Divalent metal accumulation in freshwater bivalves

The Science of the Total Environment 275 Ž2001. 27᎐41

Divalent metal accumulation in freshwater bivalves: an inverse relationship with metal phosphate solubility Scott J. MarkichU , Paul L. Brown, Ross A. Jeffree En¨ ironment Di¨ ision, Australian Nuclear Science and Technology Organisation, Pri¨ ate Mail Bag 1, Menai, NSW 2234, Australia Received 30 May 2000; accepted 26 July 2000

Abstract Whole soft tissue concentrations of Mn, Co, Ni, Cu, Zn, Pb, Cd and U were measured in two species of freshwater Žunionid. bivalves Ž Hyridella depressa and Velesunio ambiguus. from a minimally polluted site in the HawkesburyNepean River, south-eastern Australia. Although the mean concentrations of metals in the tissue were similar for each bivalve species, their patterns of accumulation were dissimilar. For each metal, positive linear relationships between tissue concentration and shell length Ž r 2 s 0.37᎐0.77; PF 0.001. and tissue dry weight Ž r 2 s 0.29᎐0.51; PF 0.01. were found in H. depressa, but not in V. ambiguus. However, for both species, positive linear relationships were found between the tissue concentration of each divalent metal and Ca tissue concentration Ž r 2 s 0.59᎐0.97; PF 0.001.. For both bivalve species, the normalised rates of accumulation of the metals relative to increasing Ca concentration andror size, were U f CdG Pb G Mn ) CoG Zn ) Cu ) Ni. The differential rates of accumulation of divalent metals are interpreted as being predominantly governed by their varying loss rates, which are controlled by the differing solubilities Žlog K sp values. of the metals in the phosphatic extracellular granules, the demonstrated major sites of metal deposition in the tissue of H. depressa and V. ambiguus. The rates of accumulation of Mn, Co, Zn, Cu and Ni were linearly and inversely related Ž r 2 s 0.91᎐0.97; PF 0.001. to their solubilities as hydrogen phosphates, a finding consistent with the bioaccumulation model previously developed for the alkaline-earth metals. However, for U, Cd and Pb, this linear inverse relationship did not continue to hold, i.e. their rates of accumulation did not increase with decreasing solubility. However, these results are still consistent with the model if U, Cd and Pb are so insoluble in the granules of H. depressa and V. ambiguus over their lifetime Žup to approx. 50 years. that there is effectively no loss of these metals, and hence, no differential between their rates of accumulation. The present results reaffirm the use of Ca tissue concentration to predict the tissue concentrations of other divalent metals by

U

Corresponding author. Tel.: q61-2-717-3592; fax: q61-2-717-9260. E-mail address: [email protected] ŽS.J. Markich.. 0048-9697r01r$ - see front matter 䊚 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 8 - 9 6 9 7 Ž 0 0 . 0 0 7 2 1 - X

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S.J. Markich et al. r The Science of the Total En¨ ironment 275 (2001) 27᎐41

explaining up to 94 and 97% of the variability between individual bivalves of H. depressa and V. ambiguus, respectively. The use of Ca tissue concentration to effectively minimise the inherent variability between individuals in their metal tissue improves the ability of an investigator to discern smaller spatial andror temporal differences in the metal tissue concentrations of these bivalves, and thus to detect metal pollution. 䊚 2001 Elsevier Science B.V. All rights reserved. Keywords: Metal; Bioaccumulation; Freshwater; Bivalve; Solubility; Granule

1. Introduction Previous investigations on the natural rates of accumulation of the alkaline-earth metals, Mg, Ca, Ba and 226 Ra, in the soft tissue of the Australian tropical freshwater bivalve, Velesunio angasi have led to the development of a mechanistic model of their differential rates of natural accumulation ŽJeffree and Simpson, 1984; Jeffree 1988a,b.. The central hypothesis of this model is that the natural rate of accumulation for each alkaline-earth metal is linearly and inversely related to its solubility when deposited in extracellular phosphate-bearing granules, the major sites of deposition of these metals in the soft tissue of V. angasi ŽJeffree and Simpson, 1984; Jeffree, 1988a.. That is, the relative rates of accumulation of the alkaline-earth metals are predominantly controlled by their loss rates, which, in turn, are directly governed by the solubility of each metal as a hydrogen phosphate salt, after deposition in the tissue ŽJeffree and Simpson, 1984; Jeffree, 1988a.. This hypothesis has been confirmed experimentally for 226 Ra and 45 Ca ŽJeffree, 1988b. and 210 Pb and 60 Co ŽJeffree and Brown, 1992., where both studies showed that the loss rates of 226 Ra and 210 Pb from the tissue were much lower than those of 45 Ca and 60 Co, which is consistent with the greater predicted solubility of Ca and Co in the extracellular granules. The assumptions and implications of the model have been discussed previously ŽJeffree and Brown, 1992.. This mechanistic and predictive model of metal accumulation has been further validated in a study on the natural rates of accumulation of the alkalineearth metals, Be, Mg, Ca, Sr, Ba and 226 Ra by the freshwater bivalves Hyridella depressa and Velesunio ambiguus from the Hawkesbury-Nepean

River in temperate south-eastern Australia ŽJeffree et al., 1993.. In this study, the natural rates of accumulation of Mn, Co, Ni, Cu, Zn, Pb, Cd and U in the soft tissue of Hyridella depressa and Velesunio ambiguus from the Hawkesbury-Nepean River are investigated, in the context of determining whether the model of metal accumulation developed for the alkaline-earth metals also holds for a range of other divalent metals. Most of the selected metals are of potential environmental significance throughout the Hawkesbury-Nepean River catchment, as a result of metal inputs from point Že.g. treated sewage effluent. and non-point sources Že.g. storm water runoff. Žsee review by Markich and Brown, 1998.. Therefore, a goal is to establish a biomonitoring system for metals using H. depressa andror V. ambiguus as indicators of metal pollution. In this context, it is important to establish accurate contemporary baseline tissue concentrations and the natural patterns of metal accumulation, so that changes induced by urbanisation of the catchment can be demonstrated and properly interpreted. However, the ability to demonstrate statistically significant changes in tissue metal concentrations can be hampered by the inherent variability between individuals. As such, this study also investigated the variability in metal tissue concentrations between individuals of both species using biotic Žsize, age. and chemical ŽCa tissue concentration . predictors.

