Environ. Sci. Technol. 2009, 43, 6822–6829
Accumulated Metal Speciation in Earthworm Populations with Multigenerational Exposure to Metalliferous Soils: Cell Fractionation and High-Energy Synchrotron Analyses J A N E A N D R E , †,§,* J O H N C H A R N O C K , ‡ ¨ RZENBAUM,| STEPHEN R. STU PETER KILLE,† A. JOHN MORGAN,† AND MARK E. HODSON§ Cardiff School of Biosciences, Cardiff University, Park Place, Cardiff, CF10 3US, U.K.
Received January 27, 2009. Revised manuscript received May 21, 2009. Accepted June 17, 2009.
Predicting metal bioaccumulation and toxicity in soil organisms is complicated by site-specific biotic and abiotic parameters. In this study we exploited tissue fractionation and digestion techniques, combined with X-ray absorption spectroscopy (XAS), to investigate the whole-body and subcellular distributions, ligand affinities, and coordination chemistry of accumulated Pb and Zn in field populations of the epigeic earthworm Lumbricus rubellus inhabiting three contrasting metalliferous and two unpolluted soils. Our main findings were (i) earthworms were resident in soils with concentrations of Pb and Zn ranging from 1200 to 27 000 mg kg-1 and 200 to 34 000 mg kg-1, respectively; (ii) Pb and Zn primarily accumulated in the posterior alimentary canal in nonsoluble subcellular fractions of earthworms; (iii) sitespecific differences in the tissue and subcellular partitioning profiles of populations were observed, with earthworms from a calcareous site partitioning proportionally more Pb to their anterior body segments and Zn to the chloragosomerich subcellular fraction than their acidic-soil inhabiting counterparts; (iv) XAS indicated that the interpopulation differences in metal partitioning between organs were not accompanied by qualitative differences in ligand-binding speciation, because crystalline phosphate-containing pyromorphite was a predominant chemical species in the wholeworm tissues of all mine soil residents. Differences in metal (Pb, Zn) partitioning at both organ and cellular levels displayed by field populations with protracted histories of metal exposures may reflect their innate ecophysiological responses to
* Corresponding author e-mail:
[email protected]; tel: +44 2920876680; fax: +44 2920874305. † Cardiff School of Biosciences, Cardiff University, Park Place, Cardiff, CF10 3US, U.K. § Department of Soil Science, School of Human and Environmental Sciences, University of Reading, Whiteknights, Reading, RG6 6DW, U.K. ‡ School of Earth, Atmospheric and Environmental Sciences, University of Manchester, Oxford Road, Manchester, M13 9PL, U.K. | King’s College London, School of Biomedical & Health Sciences, Pharmaceutical Sciences Division, London, SE1 9NH, U.K. 6822
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 17, 2009
essential edaphic variables, such as Ca2+ status. These observations are highly significant in the challenging exercise of interpreting holistic biomarker data delivered by “omic” technologies.
Introduction Metal bioavailability in soils is a dynamic process, modulated by edaphic factors, as well as the physiological and behavioral variables that characterize a receptor organism. It can be indirectly estimated by sequential chemical extractions (1) but the ecotoxicological relevance of such measures of metal mobility and speciation is questionable. Despite this, relatively simple models incorporating “total” soil metal concentrations and pH often accurately predict the equilibrated tissue metal concentrations in certain key taxa, such as earthworms (2). A truer assessment of metal bioavailability should describe an organism’s exposure. In the case of soil-dwelling invertebrates, assaying whole-organism and tissue-specific metal burdens are key steps toward this goal. For example, critical body residues (CBRs) relate the bioaccumulation of metals within tissue targets of potential toxicosis to ecologically relevant end points of the receptor organism’s function (3). Conceptually, therefore, CBRs change the focus of bioavailability assessment from processes that are essentially external to the organism (4) to processes and events occurring within the organism. Thus, the CBR paradigm is founded on the notion that it reflects metal toxico-(bio)availability, and automatically integrates the potential modulating effects of exposure route(s) and duration, as well as metabolic variables (5-7). However, the ecotoxicological application of CBRs to terrestrial invertebrates has proven problematic, being hampered by a limited understanding of the relationship between the cellular partitioning of metals into pools differing in their bioreactivity potentials and of the balance between direct and indirect metal-evoked toxic effects (5, 6, 8). The problem is exacerbated, particularly in metal macro-accumulating organisms like earthworms, because the metabolically active metal pool(s), while being the predominant toxicological fraction(s), almost certainly accounts for a minor proportion of the total accumulated body burden. Consequently, it is self-evident that expressing CBRs on the basis of total body concentrations can be misleading because the actual “critical” metal concentration causing a measurable degree of functional perturbation is that elusive fraction of the tissue burden that intrudes into, and interferes with, biochemical pathways (6). Estimating the quantity of bioreactive intracellular metal is technically challenging. Tissue fractionation methods have been introduced to address this challenge. These isolate operationally defined subcellular fractions from homogenized tissue that is subsequently differentially centrifuged. Such studies have mainly concentrated on freshwater and marine invertebrates (6, 7, 9-12). Evidence is emerging from these studies indicating that the relative insensitivity to metal toxicosis of resistant ecotypes within certain invertebrate taxa exposed to metalliferous environments over several generations is reflected in their subcellular metal partitioning profiles when compared to their counterparts inhabiting unpolluted habitats. For example, freshwater caddisfly larvae (Insecta: Trichoptera) with a population history of Cd exposure have been found with an enhanced Cd-sequestering phenotype (13). Moreover, a population of the freshwater oligochaete Lumbriculus hoffmeisteri resident in a Cd polluted sediment was observed to deposit exceptionally high con10.1021/es900275e CCC: $40.75
2009 American Chemical Society
Published on Web 08/05/2009
TABLE 1. Pb, Zn, and P Concentrations of Contaminated Mine-Site Soils and Control Soils Following an Aqua Regia Digest and Calcium Nitrate Extractiona mean soil concentration, aqua regia digest mg kg-1
mean soil concentration, 0.01 M Ca(NO3)2 extraction mg kg-1
site
pH
water holding capacity (%)
Pb
Zn
P
Pb
Zn
MCC MCS MDH CDP CP
6.5 4.8 6.8 6.9 5.3
80.4 87.5 83.7 83.1 84.6
11928 ( 659 1217 ( 51 27064 ( 9055 102.8 66 ( 10
34003 ( 3864 219 ( 9 5438 ( 971 219 165 ( 17
2454 ( 182 680 ( 75 700 ( 129 1488 1370 ( 20
3(0 2(1 2(0 0.15 0.1 ( 0
92 ( 21 5 ( 0.4 13 ( 7 0.02 0.4 ( 0
a Values are expressed as the mean ( standard error, n ) 3. The pH and water holding capacity are also given. M and C denote a metal contaminated site and control site, respectively. MCC, MDH, and CDP soils are calcareous with a circumneutral pH, whereas both MCS and CP are acidic in nature.
