Quantification of toxic and inhibitory impact of copper and zinc on mixed cultures of sulfate-reducing bacteria

The adverse effects of copper and zinc on an acetate-utilizing mixed cultures of sulfate-reducing bacteria (SRB) at concentrations below the toxic concentration (minimum metal concentration at which no sulfate reduction is observed) are reported in this paper. Mathematical models were developed to incorporate the toxic and inhibitory effects (defined as the reduction in bacterial population upon exposure to the metal and the decrease in the metabolic rate of sulfate reduction by the SRB, respectively) into the sulfate-reduction biokinetics. The characteristic toxicity and inhibition constants were obtained from the measurements of bacterial populations and dissolved metal concentrations in serum bottle studies conducted at 35 degrees C and pH 6.6. Both copper and zinc had toxic and inhibitory effects on SRB. The toxicity constants for copper and zinc were 10.6 and 2.9 mM(-1), respectively, indicating that exposure to copper resulted in a higher mortality of SRB than did exposure to zinc. The values of the inhibition constants were found to be 17.9 +/- 2.5 and 25.2 +/- 1.0 mM(-1) for copper and zinc, respectively. This implies that dissolved zinc was slightly more inhibitory to SRB than copper. The models presented in the paper can be used to predict the response of a sulfate-reduction bioreactor to heavy metals during acid mine drainage treatment.     

Effects of pH on toxicity of As,Cr, Cu,Ni and Zn to Selenastrum capricornutum Printz

Acute toxicity tests were conducted to establish the response of Selenastrum capricornutum Printz to sublethal concentrations of As, Cr, Cu, Ni and Zn at a broad range of pH levels. Cultures were incubated for a period of seven days at pH 4 in standard algal assay media containing sublethal concentrations of metals. At this low pH, growth was depressed for all metals tested. The adjustment of pH to higher levels resulted in increased growth when cultures were treated with As, Cu, or Ni and incubated for an additional 7 days. Toxicity was least at the optimum pH range for growth of the alga.The observation that the toxicity of As, Cu, and Ni to S. capricornutum decreases markedly at pH values above 4.0 may be of ecological importance in the control of acid mine pollution. If a high percentage of algae show a similar response to decreasing toxicity with increasing pH, it clearly would be of value to adopt measures which control pH as well as the levels of metals present. It was suggested that algae with a broad pH growth range, such as S. capricornutum, could benefit from the addition of highly alkaline materials to waters where certain metals are present.     

Copper resistance in Anabaena variabilis: Effects of phosphate nutrition and polyphosphate bodies

A copper-resistant Anabaena variabilis strain was obtained after repeated culturing in progressively higher concentrations of Cu(NO₃)₂. This strain maintained its resistance even after a year of repeated subculturing in copper-free medium. The resistant strain differed from the sensitive parent strain with respect to filament length, cell shape and size, and control of heterocyst formation. The resistant strain was also more resistant to cadmium, zinc, and nickel. Copper distribution studies conducted with atomic absorption spectroscopy revealed that at low copper levels the sensitive strain bound significantly more metal than the resistant strain. At higher copper levels, however, the resistant strain bound large amounts of the metal. Phosphate-loaded resistant cells could grow in higher copper concentrations than phosphate-starved resistant cells. Toluidine blue staining showed that the resistant strain contained more polyphosphate bodies than the sensitive strain; the resistant cells also had higher internal phosphate levels. X-ray microanalysis, however, did not show a direct localization of copper on polyphosphate bodies. More than one mechanism of resistance may exist in this A. variabilis strain.Correspondence to: D. J. Kushner.     

