Influence of metals and metal speciation on the growth of filamentous bacteria

Calcium, copper, nickel and zinc were examined for their effect on the growth of axenic filamentous bacteria which included strains of Thiothrix, type 021N and type 1701. All organisms grew best at the upper range of calcium concentrations tested. Heavy metals were also less toxic to Thiothrix strain A1 at higher calcium concentrations. Copper was more inhibitory than either nickel or zinc and copper-nickel and copper-zinc mixtures appeared to act synergistically in suppressing the development of Thiothrix strain A1. In contrast, nickel toxicity could be reduced by the addition of zinc to the medium. Metal toxicity could also be diminished by the chelating agent, EDTA; therefore, free metal ions were most likely responsible for the observed toxic effects. A computer model, MINEQL, was used to predict metal speciation and to determine the potential for various media components to control speciation of the metals. The suitability of the computer model was determined by comparison of predicted and analyzed free metal ion concentrations.     

An improved experimental medium for freshwater toxicity studies using Daphnia magna

Some of the difficulties involved in formulating experimental media for use in toxicity studies are pointed out, with particular reference to heavy metals and their possible chelation by agents in the media. A medium suitable for Daphnia culture with the green alga, Chlorella sp., was chosen from the literature to investigate physiological responses by Daphnia to sublethal cadmium poisoning. Like most synthetic media this included the labile Fe-EDTA complex as a source of soluble iron which is shown to be essential for growth of both Daphnia and Chlorella. Attention is drawn to the almost univeral complexing ability of EDTA with metal ions, and to the mechanisms whereby EDTA may be released from the Fe-EDTA complex under experimental conditions. Synthesis of a ferri-gluconate complex as an alternative source of soluble iron is described; this complex being theoretically more iron-specific than Fe-EDTA, whilst providing equally well for Chlorella growth and Daphnia growth and fecundity. Restriction of pH variation in the experimental medium, within its chemically stable range of 6.0–9.0, is shown to be difficult over 10 days: most buffers with a suitable pK_α value (ca. 7.5) being toxic or having some complexing ability. TES buffer (pK_α 7.5) and HEPES buffer (pK_α 7.55) were used at various concentrations, and 0.001 M HEPES is shown to restrict pH variation to within 0.5 pH units over 10 days without adversely affecting Chlorella growth or Daphnia growth and fecundity. Chemical estimation of the degree of cadmium-complexing by each iron source and each buffer under investigation was not possible since the presence of chelating agents imposes restrictions on the methods available, particularly at low cadmium concentrations. Instead, a bioassay of the reduction in 48h acute toxicity of cadmium to Daphnia in media containing each potential chelating agent in turn is used. These data indicate that 0.001 M HEPES buffer does not complex cadmium at 0.1 ppm, and that ferri-gluconate is less likely to do so than ferri-EDTA.     

Algae as a chemical speciation monitor—I A comparison of algal growth and computer calculated speciation

An alga, Selenastrum capricornutum, was used to monitor the relationship between metal speciation and toxicity. The principal effect of copper on the algae was a decrease in growth rate constant as the total concentration of copper increased. A 24-h growth experiment was used to monitor the effect of metal on the algae. Chlorophyll measurements at 17 and 23 h were used to estimate the growth rate constant. A plot of growth rate constant versus the speciation variable (total copper, pCu, pCuL, etc.) was used to determine the toxic species and the algicidal concentrations. Solutions containing EDTA, TRIEN, NTA, HIMDA and bicine became algicidal at pCu of 8.0 whereas solutions containing ethylenediamine and citric acid became algicidal at pCu 8.65 and 9.5, respectively. A comparison of growth and calculated speciation suggests that Cu en⁺² and Cu CIT OH⁻² were toxic species.     

Toxicity of lipid-soluble copper(II) complexes to the marine diatom Nitzschia closterium: Amelioration by humic substances

Algal assays, using the marine diatom Nitzschia closterium, have established that humic acid (5 mg kg⁻¹) can ameliorate the toxicity of the lipophilic complex Cu(oxine)₂ (3 × 10⁻⁸ mol l⁻¹ in unsupplemented seawater). The toxicity of Cu(PAN)₂ is not ameliorated [PAN = 1-(2-pyridylazo)-2-napthol]. In conjunction with previous visible absorption spectrophotometry and polarographic measurements it was established that humic acid sequesters copper(II) from the hydrophobic complexes, releasing a ligand molecule. The copper(II) toxicity may be ameliorated provided the ligand itself is not toxic.Fulvic acid was significantly less effective in ameliorating toxicity. Because of the significant competition from Ca(II) and Mg(II) in seawater, it is inferred that humic substances may be more effective in ameliorating toxicity of hydrophobic copper complexes in fresh water.     

