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Acid mine drainage from abandoned mines and acid mine pit lakes is an important environmental concern and usually contains appreciable concentrations of heavy metals. Because sulfate-reducing bacteria (SRB) are involved in the treatment of acid mine drainage, knowledge of acute metal toxicity levels for SRB is essential for the proper functioning of the treatment system for acid mine drainage. Quantification of heavy metal toxicity to mixed cultures of SRB is complicated by the confounding effects of metal hydroxide and sulfide precipitation, biosorption, and complexation with the constituents of the reaction matrix. The objective of this paper was to demonstrate that measurements of dissolved metal concentrations could be used to determine the toxicity parameters for mixed cultures of sulfate-reducing bacteria. The effective concentration, 100% (EC100), the lowest initial dissolved metal concentrations at which no sulfate reduction is observed, and the effective concentration, 50% (EC50), the initial dissolved metal concentrations resulting in a 50% decrease in sulfate reduction, for copper and zinc were determined in the present study by means of nondestructive, rapid physical and chemical analytical techniques. The reaction medium used in the experiments was designed specifically (in terms of pH and chemical composition) to provide the nutrients necessary for the sulfidogenic activity of the SRB and to preclude chemical precipitation of the metals under investigation. The toxicity-mitigating effects of biosorption of dissolved metals were also quantified. Anaerobic Hungate tubes were set up (at least in triplicate) and monitored for sulfate-reduction activity. The onset of SRB activity was detected by the blackening of the reaction mixture because of formation of insoluble ferrous sulfide. The EC100 values were found to be 12 mg/L for copper and 20 mg/L for zinc. The dissolved metal concentration measurements were effective as the indicators of the effect of the heavy metals at concentrations below EC100. The 7-d EC50 values obtained from the difference between the dissolved metal concentrations for the control tubes (tubes not containing copper or zinc) and tubes containing metals were found to be 10.5 mg/L for copper and 16.5 mg/L for zinc. Measurements of the turbidity and pH, bacterial population estimations by means of a most-probable number technique, and metal recovery in the sulfide precipitate were found to have only a limited applicability in these determinations. [ABSTRACT FROM AUTHOR]

The suitability of a new method to determine the minimum number of data required to stabilize a calculation is studied. Five different calculations have been computed, changing the order of data randomly. The statistical reliability of numerical values has also been calculated depending on the successive increase in the number of data included. In our study, when few data are considered, a great variation in the estimations is observed depending on the individual data included.The calculation is stabilized for a sample size between 5 and 15 data points, and the sample can therefore be considered representative enough. Results are in agreement with those presented by other authors. [ABSTRACT FROM AUTHOR]

Cesium, cadmium, cobalt, and strontium are four contaminants frequently found in soils at biotoxic levels. Introduction of certain nongenetically modified bacteria has been frequently suggested as a method for the immobilization of heavy metal contaminants in soil, thereby reducing runoff and bioavailability. In this study, we have used the polymerase chain reaction (PCR) and denaturing gradient get electrophoresis (DGGE) to track the survival of five bacterial species added tosoil microcosms with and without the addition of a mixture of these metals. The PCR primers targeted conserved regions of the 16S rDNA molecule present in all bacteria. The reaction products were shown to reflect the relative abundance of the bacteria both in mixtures of pure cultures and against a background of all the eubacterial species present in the soil following inoculation. Three of the species (Pseudomonas aeruginosa FRD-1, Shewanella putrifaciens 200, and Desulfovibrio vulgaris Hildenborough) decreased rapidly following inoculation into both soils. The proportion of Alcaligenes eutrophus CH34 remained at a constant level throughout the 8-week experiment in both soil treatments. Sphingomonas aromaticivorans B0695 showed toxic metal-dependent survival in that its relative abundance dropped rapidly in pristine soil but remained at approximately inoculation levels throughout the experiment in contaminated microcosms. [ABSTRACT FROM AUTHOR]

