Euglena gracilis as a model for the study of Cu and Zn toxicity and accumulation in eukaryotic cells

We have observed the effect of copper and zinc on the biology of Euglena gracilis. The cells displayed different sensitivities to these metals, as the apparent LC₅₀ for Cu²⁺ was 0.22 mM, and for Zn²⁺ it was 0.88 mM. While Zn²⁺ was able to increase cell proliferation even at 0.1 mM, the minimal CuCl₂ concentration tested (0.02 mM) was sufficient to impair cell division. Higher concentrations of these metals not only inhibited cell division in a concentration-dependent manner, but also interfered with the metabolism of E. gracilis. A higher accumulation of proteins and lipids per cell was observed at the DI₅₀ concentration for metal-treated cells. These results suggest that the test concentration of both metals leads to a failure in completing cell division. Ultrastructural analysis indicated a chloroplast disorganization in copper-treated cells, as well as the presence of electron dense granules with different shapes and sizes inside vacuoles. Microanalysis of these granules indicated an accumulation of copper, thus suggesting a detoxification role played by the vacuoles. These results indicate that E. gracilis is an efficient biological model for the study of metal poisoning in eukaryotic cells. They also indicate that copper and zinc (copper being more poisonous) had an overall toxic effect on E. gracilis and that part of the effect can be ascribed to defects in the structure of chloroplast membranes.     

浮游球衣菌对Pb2+、Cu2+、Zn2+、Cd2+的吸附性能研究

研究了浮游球衣菌（Sphaerotilus natans）在不同吸附条件下对溶液中Pb^2＋、Cu^2＋、Zn^2＋、Cd^2＋的吸附规律。结果表明,Sphaerotilus natans对这4种重金属离子均有一定的吸附作用,并在20min内达到吸附平衡,pH对吸附过程影响较大,pH为5.5时Sphaerotilus natans对这4种金属离子的吸附效果最好,Sphaerotilus natans对它们的吸附选择性为Pb^2＋〉Cu^2＋〉Zn^2＋〉Cd^2＋,Pb^2＋、Cu^2＋能部分置换出已被菌体吸附的Zn^2＋、Cd^2＋。HCI和EDTA溶液可有效地将金属离子从菌体上解吸下来,解吸后的菌体可重复使用。     

Oxidative stress in Scenedesmus sp. during short- and long-term exposure to Cu2+ and Zn2+

Algae are exposed to elevated levels of heavy metals in water bodies generally for a long-term, and occasionally for a short-term duration. The present study deals with oxidative stress in Scenedesmus sp., commonly found in nutrient-rich freshwaters, during short- (6h) and long-term (7d) exposure to Cu(2+) and Zn(2+). The cells accumulated almost 2- and 4-times more Cu(2+) and Zn(2+) inside the cells during long-term than during short-term exposure to these metals. But the data on photosynthetic O(2) evolution and cell viability suggest that Scenedesmus sp. experienced lesser metal stress in long-term than in short-term experiment. Although malondialdehyde content was slightly higher in the long-term experiment, the amount produced by one unit intracellular metal was significantly lower than that in the short-term experiment. Superoxide dismutase activity of Scenedesmus sp. was >30% higher during long-term than during short-term exposure to Cu(2+) and Zn(2+). But, catalase and ascorbate peroxidase activities increased only at 2.5 microM Cu(2+) and 25 microM Zn(2+) when oxidative stress was mild, but were inhibited at 10 microM Cu(2+) under intense oxidative stress. Cu(2+) and Zn(2+) reduced glutathione reductase activity and total SH content of Scenedesmus sp. in both the experiments, with greater reduction occurring in the long-term experiment. The depletion of total thiol was positively related with the intracellular level of metals. Thiols might have helped Scenedesmus sp. in overcoming metal-induced oxidative stress, but depletion of thiol pool is known to make cells vulnerable to oxidative stress. The study suggests that antioxidant enzymes play a role only under mild oxidative stress. An increased accumulation of proline seems to be an important strategy for alleviating metal-induced oxidative stress in Scenedesmus sp. The study shows that Scenedesmus sp. could acclimatize during long-term exposure to toxic concentrations of the test metals.     