Toxicity to Daphnia of the end products of wet oxidation of phenol and substituted phenols

The process of wet oxidation breaks down organic substances in aqueous solution at elevated temperatures and pressures. Experimental wet oxidations were carried out on pure solutions of phenol, 2-chlorophenol and 4-nitrophenol. After 1-h wet oxidation, final concentrations of these compounds averaged 3% of their concentrations in the starting solutions. The toxicities of the starting compounds and the residual toxicity of the end-product solutions were measured with 48-h acute toxicity tests using Daphnia magna. The solutions of end products were all less toxic than the starting solutions by factors ranging from 10 to 120. However, the end-product solutions were somewhat more toxic than would be predicted from the known concentration of initial compound remaining in the solution of end products.     

Copper toxicity to fishes and an extremely sensitive shrimp in relation to a potential Australian tropical mining‐waste seep

Ephemeral or seasonal streams tributaries to Magela Creek (Alligator Rivers Uranium province, Northern Territory), containing perennial pools, lie adjacent to a 1 km² tailings storage dam, still being filled, whose partial seepage collecting system contains an increasing concentration of copper. Acute copper toxicity to local fauna was determined in a flow‐through apparatus at 27°C and pH 6. The 4‐day LC50 values (μg L^‐1) for total copper were: rainbowfish Melanotaenia splendida inornata 170–190, pennyfish Denariusa bandata 75, and atyid shrimp Caridina sp. 3–4, and the 3‐day LC50 of the catfish Porochilus rendahli 85. Free ionic copper approximated the total copper. Test water quality was as predicted for seepage‐affected streams during a brief critical period in some years: of low hardness and alkalinity but enriched in Mg²⁺ and SO₄ ^2‐. The relative LC50 values correspond to the relative occurrence of similar species among zones of the metals‐affected Finniss River. Magela Creek system waters often had naturally low pH and ion‐concentrations (pH < 6, conductivity < 20 μS cm^‐1); the total copper concentration has repeatedly been similar to the free ionic copper LC50 of Caridina sp. The abundance and distribution of Caridina sp. are likely already influenced by local natural copper concentrations. Seepage total copper has increased to the fishes’ free ionic copper LC50.The hydraulic head of the wet tailings will increase further from 15 to 20 m.     

Acute toxicities of five nitromusk compounds in Daphnia,algae and photoluminescent bacteria

Acute toxicities of five nitromusk compounds in Daphnia, algae and photoluminescent bacteria were investigated. In order to obtain some basic data for ecotoxicological tests, physicochemical parameters were determined. The water solubility and log K_ow values were: nitromusk xylene 0.15 mg l⁻¹ and 4.4, nitromusk ketone 0.46 mg l⁻¹ and 3.8, nitromusk ambrette 0.79 mg l⁻¹ and 4.0, nitromusk moskene 0.046 mg l⁻¹ and 4.4 and nitromusk tibetene 0.052 mg l⁻¹ and 4.3. None of these compounds exhibited a toxic effect in any of the toxicity tests even at the highest concentrations achievable except for nitromusk ambrette which was toxic to Daphnia.     

Bioaccumulation and toxicity of copper as affected by interactions between humic acid and water hardness

This study evaluated the effects of water hardness and humic acid (HA) on the acute and chronic toxicity of copper to Daphnia pulex and on its accumulation by D. magna. Hardness had little effect on either the acute (3-day) or the chronic (42-day) toxicity of copper. Humic acid significantly reduced both the acute and chronic toxicity of copper when added to waters having hardnesses of 58, 115 and 230 mg l^?1 as CaCO₃. The effect, per unit of HA, on chronic toxicity was very similar for soft and medium water but less in hard water. At each of two HA concentrations, copper was chronically more toxic in hard water than in either medium or soft water. Bioaccumulation of copper varied with relative hardness and HA concentration and this was further affected by age at exposure. For 1-day-old animals, an increase in either hardness or HA- or any combination of the two, tended to decrease bioaccumulation. Results for 7-day-old animals were in general agreement except for animals exposed to copper in hard water at an intermediate HA concentration. These animals accumulated significantly more copper in the presence of HA. This agrees with the fact that this concentration of HA also increased the chronic toxicity of copper in hard water. Both of those phenomena are probably due to the displacement of Cu²⁺ from HA by competition from the increased concentrations of Ca²⁺ and Mg²⁺. The fact that HA had the opposite effect on copper accumulation by young animals in hard water could not be explained.     