In this study, we determined the conditional stability constant (logK') of copper for the gills of rainbow trout (Oncorhynchus mykiss; RBT) and brook trout (Salvelinus fontinalis; BT). Using toxicity-basedcomplexation bioassays, which measure the effect of competing organic ligands on copper toxicity, the RBT gill copper log K' range was 6.4 to 7.2. Using a Scatchard analysis of gill Cu accumulation, the RBTlog K' was 7.50 and the BT log K' was 7.25. The close agreement in RBT log K' values between these two methods suggests that measurement of gill copper accumulation is an acceptable alternative for determining a toxicity-based gill copper binding affinity. Our results also suggest that there is either a single gill copper binding component or, more realistically, multiple components with similar binding properties that function collectively to define a single toxicologically relevant copper conditional stability constant. These results suggest analytical approaches to measuring bioavailable metal concentrations, such as geochemical modeling where biological ligands are included inspeciation calculations, may adequately simulate complex biological ligands. A method to convert gill copper accumulation to a bioavailable water criterion is also discussed. [ABSTRACT FROM AUTHOR]

Currently, regulations regarding the maximum permitted concentrations of metals in soils are based on measurements of the total concentration. However, a range of chemical and biological techniques are being developed te predict the bioavailable component of these pollutants. A lux-based biosensor was tested in soil solutions extracted from two field experiments at Braunschweig, Germany, that had the same metal inputs, but differed in pH. The bioluminescence response was found to decline as the free Zn' increased, and both soils fitted the same relationship with soil solution metal concentrations. The EC25 and EC50 derived from this curve were 1.9 and 6.1 mg/L, respectively, In contrast, the response to total Zn concentrations in the bulk soil showed distinct curves for each soil, further highlighting the appropriateness of free Zn2+ as a toxicity indicator. Other metals were present in the soil, but were unlikely to be toxic, because the observed concentrations were less than their individual toxic threshold values in solution. Bioluminescence-based biosensors were concludedto possibly offer an inexpensive and rapid technique to evaluate thebioavailability of metals in soil systems. The response of these biosensors can be related to soil solution speciation measurements. and this gives a common basis for expression of toxic thresholds in different soils. [ABSTRACT FROM AUTHOR]

The relationship between benthic oxygen demand and the sediment -water flux of six heavy metals (cobalt [Co], nickel [Ni], copper [Cu], zinc [Zn], cadmium [Cd], and lead (Pb]) was examined at two locations:New Bedford Harbor and Buzzards Bay, Massachusetts, USA. Dissolved metal fluxes were measured in laboratory microcosms incubated in the dark at in situ temperatures, and microcosms were collected over different seasons to examine temporal and spatial variability in the fluxes. The net flux of all metals was generally out of the sediments. However, at low rates of benthic oxygen demand, net flux of Pb and Co was into the sediments. When data from the two sites are combined, regression of metal fluxes (normalized to sediment metal content) againstbenthic oxygen demand was significant for all metals (r2 = 0.58-0.85), with all slopes significantly different from zero. The flux values (normalized to sediment metal content) ranged from - 1.4 (mmole/m2 /d)/(mole/mole C)sediment for Zn during winter to 42 (mmole/m2 /d)/(mole/mole C)sediment for Cd during summer months. The magnitude of the fluxes generally showed the following relationship: Cd > Zn > Co, Ni, Cu > Pb, indicating higher relative mobility for Cd and lower mobility for Pb. Theestimated annual fluxes of metals to New Bedford Harbor and BuzzardsBay amount to approx. 0.06% (Pb) to 25% (Cd) of the inventory of metals in the top 2 cm of sediments and can equal the inventory of metals in the water column on a time scale as short as 7 d for Zn. [ABSTRACT FROM AUTHOR]

We performed chemical analyses of heavy metals in water and periphyton, toxicity tests with Daphnia magna and an indigenous mayfly (Deleatidium sp,), and field surveys of benthic macroinvertebrates to estimate the degree of metal pollution in three catchments in the Coromandel Peninsula of New Zealand. Good agreement was found between toxicity tests and measures of benthic community structure, particularly at stations with the highest metal levels. Responses of benthic communities at stations with low or moderate levels of metal contamination were variable and were probably confounded by factors other than heavy metals. Effects of heavy metals on benthic communities in New Zealandstreams were similar to those reported for metal-polluted streams inNorth America and Europe, suggesting that responses to metal contamination are predictable. Abundance and species richness of mayflies, number of taxa in the orders Ephemeroptera, Plecoptera, and Trichoptera, and total taxonomic richness were the best indicators of heavy metals in New Zealand streams. In contrast, the quantitative macroinvertebrate community index (QMCI), a biotic index proposed for assessing effects of organic enrichment in New Zealand streams, could not distinguish between reference and metal-polluted streams. The poor performance of the QMCI was primarily due to incorrect tolerance scores for some taxa to heavy metals, Because of concerns regarding the subjective assignment of tolerance values to species, we recommend that tolerance values for dominant species in New Zealand streams should be verified experimentally in stream microcosms. [ABSTRACT FROM AUTHOR]

