Metal concentrations in digestive gland and mantle of Sepia officinalis from two coastal lagoons of Portugal

Concentrations of both essential (Fe, Cu, Zn) and non essential (Cd, Hg and Pb) metals were measured in the digestive gland and mantle of female cephalopods Sepia officinalis captured in two distinct lagoons in Portugal: Aveiro Lagoon, with a history of anthropogenic and industrial pollution, and Formosa Lagoon receiving urban effluents. We provide evidence for the following: (1) the digestive gland is the main target organ for both essential and non essential metals, frequently containing concentrations few orders of magnitude higher as compared to mantle; the sole exception from this was the Hg that is equally distributed in the two tissues; (2) unexpectedly, the higher levels of metals were found in animals captured in the less polluted lagoon, except for Cd whose bioavailability in Aveiro lagoon might be related to industrial sources, while the influence of Cd speciation in local pray composition should not be ruled out (3) size influenced metal concentration in different way: smaller individuals accumulated significantly more Cu, while Hg concentrations showed the opposite trend; (4) Cd is positively correlated to Zn and Cu in digestive gland of specimens collected in spring in Aveiro Lagoon, and no relationship was found in Formosa Lagoon; (5) the molar ratios Cd:Zn and Cd:Cu in digestive gland increased with body weight in specimens from Aveiro area, both ratios becoming particularly higher in older individuals. Metal-specific accumulation patterns in both mantle and digestive gland at the two sites are discussed in the light of their toxicological implications.     

Subcellular and body distributions of 17 trace elements in the variegated scallop Chlamys varia from the French coast of the Bay of Biscay

Seventeen elements were analysed in the organs and tissues of the variegated scallop Chlamys varia, from the Atlantic coast of France. Concentration levels were determined in scallops of different sizes sampled in contaminated (La Rochelle Bay) and clean (Re Island) sites. Greater concentrations of Ag, Al, Ce, Cr, La, Mo, Nd, Ti, and V were found in the digestive gland while As, Cd, Co, Cu, Mn, Ni, Pb, and Zn were the highest in the kidneys. In the digestive gland, most of the metals were found in the insoluble fraction while As, Co, Cd, Mo, Ni, and V appeared to be mostly bound to soluble compounds. Among tissues, the adductor muscle always displayed the lowest trace element concentrations. According to size, Ag and Cd showed significantly higher concentrations in larger individuals, while Co and Zn were higher in the smallest ones. According to the sampling area, most of the metals, Ag, Al, Ce, Co, Cu, La, Mn, Nd, Pb, and Zn, showed significantly higher concentrations in La Rochelle Bay compared to the Re Island, reflecting differing inputs from industrial, domestic and harbour activities. However, Cr, Mo, Ni, Ti, and V concentrations did not display significant differences between sites and As and Cd were significantly higher at the Re Island. This study highlighted the ability of the variegated scallop C. varia to concentrate numerous trace elements to high levels, even those reported as poorly bioavailable for marine biota, such as rare earth elements.     

Variation of heavy metal concentrations (Ag, Cd, Co, Cu, Fe, Pb, V, and Zn) during the life cycle of the common cuttlefish Sepia officinalis

The developmental changes in the concentration of 8 essential and non-essential heavy metals (Ag, Cd, Cu, Co, Fe, Pb, V, Zn) in the tissues (digestive gland, cuttlebone and whole animal) of the common cuttlefish Sepia officinalis collected in the bay of the river Seine were monitored from the end of the embryogenesis until the adult reproductive stage. Compared to embryos, juveniles after hatching displayed much higher concentrations of Ag, Cu, Fe and Zn, suggesting an efficient incorporation from seawater. Conversely, the amounts of Cd, Pb and V in hatchlings remained constant suggesting that these metals are barely bioavailable for juveniles. Once the juveniles start to feed, the digestive gland appears to play a major role in the storage of all metals. After only one month of benthic life, the digestive gland already contains up to 90% of the total metal body burden, indicating that it plays a major role in the storage and presumed detoxification of the selected metals. Metal concentrations in the digestive gland increase in a logarithmic fashion with age during the entire life of cuttlefish, except for Ag, which decreases as soon as cuttlefish migrate to open sea. This strongly suggests that (1) Ag is excreted from the digestive gland in relation to presumably lower exposure in less contaminated environments compared to coastal waters and (2) the digestive gland of cephalopods could be a very good indicator of Ag contamination in the marine environment.     

