Cd-induced phytochelatin synthesis in Dittrichia viscosa (L.) Greuter is determined by the dilution of the culture medium

In this paper, we examined Cd accumulation and PC synthesis in two clones of Dittrichia viscosa, one with a metallicolous (DV-A) and the other with a non-metallicolous origin (DV-W). The clones were cultured in vitro with 0 and 10 mg Cd L^?1 in both short-term treatments (up to 72 h) and over 10 days. We also examined the influence of the culture medium dilution and the PC-synthesis inhibitor, l-buthionine-sulfoximine (BSO), on these parameters. Similar Cd accumulation values were found in the two clones. No synthesis of new thiolic compounds was observed in Cd-treated plants cultured in vitro in Murashige and Skoog medium up to 72 h when compared to controls. Dilution of the culture medium affected PC production, increasing it in 1/2 MS and especially in 1/4 MS. Cd uptake did not increase in the same way, but still hyperaccumulation levels were exceeded in all Cd treatments. BSO addition increased the sensitivity of D. viscosa to Cd and diminished Cd accumulation. Nevertheless, a poor correlation between PCs and Cd accumulation capacity was observed since the highest Cd content did not correspond to the highest PC levels. All these results obtained suggest that PCs are important in Cd accumulation and detoxification in D. viscosa and also that other mechanisms might be involved in these traits.     

巯基化合物在万寿菊镉解毒中的作用

采用水培实验方法研究了万寿菊体内镉积累和解毒与巯基化合物含量的关系。万寿菊植株分别在镉浓度为0、0.1、0.5、2和8 mg/L的营养液中暴露7 d,测定了根、茎、叶中镉、非蛋白巯基(NPT)、半胱氨酸(Cys)、γ-谷氨酰半胱氨酸(γ-EC)、谷胱甘肽(GSH)和植物络合素(PCs)的含量。植物根、茎、叶中镉含量都随着镉暴露浓度的增加而增加。当溶液中镉浓度较低(0.1～2 mg/L)时,茎叶中NPT、PCs、Cys和γ-EC含量随着镉浓度增加而增大;当镉浓度较高(8 mg/L)时,茎叶中PCs含量迅速降低,GSH含量大幅度增高。在根部,这些巯基化合物的含量几乎不受镉处理影响,且含量较低。以上研究结果表明:PCs在万寿菊镉的解毒机制中发挥一定的作用,暴露于高浓度的镉,GSH比PCs起着更为重要的解毒作用。     

Overexpressing GSH1 and AsPCS1 simultaneously increases the tolerance and accumulation of cadmium and arsenic in Arabidopsis thaliana

The goal of this study was to develop transgenic plants with increased tolerance for and accumulation of heavy metals and metalloids from soil by simultaneous overexpression of AsPCS1 and GSH1 (derived from garlic and baker's yeast) in Arabidopsis thaliana. Phytochelatins (PCs) and glutathione (GSH) are the main binding peptides involved in chelating heavy metal ions in plants and other living organisms. Single-gene transgenic lines had higher tolerance to and accumulated more Cd and As than wild-type. Compared to single-gene transgenic lines, dual-gene transformants exhibited significantly higher tolerance to and accumulated more Cd and As. One of the dual-gene transgenic lines, PG1, accumulated twice the amount of Cd as single-gene transgenic lines. Simultaneous overexpression of AsPCS1 and GSH1 led to elevated total PC production in transgenic Arabidopsis. These results indicate that such a stacking of modified genes is capable of increasing Cd and As tolerance and accumulation in transgenic lines, and represents a highly promising new tool for use in phytoremediation efforts.     

Variation in oxidative stress and photochemical activity in Arabidopsis thaliana leaves subjected to cadmium and excess copper in the presence or absence of jasmonate and ascorbate

We have presented changes in the photosynthetic apparatus activity of Arabidopsis thaliana plants occurring within 15-144 h of 100 microM Cu or Cd action with regard to jasmonate (JA) as well as expression of the oxidative stress and non-enzymic defense mechanisms. The inhibitory effect of both heavy metals related to developing dissipative processes and lipid peroxide formation was expressed in dark-adapted state after the longest time as a decrease in potential quantum yield of PSII. In dark- and light-adapted state the heavy metals affected the enzymic phase of photosynthesis already from the 15th hour, which was related to the lipid peroxide formation. Photochemical quenching decrease was induced after 48th hour and did not show a close correlation with the JA pathway. Blockade of endogenously formed JA by propyl gallate decreased the effect of Cu and Cd on both the whole photosynthetic apparatus starting from the 48th hour and on the primary photochemistry of PSII after 144 h. In the case of Cu the effect was related to a lipid peroxidation decrease and to an increase in glutathione and phytochelatin (PC) levels, but in the case of Cd to lipid peroxidation, O.2- and especially to PCs increase. The obtained results indicated that JA after the longest time might enhance the sensitivity of A. thaliana to Cu and Cd stress. Asc enhanced toxic action of Cu and Cd after 15 h, but after a longer time it diminished the influence of Cd (but not Cu) on photosynthetic activity.     