Agricultural, urban, industrial, and mining sources release metals into waterways. The effects of sublethal concentrations of metals on integrated physiological processes in fish, such as immunocompetency, are not well understood. The objective of this study was to determinethe physiological effects of a range of sublethal copper concentrations (6.4, 16.0, and 26.9 mu g Cu/L) on Shasta-strain rainbow trout (Oncorhynchus mykiss) exposed in soft water. Trout were sampled after 3, 7, 14, and 21 d of exposure to copper. The percentage of monocytes was consistently elevated at 26.9 mu g Cu/L, and the percentage of lymphocytes was decreased. A consistent increase in the percentage of neutrophils occurred at 26.9 and 6.4 mu g Cu/L. Respiratory burst activity was decreased for all concentrations at all sampling days, but asignificant reduction occurred only at 14 and 21 d of exposure to copper. B-like cell proliferation was decreased by exposure to the higher copper concentrations. Proliferation results, however. had high variability. T-like cell proliferation and phagocytosis were not altered. Hepatic copper concentration was consistently elevated in trout exposed to 26.9 mu g Cu/L, no correlation was found between hepatic copper concentration and the immune system responses investigated. Consistent alterations in immunological parameters suggest that these parameters could serve as indicators of chronic metal toxicity in naturalsystems. [ABSTRACT FROM AUTHOR]

The toxicity to embryo and adult zebrafish (Danio rerio) of Cu complexes with two substituted malonic acids, benzyland n-hexadecyl-, chosen as models for low-molecular-weight natural dissolved organic matter. were investigated. Toxicity test solutions at pH 6.5- 0.1 with therequired Cu speciation were designed using the chemical speciation model MINTEQA2. and the speciation was confirmed by Cu ion-specific electrode. In the absence of malonic acids, concentrations of Cu2+ up to 1. 13 mu mol/ L increased the embryo hatching time from approx. 2 din control solutions (no Cu or malonic acid) and solutions containing malonic acids without Cu to approx. 8 d. The Cu-benzylmalonic acid complex (n-octanol/water distribution coefficient [p] -0.00) in the presence of inorganic Cu species did not delay hatching beyond that attributable to Cu2+. In contrast, 0.60 mu mol/L Cu-n-hexadecylmalonic complexes (p = 6.89) delayed hatching by 5.5 d in excess of that attributable to 1.13 mu mol/L Cu2+, assuming that the hatching delays caused by the different Cu species were additive, possibly because of Cuentry into the embryo as the lipophilic Cu-n-hexadecylmalonic complex. None of the Cu-malonic acid complexes was acutely toxic to adult zebrafish at concentrations up to 1.4 mu mol/L, possibly because Cu was removed from the Cu-malonic acid complexes by stronger chelating groups at the gill surface. Substituted malonic acids with similar proton and Cu association constants can be readily prepared with a variety of similar substituents, radiolabeled if required. Our results showthat these acids could be useful ligands for investigating intracellular transport and metabolism of metal-organic complexes. [ABSTRACT FROM AUTHOR]

Juvenile winter flounder (Pleuronectes americanus) from a Long Island Sound site within the Niantic River region contain elevated levels of liver As and Cd. Previous work in mammals suggests that a reduced species of As, NaAs3+ is as potent a metallothionin (MT) inducer as CdCl2. We investigated whether this is also the case in winter flounder by subcutaneously exposing juveniles to a sublethal range of NaAs3+ (0, 0.38, 0.75, and 1.5 mu mol/g fish) or CdCl2 (0, 0.045, 0.09, and 0.18 mu mol/g fish). Of the two, CdCl2 was the more potent MT inducer, with an 8-fold lower molar equivalent causing a 3-fold higher response. Both metals rapidly induced MT protein within 24 h of exposure, but only the highest dose of CdCl2 elicited a significant increase in MT mRNA. The magnitude of the MT protein response to CdCl2 (7.25-fold) demonstrates that elevated liver As and Cd do not narrow the MT response window in juvenile flounder from the Niantic River region. These fish have relatively low liver MT protein levels (46 mu g/g) and will be a useful reference population for future metal impactstudies within Long Island Sound. [ABSTRACT FROM AUTHOR]