Sub-cellular partitioning of Zn, Cu, Cd and Pb in the digestive gland of native Octopus vulgaris exposed to different metal concentrations (Portugal)

Concentrations of Zn, Cu, Cd and Pb and their sub-cellular distributions were determined in composite samples of digestive glands of the common octopus, Octopus vulgaris caught from two areas of the Portuguese coast characterised by contrasting metal contamination. Minor contents of Zn (1%), Cu (2%), Cd (6%) and Pb (7%) were found in the insoluble fraction, consisting of nuclei, mitochondria, lysosomes and microsome operationally separated from the whole digestive gland through a sequential centrifugation. A tendency for linear relationships between metal concentrations in nuclei, mitochondria, lysosomes and whole digestive gland was observed. These relationships suggest that despite low metal content organelles responded to the increasing accumulated metals, which means that detoxifying mechanism in cytosol was incomplete. Poorer correlations between microsome and whole digestive gland did not point to metal toxicity in the analysed compartments. However, the high accumulated Cd indicated that O. vulgaris is an important vehicle of this element to its predators in the coastal environment.     

The distribution of cadmium,copper, lead,and zinc in eelgrass (Zostera marina L.)

The distribution of Cd, Cu, Pb, and Zn in eelgrass (Zostera marina L.) was studied at three locations with different heavy metal loads in the Limfjord, Denmark.The eelgrass was fractionated into roots, rhizome, stem, and leaves according to age, and the heavy metal concentrations in each fraction were determined. The distribution patterns of the four heavy metals in eelgrass were independent of the heavy metal loads at the sampling stations. The concentrations of all metals were greater in the roots than in the rhizomes. In the aerial¹ parts two different age-dependent distribution patterns were observed. The concentrations of Cd, Pb, and Zn increased with age while the opposite was true for Cu. The distribution of lead correlated with the distribution of ash content. These age-dependent distribution patterns were maintained throughout the observation period and were most pronounced for Cu and Zn in winter.The heavy metal distribution in eelgrass is discussed in relation to gross morphology, especially age-structure. It is suggested that the accumulation of Cd, Pb, and Zn is due to a slow irreversible uptake or to the existence of more binding sites in old leaves. The distribution of Cu can be explained by translocation within the plant, dilution due to growth or leakage from the older leaves.     

Elemental characterization of tissues of Octopus vulgaris along the Portuguese coast

The concentrations of V, Mn, Fe, Ni, Cu, Zn, As, Se, Mo, and Pb were measured in digestive gland (DG), branchial hearts (BH), gill (G), and muscle (M) of Octopus vulgaris Cuvier, 1797 caught in three fishing areas of the Portuguese coast, Viana do Castelo, Cascais, and Santa Luzia, for 2 years. The elemental concentrations measured for the different tissues were in accordance with values reported in the literature. The digestive gland presented high concentration levels of Fe, Cu, and Zn, while the branchial hearts showed elevated levels of V, Ni, Mo, as well as Fe and Cu. Significant variations in As, V, Cu, Mo, and Pb tissue concentrations were observed for animals originated from different sampling sites. Pb and As determined in the digestive gland and branchial hearts of animals from Cascais and Santa Luzia, can reflect local environmental characteristics. The variability observed in the elemental concentrations may be useful to further assess the species susceptibility to environmental conditions.     

Assessment of metal pollution in Onsan Bay, Korea using Asian periwinkle Littorina brevicula as a biomonitor.

Cadmium (Cd), lead (Pb), copper (Cu) and zinc (Zn) concentrations in the marine gastropod, Littorina brevicula Philippi, were determined to assess the metal pollution in Onsan Bay, Korea. Samples of L. brevicula employed as a biomonitor and seawater were collected from 12 to 20 stations of Onsan Bay in November 1997, respectively. Dissolved metal concentrations in surface seawater were highest at the station near Onsan Non-ferrous Industrial Complex: 1.15 micrograms l-1 for Cd, 2.49 micrograms l-1 for Pb, 3.75 micrograms l-1 for Cu and 23.98 micrograms l-1 for Zn. These values were 1-2 orders higher than those shown at outer regions of the Bay. Metal concentrations in the soft body of periwinkles were highly variable at different sampling locations: 0.48-27.11 micrograms g-1 for Cd, 1.41-24.91 micrograms g-1 for Pb, 57-664 micrograms g-1 for Cu and 83-246 micrograms g-1 for Zn. The values from stations near the industrial complex were higher than those expected from relationships between body sizes and metal body burdens in periwinkles collected from the whole Korean coast. Spatial distribution of metal concentrations in the periwinkle and seawater indicated that Onsan industrial complex near the Bay is the input source of these metals. Especially, Cd and Pb concentrations in the periwinkle and seawater were distinctly decreased with distance from the Onsan industrial complex. Non-essential metals such as Cd and Pb in the periwinkle showed a strong correlation with dissolved metal concentrations in seawater. Conversely, essential Cu and Zn in the periwinkle were hardly explained by those in seawater, except at the most contaminated sites.     

