Uncontaminated Sites Research Articles

Genetic analysis of Pinus banksiana and Pinus resinosa populations from stressed sites contaminated with metals in Northern Ontario (Canada)

The Sudbury region in Canada is known for the mining and smelting of high-sulphide ores containing nickel, copper, iron and precious metals. Although reports provide information of metal levels in soil and plants, knowledge of genetic effects on plants growing in contaminated areas is limited. The main objective of this study was to characterise the level of genetic diversity in Pinus banksiana and Pinus resinosa populations from the Sudbury (Ontario) region using microsatellite markers. Soil samples were analysed for concentrations of metals. High levels of metal contents in soil were observed within short distances of the smelter compared with control sites. The level of genetic diversity was very low for P. resinosa populations and moderate for P. banksiana samples. Observed heterozygosity was fivefold higher in P. banksiana populations than P. resinosa populations studied. Overall, 17 and 24% of the total genetic diversity were attributed to differences among populations for P. banksiana and P. resinosa, respectively. In general, the inbreeding was significantly higher in P. resinosa populations than P. banksiana populations and gene flows were relatively low in both species. No significant trend of the levels of genetic diversity for metal contaminated and uncontaminated sites was found.

Variation of metallothionein-like protein and metal concentrations during the reproductive cycle of the cockle Cerastoderma glaucum from an uncontaminated site: A 1-year study in the Gulf of Gabès area (Tunisia)

Natural variation of metallothionein-like protein (MTLP) concentrations in the cockle Cerastoderma glaucum were analysed over a 1-year period in specimens collected from a relatively uncontaminated site in the Gulf of Gabès (Tunisia). Sampling was carried out from June 2004 to June 2005. At each sampling time, water temperature and salinity were measured. Levels of zinc, cadmium and copper were measured in the whole soft tissues of the cockle C. glaucum. MT-like protein and subcellular metal partitioning were measured in the digestive gland. A histological study was conducted simultaneously to follow the development of the gonad in relation to the reproductive cycle of this bivalve. Results showed a significant fluctuation in MTLP concentrations throughout the whole year, with a maximum value measured in summer and in the late winter. The relationship between MTLP and metal concentrations, abiotic (temperature and salinity) and biotic (mass of target tissues) and also maturity index (MI) factors were examined. Metal levels in the tissues are likely to change during the season, reflecting variability in metabolism and/or a change in tissue weight. However, possible seasonal changes in trace metal concentration are unlikely to entirely explain the observed pattern in the MTLP levels. The physiological changes caused by gonad development and food abundance (change in tissue mass) contribute to a significant variability in MTLP levels in the cockle C. glaucum and can interfere with the estimation of level changes induced by anthropogenic sources.

Analysis of temporal variability in the concentrations of some elements in the terrestrial moss Pseudoscleropodium purum

The uptake of contaminants by moss is a complex process that depends on the nature and characteristics of the contaminants and on the physicochemical and biological processes that affect the moss. All of these factors in turn depend on environmental variables that may vary within a short time. It can therefore be assumed that the uptake of elements varies in such short periods that it is very difficult to obtain temporally representative results in biomonitoring studies with terrestrial moss. In order to investigate the temporal variability and the implications that it may have on the interpretation of the results obtained in this type of study, the tissue concentrations of 12 elements were determined in samples of the moss Pseudoscleropodium purum collected from two sampling sites in Galicia (NW Spain), during a long term survey with a high frequency of sampling. The results showed that the concentrations of the elements in the moss varied greatly within very short periods, and that the error associated with the temporal variability in the results was high. In accordance with these findings, and taking into account that the technique enabled contaminated and uncontaminated sites to be distinguished, we recommend that the results of biomonitoring studies should be regarded as qualitative or semiqualitative, rather than attempting to provide absolute data, which may not be temporally representative, and may have a high degree of uncertainty associated with them.