2. Materials and methods 2.1. Background The location and general surface water chem-


istry of the study area, together with the methods of collection and preparation of the bivalves for tissue analysis, have been described by Jeffree et al. Ž1993. and Markich and Brown Ž1998.. In summary, 24 individuals of H. depressa wshell length ŽSL., 39.90y 69.05 mmx and V. ambiguus ŽSL, 40.05y 64.55 mm., covering the widest possible size range, were sampled from abundant populations at a minimally polluted site in the Hawkesbury-Nepean River, approximately 52 km south-west of Sydney, south-eastern Australia. Specimens were maintained in a laboratory aquarium Žwithout substrate . and exposed to a synthetic Hawkesbury-Nepean River water under flow-through conditions for a period of 6 days to enable specimens to void their gut contents. The whole soft tissue was then removed from the shell and washed with deionised water ŽDIW. ŽMilli Q; 18 M⍀ cmy1 resistivity. to remove any detritus. The gender Žmalerfemale. of each individual was determined as described by Jeffree and Simpson Ž1986.. Both species were sampled during a nonreproductive period ŽJeffree et al., 1993.. Sexual dimorphism does not occur in either bivalve species. The whole soft tissue was oven-dried Ž35⬚C. to a constant weight and homogenised. Homogenised sub-samples were then solubilised using a non-boiling HNO3rH 2 O 2 digestion until a clear digest solution resulted. Digestates were evaporated to near-dryness, cooled, and then volume adjusted Ž100 ml. with DIW prior to metal analysis ŽJeffree et al., 1993.. 2.2. Tissue analysis The digest solutions were analysed for Mn, Cu and Zn by inductively coupled plasma atomic emission spectrometry ŽICPAES., as described by Jeffree et al. Ž1993., whereas Co, Ni, Pb, Cd and U were analysed by inductively coupled plasma mass spectrometry ŽICPMS., as described by Markich and Jeffree Ž1994a.. For all analytical measurements employing ICPAES and ICPMS, a multi-metal calibration standard Žmatched to the sample matrix. and a reagent blank were analysed with every eight samples to monitor signal drift. For all metals the signal typically varied by 3᎐5%,

29

although variations of 8% were noted throughout an analytical run. Additionally, where ICPMS was utilised, gallium, indium and rhenium were employed as internal standards to correct for any non-spectral interferences. 2.3. Isolation and analysis of extracellular granules Central to the developed model of metal accumulation of unionid bivalves is that the extracellular granules are the major sites of metal deposition in the soft tissue. This was tested by isolating the granules from the soft tissue of H. depressa and V. ambiguus and determining their metal concentrations relative to the whole tissue. Granules were isolated on a sucrose gradient following a procedure modified from Silverman et al. Ž1983.. Whole tissue homogenate Žpooled from 6 individuals of each species. in DIW was layered onto an equal volume of 2.5 M sucrose and centrifuged at 5200 rev. miny1 for 25 min. The resulting pellet was resuspended in DIW and the process was repeated Ži.e. two separations.. The final pellet was centrifuged Žwashed. with DIW four times and freeze-dried. The pellet was then weighed, acid digested and analysed for Mn, Co, Ni, Cu, Zn, Pb, Cd and U as described for the soft tissue. Scanning electron microscopy ŽJOEL JXA840. confirmed that the isolation procedure provided sufficient yields of tissue-free granules. The granules had a smooth surface and were similar to those observed in situ in tissue smears. The sensitivity of the energy dispersive X-ray analyser ŽNoran Voyager series 4. attached to the scanning electron microscope was insufficient to quantify the concentrations of Co, Ni, Cu, Zn, Pb, Cd and U in the granules. Qualitative analyses, however, confirmed that the granules consisted primarily of Ca and phosphate, with minor amounts of Fe, Ba and Mn. 2.4. Quality assurance The quality assurance procedures have previously been described by Jeffree et al. Ž1993. and are only briefly described here. Several DIW blanks and surrogate samples of known concen-

30


tration were taken through the digestion procedures to evaluate contamination from the reagents and sample containers. A duplicate sample and samples from two standard reference materials ŽSRMs. wCommunity Bureau of Reference ŽBCR. Mussel tissue 278 and National Institute of Standards and Technology ŽNIST. Oyster tissue 1566ax were analysed with each batch of eight samples to estimate method precision and accuracy, respectively. In addition, spiked samples were utilised to verify the absence of matrix interferences and complement the certified values. The concentrations of Mn, Cu, Zn, Pb and Cd analysed in both the BCR mussel tissue and NIST oyster tissue were within the 95% confidence intervals of the certified values. Similarly, the concentrations of Co, Ni and U were within the 95% confidence interval of the certified values for the NIST oyster tissue. The mean recoveries of metals from spiked samples ranged from 98% for U to 102% for Ni. Results were not corrected for recoveries. For duplicate, spiked and SRM samples, the mean percent coefficient of variation Ž% C.V.. was less than 8% for all metals. An interlaboratory comparison of Mn, Cu and Zn concentrations Žusing a Spectroflame ICP-AES. in the digest solutions resulted in close agreement Žmean % C.V. for all metals was - 4%. with the measured concentrations obtained from the original analyses.