Clams as pollution bioindicators in Kuwait''s marine environment: metal accumulation and depuration

This study has developed and verified clam transplantation for marine pollution monitoring in Kuwait and has assessed its reliability for monitoring pollution by heavy metals through laboratory and field transplantation experiments. In field experiments, live specimens of the clam Circenita callipyga were transplanted, either suspended in seawater or buried in sand, to 11 coastal sites in Kuwait; subsamples of transplanted clams were recovered at intervals over a 6-month period. In laboratory experiments, heavy metal accumulation for 36 days and depuration (body's release of pollutants) for 60 days in small and big clams were investigated. Clams subsampled in laboratory and field experiments were analyzed for Hg, Cu, V, Cd and Pb. Results showed the ability of clams to survive under the transplantation conditions. Accumulation of biofouling materials was a problem at only two transplantation sites and was overcome by periodic cleaning. Statistical analysis of laboratory experimental data showed significant accumulation of all test metals and significant depuration of Hg, Cu, V and Cd. Rates of metal accumulation and depuration differed in relation to clam size class. Field experiments indicated statistically significant increases in Hg and Cu concentrations in the transplanted clams at most stations, no change in Cd concentrations and an increase or, occasionally, a decrease in V and Pb concentrations.     

Heavy metal exposure, reproductive activity, and demographic patterns in white-footed mice (Peromyscus leucopus) inhabiting a contaminated floodplain wetland

We examined the concentrations of selected metals and selenium (Se) in the tissues of white-footed mice (Peromyscus leucopus) collected at a constructed wetland originally created as a retention basin for sediments dredged from Lake DePue, Illinois. These sediments were contaminated with high concentrations of cadmium (Cd), zinc (Zn), and other elements as a result of nearby smelting operations. White-footed mice inhabiting the former retention basin experienced greater exposure to Cd, Pb, and Se than those from nearby reference sites. Concentrations of Cu and Zn in livers of mice from the contaminated wetland and adjacent floodplain reference site were greater than in mice from the more-distant reference sites. Judging by concentrations in their kidneys, white-footed mice inhabiting the floodplain adjacent to the contaminated wetland had greater exposure to Cd than those from the two more-distant reference sites. Concentrations of Hg in tissues of mice did not vary appreciably among sites. Concentrations of Cd and Se in the tissues of some white-footed mice from the contaminated wetland exceeded critical concentrations observed in experimental studies of laboratory mice and rats; with few exceptions tissue Pb concentrations were below published effects levels. However, we did not detect changes in abundance, demographics, or reproductive activity that might suggest population-level effects of contaminant exposure. Mean weight of embryos expressed as a function of crown-rump length did not differ among locations sampled, and no gross lesions indicative of exposure to heavy metals were observed. Kidney and liver weight, corrected for body weight, were nominally, though not significantly, lowest in both male and female mice from areas of increased Cd and Pb exposure. Metals dredged from Lake DePue were still bioavailable 25 years after deposition. However, small mammal populations are resilient to environmental stressors and we did not detect differences in population parameters suggesting that the population of white-footed mice inhabiting the contaminated wetland was at risk from increased exposure to these contaminants.     

Spatial and seasonal variation in heavy metals in the sediments and biota of two adjacent estuaries, the Orwell and the Stour, in eastern England

A study was made of the concentrations of the elements As, Cd, Cu, Hg, Mn, Ni, Pb and Zn in the sediments and biota of two adjacent estuaries, the Orwell and Stour, in eastern England. The Orwell Estuary, with its urbanized head, was more contaminated with heavy metals than the Stour Estuary. Generally, in both estuaries, concentrations of metals were highest towards the head and the mouth. Saltmarsh sediments accumulated higher concentrations of most metals than mudflat sediments. Metal concentrations in the biota showed marked interspecific differences; Mytilus edulis had higher concentrations of Cd, Littorina littorea higher concentrations of Cu and Mn and Arenicola marina higher concentrations of Hg. Invertebrates from the Orwell had higher metal concentrations than those from the Stour. Algae had generally lower levels of metals than invertebrates. Metal concentrations were greatest and more variable in the top 10 cm of sediment. Metals were at greatest concentrations in winter and lowest in summer in sediments, algae and invertebrates. Mercury concentration increased with size in the three invertebrate species studied, but Cd and Zn generally were at higher concentrations in younger animals. Comparisons of sediments with average shale values indicated anthropogenic enrichment with several metals but it was considered that only Pb, at some sites, and possibly Hg posed potential threats to the ecology of the estuaries.     