The influence of heavy metals on a mixed bacterial population of sewage origin in the chemostat

A reproducible mixed continuous bacterial population has been cultured in the chemostat at a low dilution rate. Four species originally isolated from activated sludge were used to provide an inoculum. The influence of cadmium, chromium, copper and lead as shock doses has been examined at concentrations of 50 mg l⁻¹ and the response of the population was found to vary for each metal. The most toxic metal was copper followed by cadmium, lead and chromium. Continuous doses of chromium and lead have been studied, the latter over a range of concentrations. It appears that concentrations of lead as low as 5 mg l⁻¹ cause modifications in the individual population sizes. Some of the species studied were found to be more resistant to heavy metal toxicity than others.     

Toxic effects of cupric,chromate and chromic ions on biological oxidation

Using a completely mixed, continuously operated, lightly loaded, laboratory activated sludge system, the toxic effects of cupric, chromic and chromate ions under conditions of shock loading were observed. These were determined with the aid of a total carbon analyzer and simple mass balance techniques in terms of conversion of the organic nutrient fed. The distribution of the metal ion between aqueous solution and suspended solids was measured using atomic absorption spectrophotometry. Toxic effects were in the order: Cu²⁺ > CrO₄^2? > Cr³⁺ while the reductions in conversion were 90, 50 and 20 per cent, respectively, for concentrations of 5 ppm metal ion. Cupric ion toxicity was directly proportional to the weight of copper absorbed per unit mass of suspended matter within the total copper concentration range (0–5.5 ppm) studied. This toxicity decreased markedly with increased suspended solids concentration: an 80 per cent decrease in conversion at 210 ppm suspended solids was reduced to a negligible quantity (3 per cent) by increasing the suspended solids to 4000 ppm. At 210 ppm suspended solids, 34 per cent of the added copper was removed by the sludge in 7 h.The results of this work suggest that the toxic effect of metal ions on a sewage plant activated sludge system could be reduced by rapidly increasing the suspended solids concentration, possibly by the addition of dried sludge. It is also implied that the effect on dilute systems such as lagoons would be much greater because of the low suspended solids.This work supports a mechanism involving rapid adsorption of the cupric ion by both viable and dead sludge followed by a slower rate determining step resulting in the toxic effect. The first order rate constant for substrate utilization was found to be (1.07 ± 0.6) h^?1.     

Effects of inorganic complexing on the toxicity of copper to Daphnia magna

Effects of carbonate-bicarbonate, orthophosphate, and pyrophosphate on the toxicity of copper (II) to Daphnia magna were studied at constant pH and total hardness. Mortality rates and reciprocal survival times were directly correlated with cupric (Cu²⁺) and copper hydroxy (Cu(OH)n) ion activities as determined by equilibrium calculations. Toxicity was negatively related to activities of soluble copper carbonate (CuCO₃) and other complexes, and was found to be independent of dissolved copper or total copper concentrations.     

Toxicity of cadmium, cobalt, uranium and zinc to Zoogloea ramigera

The toxic effects of cadmium, cobalt, uranium and zinc to the bacterium Zoogloea ramigera have been studied. This organism, which is abundant in sewage-treatment plants, responded to media supplemented with heavy metals by showing a prolonged lag phase and by decreasing its growth rate. The length of the lag phase was proportional to the metal concentration when Z. ramigera was exposed to cadmium, cobalt and zinc. When exposed to uranium (uranyl ions) the organism altered its growth behavior by showing an extended lag phase almost independent of the metal concentration. The order of toxicity of the metals to Z. ramigera was Cd²⁺ = Zn²⁺ > Co²⁺ > UO₂²⁺.     

The fate of heavy metals in wastewater stabilization ponds

The distribution of toxic heavy metals was studied throughout the process of treatment of domestic wastewater by stabilization ponds. The concentrations and distribution of free and bound zinc, cadmium, lead and copper through the various stages of a treatment plant were analyzed by Anodic Stripping Voltammetry (ASV). Only a slight decrease in the total metals concentration was observed during the various stages of the wastewater treatment plant. However, the distribution among dissolved (free and chelated) and particulate fractions did change towards solubilization of most of the particulate fraction. Within the soluble fraction a significant decrease in the free cations occurred due to a proteinaceous chelating agent(s) released by the microbial population in the ponds. A similar phenomenon was found in simulated laboratory experiments which were carried out with a Chlorella strain isolated from the stabilization ponds.     