Metal accumulation in the earthworm Lumbricus rubellus. Model predictions compared to field data

The mechanistic bioaccumulation model OMEGA (Optimal Modeling for Ecotoxicological Applications) is used to estimate accumulation of zinc (Zn), copper (Cu), cadmium (Cd) and lead (Pb) in the earthworm Lumbricus rubellus. Our validation to field accumulation data shows that the model accurately predicts internal cadmium concentrations. In addition, our results show that internal metal concentrations in the earthworm are less than linearly (slope<1) related to the total concentration in soil, while risk assessment procedures often assume the biota-soil accumulation factor (BSAF) to be constant. Although predicted internal concentrations of all metals are generally within a factor 5 compared to field data, incorporation of regulation in the model is necessary to improve predictability of the essential metals such as zinc and copper.     

Age-dependent accumulation of heavy metals in a pod of killer whales (Orcinus orca) stranded in the northern area of Japan

Mercury (Hg), cadmium (Cd), iron (Fe) manganese (Mn), zinc (Zn) and copper (Cu) concentrations in the liver, kidney and muscle of nine killer whales (including three calves) that stranded together in the northern area of Japan were determined. The Hg and Cd concentrations were found at trace levels in the calf organs, and increased with age. The Fe concentration in the muscle was significantly lower in the calves than in the mature whales and also increased with age. In contrast, Mn and Cu concentrations in the muscle were significantly higher in the calves than in the mature whales, and changes in the Zn concentration relative to age were unclear. These results suggest minimal mother-to-calf transfer of the toxic metals Hg and Cd and accumulation of these metals in the organs with age, while the essential metals Mn and Cu were found at higher concentrations in the muscle of calves than in mature whales.     

Detection and localization of aluminum and heavy metals in ectomycorrhizal Norway spruce seedlings

Norway spruce seedlings colonized with Hebeloma crustuliniforme were grown in growth pouches. After formation of ectomycorrhizas, the seedlings were exposed to Al or the heavy metals Cd, Cu, Ni, or Zn at various concentrations for 5 weeks to estimate the detection limits of metals with X-ray microanalysis in the cryo-scanning electron microscope. When the lowest metal concentrations (1 mM Al(3+), 0.1 mM Cd(2+), 0.2 mM Cu(2+), 0.5 mM Ni(2+), 2 mM Zn(2+)) were applied, only Al and Zn were detected at low X-ray counts in the ectomycorrhizas. After application of 10-fold higher metal concentrations, distinct metal accumulation patterns were observed. Cd was detected predominantly in the Hartig net, Al and Ni in the Hartig net and in the cell walls of the cortex, and Zn in the Hartig net, the cortical cell walls and the fungal mantle. Cu was not detected at all. By combining X-ray microanalysis with absolute metal concentrations found in the roots, the estimated detection limits of X-ray microanalysis were: Al> or =0.86 mg g(-1), Cd> or =0.26 mg g(-1), Ni> or =1.30 mg g(-1), and Zn> or =0.54 mg g(-1), whereas Cu was not detectable even at root concentrations of 0.47 mg g(-1). Treatments with the highest metal concentrations showed high X-ray counts of metals in cells of the stele but reduced concentrations of the macronutrients K, Mg, and P in roots, indicating a possible disturbance of root and ectomycorrhizal function.     

Accumulation and partitioning of heavy metals in mangroves: a synthesis of field-based studies

We report the findings of a comparative analysis examining patterns of accumulation and partitioning of the heavy metals copper (Cu), lead (Pb) and zinc (Zn) in mangroves from available field-based studies to date, employing both species level analyses and a phylogenetic approach. Despite mangroves being a taxonomically diverse group, metal accumulation and partitioning for all metals examined were broadly similar across genera and families. Patterns of metal accumulation were also similar regardless of whether species were classified as salt secreting or non-secreting. Metals were accumulated in roots to concentrations similar to those of adjacent sediments with root bio-concentration factors (BCF; ratio of root metal to sediment metal concentration) of 1< or =. Root BCFs were constant across the exposure range for all metals. Metal concentrations in leaves were half that of roots or lower. Essential metals (Cu and Zn; translocation factors (TF; ratio of leaf metal to root metal concentration) of 0.52 and 0.53, and leaf BCFs of 0.47 and 0.51, respectively) showed greater mobility than non-essential metals (Pb; TF of 0.31 and leaf BCF of 0.11). Leaf BCFs for the essential metals Cu and Zn decreased as environmental concentrations increased. The non-essential metal Pb was excluded from leaf tissue regardless of environmental concentrations. Thus mangroves as a group tend to operate as excluder species for non-essential metals and regulators of essential metals. For phytoremediation initiatives, mangrove ecosystems are perhaps best employed as phytostabilisers, potentially aiding in the retention of toxic metals and thereby reducing transport to adjacent estuarine and marine systems.     