Concentration and fate of trace metals in Mekong River delta

In the Mekong River delta and its associated coastal zone trace elements concentrations (Cd, Cu, Ni and Pb) were measured in the dissolved phase (DP) during dry (March 1997) and wet (October 1997) seasons. As, Co, Cr, Ni, Pb, and Al were also measured in suspended matter (SM) and total and organic carbon, trace elements (Cd, Cr, Cu, Hg, Mn, Ni, Pb, Zn) and macro elements in superficial sediments (S). Trace metal concentrations in DP and SM during the contrasting hydrological conditions were generally found within the range observed for uncontaminated environment. The average DP concentrations (nM) in the river for March and October are: Cd 0.03 and 0.09, Cu 15 and 14, Ni 7.8 and 8.4, Pb 0.51 and 0.50, respectively. In general there is no significant difference between the concentrations observed during dry and wet season. The evolution of the DP trace metal concentration in the surface water within the salinity gradient suggests no noticeable exchange between the particulate and dissolved phase. This result is in good agreement with those observed in most plume structures studied so far. The average concentrations in the SM (microg/g) (March, October) at the river end-member are: As (24; 11), Co (17; 9), Cr (49; 29), Ni (32; 18), Pb (42; 19) and Al (113,000; 67,000), respectively. All trace elements show higher concentrations in March than in October, with an average increase of two times. This is essentially related to grain size effect since smaller particles were supplied during dry season. These differences are not reflected in the mixing zone, which integrates the seasonal variations. The concentration of major elements (C total, C organic, Si, Al, Ca, K, Fe, Mg, Ti), trace elements (Pb, Zn, Cu, Ni, Mn, Cr, Cd, Hg) in superficial sediments, show similar values during the two seasons and does not show any important variation with depth, indicating either a very fast sedimentation rate and/or the absence of any significant contamination.     

The effect of graded doses of cadmium on lead,zinc and copper content of target and indicator organs in rats

Eleven groups of inbred Wistar rats, five males and five females in each group, received graded doses of CdCl₂, from 0–25 ppm, in the drinking water from conception until sacrifice at the age of ten weeks. The Pb, Zn and Cu levels of various indicators and target organs were recorded by atomic absorption spectrophotometry. Cd concentrations above 5 ppm caused a significant increase in Zn content of kidneys, liver, spleen, heart and adrenal glands in most of the experimental groups, and in addition a significant positive correlation between Cd and Zn in the same tissues. With concentrations above 10 ppm, Zn contents of epiphyses and diaphyses decreased significantly, and through all groups there was a significant negative correlation between Zn and Cd in diaphysis. This local Zn deficit may cause a retarded growth of long bones. Cd supply resulted in a significant decrease in Zn content of incisors and molars, but did not significantly affect the Cu content. Neither were Zn nor Cu concentrations in hair affected. Cd supply led to a significantly increased storage of Cu in liver, spleen, heart and skeletal muscle and a positive correlation between Cd and Cu content in the same tissues. Cd administration caused a significant decrease of Pb concentrations in molars, hair, kidney cortex, heart, skeletal muscle and adrenal glands in several of the experimental groups, and also a significant negative correlation between Pb and Cd content of molars, hair, kidney cortex, liver and heart. This can be a result of decreased uptake, increased excretion or changes in the distribution pattern of Pb.     

Metals and selenium in the liver and bone of three dolphin species from South Australia, 1988-2004