Effect of some heavy metals and soil humic substances on the phytochelatin production in wild plants from silver mine areas of Guanajuato, Mexico

Phytochelatins (PCs) were determined in the wild plants, focusing on their relationship with the levels of heavy metals and humic substances (HS) in soil. Ricinus communis and Tithonia diversifolia were collected from several sites in Guanajuato city (Mexico), which had long been the silver and gold mining center. The analysis of PCs in root extracts was carried out by liquid chromatography (derivatization with monobromobimane). Total Ag, Cd, Cu and Pb in plant roots and in soil samples, as well as soil HS were determined. The association of metals with HS in soils was evaluated by size exclusion chromatography (SEC) with UV and ICP-MS detection. The results obtained revealed the induction of PCs in R. communis but not in T. diversifolia. The levels of Cd and Pb in plant roots presented strong positive correlation with PC-2 (r = 0.9395, p = 0.005; r = 0.9573, p = 0.003, respectively), indicating that these two metals promote PCs induction in R. communis. On the other hand, the inverse correlation was found between soil HS and metal levels in roots of R. communis (Cu > Pb > Cd > Ag), in agreement with the decreasing affinity of these metals to HS. Importantly, the inverse correlation between soil HS and plant PC-2 was observed (r = −0.7825, p = 0.066). These results suggest that metals strongly bound to HS could be less bioavailable to plants, which in turn would limit their role in the induction of PCs. Indeed, the SEC elution profiles showed Pb but not Cd association with HS and the correlation between metal in soil and PC-2 in plant was statistically significant only for Cd (r = 0.7857, p = 0.064). Based on these results it is proposed that the role of heavy metals in PCs induction would depend on their uptake by R. communis, which apparently is controlled by the association of metals with soil HS. This work provides further evidence on the role of environmental conditions in the accumulation of heavy metals and phytochelatin production in plants.     

Heavy metal accumulation and metallothionein concentration in the frog Rana ridibunda after exposure to chromium or a mixture of chromium and cadmium

The accumulation of two heavy metals (chromium (Cr) and cadmium (Cd)) in the liver, kidney and gut of Rana ridibunda exposed to Cr or to a mixture of Cr and Cd was investigated. The concentration of metallothioneins (MTs) in the same tissues was also studied. Both metals accumulated mainly in the kidney. Cr accumulation in the liver and gut was not affected by the presence of Cd. Furthermore, Cr concentration in the kidney was doubled when Cd was present. MT concentration did not increase after Cr treatment but it increased two- to six-fold over control values in mixture-exposed frogs, the highest value being observed in the gut. MTs in the gut could act as a barrier preventing ingested heavy metals from entering the blood stream. MT concentration correlated positively with Cd concentrations in both the liver and the gut of mixture-exposed animals.     

Cadmium-induced toxicity reversal by zinc in Ceratophyllum demersum L. (a free floating aquatic macrophyte) together with exogenous supplements of amino- and organic acids

This paper analyzes the detoxification mechanisms adopted by amino- and organic acids to alleviate Cd toxicity. In addition, with our published data on Zn–Cd interactions, the influence of Zn (200 μM) supplements on the detoxification mechanisms of amino- and organic acids have also been studied. The experimental studies on metal uptake, lipid peroxidation levels, estimation of reduced and oxidized glutathione levels as well as γ-glutamylcysteine synthetase activity in amino acid supplemented Cd treatments indicated glutathione-mediated detoxification system, which was also enhanced by Zn (200 μM) supplements. However Zn did not aid in glutathione synthesis, but maintained the ratio of reduced and oxidized forms. The supplementation of organic acids to Cd treatments indicated detoxification through the mechanism of chelation. Zn seemed to be less influential on organic acids-mediated detoxification mechanism as compared to amino acid mediated detoxification system.     

