Comparison of Subcellular Partitioning, Distribution, and Internal

Apr 18, 2008 - A population of Dendrodrilus rubidus Savigny earthworms from the Coniston Copper Mines, an area of former Cu mining, exhibit increased ...
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Environ. Sci. Technol. 2008, 42, 3900–3905

Comparison of Subcellular Partitioning, Distribution, and Internal Speciation of Cu between Cu-Tolerant and Nai1ve Populations of Dendrodrilus rubidus Savigny B E C K Y E . A R N O L D , †,| M A R K E . H O D S O N , * ,† J O H N C H A R N O C K , ‡,⊥ A N D WILLIE J.G.M. PEIJNENBURG§ Department of Soil Science, School of Human and Environmental Sciences, University of Reading, Whiteknights, Reading, Berkshire, RG6 6DW, U.K., CLRC Daresbury Laboratory, Warrington, Cheshire, WA4 4AD, U.K., and Laboratory for Ecological Risk Assessment, National Institute of Public Health and the Environment, PO Box 1, 3720 BA Bilthoven, The Netherlands

Received January 17, 2008. Revised manuscript received February 12, 2008. Accepted February 19, 2008.

When considering contaminated site ecology and ecological risk assessment a key question is whether organisms that appear unaffected by accumulation of contaminants are tolerant or resistant to those contaminants. A population of Dendrodrilus rubidus Savigny earthworms from the Coniston Copper Mines, an area of former Cu mining, exhibit increased tolerance and accumulation of Cu relative to a nearby non-Cu exposed population. Distribution of total Cu between different body parts (posterior, anterior, body wall) of the two populations was determined after a 14 day exposure to 250 mg Cu kg-1 in Cu-amended soil. Cu concentrations were greater in Coniston earthworms but relative proportions of Cu in different body parts were the same between populations. Cu speciation was determined using extended X-ray absorption fine structure spectroscopy (EXAFS). Cu was coordinated to O atoms in the exposure soil but to S atoms in the earthworms. There was no difference in this speciation between the different earthworm populations. In another experiment, earthworms were exposed to a range of Cu concentrations (200-700 mg Cu kg-1). Subcellular partitioning of accumulatedCuwasdetermined.Conistonearthwormsaccumulated more Cu but relative proportions of Cu in the different fractions (cytosol > granular > tissue fragments, cell membranes, and intact cells) were the same between populations. Results suggest that Coniston D. rubidus are able to survive in the Cu-rich Coniston Copper Mines soil through enlargement of the same Cu storage reservoirs that exist in a nearby non-Cu exposed population. * Corresponding author phone: +44 (0) 118 378 6974; fax: +44 (0) 118 378 6974; e-mail: [email protected]. † University of Reading. | Current address: Greenfinch Ltd, The Business Park, Coder Road, Ludlow, Shropshire, SY8 1XE, U.K. ‡ CLRC Daresbury Laboratory. ⊥ Current address: School of Earth, Atmospheric and Environmental Sciences, The University of Manchester, Oxford Road, Manchester, M13 9PL, U.K. § National Institute of Public Health and the Environment. 3900