Metal and selenium concentrations (wet weight) were determined in the liver (Cd, Hg, Pb, Zn, Cu and Se) and bone (Pb and Cd) of common dolphins (Delphinus delphis, N=71) and bottlenose dolphins (Tursiops truncatus, N=12, and Tursiops aduncus, N=71) stranded or by-caught in South Australia from 1988 to 2004. Differences in metal burdens existed between species, stranding location, and relative age. T. aduncus had the greatest mean tissue burdens of liver Pb (0.45 mg/kg), Cd (6.45 mg/kg), Hg (475.78 mg/kg), Se (178.85 mg/kg) and Zn (93.88 mg/kg) and bone Pb (2.78 mg/kg), probably reflecting their coastal habitat and benthic prey. Mean Cu was highest in T. truncatus (21.18 mg/kg). Bone Cd was measured only in T. aduncus and averaged 0.05 mg/kg. Stranding location impacted metal burdens. Dolphins from Spencer Gulf had higher mean levels of liver Pb (0.39 mg/kg) while Gulf St Vincent dolphins had greater liver Hg (444.64 mg/kg), liver Se (163.12 mg/kg), and bone Pb (2.85 mg/kg). This may be due to high anthropogenic inputs of Pb and Hg into Spencer Gulf and Gulf St Vincent respectively. Liver Cd, Hg, Se and Pb increased with age in all species while Cu decreased with age, in keeping with previous studies. Se and Hg were positively correlated. The possibility that metallothioneins are driving observed correlations between Zn, Cd, Hg and Cu are discussed. Future research must investigate the toxicological consequences of the metal concentrations reported.     

Assessing risk to human health from tropical leafy vegetables grown on contaminated urban soils

Fifteen tropical leafy vegetable types were sampled from farmers' gardens situated on nine contaminated sites used to grow vegetables for commercial or subsistence consumption in and around Kampala City, Uganda. Trace metal concentrations in soils were highly variable and originated from irrigation with wastewater, effluent discharge from industry and dumping of solid waste. Metal concentrations in the edible shoots of vegetables also differed greatly between, and within, sites. Gynandropsis gynandra consistently accumulated the highest Cd, Pb and Cu concentrations, while Amaranthus dubius accumulated the highest Zn concentration. Cadmium uptake from soils with contrasting sources and severity of contamination was consistently lowest in Cucurbita maxima and Vigna unguiculata, suggesting these species were most able to restrict Cd uptake from contaminated soil. Concentrations of Pb and Cr were consistently greater in unwashed, than in washed, vegetables, in marked contrast to Cd, Ni and Zn. The risk to human health, expressed as a ‘hazard quotient’ (HQ_M), was generally greatest for Cd, followed successively by Pb, Zn, Ni and Cu. Nevertheless, it was apparent that urban cultivation of leafy vegetables could be safely pursued on most sites, subject to site-specific assessment of soil metal burden, judicious choice of vegetable types and adoption of washing in clean water prior to cooking.     

Health risk assessment of heavy metal exposure to street dust in the zinc smelting district, Northeast of China

Heavy metal contamination in the street dust due to metal smelting in the industrial district of Huludao city was investigated. Spatial distribution of Hg, Pb, Cd, Zn and Cu in the street dust was elucidated. Meanwhile, noncancer effect and cancer effect of children and adults due to exposure to the street dust were estimated. The maximum Hg, Pb, Cd, Zn and Cu contents in the street dust are 5.212, 3903, 726.2, 79,869, and 1532 mg kg^− 1, and respectively 141, 181, 6724, 1257 and 77.4 times as high as the background values in soil. The trends for Hg, Pb, Cd, Zn and Cu are similar with higher concentrations trending Huludao zinc plant (HZP). The exponential equation fits quite well for the variations of Pb, Cd, Zn and Cu contents with distance from the pollution sources, but not for Hg. The biggest contribution to street dust is atmospheric deposition due to metal smelting, but traffic density makes slight contribution to heavy metal contamination. According to the calculation on Hazard Index (HI), in the case of noncancer effect, the ingestion of dust particles of children and adults in Huludao city appears to be the route of exposure to street dust that results in a higher risk for heavy metals, followed by dermal contact. The inhalation of resuspended particles through the mouth and nose is almost negligible. The inhalation of Hg vapour as the fourth exposure pathway to street dust is accounting for the main exposure. Children are experiencing the potential health risk due to HI for Pb larger than safe level (1) and Cd close to 1. Besides, cancer risk of Cd due to inhalation exposure is low.     

Trace element accumulations in 13 avian species collected from the Kanto area, Japan

In the present study, concentrations of 13 elements (Li, Cr, Mn, Cu, Zn, As, Se, Rb, Sr, Mo, Ag, Cd, Hg) were measured in the tissues of the livers, the kidneys, pectoral muscles, lungs and brains of 13 avian species collected from the Kanto area of Japan. The difference in hepatic heavy metal levels of the grey herons from the two sites was compared. Metal levels in the sediment of the Tama River estuary, situated in the Haneda area, were also measured. These results revealed that heavy metal pollution is present in an aquatic area of Haneda. The accumulation patterns of Cu and Zn in the livers of grey herons appeared to be separated into two groups. Additionally, the present study includes the properties of other metal accumulations and their relationships in avian species.     