Effects of copper, zinc and cadmium on Selanastrum Capricornutum

The algicidal and algistatic effects of copper, zinc and cadmium on Selanastrum capricornutum, a unicellular green algae were analyzed by using a modification of the Algal Assay Procedures Bottle Test.Algicidal concentrations of copper, zinc and cadmium were 0.30, 0.70, and 0.65 mg 1⁻¹. Treatment of Selanastrum with various concentrations of the metals resulted in similar growth rates characterized by extended lag growth phases.Combinations of copper, zinc and cadmium were similar in toxicity to equal concentrations of zinc. Combinations of copper and cadmium resulted in a greater growth rate than equal concentrations of copper suggesting that cadmium inhibits copper toxicity.Selanastrum was able to exist in waters from the upper South Fork and North Fork of the Coeur d'Alene River where zinc and other metals were in low concentration. However, the algae was not able to tolerate zinc concentrations greater than 0·5 mg 1⁻¹ from waters of other parts of the drainage. These observations were consistent with laboratory findings where 0.7 mg 1⁻¹ zinc was algicidal and 0.1 mg 1⁻¹ inhibited the growth of Selanastrum.     

Reduction of aquatic toxicity of linear alkylbenzene sulfonate (LAS) by biodegradation

Partial biodegradation of LAS is shown to significantly reduce the specific toxicity (i.e. per unit weight) of the remaining LAS to Daphnia magna (water fleas) and Pimephales promelas (fathead minnows). This results from the fact that the longer homologs and more terminal isomers, which are the more toxic, are also the more rapidly degraded under bacterial action. The acute aquatic LC₅₀ of LAS may range from 0.5 to 50 mg/l depending mainly upon the chain length of the particular homolog. A high molecular weight commercial type LAS with LC₅₀ around 2 mg/l before biodegradation may show Daphnia LC₅₀'s of 30–40 mg/l. for the LAS remaining after 80–85% degradation.A further contribution to this toxicity reduction may occur if the methylene blue analytical method is used to determine the amount of LAS remaining, since some of the biodegradation intermediates show methylene blue activity but no significant toxicity. For example, sulfophenylundecanoate, a model of early intermediates, shows Daphnia and fathead lc₅₀'s 200 and 75 mg/l., respectively. Sulfophenylbutyrate, modeling somewhat later intermediates, gives lc₅₀ values around 5000–10,000 mg/l. Dialkyl tetralin/indane sulfonates (the major non-linear components in commercial LAS) exhibit 1/2–1/10 the toxicity of the corresponding LAS homologs.These results re-emphasize that analysis simply for methylene blue active substances (MBAS) gives no basis for predicting the aquatic toxicity of an environmental sample. And furthermore, that meaningful water quality criteria and standards cannot be established in terms of MBAS content while based on toxicity studies on intact, undegraded LAS.     

Aspects of water quality and the toxicity of copper to rainbow trout

Studies were made of the effects of additions of a sewage effluent, an amino acid, humic substances, and suspended organic matter on the acute lethal toxicity of water containing copper sulphate to rainbow trout. In all cases the toxicity of a given total concentration of copper was quantitatively reduced. It was concluded that neither the total concentration of copper nor that of “soluble” copper in a water could be used to determine the toxicity to fish which was attributable to copper. It was also concluded that data from toxicity tests with copper in which natural surface waters are used for dilution purposes cannot define the true toxicity of copper or have application to other natural waters except when the concentrations of the toxic chemical species are known.     

The acute toxicity of vanadium and copper to eyed eggs of rainbow trout (Salmo gairdneri)

The effects of vanadium (25–595 mg l⁻¹) and of copper (0.03–4.78 mg l⁻¹) on embryonic survival and hatching of eyed eggs of rainbow trout, Salmo gairdneri, were investigated. Copper was approx. 300-fold more toxic than vanadium (96-h LC₅₀ = 0.4 and 118 mg l⁻¹, respectively) but had little effect on the timing of hatch. Vanadium induced premature hatching of eyed eggs at concentrations from 44 to 595 mg l⁻¹. Concentrations of copper required to produce lethality in eyed eggs were similar to concentrations required to produce mortality in juveniles. Vanadium concentrations approx. 15 times higher were required to produce mortality in eyed eggs than in juveniles. Therefore, acute exposure of eyed rainbow trout eggs to vanadium is not a sensitive toxicity test for use in establishing water quality criteria or maximum acceptable toxicant concentrations.     