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Introduction Earthworms have been termed “ecosystem engineers” due to their importance in developing and maintaining soil structure, incorporating and breaking down organic matter into the soil, and generating bioporosity. Because of their importance in soils earthworms are a key organism in the assessment of the health of contaminated soils. Soil dwelling organisms such as earthworms may be present in contaminated soils if the contaminant is not in a bioavailable form. Alternatively the earthworms may be tolerant (plastic physiological adaptation) or resistant (genetic modification) to the contaminants. Reasons behind the presence of soil organisms in contaminated soils are vital to accurate ecological risk assessments. Additionally if earthworms are resistant to the toxic effects of metals it may be possible to utilize them in the remediation of mine spoil and brownfield sites, as is currently the case with metal-tolerant plants (1). Several reports of metal tolerant earthworms exist in the literature in which the responses of resident and naı¨ve populations of earthworms have been compared (2-5). We have previously reported the existence of a population of the earthworm Dendrodrilus rubidus Savigny at Coniston Copper Mines, a former Cu mining area in the Lake District of the UK (2). Copper concentrations in the soil are 789 ( 4 mg kg-1 (n ) 3 ( std. error). Concentrations of Cu in the earthworms at the site are 53.34 ( 1.70 mgkg-1 compared to 4.16 ( 0.72 mg kg-1 (n ) 3 ( std. error in both cases) in earthworms from a manure heap at Crook Barn Stables with background Cu concentrations of 54 ( 1 mg Cu kg-1 (n ) 3 ( std. error). The LC50 for Cu determined using Cu nitrate amended Kettering loam soil and D. rubidus sampled from the Crook Barn Stables manure was 355 mg Cu kg-1 (95% CI 298-422 mg Cu kg-1); no LC50 was calculated for the Coniston Copper Mines earthworms as no mortality was recorded at the highest Cu exposure concentration of 700 mg Cu kg-1 (2). Earthworms may sequester metals in two broad fashions: (1) the formation of metal-bearing inclusion bodies (6) and (2) the binding of metals to proteins such as metallothionein, predominantly in the cytosol (7). The aim of this study was to determine whether the Cu accumulated in the Coniston earthworms was stored differently to that in a naı¨ve population, both in terms of location and also form. In order to achieve this aim, three complementary techniques have been used. The bulk Cu concentration in different body parts of earthworms was determined using acid digestion and solution analysis. The speciation of Cu in the different body parts was determined using X-ray absorption spectroscopy (XAS). This is the first reported work on Cu speciation in earthworms using XAS and only the third reported work on application of XAS to issues of metal speciation in earthworms, the others being concerned with As in L. rubellus (8) and Cd in D. rubidus (9). The subcellular partitioning of the Cu was determined using a chemical fractionation method (10) that has previously been applied to earthworms in only three other studies (10-12). Thus our work uses highly novel techniques and, for the first time, applies these to the same populations of earthworms.

Experimental section Earthworms. Mature D. rubidus were collected by hand sorting from an Umbric leptosol soil at Coniston Copper Mines, an area of abandoned copper mines in the Lake District, Cumbria, UK (Ordnance Survey Grid Reference SR 10.1021/es800172g CCC: $40.75

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288 987), and from a manure heap with background Cu concentrations at Crook Barn Stables, Torver, Cumbria, UK (Ordnance Survey Grid Reference SR 275 937) during March 2004. These earthworms were maintained in their native soil/manure in plastic culture boxes (36 × 27 × 20 cm) at 15 ( 1 °C for up to 3 weeks until required for experimentation. Food was applied weekly to the surface of the cultures in the form of dried and rehydrated horse manure. Approximately 0.75 g rehydrated manure was added per earthworm and any uneaten manure was removed prior to each new addition. Soils. Soil was collected from Coniston Copper Mines (pH 3.46 ( 0.01, aqua regia Cu ) 789 ( 4 mg kg-1, organic matter content 21.5 ( 0.5%) and manure from Crook Barn Stables (aqua regia Cu ) 54 ( 1 mg kg-1), at the same time as earthworms were sampled. Additionally a commercially available Kettering loam (pH 6.60 ( 0.00, aqua regia Cu ) 16.4 ( 0.3 mg kg-1, organic matter content 6.9 ( 0.1%) was purchased from Turf Management Systems, Iver Heath, Buckinghamshire, UK. All the material was dried at 30 °C, sieved to anterior > body wall, regardless of the population origin and treatment (ANOVA and Tukey test, p < 0.001). The Cu concentration associated with the posterior, anterior and body wall sections accounted for between 66-79%, 17-19% and 4-5% of the total earthworm tissue concentrations, respectively. Both the order of Cu concentrations and the relative % of total Cu found here are consistent with results obtained on Lumbricus