Cadmium-induced toxicity reversal by zinc in Ceratophyllum demersum L. (a free floating aquatic macrophyte) together with exogenous supplements of amino- and organic acids

This paper analyzes the detoxification mechanisms adopted by amino- and organic acids to alleviate Cd toxicity. In addition, with our published data on Zn–Cd interactions, the influence of Zn (200 μM) supplements on the detoxification mechanisms of amino- and organic acids have also been studied. The experimental studies on metal uptake, lipid peroxidation levels, estimation of reduced and oxidized glutathione levels as well as γ-glutamylcysteine synthetase activity in amino acid supplemented Cd treatments indicated glutathione-mediated detoxification system, which was also enhanced by Zn (200 μM) supplements. However Zn did not aid in glutathione synthesis, but maintained the ratio of reduced and oxidized forms. The supplementation of organic acids to Cd treatments indicated detoxification through the mechanism of chelation. Zn seemed to be less influential on organic acids-mediated detoxification mechanism as compared to amino acid mediated detoxification system.     

Lead detoxification by coontail (Ceratophyllum demersum L.) involves induction of phytochelatins and antioxidant system in response to its accumulation

Coontail (Ceratophyllum demersum L.) plants when exposed to various concentrations of Pb (1-100microM) for 1-7days, exhibited both phytotoxic and tolerance responses. The specific responses were function of concentration and duration. Plants accumulated 1748mugPbg(-1) dw after 7d which reflected its metal accumulation ability, however most of the metal (1222microgg(-1) dw, 70%) was accumulated after 1d exposure only. The toxic effect and oxidative stress caused by Pb were evident by the reduction in biomass and photosynthetic pigments and increase in malondialddehyde (MDA) content and electrical conductivity with increase in metal concentration and exposure duration. Morphological symptoms of senescence phenomena such as chlorosis and fragmentation of leaves were observed after 7d. The metal tolerance and detoxification strategy adopted by the plant was investigated with reference to antioxidant system and synthesis of phytochelatins. Protein and antioxidant enzymes viz., superoxide dismutase (SOD, EC 1.15.1.1), guaiacol peroxidase (GPX, EC 1.11.1.7) ascorbate peroxidase (APX, EC 1.11.1.11), catalase (CAT, EC 1.11.1.6) and glutathione reductase (GR, EC 1.6.4.2) showed induction at lower concentration and duration followed by decline. All enzymes except GPX showed maximum activity after 1d. An increase in cysteine, non-protein thiols (NP-SH) and glutathione (GSH) content was observed at moderate exposure conditions followed by decline. Phytochelatins (PC(2) and PC(3)) were synthesized to significant levels at 10 and 50microM Pb with concomitant decrease in GSH levels. Thus production of PCs seems important for the detoxification of metal, however it may lead to depletion of GSH and consequently oxidative stress. Results suggest that plants responded positively to moderate Pb concentrations and accumulated high amount of metal. Due to metal accumulation coupled with detoxification potential, the plant appears to have potential for its use as phytoremediator species in aquatic environments having moderate pollution of Pb.     

Heavy metal concentrations in soil and earthworms in a floodplain grassland

We determined accumulated heavy metal concentrations (Cd, Pb, Cu, Zn) of earthworms in moderately contaminated floodplain soils. Both soil and mature earthworms were sampled before and after flooding and earthworm species were identified to understand species specific differences in bioconcentration. Accumulated metal concentrations in floodplain earthworms differed before and after flooding. Differences in uptake and elimination mechanisms, in food choice and living habitat of the different earthworm species and changes in speciation of the heavy metals are possible causes for this observation. Regression equations taken from literature, that relate metal accumulation by earthworms in floodplains as a function of metal concentration in soil, performed well when all species specific data were combined in an average accumulation, but did not address differences in accumulation between earthworm species.     

Contrasting behaviour of cadmium and zinc in a soil-plant-arthropod system

This study investigates the transfer of Cd and Zn from a soil amended with sewage sludge at rates up to 100 t ha(-1) through a multi-trophic system consisting of barley, the aphid Sitobion avenae and the larvae of the lacewing Chrysoperla carnae. Results show marked differences in the transfer of the two metals. Cadmium was freely accumulated in barley roots, but accumulation in the shoot was restricted to a concentration of around 0.22 mg kg(-1) (dry weight). This limited the transfer of Cd to higher trophic levels and resulted in no significant accumulation of Cd in S. avenae or in C. carnae. Zinc transfer in the system was largely unrestricted, resulting in significant accumulation in roots and shoots, in S. avenae and in C. carnae. Cadmium biomagnification occurred in lacewing pupae, with concentrations up to 3.6 times greater than in aphids. S. avenae biomagnified Zn by a factor of ca. 2.5 at low sludge amendment rates, but biomagnification decreased to a factor of 1.4 at the highest amendment rate. Biomagnification of Zn did not occur in C. carnae, but concentrations were up to 3.5 time higher than in soil. Results are discussed in light of the mechanisms regulating transfer of the two metals in the system.     