2.5. E¨ aluation of metal hydrogen phosphate solubility products A detailed rationale for selection of the logarithm of the solubility products Žlog K sp values. of divalent metal hydrogen phosphates as a measure of the solubility of these metals in the extracellular granular deposits of unionid bivalves, has been reported by Jeffree et al. Ž1993. and Brown et al. Ž1996.. The nature, structure, distribution and chemical composition of the extracellular granules has been reviewed by Jeffree et al. Ž1993.. There is considerable evidence that the extracellular granules of unionid bivalves consist primarily of calcium and phosphate with minor amounts of trace metals. The solubility products Žlog K sp values. of the alkaline-earth metals ŽMg, Ca, Sr, Ba and Ra. and Cd, Co, Pb and Mn with hydrogen phosphate have been previously evaluated by Jeffree et al. Ž1993. and Brown et al. Ž1996., and are listed in Table 1. To maintain consistency with the evaluation of these solubility products, the following section reports on the critical selection of log K sp values for U, Zn, Ni and Cu hydrogen phosphate, at zero ionic strength and 25⬚C. 2.5.1. Uranium (dioxouranium) The dioxouranium Žuranyl; UO 22q . ion contains

Table 1 Selected logarithms of the solubility products Ž K sp values. for divalent metal hydrogen phosphates at 25⬚C and zero ionic strength Metal hydrogen phosphate

Log Ksp

95% confidence limits

MgHPO4 ⭈ 3H2 O NiHPO4 CuHPO4 CaHPO4 ⭈ 2H2 O ZnHPO4 CoHPO4 SrHPO4 MnHPO4 ⭈ 3H2 O BaHPO4 RaHPO4 CdHPO4 PbHPO4 UO2 HPO4 ⭈ 4H2 O

y5.75 y5.82 y6.35 y6.58 y6.74 y6.76 y6.97 y7.44 y7.46 y7.54 y8.56 y11.28 y11.86

y5.88, y5.62 y6.36, y5.28 y6.82, y5.88 y6.60, y6.55 y6.82, y6.66 y6.98, y6.54 y7.07, y6.86 y7.55, y7.33 y7.68, y7.24 y7.81, y7.28 y8.56, y8.56 y11.77, y10.80 y12.07, y11.65


two oxygen atoms bound to the central uranium atom and is divalent. The solubility products of uranyl hydrogen phosphate ŽUO 2 HPO4 ⭈ 4H 2 O. have been reported by several investigators and reviewed by Grenthe et al. Ž1992.. Vesely et al. Ž1965. and Markovic and Pavkovic Ž1983. determined the solubility product Žlog K sp . of uranyl hydrogen phosphate to be y12.17 Ž20⬚C. and y12.33 Ž25⬚C. at zero ionic strength. These values were recalculated by Grenthe et al. Ž1992. using revised uranyl phosphate stability constants, to be y11.77 and y11.88, respectively. Chukhlantsev and Stepanov Ž1956. determined the solubility product of UO 2 HPO4 ⭈ 4H 2 O at 19᎐20⬚C and various ionic strengths and obtained a log K sp value of y10.67. However, the authors did not adequately account for complex formation in their evaluation of the solubility product, and therefore, the reported value is too high ŽGrenthe et al., 1992.. Moskvin et al. Ž1967. determined the solubility products for UO 2 HPO4 ⭈ 4H 2 O from 25᎐70⬚C at zero ionic strength. These data were recalculated by Grenthe et al. Ž1992. at 25⬚C using revised uranyl phosphate stability constants, to give a log K sp value of y11.94. This study has selected a log K sp value for UO 2 HPO4 ⭈ 4H 2 O of y11.86 ŽTable 1., being the mean of the three values Žy11.77, y11.88 and y11.94. recalculated by Grenthe et al. Ž1992.. 2.5.2. Zinc The temperature dependence of the solubility of zinc hydrogen phosphate ŽZnHPO4 . has been studied by Mel’nikov et al. Ž1981.. Based on their experimental results from 25᎐75⬚C, a log K sp value of y6.74 at 25⬚C was calculated ŽTable 1.. No other studies have reported on the solubility on zinc hydrogen phosphate. 2.5.3. Nickel and copper No studies have reported on the solubility of nickel or copper hydrogen phosphate. However, Jeffree et al. Ž1993. used the electrostatic function, z 2rŽ r q 0.85., to describe the log K sp values of alkaline-earth metal hydrogen phosphates Ž r 2 s 0.997.. Fig. 1 demonstrates that the same function can be used to describe the log K sp values of the transition metals, Zn, Co, Mn, Cd, Pb and Hg,

31

Fig. 1. Log K sp values of selected divalent metal hydrogen phosphates plotted against an electrostatic function w z 2 rŽ r q 0.85.; z s ionic charge; r s ionic radiix. The simple linear regression equation is: log K sp Ž M 2q HPO4 . s y27.090q 8.191 w z 2rŽ r q 0.85.x; r 2 s 0.984, PF 0.001, n s 6.

from which an estimate of the solubility product for nickel and copper hydrogen phosphate can be obtained. The log K sp values calculated from Fig. 1 for copper and nickel hydrogen phosphate are y6.35 and y5.82, respectively. However, there are two potential shortcomings with this approach. First, the relationship shown in Fig. 1 derives from the estimation of an ionic radius for diatomic mercuryŽI.. Since mercuryŽI. forms diatomic compounds, it is difficult to determine accurately a value for the size of the ionic radius. Second, the derived log K sp values for copper and nickel hydrogen phosphate were obtained by extrapolation of the linear regression just outside its fitted range of values Žsee Fig. 1.. However, the linear regression is a very good fit of the data Ž r 2 s 0.984. over six orders of magnitude. 2.6. Statistical analyses Frequency distributions of the concentrations of each metal measured in the whole soft tissue and extracellular granules for each bivalve species were initially evaluated for departures from normality using a Shapiro-Wilk test ŽSokal and Rohlf, 1995.. The tests showed that 10 out of the 16 distributions for both whole tissue and the extracellular granules were significantly Ž PF 0.05. different from normality, all being negatively skewed.