The fish populations of some streams in Wales and northern England in relation to acidity and associated factors

Electrofishing and water quality surveys were carried out on 60 upland moorland streams in central and north Wales and the Peak District of England (south Pennines). A number of biological characteristics of the fish populations were recorded, including species representation, population density and biomass and, for brown trout, Salmo trutta L., only, growth, condition factor and diet. The stream waters were also analysed over a 2-year period for pH, calcium, aluminium and the heavy metals copper, zinc and lead.
Mean pH levels in the streams ranged from 5.0 to 7.6, with calcium concentrations in the range 0.80–27 mg 1⁻¹. Trace metal concentrations (Al, Cu, Zn, Pb) tended to be elevated in the more acid streams. The latter had fewer fish species, and a higher proportion was fishless, 73% at pH < 5.5 compared with 6% at pH >6.5. Trout and eels, Anguilla anguilla L., predominated at lower pH values, but at reduced population levels.
Dietary composition and stomach fullness of trout were similar throughout the stream pH range. More than 40% of the diet was derived from surface and terrestrial sources, supplementing autochthonous items. Growth (as back-calculated length to end of first year of life) and condition factor did not vary significantly with respect to stream acidity. It is thus concluded that the impoverishment of trout populations in the more acid streams was not a second-order effect acting through the lower trophic levels.
Water quality acting directly on fish would appear to account for the poor status of salmonid fisheries in acid streams. Absence or paucity of salmonids was related to high levels of labile monomeric aluminium (>40 μgl⁻¹) or Cu-Zn-Pb toxicity (>0.4 t-LC50 to rainbow trout, Salmo gairdneri Richardson). Limiting values in this study correspond well with published laboratory and field data.     

Adaptive acid tolerance response in Listeria monocytogenes: isolation of an acid-tolerant mutant which demonstrates increased virulence.

The ability of Listeria monocytogenes to tolerate low-pH environments is of particular importance because the pathogen encounters such environments in vivo, both during passage through the stomach and within the macrophage phagosome. In our study, L. monocytogenes was shown to exhibit a significant adaptive acid tolerance response following a 1-h exposure to mild acid (pH 5.5), which is capable of protecting cells from severe acid stress (pH 3.5). Susceptibility to pH 3.5 acid is growth phase dependent. Stationary-phase Listeria cultures are naturally resistant to the challenge pH (pH 3.5), while exponential-phase cultures require adaptation at pH 5.5 to induce acid tolerance. Adaptation requires protein synthesis, since treatment with chloramphenicol prevents the development of acid tolerance. Induction of the acid tolerance response also protects L. monocytogenes against the effect of other environmental stresses. Acid-adapted cells demonstrate increased tolerance toward thermal stress, osmotic stress, crystal violet, and ethanol. Following prolonged exposure of L. monocytogenes to pH 3.5, we isolated mutants which constitutively demonstrate increased acid tolerance at all stages of the growth cycle. These mutants do not display full acid tolerance, but their resistance to low pH can be further increased following adaptation to mild-acid conditions. The mutants demonstrated increased lethality for mice relative to that of the wild type when inoculated by the intraperitoneal route. When administered as lower inocula, the mutants reached higher levels in the spleens of infected mice than did the wild type. The data suggest that low-pH conditions may have the potential to select for L. monocytogenes mutants with increased natural acid tolerance and increased virulence.     

Effect of pH and organic matter on the toxicity of heavy metals to growth of some fungi

Increasing the pH from 5 to 9 decreased the toxicity of mercuric chloride, zinc sulfate, lead nitrate, copper sulfate and nickel chloride toward the growth ofAspergillus flavus, Penicillium chrysogenum, Cunninghamella echinulata, Myrothecium verrucaria andPhoma humicola. On the other hand, the toxicity of cadmium chloride was increased by the increasing pH. Also increasing the concentration of organic matter (peptone and yeast extract) from 0.5 to 1.5% induced a significant reduction in the toxicity of all heavy metals toward the growth of all test fungi.     

Reference ranges of copper and zinc and the prevalence of their deficiencies in an Arab population aged 15-80 years