FIGURE 1. Tissue distribution of Pb, Zn, and P in earthworms resident in the three metal contaminated sites MCS, MDH, and MCC. The diameter of each pie chart is proportional to the total worm metal concentration with the mean proportional contribution of each tissue represented by the shaded divisions with white representing the posterior (PAC), gray representing the body wall (BW), and black representing the anterior (AAC) segment of the earthworm. Numeric data from which these charts are derived and measurements for the uncontaminated site CDP are given in Table S1. centrations of Cd within intracellular metal-rich organelles, whereas the sequestration repertoire of naı¨ve counterparts inhabiting a reference site was limited to the capacity of Cd-induced metallothionein (14). Constitutive interspecific or site-specific, microevolutiondriven, intraspecific, modifications in the cellular metal partitioning patterns of chronically exposed field populations are of considerable ecotoxicological consequence. This is one conclusion that can be drawn from studies comparing the trophic transfer of Cd from two bivalve species (Macoma balthica and Potamocorbula amurensis) with differing feeding habits and metal accumulation patterns to the predatory grass shrimp Palaemon macrodatylus (15), and from Cdresistant and nonresistant individuals, respectively, of the freshwater oligochaete worm L. hoffmeisteri to the predatory shrimp Palaemonetes pugio (16). Another consequence of differential cellular metal portioning among field populations is that it can confound the interpretation of biomarkers deployed for environmental risk assessment purposes (17). Given the prominent role of earthworms in ecotoxicology (18) and in the burgeoning field of ecotoxico-genomics (19) it is surprising that only two studies (20, 21) have been conducted to describe in a relatively highly resolved manner the partitioning of metals among a number of defined subcellular fractions in these detritivorous metal macroaccumulators. Vivjer et al. (2006) confined themselves to
comparisons of the kinetics of metal uptake in populations of the endogeic species Aporrectodea caliginosa, none of which were derived from contaminated habitats. Arnold et al. (2008) used a novel combination of subcellular fractionation and X-ray absorption spectroscopy (XAS) to compare the internal partitioning and ligand speciation of Cu in the epigeic species Dendrodrilus rubidus inhabiting a mineassociated soil and in counterparts inhabiting a non Cupolluted soil but exposed to Cu-spiked standard loam soil. No differences in the partitioning and speciation of Cu were observed between the chronically exposed mine-site worms and the experimentally exposed naı¨ve worms. Arnold et al. (2008) offered the plausible interpretation that physiological plasticity may enable the mine-site worms to tolerate, as distinct from resist (17), elevated Cu exposures in their native soil without invoking “new” partitioning or sequestration strategies. However, Cu is a metal that is not strongly bioaccumulated by earthworms, displaying shallow tissue versus soil Cu concentration regression slopes (2, 22). The question of whether earthworms with different histories of metal exposures are capable of evolving different deposition and molecular-binding mechanisms remains open. The primary aim of the present study was to investigate the tissue and subcellular partitioning of Pb, Zn, and P in three distinct populations of the epigeic earthworm species Lumbricus rubellus with multigenerational histories of exposure to geochemically contrasting, metalliferous soils. A second aim was to describe and compare with XAS (both extended X-ray absorption fine structure spectroscopy, EXAFS, and X-ray absorption near edge structure, XANES) the ligand speciation of Pb and Zn in the tissues of L. rubellus with contrasting histories of metal exposure: (a) field populations chronically exposed over several generations to their native polluted soils, and (b) reference site earthworms maintained acutely on the metalliferous field soils in the laboratory for three weeks. These studies, involving an unprecedented combination of experimental design and physicochemical analytical techniques, intended to test the hypothesis that a protracted history of exposure to high concentrations of metals can result in survival-determining modifications in the subcellular partitioning and molecular sequestration of the metals.
Materials and Methods Earthworms and Soil. Soil and adult earthworms (i.e., fully clitellate) were collected from two uncontaminated sites (Dinas Powys CDP, O.S grid reference ST 146723; Pontcanna CP, ST 165779) and three contaminated disused, metalliferous mine sites (Draethen Hollow MDH, ST 217877; Cwmystwyth Stream MCS, SN 803748; and Cwmystwyth Cottage MCC, SN 806748). Soil samples (0-5 cm depth) were randomly collected, combined, and dried before being sieved to
