ? 0.02 (Cu lost from solution), 0.42 + 0.05, 0.52 ? 0.01, 0.60 f 0.02, and 0.61 2 0.03, respectively. The DOC contained some Cu, so Cu concentrations in the DOC treatments were 0.1-0.2 l.tM higher than in the Fresh Metals exposure. In this experiment, DOC concentrations in treatments without added DOC were higher (4.3-5.3 mg C 1-l) than in the previous experiment (2.5-3.0 mg C l-l), likely because mucus shed by the fish accumulated in a smaller volume of water (1 vs. 10 1). For Cd there was also no difference between Cd accumulation by gills of trout held in 3-week-old or fresh metal-DOC mixtures. Gill Cd accumulations were reduced somewhat by the addition of 5 mg C 1-l DOC compared with the Cd and Cu exposures in the absence of DOC (significantly less than Fresh Metals, but still significant Cd accumulation compared with SW), but there was no effect of aging (e.g. no significant differences between the three DOC treatments; Fig. 5). In contrast with Cu, there was no loss of Cd from solution in the Aged Metals treatment, thus there was no difference in Cd accumulation by the gills of fish exposed to aged or fresh metal solution in the absence of DOC. Total Cd (in uM, mean + 1 SEM, n = 4 for each) and DOC concentrations (in mg C 1-l) mean, n = 2 for each) in the
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soft water exposures, from left to right in Fig. 5, were: SW (Cd=0.013 2 0.004, DOC = 4.8), Aged Metals (Cd = 0.134 +.0.002, DOC=5.0), Fresh Metals (Cd = 0.139 + 0.005, DOC = 4.6), Aged DOC, Aged Metals (Cd = 0.138 & 0.003, DOC = 7.7) Fresh DOC, Fresh Metals (Cd = 0.155 + 0.002, DOC = 9.7) and Aged DOC, Fresh Metals (Cd = 0.151 + 0.003, DOC = 8.6).
4. Discussion The primary objective of our Cu, Cd, and dissolved organic carbon (DOC) experiments with trout was to determine whether the age of metal-DOC complexes affects how well DOC keeps a metal off gills of a fish. Three-week-old metal-DOC solutions did not keep Cu or Cd off trout gills better than fresh metal-DOC solutions: Cu was kept off the gills equally well in all metal exposures with the addition of 5 mg C ll’ DOC, while Cd was only partially (but equally) kept off the gills by the addition of 5 mg C ll’ DOC (Figs. 4 and 5). These results will simplify modelling of metal-gill interactions for predictive purposes (Playle et al., 1993b; Janes and Playle, 1995) because they indicate that experimentally determined metal-DOC binding constants are robust and do not need to be altered to reflect time from formation of the metal-DOC complexes. The binding constants are also robust with respect to source or size fraction of DOC (Playle et al., 1993a), which further simplifies modelling of metal-gill interactions. In other words, DOC concentration is a ‘master variable’ which needs to be considered in predictive modelling of metal binding to fish gills, but the history of the DOC and its source can be ignored. We recognize that future work using natural DOC isolated from different sources will be needed to prove the generality of this statement, but our conclusions agree with those of Cabaniss and Shuman (1988) who concluded that pH and ionic strength were more important than DOC source when considering variations in Cu-DOC binding. We thought initially that aged metal-DOC solutions might better protect fish gills against metal accumulation, on the basis of time allowing the formation of stronger metal-DOC bonds (Clark and Choppin, 1990) and on the basis of a switch to adjunctive from disjunctive metal-gill interactions (Morel and Hering, 1993). With concentrations of humic acid and Cu similar to ours, Hering and Morel (1990) found that disjunctive and adjunctive reactions were of about equal importance (terrestrial humic acid 5.4 and 10.8 mg C 1-l; Cu, about 0.05 uM). Clark and Choppin (1990) found that trivalent europium (Eu) was 100% bound by humic acid after just 15 min contact time, but only 4% of the bonds were high affinity. After 2 days contact time, 38% of the Eu-humic acid bonds were high affinity. The humic acid used by Clark and Choppin (1990) was chemically extracted from sediments of a Florida lake, and had an average molecular weight of 8000 Da. Development of higher affinity Eu-humic acid bonds over 2 days was thought to result from cation movement to sites inside the humic acid molecules and to folding of humic acid, trapping Eu inside (Clark and Choppin, 1990). Chemical shifts such as these are not discounted by our results obtained using a physically isolated DOC
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containing a mixture of fulvic and humic acids of over 400 Da. Rather, our results indicate that even if metal-DOC bonds do become stronger with time, the changes in metal-DOC binding strength are not biologically important. That is, breaking of stronger metal-DOC bonds (if they develop) is still fast relative to the time scale of even our short, 4 h metal exposures. In our experiments DOC kept Cu off gills better than it kept Cd off trout gills (Figs. 2-5). This result is a function of relatively strong binding of Cu to DOC (log &u_noC = 9.1) and relatively weak binding of Cu to gills (log Kou_gill= 7.4), and the weaker binding of Cd to DOC relative to Cd binding at gills (log &d-noo = 7.4, log KC&gill= 8.6; Playle et al., 1993b). In our experiments we did not differentiate between Cu and Cd bound to the gill surface and Cu and Cd in the interior of the gills. Gill toxicants such as Cu and Cd must first adsorb to the gills before subsequent toxic action either at the gill surface or gill interior (Simkiss and Taylor, 1989). Strong enough complexation of metals by complexing ligands such as DOC will