Organic acids in two arsenic hyperaccumulators and a non-hyperaccumulator of Pteris exposed to elevated arsenic concentrations

Six organic acids (oxalic, malonic, malic, citric, palmitic and linolenic acid) in two arsenic (As) hyperaccumulators (Pteris multifida and Pteris vittata) and a non-hyperaccumulator fern (Pteris semipinnata) exposed to different As concentrations (0, 5, 20 and 40 mg As L−1) under hydroponic conditions were determined using gas chromatography-mass spectrometry (GC-MS). Three fern species were collected from an uncontaminated site, but two As-hyperaccumulators were also collected from an As-contaminated site. Furthermore, the time-course effect of organic acid production in P. multifida and P. semipinnata collected from the uncontaminated site were also studied under 0 and 20 mg As L–1 treatments with a sampling interval at 0, 6, 12, 24 and 36 h. After esterifying (H2SO4–CH3OH), extracting (CH2Cl2), washing (saturated NaCl) and drying (anhydrous Na2SO4), these organic acids were isolated (Column: HP Ultra 2, 50 m × 0.2 mM × 0.33 µM; Carrier gas: He). There was no significant increase in the concentrations of the six organic acids in the fronds and roots of the three fern species sampled from an uncontaminated site under 5 or 40 mg As L−1 treatments. In addition, there were also no significant differences in the concentrations of organic acids among the three fern species under the same As concentration treatments. Concentrations of malic, oxalic and linolenic acids in the fronds and roots of two As hyperaccumulators collected from an As-contaminated or uncontaminated site and the non-hyperaccumulator collected from an uncontaminated site followed the sequence of malic acid > oxalic acid > linolenic acid. As for time-course effect, compared to P. semipinnata, a significant increase was observed in concentrations of oxalic and linolenic acid in P. multifida over time under 20 mg As L−1 treatment. In general, the changes of organic acid concentrations in As hyperaccumulators did not directly contribute to As accumulation.

Two strategies to live in low chronic pollution estuaries: The potential role of lifestyle

Although physiological and genetic adaptation to toxic stress has been the focus of recent research, the role of lifestyle in pollution tolerance has received less attention. In this study, copper tolerance of benthic and epi-benthic species of harpacticoid copepods was investigated. Concentration-response curves were generated for populations of both species but collected at contaminated and uncontaminated estuaries. The population of the benthic species from the contaminated site showed higher tolerance than its population from the uncontaminated site. The epi-benthic species showed no inter-specific differences in tolerance. The comparison of tolerance between field collected animals of the benthic species and animals from the same population but reared in the laboratory for two generations suggested the existence of a genetic inherited tolerance. Results revealed the importance that lifestyle may have on the generation of tolerance to toxic substances and highlight a potential unforeseen role of it in maintenance of biodiversity on contaminated habitats.

Fluoroquinolone (FQ) Contamination Does Not Correlate with Occurrence of FQ-Resistant Bacteria in Aquatic Environments of Vietnam and Thailand

Fluoroquinolone antibiotics (FQs) have been used worldwide for chemotherapy, animal husbandry, and aquaculture, and the occurrence of FQ-resistant (FQs(r)) bacteria in natural environments has been reported. Plasmid-mediated transferable quinolone resistance (PMQR) genes are suspected to originate from the chromosomes of water-dwelling bacteria. However, the occurrence of and the potential reservoir of FQs(r) bacteria and PMQR genes in aquatic environments have not been elucidated. In this study, we detected FQs(r) bacteria and PMQR genes in aquatic environments in Thailand and Vietnam, and measured FQ contamination. Levels of contamination were greater Thailand (avg. 5130, max 46100 ng L(-1)) than in Vietnam (avg. 235, max 1130 ng L(-1)); however, the occurrence of FQs(r) bacteria was higher in Vietnam (~15%) than in Thailand (~7.0%), suggesting that contamination by FQs is not directly linked to the development of FQs(r) bacteria. Diverse taxonomic groups of FQs(r)-bacteria were identified, and one of the PMQR genes, qnrB, was detected from bacteria of environmental origin, not enteric bacteria. This suggests that the environmental bacteria are a potential reservoir of antibiotic resistance determinants even at un-contaminated sites.

The definition of species richness used by species sensitivity distributions approximates observed effects of salinity on stream macroinvertebrates

The risk of chemicals for ecological communities is often forecast with species sensitivity distributions (SSDs) which are used to predict the concentration which will protect p% of species (PCp value). However, at the PCp value, species richness in nature would not necessary be p% less than at uncontaminated sites. The definition of species richness inherent to SSDs (contaminant category richness) contrasts with species richness typically measured in most field studies (point richness). We determine, for salinity in eastern Australia, whether these definitions of stream macroinvertebrate species richness are commensurable. There were strong relationships (r2≥0.87) between mean point species, family and Ephemeroptera, Trichoptera and Plecoptera species richness and their respective contamination category richness. Despite differences in the definition of richness used by SSDs and field biomonitoring, their results in terms of relative species loss from salinity in south-east Australia are similar. We conclude that in our system both definitions are commensurable.

