Tissue Distribution and Correlation Profiles of Heavy-Metal Accumulation in the Freshwater Crayfish Astacus leptodactylus

The present work details the analysis of heavy-metal and metalloid concentrations in exoskeleton, gill, hepatopancreas, and abdominal muscle tissues of 60 crayfish (Astacus leptodactylus) specimens collected from Lake Hirfanl?, a dam lake located in K?r?ehir (Turkey) with a low metal-contamination profile. Concentrations of 11 metals (aluminum [Al], chromium [Cd], manganese [Mn], cobalt [Co], nickel [Ni], copper [Cu], molybdenum [Mo], silver [Ag], cadmium [Cd], mercury [Hg], and lead [Pb]) and a metalloid (arsenic [As]) were measured by inductively coupled plasma–mass spectrometry, and the relative frequencies of the most abundant isotopes of Cr, Cu, Ag, Cd, Hg, and Pb were evaluated. Three correlation trends were evaluated between the following: (1) different elements in the each individual tissue, (2) individual elements in different tissues, and (3) different elements in different tissues. In addition, correlation rates of growth parameters (weight, cephalothorax length, and total length) with heavy-metal and metalloid concentrations in each tissue were investigated. Our results suggest that substantial differences in metal and metalloid-accumulation levels exist between male and female specimens, with stronger correlations between the heavy-metal concentrations observed in the male cohort. It is notable that correlation trends of Co, Cu, ⁵²As, Cr, and Ni in exoskeleton of the male specimens display strong similarities. Likewise, a very strong correlation is present in Ni–Cd and Ni–Pb accumulations in abdominal muscle of the male specimens; a similar trend is present between Cd and Pb concentrations in the same tissue of female specimens. For correlation rates of different heavy metals and metalloid in different tissues, the strongest positive association observed was between ⁶³Cu in gill and As in hepatopancreas, whereas the strongest negative correlation was between accumulated Ni in abdominal muscle and As in exoskeleton. Strong correlations between metals and metalloid accumulations were observed between exoskeleton and gill. In many cases, metal and metalloid accumulation was negatively correlated with growth parameters. Preferential accumulation of Cr and Cu isotopes was observed in different tissues, suggesting that significant amounts of isotope fractionation occur during heavy-metal accumulation. Relatively low correlation rates were observed between ⁵²Cr/⁵³Cr and ⁶³Cu/⁶⁵Cu concentrations in several tissue types in both male and female cohorts, whereas no such trend was observed between Cd and Pb isotopes.     

Species-specific heavy metal accumulation patterns of earthworms on a floodplain in Japan

We identified all earthworm species found on a floodplain contaminated by heavy metals (Cu, Zn, Cd, and Pb) from an old mine in central Japan and compared their abundance, biomass, and heavy metal concentrations in tissue. There were six species belonging to three families: Megascolecidae, Moniligastridae, and Lumbricidae. Earthworm community structure seemed to be influenced mostly by soil properties, especially pH and clay fraction. Despite the same endogeic characteristics, species-specific patterns of heavy metal accumulation were observed: species in Megascolecidae and Lumbricidae had relatively lower concentrations compared to those in Moniligastridae. Within Moniligastridae, Drawida sp. accumulated Cu and Pb markedly higher than Drawida japonica. Based on heavy metal concentrations in extracts of CaCl(2) and diethylenetriaminepentaacetic acid, the aging caused remarkably low concentrations in pore water, indicating low availability by dermal uptake. Therefore the different patterns of heavy metal accumulation among species would partly result from species-specific gut process.     

Effect of Cd or Pb Addition to Cu-Contaminated Soil on Tissue Cu Accumulation in the Earthworm,Dendrobaena veneta

Generally, soil heavy metal contamination consists of a mixture of heavy metals. Soil chemical properties and interaction with other pollutants in soil affect the external heavy metal bioavailability. Moreover, interaction with other pollutants accumulated in organisms may change the toxicity of each pollutant. Therefore, the hypotheses was tested that addition of Cd or Pb to Cu-contaminated soil would lead to an increase in tissue Cu accumulation in the earthworm,Dendrobaena veneta,caused by (i) induction of metallothionein by Cd, or (ii) an increase in Cu concentration in soil solution due to the exchange of adsorbed Cu for Pb. Tissue heavy metal concentrations were determined after exposure in contaminated soils for 3 or 21 days. Considerable amounts of Cu, Cd, and Pb were accumulated, indicating that these heavy metals were available for uptake byD. veneta.Both Cd and Pb, however, did not significantly affect tissue Cu accumulation.     