Trace elements in organs and tissues of striped dolphins (Stenella coeruleoalba) from the Mediterranean sea (Southern Italy)

The distribution of metals (Cd, Cu, Zn, Fe, Cr, Pb and Sn) were investigated in various tissues and organs obtained from striped dolphins (Stenella coeruleoalba) stranded along the Apulian coasts (Southern Italy) during April-July 1991. Metals were determined by atomic absorption spectrophotometry. Metal concentrations were generally high in the liver, and low in brain and melon. Some metals showed organ-specific accumulations: copper, tin and zinc exhibited high concentrations in liver, the highest cadmium concentration was observed in kidney. Pathological, microbiological and parasitological surveys were performed on the animals. It was not possible to relate dolphin death to a specific cause, or to contaminants; however, the accumulation of metals may contribute to certain pathological alterations.     

Response of kidney bean to concentration and chemical form of cadmium, zinc, and lead in polluted soils

Seven soils which had been polluted with heavy metals from a zinc smelter were sequentially extracted so that Cd, Zn, and Pb could be partitioned into five operationally defined geochemical fractions: exchangeable, carbonate, Fe-Mn oxide, organic, and residual fractions. Kidney beans were planted in the soils to examine the effect of concentration and chemical form of the metals in soil on the growth and metal uptake of the plants. The growth of kidney bean was restricted in heavy metal polluted soils compared with controls. Metal concentration and metal uptake by plants were correlated. The highest relationship was found between amount of metal uptake and the metal concentration in exchangeable + carbonate forms. The uptake of metals was according to their solubility sequence, i.e. Cd > Zn > Pb. The uptake rate of exchangeable + carbonate forms was the same for the three elements.     

Change of PCBs and forms of heavy metals in sewage sludge during thermophilic anaerobic digestion

Determination of seven congeners of PCBs was carried out for sewage sludge before, during and after thermophilic digestion. The overall content of heavy metals (Zn, Cu, Ni, Cd, Pb, Cr) in sludge before and after digestion was determined. Moreover the concentration of heavy metals in particular chemical fractions of the sludge was analyzed. After the thermophilic digestion total concentration of seven PCBs was reduced by 47%, which suggests that thermophilic digestion affects PCB reduction positively. On the 10th d of the process, concentration of lower chlorinated PCBs increased, whereas those of higher chlorinated PCBs decreased. The thermophilic digestion process showed no accumulation of the studied heavy metals in the mobile fractions (exchangeable and carbonate) of the stabilized sewage sludge, except for nickel. The highest increase in zinc, copper, cadmium, and chromium concentration was observed in the organic-sulfide fraction, whereas the highest increase in lead was found in the residual fraction of the sludge. In case of nickel both fractions of organic-sulfide and exchangeable-carbonate fractions were enriched.     

Sensitivity of Mediterranean woody seedlings to copper, nickel and zinc

The restoration of heavy metal contaminated areas requires information on the response of native plant species to these contaminants. The sensitivity of most Mediterranean woody species to heavy metals has not been established, and little is known about phytotoxic thresholds and environmental risks. We have evaluated the response of four plant species commonly used in ecological restoration, Pinus halepensis, Pistacia lentiscus, Juniperus oxycedrus, and Rhamnus alaternus, grown in nutrient solutions containing a range of copper, nickel and zinc concentrations. Seedlings of these species were exposed to 0.048, 1 and 4 microM of Cu; 0, 25 and 50 microM of Ni; and 0.073, 25 and 100 microM of Zn in a hydroponic silica sand culture for 12 weeks. For all four species, the heavy metal concentration increased in plants as the solution concentration increased and was always higher in roots than in shoots. Pinus halepensis and P. lentiscus showed a higher capacity to accumulate metals in roots than J. oxycedrus and R. alaternus, while the allocation to shoots was considerably higher in the latter two. Intermediate heavy-metal doses enhanced biomass accumulation, whereas the highest doses resulted in reductions in biomass. Decreases in shoot biomass occurred at internal concentrations ranging from 25 to 128 microg g-1 of Zn, and 1.7 to 4.1 microg g( -1) of Cu. Nickel phytoxicity could not be established within the range of doses used. Rhamnus alaternus and J. oxycedrus showed higher sensitivity to Cu and Zn than P. halepensis and, especially, P. lentiscus. Contrasted responses to heavy metals must be taken into account when using Mediterranean woody species for the restoration of heavy metal contaminated sites.     