rubellus and Aporrectodea caligninosa sampled from former mine sites (22, 23) and are due to the posterior section containing the chloragogenous tissue, the major metal-sequestering tissue in earthworms (24). The higher concentrations of Cu in the different body sections of the Coniston earthworms maintained in 250 mg kg-1 Cu amended Kettering loam compared to those kept in their native soil (789 mg Cu kg-1) indicates that the Cu in the Cu-amended soil was more bioavailable. This result is again consistent with other studies (25-28) where availability of metals in contaminated soil is controlled by the form of metal and particularly the relative solubilities of those forms. The dominant Cu bearing phases at Coniston Copper Mine are malachite (Cu2CO3(OH)2), azurite (2CuCO3Cu(OH)2), and chalcopyrite (CuFeS2) and also less abundantly bornite (Cu5FeS4) and chrysocolla, (Cu5SiO3.2H2O) (29, 30). Thus much of the Cu in the Coniston soil will be present as relatively unavailable, insoluble solid form. EXAFS. EXAFS is a powerful technique that provides direct information on the molecular environment surrounding an element of interest. Results of the EXAFS carried out in this study are given in Table 1. χ(k)k3 EXAFS spectra and their corresponding radial structure-functions (derived from the phase shift-adjusted Fourier transforms) are given as Figures A1 and A2 in the Supporting Information. There were no significant differences in the Cu speciation between the whole earthworm, posterior section, and body wall samples of the Coniston D. rubidus that was cultured in its native soil. There were no significant differences between the Cu speciation of the whole earthworm samples of the Coniston D. rubidus cultured in its native soil and the Coniston and Crook Barn Stables D. rubidus cultured in the Cu-amended Kettering loam. All the earthworm samples indicated that Cu was coordinated by three S atoms with a CusS bond length of c. 2.2 Å. Attempts to model a second shell of atoms slightly improved the R value but were not statistically justified as indicated by a reduced chi-squared test result (data not shown). There are no published earthworm Cu EXAFS data with which to compare our data but EXAFS-based studies of As and the earthworm L. rubellus (8) and Cd and the earthworm D. rubidus (9) indicate that these metals are coordinated to S atoms, most likely from metallothioneintype ligands. In the current experiments it was impossible to determine whether the CusS coordination evident in the D. rubidus native to Coniston and Crook Barn Stables was metallothionein complexation because it was not possible to undertake XANES analysis due to lack of appropriate model compound data; furthermore, the signal-noise ratio meant that only one shell of coordination could be resolved. Copper is a poor inducer of metallothionein (31-33), although the metal will avidly bind to the protein if the latter is induced by an alternative metal, particularly cadmium (31, 34, 35). Metalliferous mine sites are frequently contaminated with more than one element (8, 22, 36), however, the aqua regia extractable concentrations of Pb, Zn, Fe, and Ni in the Coniston soils were low (171 ( 2 mg kg-1, 100 ( 2 mg kg-1, 36 900 ( 900 mg kg-1, 9 ( 0 mg kg-1, respectively, n ) 3 ( std. error), and the concentration of Cd was below the ICP-OES detection limit (0.18 mg kg-1). Furthermore, the presence of metallothionein has primarily been identified in the chloragogenous tissue associated with the chloragocytes, surrounding the blood vessels, within the typhlosolar fold and within the peri-intestinal region (8). Therefore, if Cu was sequestered within metallothionein it would be expected that a clear edge step within the EXAFS spectra of the chloragogenous sample would be evident. The high level of noise prevented a useable EXAFS spectra being