Exotic Earthworms Decrease Cd,Hg, and Pb Pools in Upland Forest Soils of Vermont and New Hampshire USA

Exotic earthworms are present in the forests of northeastern USA, yet few studies have documented their effects on pollutant metals in soil. The objective of this study was to identify if Cd, Hg, and Pb strong-acid extractable concentrations and pools (bulk inventories) in forest soils decreased with the presence of exotic earthworms. We compared ‘Low Earthworm Abundance’ (LEA) sites (≤10 g m^?2 earthworms, n?=?13) and ‘High Earthworm Abundance’ (HEA) (>10 g m^?2 earthworms, n?=?17) sites at five watersheds across Vermont and New Hampshire. Organic horizon Cd, Hg, and Pb concentrations were lower at HEA than LEA sites. Organic horizon and total soil pools of Cd and Hg were negatively correlated with earthworm biomass. Soil profile Cd and Hg concentrations were lower at HEA than LEA sites. Our results suggest earthworms are decreasing accumulation of Cd, Hg, and Pb in forest soils, potentially via greater mobilization through organic matter disruption or bioaccumulation.     

Effect of pH and calcium on lead and cadmium uptake by earthworms in water

pH and calcium are important as factors determining heavy metal bioavailability to soil organisms. To study their intrinsic effect without the influence of soil adsorption, earthworms (Lumbricus rubellus) were exposed to lead and cadmium in reconstituted water adjusted to various levels of pH and calcium chloride. Concentration of cadmium in worms increased linearly with increasing concentration of exposure independent of pH. Concentration of Pb in worms, however, showed a pH-dependent increase with increasing concentration of exposure. The uptake of both metals was positively influenced by increasing pH. Calcium reduced the uptake of Cd, but had no significant effect on Pb uptake. It is suggested that Ca2+ and H+ compete with lead and cadmium ions for binding sites in the earthworm, and that the affinity for Pb is much greater than that for Cd. The results are discussed in relation to available data on Pb and Cd accumulation by earthworms in soil, showing the predominant importance of soil adsorption processes.     

Heavy Metal Contamination in the Cultivated Oyster <Emphasis Type="Italic">Crassostrea rivularis</Emphasis> and Associated Health Risks from a Typical Mariculture Zone in the South China Sea

With the rapid development of mariculture in potentially contaminated regions in China and the world, food safety, is a growing concern. To evaluate heavy metals and their associated health risks in the cultivated oyster Crassostrea rivularis, the concentrations of heavy metals (Cd, Cr, Pb, Zn) in oysters and water/sediment were examined in a typical mariculture environment (Kaozhou Bay, South China Sea). Trends in the seasonal dynamics of heavy metals in oysters revealed a potential synergistic effects among the concentrations of Cd, Cr, and Zn; trends associated with Pb were less clear, although the ability of oysters to bioaccumulate and depurate Pb was excellent. Bioconcentration factors (BCF) indicated that C. rivularis has a strong ability to accumulate heavy metals, and the BCF was the highest for Zn (2.32?×?10⁵), followed by Cd (6.84?×?10⁴), Pb (2.77?×?10⁴) and Cr (1.23?×?10³) through the four seasons. Results showed that Cd concentrations in oysters could pose a risk to human health (HQ?>?1). This study, therefore, suggests that there are potential human health risks due to heavy metal exposure through the consumption of C. rivularis from mariculture zones in South China Sea.     

Effect of Chronic Sublethal Exposure of Major Heavy Metals on Filtration Rate, Sex Ratio, and Gonad Development of a Bivalve Species

The chronic toxic effects of major heavy metals including copper (Cu), zinc (Zn), lead (Pb), and cadmium (Cd) on the filtration rate (FR), sex ratio, and gonad development of immature blood clams, Tegillarca granosa, were investigated. The FRs were significantly inhibited by Cu, Pb and Cd, with rates generally decreasing with both increasing metal concentrations and exposure time. EC₅₀ values for FR after 28 days of exposure were 12.9, 12.7 and 14.4 μg/L for Cu, Pb and Cd, respectively. Zn exposure had no effect on FR. Sex ratios were significantly altered from controls in favor of an increased proportion of males at metal concentrations of ≥14.2, ≥86 and ≥110 μg/L for Cu, Pb and Cd, respectively; and at ≥1.68 mg/L for Zn. The gonado-somatic index was significantly reduced in clams at all metal exposures, except for the lowest concentration of Cu (7.1 μg/L).     