The behavior of heavy metal release from sulfide waste rock under microbial action and different environmental factors

The dissolution of heavy metals from the waste rock is controlled by many factors. Herein, we investigated the release behavior of iron (Fe), chromium (Cr), copper (Cu), and zinc (Zn) from sulfide waste rock under the actions of microorganisms and different environmental factors (solution pH value, particle size of waste rock, temperature, Fe³⁺ concentration). The release quantity of heavy metals was negatively correlated with pH and particle size and positively correlated with ambient temperature and Fe³⁺ concentration. Under the experimental conditions of pH value of 3.0, temperature of 35°C, and waste stone particle size of less than 0.075 mm,, the release quantity of Fe, Cr, Cu, and Zn reached 3680, 18.32, 132.20, 26.60 mg·kg^?1 after 20 days of leaching, respectively. Rising the temperature to 45 °C, Fe, Cr, Cu, and Zn release quantities increased to 89.30, 5.81, 105.08, and 28.00 mg·kg^?1. Six hundred milligrams per liter Fe³⁺ increased the release of heavy metals considerably (2.63–65.48 folds). The presence of microorganisms can significantly facilitate the release of heavy metals. Compared to the control group, the release quantities of Fe, Cr, Cu, and Zn increased 4.29, 3.17, 1.54, and 2.39 times, respectively. In addition, the waste rock under microbial action was more seriously corroded than that under chemical factors. The release behavior of these four heavy metals was consistent with the interfacial chemical reaction control model, indicating that the reactions mainly occurred on the surface of the waste rock. This study provides an essential reference for the study of heavy metal leaching behavior.

     

Dynamics of Cd, Cu and Zn accumulation in organs and sub-cellular fractions in field transplanted juvenile yellow perch (Perca flavescens)

Juvenile yellow perch (Perca flavescens) were caught in a reference lake and transplanted to cages held within a lake impacted by mining activities, with elevated levels of aqueous bioavailable copper (Cu(2+)), zinc (Zn(2+)) and cadmium (Cd(2+)). Fish were sampled from the cages over 70 d and changes in metal concentrations were followed over time in the gills, gut, liver and kidney. In addition, the hepatic sub-cellular partitioning of the three metals was determined by differential centrifugation of liver samples, yielding the following fractions: cellular debris; organelles; heat-denaturable proteins (HDP); and heat-stable proteins (HSP) (including metallothionein). In transplanted fish, Cd concentrations increased in all the organs sampled, whereas Cu mainly increased in the gills, gut and liver but not the kidney; some slight accumulation of Zn occurred in the kidneys and gills of the transplanted fish. The sub-cellular partitioning results demonstrated that metal-handling strategies in juvenile yellow perch differed among metals. Cellular sequestration in the HSP fraction was an important strategy used by these fish in response to increased ambient Cd. Accumulation of Zn was not seen in the organs examined, indicating that transplanted perch were able to either reduce influx, or increase efflux rates of this metal. The response of yellow perch to elevated ambient Cu appeared to combine the strategies used for Cd and Zn, as both cellular sequestration and reduced accumulation were observed in transplanted fish.     

Effect of Zn toxicity on root morphology,ultrastructure, and the ability to accumulate Zn in Moso bamboo (Phyllostachys pubescens)

The effects of zinc (Zn) on seed germination and growth of Moso bamboo (Phyllostachys pubescens) were investigated. Under zinc stress, the seed germination rate did not show significant difference from that of the control. Hydroponics experiments indicated that Moso bamboo had a strong ability to accumulate Zn in the shoot and it reached its maximum value in the shoot at 100 μM Zn. The root Zn concentration ranged from 2,329.29 to 8,642.51 mg kg^?1, with the root Zn concentration at 10 μM Zn being 58.23 times that of the control. The root morphology parameters slightly increased at the lower Zn treatments, while growth restriction was evident at higher Zn treatments. Root ultrastructural studies revealed that the cell structure, root tips, and organelles were significantly changed under Zn stress as compared to those of the control. Some abnormalities were evident in the cell walls, vacuoles, mitochondria, plasmalemma, tonoplast, and xylem parenchyma of root cells. While Moso bamboo seems a suitable candidate for phytoremediation, its metal remediation ability should be further explored in future investigations.     

