Metal compartmentation and speciation in a soil sentinel: the earthworm, Dendrodrilus rubidus

Earthworms are well-studied organisms in ecotoxicology because of their keystone ecological status and metal-accumulating capacity. However, the direct estimation of the bioreactive fractions of accumulated metal burdens remains technically elusive. In this study we exploited two physical techniques, electron probe X-ray microanalysis (EPXMA) and X-ray absorption spectroscopy (XAS), to improve understanding of the subcellular spatial distributions, ligand affinities, and coordination chemistries of Cd, Pb and Zn in a field population of the epigeic earthworm, Dendrodrilus rubidus. EPXMA and XAS analyses were performed on cryopreparations to maintain compositional fidelity; EPXMA data were analyzed by multivariate statistics. XAS provided whole-worm insights; EPXMA provided in situ, subcellular data from the major metal-sequestering tissue, the chloragog. Both techniques showed that Cd is coordinated with S; the measured Cd-S bond distance in XAS suggests a metallothionein-type ligand. The mean Cd:S molar ratio (EPXMA) of 0.36 is higher than the ratio of 0.29 estimated from published biochemical data. EPXMA and XAS data also found that Ca, Pb, and Zn are predominantly bound to one or more O-donating, probably phosphate-rich, ligands. X-ray distribution maps (EPXMA) of the hepatocyte-resembling chloragocytes revealed that the O-seeking (Ca, Pb, Zn) metals and S-seeking Cd bioaccumulate in distinct organelles. Extended X-ray absorption fine structure showed that the Pb complex is not biogenic pyromorphite, although X-ray absorption near edge structure did not eliminate the possibility. XAS provided no evidence of Pb spillage from the "sequestration compartment" within D. rubidus. However, the correspondence of Pb with Ca and P in EPXMA is not as strong as that of Zn. This is indicative either of spillover or of a second, hitherto unidentified, sequestered-Pb pool. By exploiting the complimentary techniques of EPXMA and XAS,we are closer to describing the mechanistic link between equilibrated body burdens and biomarker responses in earthworms.