Growth and Reproduction of Earthworms in Ultramafic Soils

Ultramafic soils are characterized by high concentrations of heavy metals of natural origin—such as chromium, cobalt, manganese, and nickel—as well as a shortage of primary nutrients. This can result in extremely disadvantageous living conditions for all soil-dwelling organisms. Responses to these conditions were addressed by studying growth, cocoon production, and fecundity of earthworms as endpoints of sublethal effects and how this influences the reproductive system and, consequently, population development. Mature specimens of two ecophysiologically different species of earthworms, Eisenia fetida (Savigny) and Aporrectodea caliginosa (Savigny), were exposed for 56 days to an uncontaminated soil and ultramafic soils collected from six ultramafic sites in the Barberton greenstone belt. In all ultramafic soil samples, the specimens of both species grew slower than those in the control soil. In A. caliginosa, an autotomization of the tail section was observed at higher concentrations of heavy metals. At high levels of heavy metals such as manganese, chromium, nickel, and cobalt, a significantly lower cocoon production was recorded for E. fetida, and at medium levels, a time delay in cocoon production was found. A. caliginosa showed an increase in production at medium levels and a decrease at high levels of heavy metals. In both species, no correlation between growth and cocoon viability was found, indicating different target levels for toxicants present in ultramafic soils. To determine effects of these soils on population dynamics, hatching success may be a more useful endpoint of reproduction.     

Heavy Metal Contamination and Ecological Risk Assessment of Swine Manure Irrigated Vegetable Soils in Jiangxi Province,China

Heavy metal are often added to animal fodder and accumulate in the soils with swine manure. In this study, heavy metal (Cu, Pb, Cd, Zn, As and Cr) concentrations were determined in agricultural soils irrigated with swine manure in Jiangxi Province, China. Results showed that the average concentrations of Cu, Zn, As and Cr (32.8, 93.7, 21.3 and 75.8 mg/kg, respectively) were higher than the background values, while Pb and Cd (15.2 and 0.090 mg/kg, respectively) were lower than the background values. Contamination factors \(\left( {C_{f}^{i}} \right)\) indicated that they were generally moderate for Cu, Zn, As and Cr and generally low for Pb and Cd. The contamination degree (C _d ) was calculated to be 7.5–10.0 indicating a moderate degree of contamination. The geoaccumulation index (I_geo) indicated that the soils were unpolluted with Zn, Cd and Pb, while unpolluted to moderately pollute with Cr, Cu and As. The single ecological risk factor \(\left( {E_{r}^{i}} \right)\) revealed that the six heavy metals all belonged to low ecological risk. The ecological risk indices suggested that all the sampling sites were at low risk level.     

Heavy metal induced physiological alterations in Salvinia natans

Salvinia possess inherent capacity to accumulate high levels of various heavy metals. Accumulation of Cr, Fe, Ni, Cu, Pb and Cd ranged between 6 and 9 mg g⁻¹ dry wt., while accumulation of Co, Zn and Mn was ∼4 mg g⁻¹ dry wt. Heavy metal accumulation affected the physiological status of plants. Photosystem II activity noted to decline in Ni, Co, Cd, Pb, Zn and Cu exposed plants, while Photosystem I activity showed enhancement under heavy metal stress in comparison to control. The increase in PS I activity supported build up of transthylakoidal proton gradient (ΔpH), which subsequently helped in maintaining the photophosphorylation potential. Ribulose 1,5 dicarboxylase/oxygenase (Rubisco) activity noted a decline. Alterations in photosynthetic potential of Salvinia result primarily from changes in carbon assimilation efficiency with slight variations in primary photochemical activities and photophosphorylation potential. Studies suggest that Salvinia possess efficient photosynthetic machinery to withstand heavy metal stress.     