Serum concentrations of copper and zinc were measured by flame atomic absorption spectrophotometry in 560 Kuwaitis aged from 15 to 80 yr who were in apparent good health to establish reference ranges and determine the prevalence of the deficiency of the trace metals. Zinc/copper ratios were derived by calculation. Because the mean +/- SD (microM) for copper in females (24.9 +/- 7) was significantly higher (p<0.0001) than in males (21.0 +/- 5.8) and vice versa for zinc (17.0 +/- 3.5 [males] vs 15.5 +/- 3.4 [females]) and zinc/copper ratios (0.87 +/- 0.28 [males] vs 0.67 +/- 0.27 [females]), gender-specific reference ranges were established in addition to reference ranges for the total population. The reference range for zinc was closer to those reported for other populations than was copper. Body mass index (BMI) and copper values were lower and the zinc/copper ratio was higher in the young (15-24 yr) compared to the older subjects. Copper concentrations were positively associated with BMI values (r=0.302, p<0.0001). Smokers had significantly lower (p=0.011) BMI than nonsmokers. The prevalence of copper and zinc deficiency, 0.36% and 0.53%, respectively, was low. Generally, the values for serum copper and zinc obtained for the Kuwaiti population studied suggest adequate dietary intake of the trace metals.     

Leaching of copper ore of the Udokanskoe deposit at low temperatures by an association of acidophilic chemolithotrophic microorganisms

Pure cultures of indigenous microorganisms Acidithiobacillus ferrooxidans strain TFUd, Leptospirillum ferrooxidans strain LUd, and Sulfobacillus thermotolerans strain SUd have been isolated from the oxidation zone of sulfide copper ore of the Udokanskoe deposit. Regimes of bacterial-chemical leaching of ore have been studied over a temperature range from −10 to +20°C. Effects of pH, temperature, and the presence of microorganisms on the extraction of copper have been shown. Bacterial leaching has been detected only at positive values of temperature, and has been much more active at +20 than at +4°C. The process of leaching was more active when the ore contained more hydrophilic and oxidized minerals. The possibility of copper ore leaching of the Udokanskoe deposit using sulfuric acid with pH 0.4 at negative values of temperature and applying acidophilic chemolithotrophic microorganisms at positive values of temperature and low pH values was shown.     

A field investigation of water quality, fish and invertebrates in the Mawddach river system, Wales

A 3-year study was made of the soft, acid waters of the Rivers Mawddach, Wen and Gain, which contained copper, zinc and iron from natural outcrops and abandoned mines. Invertebrates, typical of acid to neutral streams, were present but there was no pattern in overall numbers or in biomass between stations. However, the abundance and distribution of Ephemeroptera were related to pH and water hardness although not to copper and zinc. Brown trout, Salmo trutta , were present and there was little difference in rate of growth between stations, whereas biomass and population density varied considerably between stations, being related to water quality such that, where annual median and 95-percentile of the summed proportions ( p ) of the threshold LC50 concentrations of copper and zinc to rainbow trout, S. gairdneri were 0·3 and 0·7 respectively, biomass was half that in the absence of the metals and, where they were 0·6 and 1·3 respectively, no fish would be expected to occur. Low pH was an additional adverse factor at some locations. Salmon, S. salar , failed to populate a tributary where the sum of the p t LC50 values of copper and zinc was 0·45. High aqueous concentrations of copper resulted in elevated concentrations of copper in the liver of trout, especially in older fish, but not in the muscle. Concentrations of zinc in fish liver and muscle were low despite high ambient concentrations.     

Toxicity of common biocides used in aquaculture to embryos and larvae of Brachymystax tsinlingensis Li