Exposure to sublethal concentrations of Zn(II) and Cu(II) changes biochemical parameters in Leporinus obtusidens

The aim of the present study was to assess the effect of the exposure of Leporinus obtusidens (Piava) to zinc and copper on catalase activity in the liver, delta-aminolevulinate dehidratase (delta-ALA-D) activity in liver, muscle, brain and kidney, and thiobarbituric reactive species (TBARS) in brain, muscle and liver. In addition, hematological parameters were measured in blood. The fish were exposed to 10% and 20% of the derived LC(50) values, 2.3 and 4.6 mg Zn l(-1) and 0.02 and 0.04 mg Cu l(-1), and sampled on days 30 and 45. Exposure to Zn(II) and Cu(II) decreased hematological parameters and also delta-ALA-D activity mainly in liver and kidney at all concentrations tested. Liver catalase activity increased after zinc or copper exposure at all concentrations and exposure times tested. Thiobarbituric reactive substances (TBARS) increased in the brain and liver of the fish exposed to zinc(II) for 45 days at both metal concentrations. In muscle, zinc(II) increased TBARS production at both exposure times and concentrations tested. Copper(II) exposure reduced the TBARS levels in liver at both concentrations and times tested. In brain, there was a decrease in TBARS levels only after 45 days of exposure. In muscle, this decrease was observed after 30 days of exposure at both concentrations. Although zinc and copper are required as microelements in the cells, our results showed that the sublethal concentrations of these metals can change biochemical parameters which may alter normal cellular function. These results pointed out the differential sensitivity of fish tissues to essential, but also toxic and environmentally relevant metals. The alterations of distinct biochemical parameters in fish tissues certainly contribute to the toxicity of Zn and Cu, and are of importance for an area that has been growing and has still been poorly explored in the literature.     

Comparative tolerance of Pinus radiata and microbial activity to copper and zinc in a soil treated with metal-amended biosolids

A study was conducted to evaluate the effects of elevated concentrations of copper (Cu) and zinc (Zn) in a soil treated with biosolids previously spiked with these metals on Pinus radiata during a 312-day glasshouse pot trial. The total soil metal concentrations in the treatments were 16, 48, 146 and 232 mg Cu/kg or 36, 141, 430 and 668 mg Zn/kg. Increased total soil Cu concentration increased the soil solution Cu concentration (0.03–0.54 mg/L) but had no effect on leaf and root dry matter production. Increased total soil Zn concentration also increased the soil solution Zn concentration (0.9–362 mg/L). Decreased leaf and root dry matter were recorded above the total soil Zn concentration of 141 mg/kg (soil solution Zn concentration, >4.4 mg/L). A lower percentage of Cu in the soil soluble?+?exchangeable fraction (5–12 %) and lower Cu²⁺ concentration in soil solution (0.001–0.06 μM) relative to Zn (soil soluble?+?exchangeable fraction, 12–66 %; soil solution Zn²⁺ concentration, 4.5–4,419 μM) indicated lower bioavailability of Cu. Soil dehydrogenase activity decreased with every successive level of Cu and Zn applied, but the reduction was higher for Zn than for Cu addition. Dehydrogenase activity was reduced by 40 % (EC₄₀) at the total solution-phase and solid-phase soluble?+?exchangeable Cu concentrations of 0.5 mg/L and 14.5 mg/kg, respectively. For Zn the corresponding EC₅₀ were 9 mg/L and 55 mg/kg, respectively. Based on our findings, we propose that current New Zealand soil guidelines values for Cu and Zn (100 mg/kg for Cu; 300 mg/kg for Zn) should be revised downwards based on apparent toxicity to soil biological activity (Cu and Zn) and radiata pine (Zn only) at the threshold concentration.     

Effects of Cd and Zn waterborne exposure on the uptake and depuration of 57Co, 110mAg and 134Cs by the Asiatic clam (Corbicula fluminea) and the zebra mussel (Dreissena polymorpha)--whole organism study

Groups of zebra mussels (Dreissena polymorpha) and asiatic clams (Corbicula fluminea) were exposed to cadmium and zinc with the aim of studying the effect of these metals on the 57Co, 110Ag and 134Cs uptake and depuration by these freshwater bivalves. In the presence of zinc, the 57Co concentration factor for the whole organism of the two species was halved, notably because of a decrease of the uptake parameter. Conversely, Zinc and the Cd + Zn mixture increased the 110mAg uptake process by clams and mussels. The two metals also increased the depuration of this radionuclide in mussels, whereas this phenomenon was only observed in clams exposed to cadmium. In comparison with 57Co and 110mAg, the 134Cs bioconcentration was 5-10 times lower in D. polymorpha and not detected in C. fluminea. This weak contamination by this radionuclide resulted from a lower uptake and a higher depuration parameters.     