Heavy Metal Concentration in Largehead Hairtail (<Emphasis Type="Italic">Trichiurus lepturus</Emphasis> Linneaus, 1758) and Savalai Hairtail (<Emphasis Type="Italic">Lepturacanthus savala</Emphasis> (Cuvier, 1829)) Obtained from Karachi Fish Harbour,Pakistan

In this study, muscle samples gathered from Largehead hairtail (Trichiurus lepturus Linneaus, 1758) and Savali hairtail [Lepturacanthus savala (Cuvier, 1829)] from the Karachi Fish Harbour, Pakistan were analyzed to determine heavy metal concentrations (iron [Fe], zinc [Zn], copper [Cu], manganese [Mn], lead [Pb] and cadmium [Cd]) between January and December 2016. All samples were analyzed using the AAnalyst 700 Flame Atomic Absorption Spectrophotometer. It was observed that the average measured level of Fe, Zn, Cu, Mn, Pb and Cd in muscle were 77.72?±?47.84 µg/g, 20.34?±?8.49 µg/g, 2.23?±?1.16 µg/g, 0.57?±?0.36 µg/g, 0.20?±?0.16 µg/g and 0.42?±?0.19 µg/g for T. lepturus, respectively. Besides, the average level of the same metal concentrations in muscle for L. savala were 85.11?±?57.64 µg/g, 16.63?±?9.25 µg/g, 2.53?±?1.90 µg/g, 0.47?±?0.27 µg/g, 0.23?±?0.18 µg/g and 0.47?±?0.20 µg/g, respectively. The correlation between size groups and metal accumulation in muscle tissues were investigated for both fish. In terms of public risk assessment, the provisional tolerable weekly intake’s of various heavy metals were compared with the consumption of both fish. As a result of the analysis, Fe, Zn, Cu, Mn, Pb and Cd accumulations in muscle tissues of T. lepturus and L. savala collected from Karachi Fish Harbour Pakistan did not exceed limit values.     

Growth and metal accumulation in Porcellio scaber exposed to poplar litter from Cd-, Pb-, and Zn-contaminated sites

This study aimed at determining, in the laboratory, the effects of poplar litter collected in woody habitats contaminated by heavy metals on growth and metal accumulation in the terrestrial isopod Porcellio scaber. Cd, Pb, and Zn pseudototal and CaCl₂-extractable concentrations in litter types were determined using AAS. Juveniles were fed ad libitum, individually, for 28 days with four litter types presenting an increasing gradient of metal contamination. Individuals were weighed every week and metal body burdens were determined at the end of the experiment. From the first week until the end of the experiment, a decrease in P. scaber growth related to the increase of metal concentration in litter types was recorded. Significant correlations were observed between metal body burdens and metal concentrations in litter types. However, Cd accumulation in woodlice appeared to be related to the Cd/Zn concentration ratio in litter types. All these results showed the potential of weight gain in P. scaber as a suitable indicator for litter quality assessment with ecological relevance.     

Determination of selected heavy metal concentrations and distribution in a southwestern stream using macrophytes

Since the reduction of the arsenic standard from 50 to 10 μg L⁻¹ by the US Environmental Protection Agency in 2006 many small town and rural water municipalities were left with the task of preventing or mitigating arsenic contamination of drinking water supplies. In this study macrophytes and sediments were used to determine the concentration and distribution of heavy metals (As, Cd, Cu, Pb, and Zn) within the primary source of drinking water (Gallinas River watershed) to the residents of Las Vegas, New Mexico. Sampling was done in the spring and fall at four sites, two above the city and two below, and samples were analyzed using ICP-MS. Results showed significantly higher (p<.05) metal concentrations in plant roots than shoots for most metals. Spearman’s correlation showed positive correlations (r>.3) between plant and sediment concentrations of Cd, Pb, Zn, As, and a negative correlation for Cu. The site above waste water treatment plant (AWWTP) had the highest plant tissue concentrations of Cd, Pb, Zn, and As. All of these concentrations attained critical toxicity levels exceeding sediment quality guidelines. High concentration factor values and levels of metals detected in macrophyte tissues indicate that heavy metals within sediments in the Gallinas River occur in bioavailable forms. Correlations between plant and sediment metal concentrations indicate that metal concentrations in macrophyte tissues are a good reflection of metal concentrations within the sediment in the Gallinas River.     