Microbial toxicity of metal oxide nanoparticles (CuO, NiO, ZnO, and Sb2O3) to Escherichia coli, Bacillus subtilis, and Streptococcus aureus

In this study, the microbial toxicities of metal oxide nanoparticles were evaluated for Escherichia coli, Bacillus subtilis, and Streptococcus aureus in laboratory experiments. The nanoparticles tested were CuO, NiO, ZnO, and Sb₂O₃. The metal oxide nanoparticles were dispersed thoroughly in a culture medium, and the microorganisms were cultivated on Luria-Bertani agar plates containing different concentrations of metal oxide nanoparticles. The bacteria were counted in terms of colony forming units (CFU). The CFU was reduced in a culture medium containing metal oxide NP, and the dose-response relationship was characterized. CuO nanoparticles were found to be the most toxic among the tested nanoparticles, followed by ZnO (except S. aureus), NiO, and Sb₂O₃ nanoparticles. We determined that the intrinsic toxic properties of heavy metals are also associated with the toxicity of metal oxide nanoparticles. Ion toxicity was also evaluated to determine the effects of metal ions dissolved from metal oxide NPs, and the toxicity induced from the dissolved ions was determined to be negligible herein. To the best of our knowledge, this is the first study of the toxicity of NiO and Sb₂O₃ NPs on microorganisms. We also discuss the implications of our findings regarding the effects of the intrinsic toxic properties of heavy metals, and concluded that the apparent toxicities of metal oxide NPs can largely be understood as a matter of particle toxicity.     

Determination of the complexing properties of drinking waters toward copper(II) and aluminium(III) by ligand titration

The complexing capacity of some drinking waters for aluminium(III) and copper(II) is determined by a ligand titration with metal ions based on the use of complexing resins. The resins used in the titration are the iminodiacetic resin Chelex 100, the carboxylic resin Amberlite CG50 and the anionic exchange resin AG1X8. They allow the detection of ligands forming complexes of different stability with the metal ions used for the titration, since they have different sorbing properties. After equilibration with the resin, the concentration of the free metal ion in solution is evaluated from the concentration of sorbed metal ion and from the quantity K^*, which is the ratio of the concentration of the metal ion sorbed on the resin to the free metal ion in solution. It strongly depends on the conditions, but it can be evaluated, at the considered conditions, from the sorption equilibria of the metal ion on the resin. The concentration of the ligands in solution and the conditional stability constant are obtained from the Ruzik linearization procedure. Very strong ligands of copper(II) and aluminium(III) were detected in a tap water sample at concentrations ranging from 10⁻⁷ to 10⁻⁶ mol kg⁻¹, and forming complexes having conditional complexation constants K_cI=2.3×10¹⁷ (pH=6.77) and 4.5×10¹⁶ (pH=6.24), respectively, for copper(II) and aluminium(III). Weaker ligands were detected using the less strongly sorbing resins Amberlite CG50 and AG1X8, but at a concentration equal to that of the strong ligands. This was ascribed to the presence of competing metals in solution, not sorbed by the weak resins. Two other drinking waters had completely different complexing properties both towards copper(II) and aluminium(III), containing much weaker ligands.     

The use of hydrobia jenkinsi to detect intermittent toxic discharges to a river

Heavy metals and dieldrin concentrations were measured during 1976 and 1977 in the River Holme and some of its tributaries, West Yorkshire, England. The concentration of dieldrin and certain heavy metals were thought to be high enough to have a biological effect. The survival of the operculate gastropod H. jenkinsi in simulated River Holme water artificially spiked with dieldrin and heavy metals was assessed. The toxic responses noted during these experiments correlated closely with the response found when H. jenkinsi was exposed to collected polluted river samples. In situ toxicity of caged H, jenkinsi sited below pollutant discharges appeared to be capable of detecting intermittent discharges contaminated with certain heavy metals notably copper and hexavalent chromium. Deaths in situ could not, however, be solely attributed to these poisons. The lack of toxic response to dieldrin at concentrations in excess of 30,000 ng 1⁻¹ suggests a dual indicator system using H. jenkinsi and an organism suscpetible to pesticides would be needed to monitor pollution in the River Holme. This aspect is briefly discussed.