32


Accordingly, data from such distributions were log 10 transformed to approximate normality. Mean values and their 95% confidence intervals ŽSokal and Rohlf, 1995. were calculated and back-transformed to determine the arithmetic metal concentrations, taking into account any bias ŽNewman, 1993.. Arithmetic mean values and their 95% confidence intervals were also calculated for the other sets of metal concentrations whose frequency distributions did not deviate significantly Ž P) 0.05. from normality. Simple linear regression analysis was employed to investigate the relationships between Mn, Zn, Cu, Pb, Cd, Co, Ni and U tissue concentrations and Ca tissue concentrations in H. depressa and V. ambiguus, as well as the relationships of all the above-mentioned metal tissue concentrations with shell length and tissue dry weight. Simple linear regression analysis was also used to investigate the relationship between the log K sp of each metal as a hydrogen phosphate and the factor of increase in each metal over the range of Ca tissue contents and concentrations for both species. The assumptions of simple linear regression analysis ŽSokal and Rohlf, 1995. were tested and model adequacy was confirmed in all cases. The effect of mussel gender Ž13 female, 11 male. on the tissue concentrations of each metal was evaluated by its inclusion as a dummy variable in multiple linear regression analysis with Ca tissue concentration, shell length and tissue dry weight, as described in Jeffree and Simpson Ž1986.. Significance levels were tested at the Ps 0.05 level, unless otherwise indicated.

3. Results and discussion 3.1. Whole soft tissue concentrations of Mn, Zn, Cu, Pb, Cd, Co, Ni and U in H. depressa and V. ambiguus The mean soft tissue concentrations of Mn, Zn, Cu, Pb, Cd, Co, Ni and U in H. depressa were on average 32% Žrange, 14᎐81%. higher than in V. ambiguus ŽTable 2., but were not significantly Ž P) 0.05. different Žoverlapping 95% confidence limits., due to the high inherent variability

between individuals w22᎐86% coefficient of variation ŽCOV. for H. depressa and 25᎐51% COV for V. ambiguusx. This is consistent with experimental results ŽMarkich and Jeffree, 1994a. which showed that H. depressa has a 59% greater short-term Ž10 days. accumulation rate, relative to V. ambiguus, for isotopes of Cd, Co, Mn and Pb. The relative difference between the two bivalve species in their mean tissue concentrations is similar for Co, Ni, Cu and Zn ŽTable 2., but becomes proportionately greater in H. depressa for Mn, Pb, Cd and U, the least soluble metals ŽTable 1.. For both species, Ni has the lowest ratio of mean tissue concentration relative to its mean water concentration and Zn has the highest, being at least seven times greater than the ratios of Mn, Co, Cu, Pb, Cd and U ŽTable 2.. The mean tissue concentrations of Mn, Co, Cu, Pb, Cd, Pb, Zn and U in V. ambiguus are consistent with those reported previously for this species, and V. angasi, from minimally polluted sites ŽCroome et al., 1974; Jones and Walker, 1979; Atkins, 1981; Millington and Walker, 1983; Allison and Simpson, 1989; Markich and Jeffree, 1994b.. The tissue concentrations of Cu, Cd, Pb, and Zn in H. depressa are also consistent with those previously reported for this species ŽEaston, 1997. and other Hyridella species, including H. drapeta and H. narracaensis ŽWood, 1975; Atkins, 1981; Markich, 1996; Tran, 1998.. Furthermore, for both species, the tissue concentrations of all eight divalent metals are similar to those reported for unionid bivalve species inhabiting minimally polluted sites in North America and Western Europe ŽSeagar et al., 1971; Gardner et al., 1981; Caldwell and Buhler, 1983; Tessier et al., 1984; Metcalfe-Smith, 1994; Makela ¨ ¨ et al., 1996.. 3.2. Metal concentrations in the extracellular granules of H. depressa and V. ambiguus The extracellular granules contain 74᎐96% of the selected metals measured in the whole tissue of both H. depressa and V. ambiguus ŽTable 2.. The mean concentrations of Mn, Zn, Ni and U in the granules of both species are ) 90% of their mean whole-tissue concentrations. Analyses of granules for the alkaline-earth metals, Ca, Mg, Sr


33

Table 2 Metal concentrations in the whole soft tissue and extracellular granules of H. depressa and V. ambiguus and in the surface waters from the Hawkesbury-Nepean River Metal

Watera Ž␮g ly1 .

Caf

4300

Mn

36

Zn

0.56

Cu

0.50

Pb

0.051

Cd

0.029

Co

0.19

Ni

0.22

U

0.074

H. depressa

V. ambiguus b

c

Whole tissue Ž␮g gDWy1 .

Granule Ž␮g gDWy1 .

17900 Ž14300᎐21500. 2990g Ž2260᎐3750. 267 Ž212᎐322. 18.3g Ž15.0᎐21.9. 4.49g Ž2.98᎐6.18. 1.39g Ž0.89᎐1.96. 0.98 Ž0.73᎐1.19. 0.45 Ž0.41᎐0.49. 0.12g Ž0.08᎐0.17.