Lead,zinc and copper accumulation and tolerance in populations of Paspalum distichum and Cynodon dactylon

Both Fankou and Lechang lead/zinc (Pb/Zn) mine tailings located at Guangdong Province contained high levels of total and DTPA-extractable Pb, Zn and Cu. Paspalum distichum and Cynodon dactylon were dominant species colonized naturally on the tailings. Lead, zinc and copper accumulation and tolerance of different populations of the two grasses growing on the tailings were investigated. Tillers of these populations including those from an uncontaminated area were subjected to the following concentrations: 5, 10, 20, 30 and 40 mg l(-1) Pb, 2.5, 5, 10, 20 and 30 mg (l-1) Zn, or 0.25, 0.50, 1 and 2 mg l(-1) Cu for 14 days, respectively, then tolerance index (TI) and EC50 (the concentrations of metals in solutions which reduce 50% of normal root growth) were calculated. The results indicated that both Lechang and Fankou populations of the two grasses showed a greater tolerance to the three metals than those growing on the uncontaminated area, which suggested that co-tolerant ecotypes have evolved in the two grasses. P. distichum collected from Fankou tailings had the highest tolerance to Cu while Lechang population the highest tolerance to Pb and Zn among the tested populations, and tolerance levels in P. distichum were related to metal concentrations in the plants. P. distichum had a better growth performance than C. dactylon when both of them were grown on the tailings sites. Tolerant populations of these species would serve as potential candidates for re-vegetation of wastelands contaminated with Pb, Zn and Cu.     

Assessment of copper and zinc levels in fish from freshwater ecosystems of Moldova

Purpose

The aim of this work was to assess the levels of copper and zinc in fish from the main freshwater ecosystems of Moldova, in relation with species, habitat, age, sex, season, and development stage.

Methods

Fish from Cyprinidae and Percidae families (Cyprinus carpio, Carassius auratus gibelio, Rutilus rutilus heckeli, Abramis brama, Aristichthys nobilis, Hypophtalmichthys molitrix, Sander lucioperca) were collected from Prut and Dniester rivers, Cuciurgan, Dubasari, and Costesti-Stanca reservoirs, and ponds of farms in the Dniester delta. The Cu and Zn content of fish tissues (skeletal muscles, liver, gonads, gills, skin, and scales) was determined by flame atomic absorption spectrophotometer AAS-3, of water by graphite furnace HGA 900 of AAnalist 400.

Results

The level of heavy metals accumulation in muscles of immature fish follows their dynamics in water. The highest concentration of zinc was registered in the gonads of mature fish, and of copper??in the liver. The lowest Cu and Zn contents were recorded in the muscles and are in the United Nations Food and Agriculture Organization safety-permissible levels for human consumption. Cu and Zn contents in muscles of fish depend on specimen age. Their level in fish gonads was sharply increasing during pre-spawning period. During the early developmental stages, the metal concentration in fish eggs and larvae varies within wide limits, but the accumulation pattern is similar in the investigated species.

Conclusions

The fish represent one of the most indicative factors for the estimation of trace metals pollution in freshwater systems and this is important not only for monitoring purposes, but also for the fish culture ones.     

Bacterial leaching of heavy metals from sewage sludge-bioreactors comparison

More than 50% of municipal sewage sludges cannot be used on agricultural land because of their heavy metals content. Therefore, microbial leaching of heavy metal from municipal sludge was studied in a continuously stirred tank reactor without recycling (CSTR) or with sludge recycling (CSTRWR) at residence times of 1, 2, 3 and 4 days. The reactor CSTRWR is supposed to be more efficient for bacterial process due to the recycling of active bacteria from the settling tank to the reactor. The CSTRWR and the CSTR with 1 g litre(-1) FeSO(4).7H(2)O addition were equally efficient because of copper reprecipitation or recomplexation in the settling tank of the CSTRWR. In the CSTR, about 62% of copper and about 77% of zinc were dissolved in 3 days residence time compared to 50% of copper and 64% of zinc in the CSTRWR, if 3 g litre(-1) FeSO(4).7H(2)O was added. Thus with larger amount of substrate, the CSTR was more efficient than the CSTRWR. Residence time and pH were the main factors for zinc solubilization while for copper, the redox potential was also a major factor. The effect of FeSO(4).7H(2)O concentration on bacterial activity to solubilize heavy metals was also studied, increased concentration of FeSO(4).7H(2)O yielded better copper solubilization while it had no effect or a negative effect on zinc. This supports the hypothesis of a direct mechanism for zinc solubilization and of an indirect mechanism for copper solubilization.