Multi-generation cadmium acclimation and tolerance in Daphnia magna Straus

The cladoceran Daphnia magna was acclimated for seven generations to cadmium concentrations ranging from 0 (control) to 250 microg/l Cd (corresponding to a free ion activity of 4.60 nM Cd2+). Acute and chronic cadmium tolerance as well as cadmium accumulation were monitored as a function of acclimation time. After two to three generations of acclimation to concentrations ranging from 0.23 to 1.11 nM Cd2+ increases in acute tolerance were maximal (factor 7.2) and significant. Acclimation for seven generations to the same acclimation concentrations did result in an increased chronic cadmium tolerance (21 days EC50 values increased). Organisms acclimated to 1.93 nM Cd2+ were equally or more sensitive than non-acclimated daphnids in acute and chronic toxicity tests. Cadmium contents in D. magna increased significantly as a function of the acclimation concentration. Maximum body burdens of 236+/-30 microg Cd/g dry weight were measured in organisms exposed to 4.60 nM Cd2+, but detoxification mechanisms were only successful up to 82+/-20 microg Cd/g dry weight as this concentration did not cause major decreases in survival and reproduction in chronic toxicity tests. As the potential positive effect of acclimation on cadmium tolerance disappeared with successive acclimation generations and increasing acclimation concentrations, it is concluded that multi-generation acclimation studies are important for the evaluation of the long-term effects of environmental toxicants.     

Growth and metal accumulation in vetiver and two Sesbania species on lead/zinc mine tailings

The lead (Pb)/zinc (Zn) tailings contained high concentrations of heavy metals (total Pb, Zn, Cu and Cd concentrations 4164, 4377, 35 and 32 mg kg(-1), respectively), and low contents of major nutrient elements (N, P, and K) and organic matter. A field trial was conducted to compare growth performance, metal accumulation of Vetiver (Vetiveria zizanioides) and two legume species (Sesbania rostrata and Sesbania sesban) grown on the tailings amended with domestic refuse and/or fertilizer. It was revealed that domestic refuse alone and the combination of domestic refuse and artificial fertilizer significantly improved the survival rates and growth of V. zizanioides and two Sesbania species, especially the combination. However, artificial fertilizer alone did not improve both the survival rate and growth performance of the plants grown on tailings. Roots of these species accumulated similar levels of heavy metals, but the shoots of two Sesbania species accumulated higher (3-4 folds) concentrations of Pb, Zn, Cu and Cd than shoots of V. zizanioides. Most of the heavy metals in V. zizanioides were accumulated in roots, and the translocation of metals from roots to shoots was restricted. Intercropping of V. zizanioides and S. rostrata did not show any beneficial effect on individual plant species, in terms of height, biomass, survival rate, and metal accumulation, possibly due to the rather short experimental period of 5 months.     

Environmental distribution of uranium and other trace elements at selected Kosovo sites

This paper reports the results of a study using lichens as biomonitors to investigate the environmental distribution of uranium and other trace elements at selected Kosovo sites. The results suggested that the use of depleted uranium (DU) ammunitions in Kosovo did not cause a diffuse environmental contamination in such a way to have caused a detectable U enrichment in lichens. Also isotopic (235)U/(238)U measurements did not indicate the presence of DU particles in lichens. The present results also provided no indication of intense environmental contamination by the other trace elements analyzed, with the exception of Kosovska Mitrovica, where a diffuse environmental contamination by several heavy elements such as Pb, Zn, As and Cd was found.     