17300 Ž13800᎐20800. 2870g Ž1920᎐3920. 246g Ž177᎐321. 13.9g Ž10.5᎐17.7. 4.00 Ž2.16᎐5.84. 1.14g Ž0.59᎐1.78. 0.91g Ž0.62᎐1.25. 0.40 Ž0.35᎐0.45. 0.11g Ž0.08᎐0.16.

Concentration ratiod 871 17400 99800 7660 18400 10030 1080 428 339

Whole tissuee Ž␮g gDWy1 .

Granulec Ž␮g gDWy1 .

13000 Ž11200᎐14800. 2210 Ž1850᎐2570. 235g Ž202᎐272. 14.4g Ž12.3᎐16.8. 3.17g Ž2.51᎐3.90. 0.77g Ž0.60᎐0.97. 0.79 Ž0.67᎐0.91. 0.39 Ž0.35᎐0.43. 0.072g Ž0.058᎐0.089.

12500 Ž10300᎐14700. 2130 Ž1650᎐2630. 215g Ž174᎐261. 10.7g Ž8.6᎐13.2. 2.76 Ž1.98᎐3.65. 0.62 Ž0.57᎐0.67. 0.72g Ž0.58᎐0.90. 0.34 Ž0.29᎐0.39. 0.066g Ž0.049᎐0.087.

Concentration ratiod 632 12800 87800 6030 13000 5550 870 371 204

a

Mean total concentrations Ž n s 48. for surface water samples collected monthly from four sites in the upper HawkesburyNepean River from January to December, 1991 ŽMarkich and Brown, 1998.. b Mean Žand 95% confidence interval. concentrations Ž n s 24. for individuals of varying size wshell length ŽSL.: 39.90᎐69.05 mm, median s 55.85 mm; dry tissue weight ŽDTW.: 0.278᎐1.227 g, median s 0.728 gx. c Mean Žand 95% confidence interval. concentrations Ž n s 6. for individuals of varying size ŽSL. Ž H. depressa: 42.10᎐66.05 mm, median s 54.25 mm; V. ambiguus: 38.90᎐62.20 mm, 56.90 mm.. d Concentration ratio was calculated by dividing the mean tissue concentration Ž␮grg wet wt.; i.e. dry wt. values above were divided by 4.78, the wetrdry wt. ratio. by the mean total water concentration Ž␮grg. of each metal. e Mean Žand 95% confidence interval. concentrations Ž n s 24. for individuals of varying size ŽSL: 40.05᎐64.55 mm, median s 58.70 mm; DTW: 0.213᎐1.722 g, median s 1.28 g.. f Whole tissue concentrations for both bivalve species were taken from Jeffree et al. Ž1993. for comparison with Mn, Zn, Cu, Pb, Cd, Co, Ni and U. Calcium content in the granules was 96᎐97% of that in the whole tissue. g Geometric mean values.

and Ba, showed that they comprised 92᎐97% of the whole-tissue concentrations ŽJeffree et al., 1993; Table 2. of these metals. In contrast, 76 and 74% of the Cu concentration in the whole tissue of H. depressa and V. ambiguus, respectively, is accounted for by the extracellular granules. The Carmetal ratios in the granules of H. depressa are consistent with those from in vitro qualitative studies on this species by Byrne and Vesk Ž2000. at minimally polluted sites in the HawkesburyNepean River system. Similarly, the Carmetal ŽMn, Mg, Zn. ratios for H. depressa and V. ambiguus in this study are similar to those from

quantitative or qualitative studies on North American and European species of unionids ŽHarrison, 1969; Roinel et al., 1973; Pynnonen et ¨ al., 1987; Whyte, 1991.. These results clearly demonstrate that the extracellular granules are the major sites of metal deposition in the soft tissues of H. depressa and V. ambiguus, at least from a minimally polluted site, and underpins the developed model of metal bioaccumulation for unionid bivalves. Pynnonen ¨ et al. Ž1987. reported increased Cd concentrations in the extracellular granules of three species of unionids experimentally exposed to sublethal lev-

34


els of Cd Ž40 ␮g ly1 . in water for 21 days. However, at the end of the exposure period, the extracellular granules comprised just 20% of the whole-tissue Cd concentration. The majority of Cd was associated with intracellular thiol Žsulfur. groups, which increased in proportion over the exposure period. Moura et al. Ž1999. reported increased Mn concentrations in the extracellular granules of Anodonta cygnea following experimental exposure Ž16 h. to elevated Ž1000᎐2000 ␮g ly1 ., sublethal, levels of Mn in water. In contrast to the findings of Pynnonen et al. Ž1987. ¨ for Cd, Mn was confined to the granules. The results of these two studies can be explained in terms of differential binding of metals to ligands on the granule surface. Following the hard and soft acid and base concept of Pearson Ž1963., hard metals Žincluding Ca, Mg, Ba, Sr, Co, Ni, U, Mn and Zn. have a high affinity with oxygen donor ligands Žsuch as phosphate, the major anion in the granules., whereas soft metals Žincluding Cu and Cd. have a higher affinity with sulfur or nitrogen donor ligands Žsuch as thiol groups or metallothioneins .. This may also assist in explaining the lower percentages of Cu Ž74 and 76%. and Cd Ž80 and 81%. in the granules, relative to the hard metals Žtypically ) 90%. under ‘background’ metal levels. The chemical mechanism of metal deposition on the granules is discussed in detail by Brown et al. Ž1996.. 3.3. Predictors of metal tissue concentration in H. depressa and V. ambiguus Table 3 summarises the results of simple linear regression analyses, where tissue concentrations of Mn, Co, Ni, Cu, Zn, Pb, Cd and U in both bivalve species are regressed against shell length and tissue dry weight. As shown for the alkalineearth metals ŽJeffree et al., 1993., positive linear relationships Ž P F 0.01. were found between metal tissue concentrations in H. depressa and shell length or tissue dry weight. These predictors typically explained only a small amount of the variability Žshell length: 37᎐77%; dry tissue wt.: 26᎐51%. in tissue concentrations between indi-