obtained for the chlorogogenous tissue, but there was no evidence for the presence of Cu. Additionally, the low mass of the chloragogenous tissue prevented the analysis of the concentration of Cu by tissue digestion. Therefore, while the CusS coordination is consistent with bonding to metallothionein this is unproven. However the EXAFS data give clear evidence that the speciation of the Cu changes when the soil is ingested by the earthworms and Cu is incorporated into the earthworm tissues. The Cu in the Coniston soil and Kettering loam soils was most likely coordinated by four O atoms with a CusO bond length of c. 1.9 Å. For the Coniston soil, this is consistent with the CusO coordination and bond distances of malachite (4 O at 1.89 -1.99 Å; (37)) and azurite (four O at 1.93 - 1.94 Å; (38)) and thus the known mineralogy of the site (29, 30). In addition to the four O atoms at 1.9 Å the fit to Cu in the Kettering loam spectrum was improved by additional coordination by two longer axial O atoms with a bond distance of 2.3 Å. This is indicative of the Jahn-Teller effect; a distorted octahedral coordination of atoms around a central copper atom, which is frequently observed in inorganic compounds, for example in the ion [Cu(H2O)6]2+ (39). Thus the coordination of Cu atoms with O in the Kettering loam soil is consistent with the application of 250 mg Cu kg-1 applied as copper nitrate solution, with the copper being present as a hydrated copper ion. Analysis of a CuSO4.5H2O standard gave CusO bond distances in agreement with published crystal structure (40). Subcellular Partitioning of Cu. The subcellular partitioning of Cu between the C, D, and E fractions of D. rubidus native to Coniston Copper Mine and Crook Barn Stables following their exposure to Kettering loam soils treated with concentrations of Cu nitrate is shown in Figures 1 and 2, respectively. The concentrations of Cu associated with the C, D, and E fractions of D. rubidus native to Coniston Copper Mine were significantly greater than those of the Crook Barn Stables population (t test, p < 0.05, C and E fractions; p < 0.01, D fraction). However, when expressed as a proportion of the total earthworm tissue concentrations, the concentrations of copper associated with the C, D, and E fractions were not significantly different between the two earthworm populations (ANOVA, p < 0.005). Within both earthworm populations the largest proportion of the total earthworm tissue concentration (59-80%) was associated with the C fraction (ANOVA, p < 0.001) and the concentration of Cu in the C fraction showed a linear increase when regressed against soil Cu concentration (R2 ) 0.55 and 0.59, p < 0.005 for Coniston and Crook Barn Stables earthworms, respectively). This result is consistent with studies in which Cu has been found to concentrate in the C fraction of the earthworm Aporrectodea caliginosa (10, 11), and indeed, studies investigating other metals such as Cd and Zn in earthworms (11, 12) and aquatic organisms (41, 42). Mechanism of Tolerance/Resistance of D. rubidus to Elevated Cu Concentrations at Coniston Copper Mines. Langdon et al. (3) reported Cu tolerant D. rubidus and L. rubellus from Devon Great Consols and Cu tolerant L. rubellus from Carrock Fell. Both sites are former mine sites. They have suggested that as Cu is a poor inducer of metallothenein the Cu tolerance demonstrated by these earthworms could be a consequence of the mechanism responsible for As tolerance in the earthworms inhabiting these soils. Concentrations of As and Cu at Devon Great Consols and Carrock Fell are 8983 ( 43 mg As kg-1 and 1732 ( 350 mg Cu kg-1 and 10277 ( 270 mg As kg-1 and 725 ( 30 mg Cu kg-1, respectively, i.e. the concentration of As is far greater than that of Cu. In contrast at the Coniston site concentrations of As and Cu are 267 ( 6.3 mg kg-1 and 789 ( 4 VOL. 42, NO. 10, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Subcellular partitioning of copper in D. rubidus native to Coniston Copper Mine following a 14 day exposure to Kettering loam soils treated with copper nitrate/mg kg-1 (mean ( s.e., n ) 3 at each soil copper concentration except at 700 mg kg-1 where n ) 2). The concentrations are expressed in terms of mass of copper per mass of subcellular fraction on a (frozen and defrosted) wet weight basis. For a given fraction columns labelled with the same letter are not significantly different (p < 0.05).

FIGURE 2. Subcellular partitioning of copper in D. rubidus native to Crook Barn Stables following a 14 day exposure to Kettering loam soils treated with copper nitrate/mg kg-1 (mean ( s.e., n ) 3 at each soil copper concentration). The concentrations are expressed in terms of mass of copper per mass of subcellular fraction on a wet weight basis. For a given faction columns labelled with the same letter are not significantly different (p e 0.05). mg kg-1, respectively. Thus it seems unlikely that As concentrations at the Consiton site are playing a role in the development of Cu tolerance in the Coniston earthworms. Previous work has reported higher bulk body burden Cu and an ability to survive at higher Cu concentrations for the Coniston D. rubidus compared to the Crook Barn Stables earthworms (2). This work has further refined this finding. There is no difference between the location of the highest concentration of Cu (posterior section, C fraction-proteins, and microsomes), the relative percentages of Cu present in these locations or the speciation of the Cu (coordinated by three S atoms with a CusS distance of 2.2 Å) between these two earthworm populations. Thus it would appear that Cu is processed in the same manner by both earthworm populations and that the only “special” thing about the Coniston population is its ability to process and accumulate more Cu than the Crook Barn Stables population. This suggests that the Coniston earthworms are tolerant to high Cu levels (a reversible change in physiology) rather than resistant (a change in the genetic structure of the organism) however this must remain speculative until either F1 and F2 generational studies are 3904

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performed or the genetic makeup of the different earthworm populations is determined.

Acknowledgments This work was carried out as part of a PhD study by R.E.A. entitled “Earthworm-Copper Interactions” which was sponsored by the NERC (NER/S/A/2001/06327). Confirmation of earthworm identification was kindly carried out by Dr T. Piearce, University of Lancaster. Dr C.J. Langdon provided assistance in the field. We are grateful to the NERC and CCLRC for provision of beamtime at the Daresbury Synchrotron Radiation Source and would like to thank Mr Bob Bilsborrow for his help setting up the experiment.

Supporting Information Available Tables of dissection fractions and masses used for EXAFS study. Figures of Cu-K edge k3 weighted EXAFS and the best model fits discussed in the paper. This information is available free of charge via the Internet at http://pubs.acs.org.

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