Municipal sewage sludge application on Ohio farms: tissue metal residues and infections

Transmission of infectious agents and translocation of Cd, Cu, Pb, and Zn from anaerobically digested sludge to the tissues of farm animals grazing on pastures to which sludge was applied (2-10 metric tons per hectare) were studied on eight farms. No significant health risk associated with the possible presence in sludge of Salmonella spp., or of common animal parasites including Nematodirus spp., Strongylus spp., Strongyloides spp., Trichuris spp., Eimeria spp., Ascaris spp., and Ancylostomum spp. was noted. Caudal fold as well as cervical tuberculin testing indicated no conversions from negative to positive following exposure of cattle to sludge. Significantly higher fecal Cd concentrations were detected in samples collected from cattle soon after being placed on sludge-treated pastures compared to preexposure values in the same animals. Significant Cd and Pb accumulations were found in the kidneys of calves grazing sludge-treated pastures compared to control calves. Although older cows grazing sludge-treated pastures had significantly higher blood Pb levels, no metal accumulation was observed in other tissues. Statistically significant accumulations of Cd and Pb in the kidney of calves grazing these pastures for a relatively short period suggest that caution should be exercised to avoid prolonged grazing of cattle on pastures receiving heavy sludge applications, especially with sludges containing high concentrations of heavy metals.     

Appraisal of Heavy Metals in Groundwater in Chennai City Using a HPI Model

Heavy metal contamination in Chennai city was evaluated using a heavy metal pollution index (HPI) model in conjunction with the spatial distribution maps. Metals such as Cd, Cr, Cu, Pb and Zn in the groundwater were determined using standard methods and the resultant data was utilized in the development of a HPI model. The metal concentrations showed a dominance in the order of Zn > Cu > Cr > Pb > Cd respectively. About 14.3 % of the samples (n = 2), exhibited high HPI (>38) and the highest value, HPI = 97.55, has been recorded from Thiruvanmiyur area. Statistical analysis revealed a positive correlation between metals such as Cd and Cr (r = 0.606), Cd and Cu (r = 0.601), Cr and Cu (r = 0.464) and Pb and Zn (r = 0.416), suggested their common origin. The spatial distribution maps of heavy metals and the HPI suggested that the SW region, especially Adyar and Thiruvanmiyur regions are highly contaminated with the metals. Industrialisation and improper waste dumping were identified as the major cause for the accumulation of metals in the groundwater of Chennai city.     

Influence of traffic activity on heavy metal concentrations of roadside farmland soil in mountainous areas

Emission of heavy metals from traffic activities is an important pollution source to roadside farmland ecosystems. However, little previous research has been conducted to investigate heavy metal concentrations of roadside farmland soil in mountainous areas. Owing to more complex roadside environments and more intense driving conditions on mountainous highways, heavy metal accumulation and distribution patterns in farmland soil due to traffic activity could be different from those on plain highways. In this study, design factors including altitude, roadside distance, terrain, and tree protection were considered to analyze their influences on Cu, Zn, Cd, and Pb concentrations in farmland soils along a mountain highway around Kathmandu, Nepal. On average, the concentrations of Cu, Zn, Cd, and Pb at the sampling sites are lower than the tolerable levels. Correspondingly, pollution index analysis does not show serious roadside pollution owing to traffic emissions either. However, some maximum Zn, Cd, and Pb concentrations are close to or higher than the tolerable level, indicating that although average accumulations of heavy metals pose no hazard in the region, some spots with peak concentrations may be severely polluted. The correlation analysis indicates that either Cu or Cd content is found to be significantly correlated with Zn and Pb content while there is no significant correlation between Cu and Cd. The pattern can be reasonably explained by the vehicular heavy metal emission mechanisms, which proves the heavy metals' homology of the traffic pollution source. Furthermore, the independent factors show complex interaction effects on heavy metal concentrations in the mountainous roadside soil, which indicate quite a different distribution pattern from previous studies focusing on urban roadside environments. It is found that the Pb concentration in the downgrade roadside soil is significantly lower than that in the upgrade soil while the Zn concentration in the downgrade roadside soil is marginally higher than in the upgrade soil; and the concentrations of Cu and Pb in the roadside soils with tree protection are significantly lower than those without tree protection. However, the attenuation pattern of heavy metal concentrations as a function of roadside distance within a 100 m range cannot be identified consistently.     