vidual bivalves ŽTable 3.. For V. ambiguus, no significant Ž P) 0.05. relationships were found between metal tissue concentration and shell length or tissue dry weight ŽTable 3.. Table 3 also summarises the results of simple linear regression analyses, where the tissue concentrations of each divalent trace metal are regressed against Ca tissue concentration. This was found to be a significant Ž PF 0.001. positive predictor of the tissue concentrations of the alkaline-earth metals in a previous study ŽJeffree et al., 1993.. Similarly, in this study, and for both bivalve species, Ca tissue concentration is a highly significant Ž PF 0.001. positive predictor of divalent metal concentrations, explaining between 59 and 97% of the variability between individuals. For both species, Ca tissue concentration explains the least variability in Cu tissue concentration and the most in Mn tissue concentration. These results are also consistent with those reported by Markich and Jeffree Ž1994b. for V. ambiguus in South Creek Ž53᎐84% of variability., a tributary of the Hawkesbury-Nepean River. The inclusion of gender as a predictor did not significantly Ž P) 0.05. explain any additional variability in the tissue concentrations of the divalent metals in either species, as was shown for the alkaline-earth metals ŽJeffree et al., 1993.. The ability of Ca tissue concentration to explain a large amount of the inherent variability in the concentrations of other divalent metals in the individuals of H. depressa and V. ambiguus has important implications for environmental monitoring. Since the 95% confidence limits around a fitted linear regression of Ca tissue concentration plotted against divalent metal concentration are relatively small, the capacity to demonstrate spatial andror temporal increases in the tissue concentration of a divalent metal above ‘background’ Žminimally polluted sites. is improved. Previous investigations ŽJeffree and Simpson, 1986; Jeffree, 1988a. have indicated that it is the ratio of divalent metal to Ca water concentration that determines, and is positively related to, the elevation of the regression line where the slope remains constant. Accordingly, the exposure of individuals to waters containing elevated concentra-


35

Table 3 Simple linear regressions where Ca tissue concentration and parameters of size Žshell length and tissue dry weight. predict tissue concentrations of Mn, Zn, Cu, Pb, Cd, Co, Ni and U in H. depressa and V. ambiguusa Metal

H. depressa Equation

V. ambiguus 2

r

Equation UUU

Mn Zn Cu Pb Cd Co Ni U

0.143 ŽSL. y 4.846b 0.011 ŽSL. y 0.349 0.549 ŽSL. y 10.912 0.270 ŽSL. y 9.634 0.098 ŽSL. y 3.676 0.051 ŽSL. y 1.795 0.006 ŽSL. q 0.092 0.008 ŽSL. y 0.301

0.59 UUU 0.64 UU 0.37 UUU 0.46 UUU 0.56 UUU 0.77 UUU 0.39 UUU 0.51


3.572 ŽDW. q 0.279c 0.270 ŽDW. q 0.077 16.400 ŽDW. q 7.032 6.709 ŽDW. q 0.283 2.597 ŽDW. y 0.230 1.299 ŽDW. q 0.075 0.210 ŽDW. q 0.292 0.154 ŽDW. y 0.020

0.41 UUU 0.39 UU 0.35 UU 0.29 UU 0.41 UUU 0.51 UU 0.39 UU 0.26


0.233 ŽCa. y 0.835d 0.016 ŽCa. q 0.011 0.819 ŽCa. q 5.251 0.423 ŽCa. y 2.021 0.135 ŽCa. y 0.615 0.066 ŽCa. y 0.078 0.010 ŽCa. q 0.265 0.012 ŽCa. y 0.056

0.94 UUU 0.87 UUU 0.59 UUU 0.79 UUU 0.77 UUU 0.88 UUU 0.74 UUU 0.80

UU

UUU

r2

NSe NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS 0.197 ŽCa. y 0.360 0.017 ŽCa. q 0.028 0.905 ŽCa. q 3.218 0.313 ŽCa. y 0.645 0.081 ŽCa. y 0.213 0.060 ŽCa. y 0.047 0.014 ŽCa. q 0.214 0.007 ŽCa. y 0.022

UUU

0.97 UUU 0.85 UUU 0.60 UUU 0.73 UUU 0.72 UUU 0.84 UUU 0.71 UUU 0.80

a Tissue concentrations of Mn and Zn are reported in mg gDWy1 , whilst Cu, Pb, Cd, Co, Ni and U are reported in ␮g gDWy1 . n s 24. b SL, shell length Žmm.. c DW, tissue dry weight Žg.. d Ca, calcium tissue concentration Žmg gDWy1 .. e NS: not significant, i.e. P) 0.05. UUU PF 0.001. UU 0.001- PF 0.01.

tions of these metals would result in elevated metal tissue concentrations, and hence, an elevated regression line, relative to individuals from a minimally polluted site. This approach has been used to statistically demonstrate small changes in the tissue concentrations of selected divalent metals ŽCu, Co, Pb, Mn, U and Zn., relative to the variability between individuals, in V. angasi from the Finniss River, downstream of the Rum Jungle UrCu mine in northern Australia ŽS. Markich, unpublished..