Effects of exogenous organic chelators on phytochelatins production and its relationship with cadmium toxicity in wheat (Triticum aestivum L.) under cadmium stress

Phytochelatins (PCs) have been proposed as a potential biomarker for metal toxicity. In this study, cadmium (Cd) toxicity, PCs production and their relationship in wheat under Cd stress were examined using various exogenous organic chelator-buffered nutrient solutions. Single Cd stress produced strong toxic effects, as indicated by decreases of growth parameters, high level of lipid peroxidation in leaf and overproduction of PCs in root. Exogenous organic chelators with proper dose more or less reduced Cd toxicity by increasing growth parameters and decreasing lipid peroxidation in leaves. Of organic chelators (EDTA, DTPA, citric acid, malic acid and oxalic acid), EDTA was the most effective in decreasing Cd toxicity in plants, followed by DTPA and citric acid. Simultaneously, the concentrations of Cd-induced PCs in roots decreased, and the greatest decrease was caused by application of EDTA and DTPA. Linearly positive relationships were observed between Cd toxicity and root PCs concentrations under the influences of organic chelators, particularly EDTA, DTPA and citric acid. Furthermore, present results provide stronger evidence that PCs synthesis in plant cells was related to free Cd ion concentrations, not total Cd, and demonstrate that the levels of PCs production in plants correlated well with toxic effects caused by the bioavailable Cd levels.     

Strategies of heavy metal uptake by three plant species growing near a metal smelter

Some higher plant species have developed heavy metal tolerance strategies which enable them to survive and reproduce in highly metal-contaminated soils. We have investigated such heavy metal uptake and accumulation strategies of two absolute metallophyte species (Armeria maritima ssp. halleri and Cardaminopsis halleri) and one pseudometallophyte (Agrostis tenuis) growing near a former metal smelter. Samples of plant parts and soil were analysed for Zn, Cd, Pb, and Cu. In soil, there were two dominant types of metal concentration gradients with depth. Under the absolute metallophytes, extremely high metal contents were measured in the surficial Ah horizon, followed by a strong decrease in the underlying soil horizons (L(11) and L(12)). Under the pseudometallophyte, metal concentrations in the Ah horizon were much lower and fewer differences were observed in metal concentrations among the Ah, L(11), and L(12) horizons. The concentrations of Zn, Cd, Pb, and Cu in Agrostis tenuis roots were greater than concentrations in leaves, indicating significant metal immobilisation by the roots. For C. halleri, Zn and Cd concentrations in leaves were >20,000 and >100 mg kg(-1), respectively, indicating hyperaccumulation of these elements. Armeria maritima ssp. halleri exhibited root concentrations of Pb and Cu that were 20 and 88 times greater, respectively, than those in green leaves, suggesting an exclusion strategy by metal immobilisation in roots. However, Zn, Cd, Pb, and Cu concentrations in brown leaves of Armeria maritima ssp. halleri were 3-8 times greater than in green leaves, suggesting a second strategy, i.e. detoxification mechanism by leaf fall.     

Arsenite tolerance in rice (Oryza sativa L.) involves coordinated role of metabolic pathways of thiols and amino acids

Thiolic ligands and several amino acids (AAs) are known to build up in plants against heavy metal stress. In the present study, alteration of various AAs in rice and its synchronized role with thiolic ligand was explored for arsenic (As) tolerance and detoxification. To understand the mechanism of As tolerance and stress response, rice seedlings of one tolerant (Triguna) and one sensitive (IET-4786) cultivar were exposed to arsenite (0–25 μM) for 7 days for various biochemical analyses using spectrophotometer, HPLC and ICPMS. Tolerant and sensitive cultivars respond differentially in terms of thiol metabolism, essential amino acids (EEAs) and nonessential amino acids (NEEAs) vis-á-vis As accumulation. Thiol biosynthesis-related enzymes were positively correlated to As accumulation in Triguna. Conversely, these enzymes, cysteine content and GSH/GSSG ratio declined significantly in IET-4786 upon As exposure. The level of identified phytochelatin (PC) species (PC₂, PC₃ and PC₄) and phytochelatin synthase activity were also more pronounced in Triguna than IET-4786. Nearly all EAAs were negatively affected by As-induced oxidative stress (except phenylalanine in Triguna), but more significantly in IET-4786 than Triguna. However, most of the stress-responsive NEAAs like glutamic acid, histidine, alanine, glycine, tyrosine, cysteine and proline were enhanced more prominently in Triguna than IET-4786 upon As exposure. The study suggests that IET-4786 appears sensitive to As due to reduction of AAs and thiol metabolic pathway. However, a coordinated response of thiolic ligands and stress-responsive AAs seems to play role for As tolerance in Triguna to achieve the effective complexation of As by PCs.     