Assessing of Heavy Metal Concentrations in the Tissues of Rutilus rutilus caspicus and Neogobius gorlap from Miankaleh International Wetland

The concentrations of four heavy metals (Pb, Cd, Ni and Cr) were measured in tissues of pelagic (Rutilus caspicus) and benthic (Neogobius gorlap) fishes from the Miankaleh international wetland. The maximum concentrations of Pb, Cd, Ni, and Cr (2.06, 1.20, 1.14 and 0.70 μg g^?1, respectively) were measured in the liver of N. gorlap, while the lowest concentrations were measured in muscle tissue collected from R. caspicus (Pb 0.67; Cd 0.25; Ni 0.21 and Cr 0.08 μg g^?1). The mean concentrations of metals in liver of R. caspicus and N. gorlap followed a trend where Pb > Cd > Ni > Cr, whereas in R. caspicus and N. gorlap gills and N. gorlap muscle the following trend was observed Pb > Ni > Cd > Cr. The data of presented study show that the liver and gill tissues have higher metal concentrations than muscle and also the metal concentrations in tissues of N. gorlap (benthic) were higher in comparison with R. caspicus (pelagic).     

Increased cadmium and lead uptake of a cadmium hyperaccumulator tomato by cadmium-resistant bacteria

Two cadmium (Cd)-resistant strains Pseudomonas sp. RJ10 and Bacillus sp. RJ16 were investigated for their effects on the soil Cd and lead (Pb) solubilization and promotion of plant growth and Cd and Pb uptakes of a Cd-hyperaccumulator tomato. In the heavy metal-contaminated inoculated soil, the CaCl₂-extractable Cd and Pb were increased by 58–104% and 67–93%, respectively, compared to the uninoculation control. The bacteria produced indole acetic acid, siderophore and 1-aminocyclopropane-1-carboxylate deaminase. Root elongation assay conducted on tomato under gnotobiotic conditions demonstrated increase in root elongation of inoculated tomato seedlings compared to the control plants. An increase in Cd and Pb contents of above-ground tissues varied from 92% to 113% and from 73% to 79% in inoculated plants growing in heavy metal-contaminated soil compared to the uninoculation control, respectively. These results show that the bacteria could be exploited for bacteria enhanced-phytoextraction of Cd- and Pb-polluted soils.     

Effects of heavy metals on production of thiol compounds and antioxidant enzymes in Agaricus bisporus

In a pre-experiment, Agaricus bisporus mycelia grown in PDL medium were found to have a substantial ability to tolerate and accumulate heavy metals. In the study, we investigated changes in the contents of soluble protein and thiol compounds as well as the activities of antioxidant enzymes caused by copper, zinc, lead, and cadmium (nitrate salts) in mycelia of A. bisporus during short-and long-term exposure. Results showed that high-level metal concentrations significantly decrease the contents of soluble protein after long-term exposure, Cu and Zn concentrations significantly increase the thiol compounds levels after long-term exposure, while high-level Cd significantly decrease thiol compounds after long-term exposure. Additionally, SOD activities were significantly increased after long-term exposure to metals, especially to Cd. The CAT activities were enhanced after long-term exposure to low-level Cu and high-level Zn, and enhanced after short-and long-term exposure to high-level Pb. The POD activities were significantly increased after long-term exposure to metals, and increased after short-term exposure to Cd and high-level Pb.     

Effects of Soil Properties and Land Use Types on the Bioaccessibility of Cd,Pb, Cr,and Cu in Dongguan City,China

In order to determine the potential heavy metal contamination in soil across Dongguan City, 124 soil samples from seven land use types were collected, four heavy metals (Cd, Pb, Cr, and Cu) were analyzed. Total Cd, Cr, and Cu contents were significantly higher than the background values for Guangdong Province. Lead bioaccessibility in urban green land was lower than that in industrial and abandoned districts. The bioaccessibility of heavy metals was affected by total metal concentrations, soil properties, and land use types. The results showed that there was a negative correlation between the bioaccessibility of heavy metals (except for Cu) and their total concentrations. Soil pH and organic matter were the main factors affecting the bioaccessibility of Cd, Cr, Pb, and Cu in most land use types. Furthermore, sand, P, and clay also affected Pb, Cr, and Cu bioaccessibility. With the exception of the industrial zone periphery and urban green land, the bioaccessibility of heavy metals was mainly affected by clay.