Effects of diesel and lubricant oils on Antarctic benthic microbial communities over five years

A 5 yr field experiment was carried out comparing the effects of Special Antarctic Blend (SAB) diesel, synthetic lubricant (Mobil 0W40), used synthetic lubricant (used Mobil 0W40) and a biodegradable lubricant (Titan GT1) on Antarctic benthic microbial communities. Sediment from an uncontaminated site was collected, spiked with one of the oils and deployed in O’Brien Bay, East Antarctica. Sediment samples were then collected over 5 yr. All the oils caused changes in the microbial community in the top 1 cm of sediment, as determined by 16S ribosomal ribonucleic acid (rRNA) gene-based terminal restriction fragment length polymorphism analysis. The greatest effect was observed in the SAB diesel treatment. The biodegradable oil did not have a significant effect on the communities initially, but the communities present at 2 and 5 yr were significantly different to the controls. The unused and used lubricants were both significantly different to the control treatment. All the oils degraded, with 39% of the SAB, 15% of the unused lubricant, 15% of the used lubricant and 12% of the biodegradable oil, remaining after 5 yr. These results show that the effects of all these oils will be evident in the Antarctic benthic ecosystem for longer than 5 yr. This highlights the need for studies into the longevity and ecotoxicology of oil products in the Antarctic environment.

Mechanism of uranium (VI) removal by two anaerobic bacterial communities

The mechanism of uranium (VI) removal by two anaerobic bacterial consortia, recovered from an uncontaminated site (consortium A) and other from an uranium mine (consortium U), was investigated. The highest efficiency of U (VI) removal by both consortia (97%) occurred at room temperature and at pH 7.2. Furthermore, it was found that U (VI) removal by consortium A occurred by enzymatic reduction and bioaccumulation, while the enzymatic process was the only mechanism involved in metal removal by consortium U. FTIR analysis suggested that after U (VI) reduction, U (IV) could be bound to carboxyl, phosphate and amide groups of bacterial cells. Phylogenetic analysis of 16S rRNA showed that community A was mainly composed by bacteria closely related to Sporotalea genus and Rhodocyclaceae family, while community U was mainly composed by bacteria related to Clostridium genus and Rhodocyclaceae family.

Elucidating cellular and behavioural effects of contaminant impact (polycyclic aromatic hydrocarbons, PAHs) in both laboratory-exposed and field-collected shore crabs, Carcinus maenas (Crustacea: Decapoda)

Monitoring effects of contaminants at lower levels of biological organisation (e.g. biochemical and cellular) allows for mechanistic evaluation of effects of contaminant exposure through laboratory exposures. However, higher level organism effects (e.g. physiological and behavioural) are deemed more ecologically-relevant. In the present study, cellular (cell viability and immune function), physiological (cardiac activity) and behavioural (foraging behaviour) responses were evaluated in field-collected shore crabs Carcinus maenas from three estuaries [a ‘relatively’ (polycyclic aromatic hydrocarbon) PAH-contaminated site (Plym Estuary) and two ‘comparatively clean’ field sites (Avon and Yealm Estuaries)] and compared with responses of crabs exposed in the laboratory to a model organic contaminant (PAH) pyrene (200 μg l −1 for 28 days). The hypothesis that behavioural indicators may be more sensitive than other more traditional methods was tested. No significant impacts were observed at the cellular or physiological level in Plym-collected crabs (the ‘relatively’ contaminated site), but foraging behaviour was significantly altered (increased prey handling time) compared to individuals collected from the ‘relatively’ uncontaminated sites (Avon and Yealm). When given a cockle as a prey item, both Plym-collected and laboratory-exposed crabs took longer to handle and break into cockle shells. Therefore, ecologically-relevant behavioural observations may serve as valuableindicators of environmental quality.