Brachymystax tsinlingensis Li is a threatened fish species endemic to China. With the problems of environmental factors and seeding breeding diseases, it is important to further improve the efficiency of seeding breeding and the basis of resource protection. This study investigated the acute toxicity of copper, zinc and methylene blue (MB) on hatching, survival, morphology, heart rate (HR) and stress behaviour of B. tsinlingensis. Eggs (diameter: 3.86 ± 0.07 mm, weight: 0.032 ± 0.004 g) of B. tsinlingensis were selected randomly from artificial propagation and developed from eye-pigmentation-stage embryos to yolk-sac stage larvae (length: 12.40 ± 0.02 mm, weight: 0.03 ± 0.001 g) and exposed to different concentrations of Cu, Zn and MB for 144 h in a series of semi-static toxicity tests. The acute toxicity tests indicated that the 96-h median lethal concentration (LC₅₀) values of the embryos and larvae were 1.71 and 0.22 mg l⁻¹ for copper and 2.57 and 2.72 mg l⁻¹ for zinc, respectively, whereas the MB LC₅₀ after 144-h exposure for embryos and larvae were 67.88 and 17.81 mg l⁻¹, respectively. The safe concentrations of copper, zinc and MB were 0.17, 0.77 and 6.79 mg l⁻¹ for embryos and 0.03, 0.03 and 1.78 mg l⁻¹ for larvae, respectively. Copper, zinc and MB treatments with concentrations greater than 1.60, 2.00 and 60.00 mg l⁻¹, respectively, led to a significantly low hatching rate and significantly high embryo mortality (P < 0.05), and copper and MB treatments with concentrations greater than 0.2 and 20 mg l⁻¹ led to significantly high larvae mortality (P < 0.05). Exposure to copper, zinc and MB resulted in developmental defects, including spinal curvature, tail deformity, vascular system anomalies and discolouration. Moreover, copper exposure significantly reduced the HR of larvae (P < 0.05). The embryos exhibited an obvious change in behaviour, converting from the normal behaviour of emerging from the membrane head first to emerging tail first, with probabilities of 34.82%, 14.81% and 49.07% under copper, zinc and MB treatments, respectively. The results demonstrated that the sensitivity of yolk-sac larvae to copper and MB was significantly higher than that of embryos (P < 0.05) and that B. tsinlingensis embryos or larvae might be more resistant to copper, zinc and MB than other members of the Salmonidae family, which benefits their resource protection and restoration.     

Submersed macrophyte growth at low pH

Summary Vallisneria americana was grown for six weeks in a greenhouse on relatively fertile sediment to test for factors other than nutrient limitation which may slow growth of this submersed macrophyte at pH 5. On the basis of dry mass accumulated, (1) low pH significantly depressed Vallisneria growth at constant free CO₂ levels; (2) free CO₂ enrichment, however, greatly stimulated Vallisneria growth at pH 5, by 2.8-fold and 10-fold at 3.2 times and 10 times air-equilibrated CO₂ levels, respectively; and (3) growth was greater by far at pH 5 than at higher pH with constant total dissolved inorganic carbon (DIC). Free CO₂ availability was thus an important controller of growth at low pH by Vallisneria americana on fertile sediment, and low pH was not directly deleterious. Field surveys of acidic lakes in the Adirondack Mountains of New York state revealed that DIC levels in low pH lakes were often well above equilibrium values and could potentially support vigorous macrophyte growth. Aluminum and/or iron toxicity did not appear to impair growth at low pH, and aluminum concentrations in Vallisneria shoots significantly decreased with increasing free CO₂ concentrations at pH 5.0, perhaps due to growth dilution. Rosette production (a measure of asexual reproduction), maximum leaf length, and extent of flowering within treatments were positively correlated with plant biomass, rather than with pH or free CO₂ levels per se.     

Influence of zinc on lotic plants

SUMMARY. The toxicity of zinc to Hormidium rivulare Kütz. in laboratory culture media is decreased by rises in the levels of magnesium, calcium and phosphate, and increased by rises in pH and cadmium. The effects of all these are sufficiently marked that they may be expected to have considerable importance in the field. In contrast, assays with sodium, chloride and sulphate showed no detectable influence of these ions on zinc toxicity.
When applied at higher concentrations, calcium was always more effective than magnesium, but the reverse was sometimes true at lower concentrations. Both magnesium and phosphate were more effective in reducing zinc toxicity with zinc-tolerant populations than with zinc-sensitive ones. Cadmium was highly toxic, either alone or in combination with zinc; however, calcium had a proportionately greater effect in reducing cadmium toxicity than zinc toxicity. The toxic effects of zinc and cadmium were synergistic, and it seems probable that the presence of cadmium at levels of 0.01 mgl⁻¹ and above will usually lead to a significant increase in the toxicity of any zinc also present. Cadmium (in the absence of zinc) was 34 times more toxic than zinc to a zinc + cadmium sensitive population, and 15.5 times more toxic to a zinc + cadmium tolerant population. Because of the synergistic response, cadmium had an even greater effect in the presence of zinc.     