Cadmium and zinc bioaccumulation and metallothionein response in two freshwater bivalves (Corbicula fluminea and Dreissena polymorpha) transplanted along a polymetallic gradient

This study was designed to compare the metallothionein (MT) response capacity of two freshwater bivalves, Corbicula fluminea and Dreissena polymorpha, along an environmental gradient of polymetallic pollution. The bivalves were transplanted into 4 stations in southwestern France, with a cadmium and zinc pollution gradient. MT and metal concentrations were measured in the soft bodies of the clams and mussels over 2.5 months. In the zebra mussels, variations in MT concentrations were strictly correlated to progressive Cd and Zn bioaccumulation. In contrast, the faster response in the clams appeared positively correlated to Cd accumulation only, with the activation of efficient detoxification mechanisms which limited Cd bioaccumulation and reduced Zn concentrations over time. Nevertheless, despite stronger metal accumulation factors in D. polymorpha, C. fluminea revealed higher sensitivity of MT response along the pollution gradient.     

Sub-cellular partitioning of Cd, Cu and Zn in tissues of indigenous unionid bivalves living along a metal exposure gradient and links to metal-induced effects

We studied organ and sub-cellular distributions of several trace metals in a freshwater bivalve that has been proposed for use as a metal biomonitor. Specimens of Pyganodon grandis were collected from nine lakes located along a Cd, Cu and Zn concentration gradient (Rouyn-Noranda area, Quebec). Gills and digestive gland were isolated, homogenized and six sub-cellular fractions were separated by differential centrifugation and analysed for their Cd, Cu and Zn content. Metallothionein was quantified independently. Gill tissues contained abundant calcium concretions that accounted for over 60% of the total gill burden of each metal. Cadmium and Zn concentrations in this granule fraction reflected ambient metal concentrations. Metal concentrations in the digestive gland also responded to the metal contamination gradient, but to a lesser extent than the gills, reflecting the lower abundance of granules in the digestive gland. Metals (Cd, Cu) in this organ were present largely in the "heat-stable proteins" fraction, and metal concentrations in this fraction were strongly correlated with those of both metallothionein and, to a lesser extent, the "lysosomes+microsomes" and "mitochondria" fractions. In both organs, Cd concentrations in the "heat-denaturable protein" fraction remained low and constant, suggesting reasonably effective metal detoxification. Some evidence for oxidative stress was noted in the gills but not in the digestive gland. Overall, we conclude that in nature metals in P. grandis are bound differently in the gills and in the digestive gland and that metal detoxification in the former organ may be less effective than in the latter.     

The distribution and intracellular compartmentation of metals in the endogeic earthworm Aporrectodea caliginosa sampled from an unpolluted and a metal-contaminated site

The tissue distribution of Cd, Cu, Pb, Zn and Ca in the endogeic earthworm Aporrectodea caliginosa living in a non-polluted and a heavy metal polluted soil was investigated. The tissues of animals from the contaminated soil contained greater concentrations of Cd, Pb and Zn than the corresponding tissues of animals from the unpolluted soil. The greatest concentrations of Cd, Pb, Zn, and Ca were primarily accumulated within the posterior alimentary canal (PAC), a tissue fraction which contained the greatest proportion of the whole-worm burdens of the respective metals. Cu was distributed fairly evenly in the tissue fractions investigated. The pattern of accumulation for the 'heavy' metals is broadly similar to that for epigeic earthworms; in contrast, a different pattern of tissue accumulation was found for Ca. In animals from the uncontaminated site, the major elemental constituents of the chloragosomes were P, Ca, Zn and S. A significant positive correlation exists between P and Ca within the chloragosomal matrix. These intracellular vesicles are major foci for Pb and Zn accumulation within the PAC, with 'excess' metals associated with P ligands within the chloragosome matrix. The incorporation of Pb and Zn appears to involve the cationic displacement of Ca. Such compartmentation appears to prevent dissemination of large concentrations of these metals into other earthworm tissues, and may thus represent a detoxification strategy based on accumulative immobilization. No intracellular localization of Cd was identified in the study, although the Cd concentration in the metalliferous soils examined was not exceptionally high. The observations are discussed in the context of a contribution to enhanced understanding of metal ecotoxicology in earthworms by providing baseline data on a little investigated ecophysiological group of earthworms. Comparisons of metal distribution and mechanisms of metal sequestration are made with other ecophysiological groups of earthworms, and the significance of the findings to biomonitoring and toxicity-testing programmes is considered.