Diversity of Benzyl- and Alkylsuccinate Synthase Genes in Hydrocarbon-Impacted Environments and Enrichment Cultures

Hydrocarbon-degrading microorganisms play an important role in the natural attenuation of spilled petroleum in a variety of anoxic environments. The role of benzylsuccinate synthase (BSS) in aromatic hydrocarbon degradation and its use as a biomarker for field investigations are well documented. The recent discovery of alkylsuccinate synthase (ASS) allows the opportunity to test whether its encoding gene, assA, can serve as a comparable biomarker of anaerobic alkane degradation. Degenerate assA- and bssA-targeted PCR primers were designed in order to survey the diversity of genes associated with aromatic and aliphatic hydrocarbon biodegradation in petroleum-impacted environments and enrichment cultures. DNA was extracted from an anaerobic alkane-degrading isolate (Desulfoglaeba alkenexedens ALDC), hydrocarbon-contaminated river and aquifer sediments, a paraffin-degrading enrichment, and a propane-utilizing mixed culture. Partial assA and bssA genes were PCR amplified, cloned, and sequenced, yielding several novel clades of assA genes. These data expand the range of alkane-degrading conditions for which relevant gene sequences are available and indicate that considerable diversity of assA genes can be found in hydrocarbon-impacted environments. The detection of genes associated with anaerobic alkane degradation in conjunction with the in situ detection of alkylsuccinate metabolites was also demonstrated. Comparable molecular signals of assA/bssA were not found when environmental metagenome databases of uncontaminated sites were searched. These data confirm that the assA gene is a useful biomarker for anaerobic alkane metabolism.

Allozyme polymorphisms in horseshoe crabs, Carcinoscorpius rotundicauda, collected from polluted and unpolluted intertidal areas in Peninsular Malaysia

It has been widely reported that allozyme frequency variation is a potential indicator of heavy metal-induced impacts in aquatic populations. In the present study, wild populations of horseshoe crab (Carcinoscorpius rotundicauda) were collected from contaminated and uncontaminated sites of Peninsular Malaysia. By adopting horizontal starch gel electrophoresis, seven enzyme systems were used to study allozyme polymorphisms. Nine polymorphic loci were observed in C. rotundicauda. The relationships of allozyme variations with the concentrations of Cd, Cu, Ni, and Zn in sediments and in muscle tissues of horseshoe crabs were determined. Based on genetic distance, the lower mean value of Nei's D (0.017) indicated that both of the contaminated populations of Kg. Pasir Puteh and Kuala Juru were very closely related when compared to the relatively uncontaminated Pantai Lido population. Higher heterozygosities were shown by the contaminated populations when compared to the uncontaminated population. Different allelic frequencies could be observed for the aldolase (ALD; E.C. 2.7.5.1) locus between the contaminated and uncontaminated populations of C. rotundicauda. The dendrogram of genetic relationships of the three populations of C. rotundicauda showed the same clustering pattern as the dendrograms are based on heavy metals in the sediments and in the horseshoe crabs' abdominal muscles. From the F statistics, the present study showed that the three populations of horseshoe crabs were considered to have undergone moderate genetic differentiation with a mean F (ST) value of 0.092 .The current results suggest that allozyme polymorphism in horseshoe crabs is a potential biomonitoring tool for metal contamination, although further validation is required.

Accumulation of As, Pb, Zn, Cd and Cu and arbuscular mycorrhizal status in populations of Cynodon dactylon grown on metal-contaminated soils

Metal(loid) accumulation and arbuscular mycorrhizal (AM) status of the dominant plant species, Cynodon dactylon, growing at four multi-metal(loid)s-contaminated sites and an uncontaminated site of China were investigated. Up to 94.7 As mg kg −1, 417 Pb mg kg −1, 498 Zn mg kg −1, 5.8 Cd mg kg −1 and 27.7 Cu mg kg −1 in shoots of C. dactylon were recorded. The plant was colonized consistently by AM fungi (33.0–65.5%) at both uncontaminated site and metal-contaminated sites. Based on morphological characteristics, fourteen species of AM fungi were identified in the rhizosphere of C. dactylon, with one belonging to the genus of Acaulospora and the other thirteen belonging to the genus of Glomus. Glomus etunicatum was the most common species associated with C. dactylon growing at metal-contaminated sites. Spore abundance in the rhizosphere of C. dactylon growing at the metal-contaminated soils (22–82 spores per 25 g soil) was significantly lower than that of the uncontaminated soils (371 spores per 25 g soil). However, AM fungal species diversity in the metal-contaminated soils was significantly higher than that in the uncontaminated soils. This is the first report of AM status in the rhizosphere of C. dactylon, the dominant plant survival in metal-contaminated soils. The investigation also suggests that phytorestoration of metal-contaminated sites might be facilitated using the appropriate plant with the aid of tolerant AM fungi.