Biomethanation of spent wash: Heavy metal inhibition of methanogenesis in synthetic medium

Five heavy metals detected in distillery waste were lead (1.0–8.8 μg/ml), copper (1.7–15.7 μg/ml), zinc (3.1–11.8 μg/ml), iron (36.0–43.5 μg/ml), and manganese (3.0–5.1 μg/ml). Their toxicity to biomethanogenesis in a synthetic medium containing 1% sodium acetate, propionate, or butyrate was measured by batch fermentation, after cultivating the bacterial biomass semicontinuously. Lead, copper, and zinc in decreasing order were found to be toxic to biomethanogenesis. Lead at the concentration of 10 μg/ml completely stopped methane production. Iron did not produce any notable change in the process while manganese stimulated the rate of methane production. The toxicity of lead, copper, and zinc to methanogenic bacteria and methane production was dose-dependent but the growth of acetogenic bacteria was impaired at higher concentrations (2.5–10.0 μg/ml) of lead, copper, and zinc. Manganese stimulated the growth of only methanogenic bacteria, but not that of non-methanogenic bacteria or acetic acid production. The reduction in the synthesis of acetic acid via butyrate was more in the presence of these three metals than the synthesis of this acid via propionate.     

Tolerance to copper and zinc of Acidithiobacillus thiooxidans isolated from sewage sludge

The effect of copper and zinc ions on sulphur oxidation by Acidithiobacillus thiooxidans, strain SFR01, isolated from anaerobic sewage sludge was assessed, resulting in tolerance levels up to 20 and 200 mmol l^–1 for copper and zinc, respectively. The tolerance levels obtained were higher than the concentration of copper and zinc usually found in the collected sewage sludge. The tolerance levels obtained indicate no constraints for sludge bioleaching of those metals due to their toxicities to the indigenous A. thiooxidans.     

Functional stability of microbial communities in contaminated soils

Functional stability, measured in terms of resistance and resilience of respiration and growth rate of bacteria and fungi, was studied in soils that have been exposed to copper and low pH for more than twenty years. We used treatments, consisting of soil with no or high copper load (0 or 750 kg ha^?1) and low or neutral pH (4.0 or 6.1). Stability was examined by applying an additional stress in the form of lead or salt. After addition of lead, respiration (decomposition of freshly added lucerne meal) showed lower resistance at low than at neutral pH and at high copper than at low copper. The most acid and contaminated soil was the least resistant. Respiration showed no resilience after addition of lead. Bacterial growth rate (thymidine incorporation) also showed resistance at low pH but only in soils that were not contaminated with copper. After addition of salt, respiration showed no differences in resistance but the soils without copper contamination showed higher resilience. Bacterial growth rate showed lower resistance at low pH than at neutral pH, the latter in which the growth rate increased by on average 123%. This increase at high pH was faster in soil without copper than in soil with copper contamination in which the growth rate initially decreased and then increased. The effects of secondary stress depended on the nature of the stress (lead or salt) and on the parameter measured (respiration or bacterial growth rate). In general the highest resistance and/or resilience were found in the least contaminated soils with neutral pH and/or no copper contamination. Thus, the microbial communities in the cleaner soils showed the highest functional stability. The results seem to confirm the notion that environmental stress alters ecosystems such that supplementary stress will have stronger impacts than in an unstressed system. The results may also confirm the insurance‐hypothesis that reduced biodiversity due to the first stress negatively affected community stability. As an alternative, we discuss the observed effects in terms of altered energy budget.     

Effect of Copper on Growth of Yeast

The effect of copper was tested on the growth of many strains of yeast. Plate culture on density gradient agar of copper was used for estimating the growth response to copper. Growth in many strains was more strongly inhibited by the copper-aquo complex than by the copper-amino acid complex. Debaryomyces hansenii IFO 023 was found a suitable strain for the present study, because it was not resistant, not producing H₂S, and copper absorption by this strain was similar to that of the resistant strain. Growth of yeast cells in medium containing copper was affected by pH and concentration of amino acid in medium. Absorption of copper into intact cells was almost saturated for the initial few minutes. It was also affected by the addition of amino acid to copper solution. Our results indicated that the growth response of yeast to copper was closely related to copper absorption into cells. About 60 percent of copper absorbed into cells was distributed in the soluble fraction of the cell homogenate which was obtained by centrifugation at 105,000 g for 60 min.     