reduce or eliminate metal adsorption on the gill surface and therefore subsequent internalization of a metal. Thus, for the purposes of this study of changes in metal-DOC binding relative to metal-gill binding, it was not necessary to treat extracted gills with a complexing agent to separate surface bound from interior metal (e.g. with EDTA; Verbost et al., 1989). Dissolved organic carbon reduced the toxicity of Cu and Cd to trout over 2 weeks (Fig. l), but it is not clear through what mechanism the protection arose. Cadmium accumulated on or in trout gills even in the presence of DOC (Fig. 2), in accord with the relatively strong binding of Cd to gills and its weak binding to DOC. This Cd accumulation by trout gills does not correlate with toxicity of the metal mixture (Fig. 1). Although the Cu accumulation results are not definitive, by the end of the experiment the only significant accumulation of Cu by the gills was from trout exposed to the metal mixture in the absence of added DOC, and the amount of Cu on or in the gills (although not significant) decreased as DOC concentration increased (Fig. 3, 15 days). The relative lack of Cu accumulation on or in the gills in the more toxic solutions might be related to mucus sloughing of Cu; histological examination of the gills via light or electron microscopy might have indicated damage to the gills in the absence of significant Cu accumulation. However, in the absence of histological work it appears, for these concentrations of Cu, Cd, and DOC in our synthetic soft water system, that Cu was more likely the toxic agent to the trout. About one-third of the added Cu in the Aged Metals mixture was lost from solution during aging, presumably through reactions of Cu with the sides of the polyethylene containers. Thus, little Cu bound to trout gills exposed 4 h to the Aged Metals solution (Fig. 4). In contrast, Cu was not lost from solution during aging with DOC. This property of DOC is well known: DOC keeps metals in solution by binding them, preventing them from precipitating from solution (Urban et al., 1990; Livens, 1991; Watras et al., 1995). In this manner, DOC tends to elevate total metal concentrations in water at the same time as it tends to detoxify metals in solution. In conclusion, we showed that the protective effect of DOC against Cu and Cd
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accumulation by trout gills is not affected by age of the metal-DOC complexes. Earlier results of ours indicated that source and size fraction of DOC do not influence the protective effects of DOC. Together, these results make modelling of metal-gill interactions for predictive purposes simpler because experimentally determined metal-DOC equilibrium constants are robust values which do not need to be altered to reflect DOC history or source.
Acknowledgements
We thank Dr. Chris Wood and Dr. Gord McDonald, McMaster University, Hamilton, Ontario, Canada, for the use of their graphite furnace for metal analyses, and Donna Nuttley for technical assistance and DOC analyses. This research was supported by grants from the Canadian Network of Toxicology Centers and from the Natural Sciences and Engineering Research Council of Canada. References Cabaniss, S.E. and Shuman, MS., 1988. Copper binding by dissolved organic matter: II. Variation in type and source of organic matter. Geochim. Cosmochim. Acta, 52: 195-200. Clair, T.A., Kramer, J.R., Sydor, M. and Eaton, D., 1991. Concentration of aquatic dissolved organic matter by reverse osmosis. Water Res., 25: 1033-1037. Clark, S.B. and Choppin, G.R., 1990. Kinetics of rare earth metal binding to aquatic humic acids. In: D.C. Melchior and R.L. Bassett (Editors), Chemical Modeling of Aqueous Systems II. American Chemical Society, Washington, DC. Hering, J.G. and Morel, F.M.M., 1990. Kinetics of trace metal complexation: ligand-exchange reactions. Environ. Sci. Technol., 24: 242-252. Janes, N. and Playle, R.C., 1995. Modeling silver binding to gills of rainbow trout (Oncorhynchus mykiss). Environ. Toxicol. Chem., 14: 1847-1858. Laurkn, D.J. and McDonald, D.G., 1986. Influence of water hardness, pH, and alkalinity on the mechanisms of copper toxicity in juvenile rainbow trout, Salmo gairdneri. Can. J. Fish. Aquat. Sci., 43: 1488-1496. Livens, F.R., 1991. Chemical reactions of metals with humic material. Environ. Pollut., 70: 183-208. Morel, F.M.M. and Hering, J.G., 1993. Principles and Applications of Aquatic Chemistry. John Wiley and Sons, New York, 588 pp. Playle, R.C., Dixon, D.G. and Burnison, K., 1993a. Copper and cadmium binding to fish gills: modification by dissolved organic carbon and synthetic ligands. Can. J. Fish. Aquat. Sci., 50: 2667-2677. Playle, R.C., Dixon, D.G. and Burnison, K., 1993b. Copper and cadmium binding to fish gills: estimates of metal-gill stability constants and modelling of metal accumulation. Can. J. Fish. Aquat. Sci., 50: 2678-2681. Simkiss, K. and Taylor, M.G., 1989. Metal fluxes across the membranes of aquatic organisms. Rev. Aquat. Sci., 1: 173-188. Spry, D.J. and Wiener, J.G., 1991. Metal bioavailability and toxicity to fish in low-alkalinity lakes: a critical review. Environ. Pollut., 71: 243-304. Urban, N.R., Gorham, E., Underwood, J.K., Martin, F.B. and Ogden, J.G. III, 1990. Geochemical processes controlling concentrations of Al, Fe, and Mn in Nova Scotia Lakes. Limnol. Oceanogr., 35: 1516-1534. Verbost, P.M., VanRooij, J., Flik, G., Lock, R.A.C. and Wendelaar Bonga, SE., 1989. The movement of cadmium through freshwater trout branchial epithelium and its interference with calcium transport. J. Exp. Biol., 145: 185-197.
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