Differences in EDTA-assisted metal phytoextraction between metallicolous and non-metallicolous accessions of Rumex acetosa L.

Two common sorrel ( Rumex acetosa) accessions, one from a Zn–Pb contaminated site (CS accession) and the other from an uncontaminated site (UCS accession), were hydroponically exposed to a mixture of heavy metals (Pb 2+ + Zn 2+ + Cd 2+) with and without EDTA at an equimolar rate. The metallicolous CS accession showed a higher tolerance to metal treatment in the absence of the chelating agent, whereas the UCS accession was especially tolerant to EDTA treatment alone. Combination of metal and EDTA treatment resulted in a higher Pb accumulation in shoots of both accessions although plants hardly showed phytotoxic symptoms. Cd and Zn uptake was not augmented by EDTA addition to the polymetallic medium. Chelant-assisted Pb accumulation was 70% higher in the CS accession than in the UCS accession, despite the fact that the former accession evapotranspired less water than the UCS accession. These results support the existence of a non-selective apoplastic transport of metal chelates by R. acetosa roots, not related to transpiration stream.

Cadmium accumulation and distribution in populations of Phytolacca americana L. and the role of transpiration

The concentrations of heavy metals in Phytolacca americana L. and corresponding soil samples from three contaminated sites and an uncontaminated site were studied. Hydroponic experiments were also conducted to investigate the Cd uptake ability and mechanism of P. americana. The field results showed that the average Cd concentration was 42 mg kg −1 in P. americana leaves, with the highest concentration of 402 mg kg −1 found at Datianwan. A significant relationship was observed between the concentrations of Cd in leaves and those of corresponding soils on a logarithmic scale. Under laboratory hydroponic conditions, the maximum Cd concentration in aerial tissues of P. americana was 637 mg kg −1, under treatment with 100 μM Cd. The population from the uncontaminated site (Zijinshan) also had a remarkable ability to accumulate Cd in shoots to concentrations well in excess of 100 μM in the hydroponic experiment, similar to the population from contaminated site, suggesting that Cd accumulation is a constitutive trait of P. americana. In the presence of 100 μM Cd, the addition of polyethylene glycol decreased leaf transpiration, the shoot Cd concentration, and the shoot/root Cd concentration ratio. There was a significantly positive relationship between the shoot Cd concentration and the leaf transpiration of P. americana. A similar significant positive correlation was also obtained between the shoot/root Cd concentration and leaf transpiration. Moreover, pretreatment with 5 μM abscisic acid or 5 μM HgCl 2 significantly decreased the Cd concentration in P. americana shoots. These results suggest that transpiration has an important role in Cd accumulation in shoots of P. americana.

DNA damage in earthworms from highly contaminated soils: Assessing resistance to arsenic toxicity by use of the Comet assay

Earthworms native to the former mine site of Devon Great Consols (DGC), UK reside in soils highly contaminated with arsenic (As). These earthworms are considered to have developed a resistance to As toxicity. The mechanisms underlying this resistance however, remain unclear. In the present study, non-resistant, commercially sourced Lumbricus terrestris were exposed to a typical DGC soil in laboratory mesocosms. The earthworms bio-accumulated As from the soil and incurred DNA-damage levels significantly above those observed in the control mesocosm (assessed using the Comet assay). A dose response was observed between DNA damage (% tail DNA) and As concentration in soil (control, 98, 183, 236, 324 and 436 mg kg −1). As-resistant earthworms ( Lumbricus rubellus, Dendrodrilus rubidus and L. terrestris) collected from contaminated soils at DGC (203 to 9025 mg kg −1 As) had also bio-accumulated high levels of As from their host soils, yet demonstrated low levels of DNA damage compared with earthworms from uncontaminated sites. The results demonstrate that the As-contaminated soils at DGC are genotoxic to non-native earthworms and much less so to earthworms native to DGC, thus providing further evidence of an acquired resistance to As toxicity in the native earthworms.