Sodium vanadate toxicity in adult and developing rats

The toxic effect of vanadium (sodium metavanadate) during pregnancy and lactation was studied by feeding vanadium to pregnant, Sprague-Dawley rats at levels of 1 (control) or 75 μg V/g diet through d 21 postpartum, at which time they were killed. Vanadium-fed dams had lower food intakes and weight gains than controls during pregnancy. Survival until d 21 postpartum was significantly lower in the vanadium pups compared to controls. In addition, the surviving pups gained less weight than control pups, despite similar birth weights. On a relative body weight basis, vanadium pups had larger livers, brains, and testes than controls, suggesting that these animals were developmentally delayed. Vanadium dams and pups had higher concentrations of hepatic vanadium than controls. Vanadium pups also had higher concentrations of hepatic zinc than control pups. Maternal hepatic zinc concentrations were not affected by diet. Also, no significant differences in hepatic iron, copper, or manganese concentrations were observed for either dams or pups. Hepatic thiobarbituric acid reactivity was higher in whole cell and isolated mitochondria for vanadium dams and pups than for control dams and pups, indicating that these animals may have had higher levels of lipid peroxidation. This idea was supported by the observation of lower concentrations of reduced glutathione in the livers of vanadium pups compared to controls. In contrast, kidney and brain glutathione levels were not affected by diet. In conclusion, animals during periods of rapid growth are susceptible to vanadium toxicity, and increased lipid peroxidation may be one factor underlying this toxicity.     

Sorption and desorption of copper by and from clay minerals

Summary The sorption of Cu by different clay minerals from dilute CuSO₄ solutions was studied as a function of pH. It was found that Cu sorption increased with increasing the equilibrium pH. The low sorption at low pH values was attributed to the competition effect of H⁺ ions and the release of octahedral Mg, Fe and Al from the 2: 1 minerals in acid solutions. The higher sorption of copper at higher pH values was related to the absence of H⁺ ions and to the increase in the pH-dependent negative charge in kaolinite and gibbsite.The desorption of copper using solutions of 1 N NaCl at different pH values indicated that kaolinite and montmorillonite released large amounts of their adsorbed Cu even at high pH values. Vermiculite, on the other hand, exhibited a strong retention of Cu against extraction with NaCl. Oven-drying had no significant effect on the desorption characteristics of the Cu-saturated clays.The Ca-Cu exchange isotherms on montmorillonite were studied at two different pH values. The isotherms indicated a preference of Ca at the lower pH (pH 3.5), with K = 0.931 and G = +41.0 cal./mole. At the higher pH (5.2) the isotherms indicated a preference of Cu with K = 1.282 and G = –141.0 cal./mole. The difference was attributed to the competition of H⁺ at low pH.     

Superoxide dismutase activity and zinc and copper concentrations in growth retardation

The aim of this study was to determine the effects of oral zinc treatment on red cell copper/zinc-superoxide dismutase (Cu/Zn-SOD) activity and zinc and copper concentrations in growth retardation. Thirty-five patients, average age of 11 yr, were selected. The control group consisted of 10 healthy children whose average age was 10 yr. Superoxide dismutase activity was determined by spectrophotometer. Copper and zinc concentrations were measured by an atomic absorption spectrophotometer. The activity of Cu/Zn-SOD before zinc treatment was higher than the controls (p<0.001). There was a decrease in Cu/Zn-SOD activity after zinc treatment (p<0.001) and the values after treatment were still higher than the controls (p<0.001). Plasma zinc concentrations before zinc treatment were lower than controls (p<0.01). After treatment, there was an increase in plasma zinc concentrations compared to controls and the patients' values before zinc treatment, respectively (p<0.001, p<0.001). After zinc treatment, plasma copper concentrations were decreased significantly (p<0.01). An increase in red cell zinc concentration (p<0.01) and a decrease in copper concentration (p<0.001), which were statistically significant, were seen after zinc treatment. The results suggested that there were significant alterations in Cu/Zn-SOD activity and zinc and copper concentrations during growth retardation. With zinc treatment, these parameters appeared to approach normal values.