3.4. Calcium tissue concentration as a measure of the kinetics of di¨ alent metal bioaccumulation Jeffree et al. Ž1993. concluded that the theoretical basis for the selection of Ca tissue concentration as a predictive variable is that the model of the bioaccumulation of non-essential alkalineearth metals interprets their uptake by mistaking them for Ca, a biologically essential metal, because of their chemical similarity. This conclusion has been validated for the alkaline-earth metals

36


in several experimental and field investigations ŽJeffree and Simpson, 1986; Jeffree, 1988a; Jeffree et al., 1993.. Although the divalent transition metals investigated in the present study are not as chemically similar to Ca as the alkaline-earth metals, the results still demonstrate highly significant Ž PF 0.001. positive linear relationships between the tissue concentrations of each divalent trace metal and Ca. Furthermore, the selected divalent metals are predominantly colocated with Ca in the granules, suggesting that they are treated in a metabolically similar manner to Ca by both bivalve species. Previous studies using V. angasi ŽJeffree, 1988b; Brown et al., 1996. have indicated that the measured Ca tissue concentration is related to the total influx and efflux of Ca through the tissue over the life of the individual bivalve. Moreover, the measured tissue concentration of other metals will be dependent on three separate entities, namely: 1. the concentration of the particular metal in the aqueous medium to which the bivalves are exposed; 2. the total influx of Ca into the bivalve, since each metal is accumulated as a metabolic analogue of Ca; and 3. the total efflux of each individual metal from the tissue over the life of the individual bivalve. This latter process is governed by the differential loss rates of individual metals as a result of their differing solubilities in the extracellular granules. 3.5. Natural rates of accumulation of Mn, Co, Ni, Cu, Zn, Pb, Cd and U in H. depressa and V. ambiguus Fig. 2 shows the relative natural rates of accumulation of Mn, Co, Ni, Cu, Zn, Pb, Cd and U in H. depressa and V. ambiguus, where metal tissue concentrations are normalised and plotted as a function of increasing Ca tissue concentration. Fig. 2 also shows the relative natural rates of metal accumulation as a function of shell length and tissue dry weight for H. depressa. For each

metal, the fitted regression line is positive and highly significant ŽTable 2.. For H. depressa, the rates of accumulation follow the sequence U f Pb f Cd) Mn ) CoG Zn ) Cu ) Ni for each of the three plots. For V. ambiguus, the single plot follows a similar sequence, U f Cd) Mn f Pb ) Cof Zn ) Cu ) Ni. Jeffree et al. Ž1993. found an inverse linear relationship Ž PF 0.05. between the relative rates of accumulation of the alkaline-earth metals Ž 226 Ra ) Ba G Sr ) Ca ) Mg. and their solubility as hydrogen phosphates. For example, Mg is the most soluble metal as a hydrogen phosphate Ži.e. lowest log K sp value. and has the lowest natural rate of accumulation. The rates of accumulation of each alkaline-earth metal were calculated over the range of Ca tissue contents or concentrations, using a factor of increase in terms of metal tissue content or concentration. Subsequently, a plot of log K sp values for the alkaline-earth metal hydrogen phosphates against the factor of increase in each metal, indicated a significant Ž PF 0.05. inverse relationship between log K sp and normalised metal tissue content or concentration ŽJeffree et al., 1993.. Similar plots for the divalent transition metals are shown in Fig. 3 for H. depressa and V. ambiguus, with the values determined by Jeffree et al. Ž1993. for the alkalineearth metals also included. In accord with the results of Jeffree et al. Ž1993., regressions using Ca tissue content as a predictor of divalent trace metal content mirror the results for Ca tissue concentration. Calcium tissue concentration Žor content., rather than a parameter of size, was selected because of the higher percentage of variability that it could explain in H. depressa, and because it is the only significant Ž PF 0.05. predictor of divalent trace metal tissue concentration Žor content. in V. ambiguus. The highly linear patterns of accumulation of each metal ŽFig. 2., when regressed against Ca, indicate a consistent metabolism Žsee Section 3.4.. The model developed in previous studies ŽJeffree, 1988a,b; Jeffree et al., 1993. interprets the differential rates of accumulation of the alkalineearth metals as being predominantly governed by their varying loss rates. These loss rates are, in turn, controlled by the differing solubilities Žlog


37

Fig. 2. Normalised tissue concentrations of Mn, Zn, Cu, Pb, Cd, Co, Ni and U Ždetermined by the regression equations in Table 3. plotted against Ca tissue concentration for H. depressa and V. ambiguus, and shell length and tissue dry wt. for H. depressa.

K sp values. of the metals in the extracellular granular deposits, the major sites of alkaline-earth metal deposition in the tissue of V. angasi ŽJeffree and Simpson, 1984; Jeffree 1988a.. This study has demonstrated that the extracellular granules are also the major sites of deposition for Mn, Co, Ni, Cu, Zn, Pb, Cd and U in H. depressa and V. ambiguus, at least for individuals from a minimally polluted site. The concentration of a metal in the granules, at any given point in time, will be equivalent to the concentration of metal hydrogen phosphate wsince each molecule of M HPO4 contains one metal Ž M . ionx. Fig. 3 shows that an inverse linear relationship holds for Mn, Co, Ni, Cu, Zn, Pb, Cd and U for log K sp values between

y7.54 Ž 226 Ra. and y5.75 ŽMg. with both bivalve species. For Cd, Pb and U, with solubilities Ži.e. log K sp values. less than that of 226 Ra, there is a discontinuity in the relationship Ži.e. a plateau effect., where there is no further rise in the factor of increase in metal tissue concentration or content with a decrease in solubility Žlog K sp value.. Indeed, it would appear that Pb, Cd and U all have a similar factor of increase, being only marginally higher than that determined for 226 Ra. Divalent metals with log K sp values between Mg and 226 Ra Ži.e. Ni, Cu, Zn, Co and Mn. conform to the developed model of metal bioaccumulation. At first glance, it would appear that there is a departure from the model for divalent