Assessment of soil-pollution by slag from an automobile battery manufacturing plant in Nigeria

This study assessed heavy metals pollution of soil by slag from an automobile battery manufacturing plant in Nigeria to ascertain the potential danger they pose on the environment. 84 soil samples were randomly collected at 0 - 15 cm and 15 - 30 cm depths from the premises of the plant and an uncontaminated site as control for both dry and wet seasons. Samples were analyzed for lead, cadmium, chromium and nickel using standard analytical methods. Lead level in soil ranged from 243 to 126000 mg/kg on the premises of the manufacturing plant with about 98% of all soil samples analyzed having levels higher than 400 mg/kg permissible level for Pb in soil. Lead levels for both seasons and depths were statistically comparable (p > 0.05). Cadmium, chromium and nickel were within the permissible limits in soil. Speciation of Pb showed about 89% Pb in non residual phases implying high availability to ecological materials. Soil within the company’s premises and the environs needs to be urgently cleaned up. Key words: Heavy metals, automobile-battery, speciation, slag, pollution.

Mercury accumulation in caged Corbicula: rate of uptake and seasonal variation

The uptake and seasonal fluctuations of total mercury were followed in caged and uncaged Asiatic clams, Corbicula fluminea, over a 1-year period in South River, Virginia. Mercury was rapidly accumulated in clams transplanted from a nominally uncontaminated site into cages on the contaminated South River, reaching 0.99 microg g(-1) dry mass within the first month. Resident clams moved to cages had higher mercury contents after the first month (2.04 microg g(-1) dry mass) and at all subsequent times in the study. Large monthly fluctuations in mercury were noted for both resident caged and transplant caged clams with a notable peak occurring in early spring (4.31 microg g(-1) dry mass in resident caged clams). Tissue mass of caged clams steadily increased through the winter and early spring. Adjustment of mercury concentrations for tissue mass changes indicated that the changes in mercury contents were primarily due to uptake/release rather than changes in tissue mass (concentration/dilution). The present study demonstrates the utility of using caged Corbicula as mercury biomonitors and illustrates the importance of accounting for large, short-term changes of mercury content in Corbicula when designing long-term biomonitoring studies.

Intraspecific differences of arbuscular mycorrhizal fungi in their impacts on arsenic accumulation by Pteris vittata L.

It has been shown that Pteris vittata, an arsenic hyperaccumulator, could be colonized by arbuscular mycorrhizal (AM) fungi either in controlled conditions or at field sites. However, physiological mechanisms of AM fungi influencing As accumulation and tolerance in the plant are not fully elucidated. Two predominant fungal species, Glomus mosseae and Glomus geosporum, and a rapidly sporulating fungal species, Glomus etunicatum, associated with P. vittata were isolated from As-contaminated soils. Two uncontaminated isolates, G. mosseae and G. etunicatum, served as reference isolates. Based on germination of spores exposed to elevated As, Pb and Zn concentrations, two contrasting isolates of G. mosseae were selected to investigate As accumulation in two populations of P. vittata [from an uncontaminated site of Hong Kong (HK) and an As-contaminated site located in Jinchuantang (JCT) of Hunan Province, China, respectively] under hydroponic culture and pot trials. At lower levels of As exposure (50–200 μM), both uncontaminated and metal-contaminated isolates of G. mosseae significantly increased short-term As influx into roots of P. vittata. However, at higher levels of As exposure (400–1000 μM), only uncontaminated isolates significantly increased short-term As influx into roots. When growing on 100 mg As kg −1 soils, uncontaminated isolates exhibited a higher level of colonization in roots of P. vittata than metal-contaminated isolates and only the former significantly increased As accumulation in roots of HK population and in fronds of JCT population. It was concluded that there were intraspecific differences of AM fungi in their impacts on As accumulation by P. vittata.

As-resistance in laboratory-reared F1, F2 and F3 generation offspring of the earthworm Lumbricus rubellus inhabiting an As-contaminated mine soil

Previous studies provided no unequivocal evidence demonstrating that field populations of Lumbricus rubellus Hoffmeister (1843), exhibit genetically inherited resistance to As-toxicity. In this study F1, F2 and F3 generation offspring derived from adults inhabiting As-contaminated field soil were resistant when exposed to 2000 mg kg −1 sodium arsenate. The offspring of uncontaminated adults were not As-resistant. Cocoon viability was 80% for F1 and 82% for F2 offspring from As-contaminated adults and 59% in the F1 control population. High energy synchrotron analysis was used to determine whether ligand complexation of As differed in samples of: resistant mine-site adults, the resistant F1 and F2 offspring of the mine-site earthworms exposed to the LC 25 sodium arsenate (700 mg kg −1) of the F1 parental generation; and adult L. rubellus from an uncontaminated site exposed to LC 25 concentrations of sodium arsenate (50 mg kg −1). XANES and EXAFS indicated that As was present as a sulfur-coordinated species.