Biomonitoring of trace metals in the Black Sea (Romania) using mussels Mytilus galloprovincialis

In a preliminary biomonitoring study, accumulated trace metal concentrations (Cd, Cu, Zn, Hg, Fe, Mn) have been measured in the mussel Mytilus galloprovincialis collected in the Black Sea, at 100 km from the Danube Delta in February 2001 and February 2002. Mussels were collected from four sites. In 2001 metal concentrations were determined in the whole soft body whereas, in 2002, the distribution of metals was evaluated in three different organs (gills, visceral mass and remaining tissues). The results obtained in 2002 confirmed those of 2001. For a given site, concentrations were always higher in the gills and visceral mass of mussels than in the remaining tissues. Principal component analysis allowed separating stations as a function of the metal concentrations in the organs. It is concluded that the mussels M. galloprovincialis are suitable biomonitors to assess changes in metal pollution in this coastal area of the Black Sea.     

The spatial and temporal trends of Cd, Cu, Hg, Pb and Zn in Seine River floodplain deposits (1994-2000)

Fresh floodplain deposits (FD), from 11 key stations, covering the Seine mainstem and its major tributaries (Yonne, Marne and Oise Rivers), were sampled from 1994 to 2000. Background levels for Cd, Cu, Hg, Pb, and Zn were established using prehistoric FD and actual bed sediments collected in small forested sub-basins in the most upstream part of the basin. Throughout the Seine River Basin, FD contain elevated concentrations of Cd, Cu, Hg, Pb and Zn compared to local background values (by factors>twofold). In the Seine River Basin, trace element concentrations display substantial downstream increases as a result of increasing population densities, particularly from Greater Paris (10 million inhabitants), and reach their maxima at the river mouth (Poses). These elevated levels make the Seine one of the most heavily impacted rivers in the world. On the other hand, floodplain-associated trace element levels have declined over the past 7 years. This mirrors results from contemporaneous suspended sediment surveys at the river mouth for the 1984-1999 period. Most of these temporal declines appear to reflect reductions in industrial and domestic solid wastes discharged from the main Parisian sewage plant (Seine Aval).     

Submersible integrating water sampler for heavy metals

A simple submersible apparatus for the collection of a continuous composite water sample, suitable for heavy metal determination, is described. A comparison between results obtained for Cd, Pb, Cu, Zn and Hg using this sampler and a series of hand collected samples showed a satisfactory level of agreement. The apparatus is suitable for pollution monitoring purposes in relatively shallow (<20 m) waters where the concentrations of Cd, Pb, Cu, Zn and Hg are expected to exceed 0.05, 0.2, 0.2, 0.4 and 0.02 μg l⁻¹ respectively.     

A field survey of metal binding to metallothionein and other cytosolic ligands in liver of eels using an on-line isotope dilution method in combination with size exclusion (SE) high pressure liquid chromatography (HPLC) coupled to Inductively Coupled Plasma time-of-flight Mass Spectrometry (ICP-TOFMS)

The effect of metal exposure on the accumulation and cytosolic speciation of metals in livers of wild populations of European eel with special emphasis on metallothioneins (MT) was studied. Four sampling sites in Flanders showing different degrees of heavy metal contamination were selected for this purpose. An on-line isotope dilution method in combination with size exclusion (SE) high pressure liquid chromatography (HPLC) coupled to Inductively Coupled Plasma time-of-flight Mass Spectrometry (ICP-TOFMS) was used to study the cytosolic speciation of the metals. The distribution of the metals Cd, Cu, Ni, Pb and Zn among cytosolic fractions displayed strong differences. The cytosolic concentration of Cd, Ni and Pb increased proportionally with the total liver levels. However, the cytosolic concentrations of Cu and Zn only increased above a certain liver tissue threshold level. Cd, Cu and Zn, but not Pb and Ni, were largely associated with the MT pool in correspondence with the environmental exposure and liver tissue concentrations. Most of the Pb and Ni and a considerable fraction of Cu and Zn, but not Cd, were associated to High Molecular Weight (HMW) fractions. The relative importance of the Cu and Zn in the HMW fraction decreased with increasing contamination levels while the MT pool became progressively more important. The close relationship between the cytosolic metal load and the total MT levels or the metals bound on the MT pool indicates that the metals, rather than other stress factors, are the major factor determining MT induction.