38


Fig. 3. The factor of increase in Mn, Zn, Cu, Pb, Cd, Co, Ni and U Žin addition to the alkaline-earth metals; Jeffree et al ., 1993. tissue content and tissue concentration plotted against the logarithm of their respective solubility product Žlog K sp . with HPO4 for H. depressa and V. ambiguus. The factor of increase was calculated by dividing each metal’s tissue contentrconcentration at the largest Ca tissue contentrconcentration by its tissue contentrconcentration at the smallest Ca tissue contentrconcentration, respectively, as predicted from the regression equations in Table 3. Simple linear regression analyses were performed for the linear part of the relationship Ži.e. metals ranging from Mg to 226 Ra, n s 10.. UUU P F 0.001.

metals less soluble than 226 Ra Ži.e. Cd, Pb and U.. However, the natural accumulation behaviour of these insoluble metals can be explained as follows. Cadmium, Pb and U are so insoluble in the tissue Žgranules., that over the lifetime of a individual Žup to approx. 50 years for V. angasi, H. depressa and V. ambiguus. there is effectively no loss of these metals, as indicated experimentally ŽBrown et al., 1996. and under field conditions ŽS. Markich, unpublished., where no loss of 109 Cd, 210 Pb and 232 U was detected after 160 days Žlaboratory. or 370 days Žfield. exposure to radionuclide-free water. As a consequence, there would also be no differential between the rates of

accumulation of Cd, Pb and U since, as already stated, differential rates of accumulation require differential rates of loss. The highly linear natural rates of accumulation of divalent metals by H. depressa, V. ambiguus and V. angasi over their lifetime ŽJeffree, 1988a; Jeffree et al., 1993; Markich and Jeffree, 1994b. is paradoxical, given that metal loss follows first order Žexponential. loss kinetics ŽJeffree and Simpson, 1986; Jeffree, 1988b; Brown et al., 1996.. That is, for those metals where loss occurs, a curvilinear Ži.e. concave to the sizerage axis. relationship should exist ŽBrown et al., 1996.. This paradox has been interpreted by Jeffree et al.


Žsubmitted., as follows, using a model of the exchangeable pool and sink kinetics of metals. The most recently deposited concentric lamination Žlayer. of the extracellular granules, which is in direct contact with the body fluids, acts as an exchangeable pool, where metals accumulate according to their differential solubilities as hydrogen phosphates. As each lamination is covered by a newly deposited one, it becomes more remote from the body fluids and functions more as a metal sink Žnon-exchangeable pool.. Therefore, as the granules increase in size with increasing age of the bivalve, their surface area-to-volume ratio decreases and accumulation approximates linearity, reflecting sink kinetics. A more detailed discussion is provided by Jeffree et al. Žsubmitted..

4. Conclusions The results of this work extend the previously developed model of metal accumulation for alkaline-earth metals Ž 226 Ra, Ba, Sr, Ca, and Mg. to include a range of divalent transition metals ŽMn, Co, Ni, Cu, Zn, Pb, Cd and U.. The central hypothesis of this model is that the natural rate of accumulation of a metal is linearly and inversely related to its solubility when deposited in extracellular phosphate-bearing granules, the major sites of deposition of these metals in the soft tissue. This study confirmed that the extracellular granules of H. depressa and V. ambiguus were the major sites of deposition for Mn, Co, Ni, Cu, Zn, Pb, Cd and U. The ‘hard’ metals, such as Mn, Co, Ni, Zn and U, appear to have a higher affinity for the granules than the ‘softer’ metals, Cd and Cu. Further work should evaluate this notion, particularly where bivalves are naturally exposed to sublethal metal concentrations. Nickel, Cu, Zn, Co and Mn were found to conform to the developed model of metal bioaccumulation. That is, their natural rates of accumulation were linearly and inversely related to their solubility in the granules Žas hydrogen phosphates.. Conversely, the natural rates of accumulation of the least soluble metals, Pb, Cd and U did not increase with decreasing solubility, and thus, do not appear to fit the model. However, if

39

U, Cd and Pb are so insoluble in the tissue Žgranules. of H. depressa and V. ambiguus over their lifetime Žup to approx. 50 years. that there is effectively no loss of these metals, then the relative rates of accumulation of U, Cd and Pb should not differ. If this interpretation is correct, then their accumulation is consistent with the proposed model. The present results reaffirm the use of Ca tissue concentration to predict the tissue concentrations of other divalent metals by explaining up to 94% and 97% of the variability between individual bivalves of H. depressa and V. ambiguus, respectively. The use of Ca tissue concentration to minimise the inherent variability between individuals in their metal tissue concentrations improves the ability of an investigator to statistically demonstrate smaller spatial andror temporal differences in the metal tissue concentrations of bivalves, and thus, detect metal pollution. The use of H. depressa and V. ambiguus in transplant experiments to gauge improvements in water quality in the Hawkesbury-Nepean catchment Ži.e. by placing individuals from polluted sites to minimally polluted sites., will be of no value for elements such as U, Pb, Cd and 226 Ra, which have very long biological half-lives Ži.e. no demonstrated loss. in the tissues Žgranules.. Consequently, individuals transplanted from contaminated sites to minimally polluted sites will continue to reflect their ‘source’ location.

Acknowledgements We thank Ms Maree Emett, Ms Elizabeth Keegan, Ms Julie Weir ŽEnvironment Division, ANSTO. and thank Mr John Buchanan ŽCSIRO Centre for Advanced Analytical Chemistry. for assistance with the metal analyses. Sammy Leung ŽMaterials Division, ANSTO. provided assistance with the scanning electron microscopy. References Allison HE, Simpson RD. Element Concentrations in the Freshwater Mussel, Velesunio angasi, in the Alligator Rivers Region. Canberra: Australian Government Publishing Ser-

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