Copper sulphate and DCMU-herbicide treatments increase asymmetry between sister cells in the toxic cyanobacteria Microcystis aeruginosa: implications for detecting environmental stress

Works correlating fluctuating asymmetry with environmental stress or genetic damages have been largely reported in multicellular organisms but not in single-celled ones. We hypothesize that asymmetry analysis could also be applied to single-celled organisms, because the asymmetry between two sister cells originated from a cellular division event (same genotype in similar environment) must tend to zero in the absence of environmental or genetic perturbations. Laboratory experiments with copper sulphate and DCMU-herbicide treatments as well as experiments in a water reservoir after treatment with copper sulphate algaecide show that environmental stress increases asymmetry between sister cells of Microcystis aeruginosa (Cyanobacteria). Even low Cu(2+) or DCMU doses, which were unable to reduce growth rate, considerably enlarge asymmetry with respect to untreated controls. Asymmetry between sister cells of cyanobacteria seems to be a reliable indicator of environmental perturbation. Analysis of asymmetry in single-celled organisms could become as important as fluctuating asymmetry of multicellular organisms is today.     

Evaluation of the mechanisms of the effect of ultrasound on Microcystis aeruginosa at different ultrasonic frequencies

Blooms of cyanobacteria are now considered to be a common environmental issue. They are hazardous to both domestic and wild animals and humans. Current treatments are unable to effectively control such blooms as they become tolerant to biocides and it is difficult to degrade cyanobacterial toxins in water. Alternative methods for control are currently under investigation. One potential effective method is ultrasonic irradiation. Ultrasound inactivates algal and cyanobacteria cells through cavitation by generating extreme conditions, resulting in a number of physical, mechanical and chemical effects. The aim of this study was to investigate the effect of ultrasound at different frequencies on Microcystis aeruginosa. Flow cytometry was used to measure cyanobacterial metabolic cell viability in addition to the more commonly used haemocytometry, optical density and fluorimetry. Results indicate low frequency 20 kHz ultrasound with high intensity (0.0403 W cm⁻³) is effective for the inactivation of cyanobacterial cells. Higher frequencies of 580 kHz (0.0041 W cm⁻³) also resulted in an inactivation effect, but 1146 kHz (0.0018 W cm⁻³) showed a declumping effect as evidenced by flow cytometry. Ultrasonic treatment over time under different sonication conditions demonstrates the following:1. Acoustic cavitation via mechanical effects can induce sufficient shear forces to directly rupture cyanobacteria cells.2. At higher ultrasonic frequencies the mechanical energy of cavitation is less but a larger proportion of free radicals are produced from the ultrasonic degradation of water, which chemically attacks and weakens the cyanobacteria cell walls.3. At higher frequencies free radicals also damage chlorophyll a leading to a loss in photosynthetic cell viability.4. At low powers ultrasonic energy results in declumping of cyanobacteria.     

Evaluating the effectiveness of copper sulphate,chlorine, potassium permanganate,hydrogen peroxide and ozone on cyanobacterial cell integrity

Cyanobacterial blooms are continuously critical challenges in drinking water systems which can have various negative impacts such as production of taste, odour and toxic compounds. Furthermore, the intracellular metabolites could be released into surrounding waters when the cyanobacterial membranes are destroyed. Although a variety of techniques have been developed to control cyanobacterial blooms and remove cyanobacterial cells or metabolites in water treatment processes, the effect of these treatments on the membrane integrity of cyanobacterial cells have not been systematically studied and compared. This study evaluated the effectiveness of copper sulphate (CuSO₄), chlorine, potassium permanganate (KMnO₄), hydrogen peroxide (H₂O₂) and ozone on the cell integrity and densities of Microcystis aeruginosa. All of these technologies can compromise the cell membrane of cyanobacteria to varying degrees. Chlorine showed the strongest ability to impair the cell integrity with a majority (≥88%) of the cells compromised within the first minute and with the cell lysis rates ranging of 0.640–3.82 h⁻¹ during 1–60 min. Ozone dose of 6 mg L⁻¹ also could induce 90% lysis of the cyanobacterial cells in 5 min and the cell lysis rate of KMnO₄ (10 mg L⁻¹) was 0.829 h⁻¹. CuSO₄ and H₂O₂ could not only destroy the viability of cyanobacterial cells but also showed algistatic potential over the 7 day treatment. The potential of all the oxidants (chlorine, KMnO₄, H₂O₂ and ozone) considered as algicides were discussed in this study. The benefits and drawbacks of these control and water treatment options were assessed as well.     

Studies on the toxicity of copper sulphate to stone loach Noemacheilus barbatulus (L.) in hard water

The toxicity of copper sulphate to Noemacheilus barbatulus was studied for 64 days in a water of total hardness 249 mg l⁻¹ as CaCO₃. The 63-day lc₅₀ was approximately 0.25 mg Cu l⁻¹. Larger fish survived longer, and at concentrations greater than 0.29 mg l⁻¹ fish hid less during daylight. Noemacheilus surviving 0.12 mg Cu l⁻¹ for 64 days shed copper when placed in clean water for 7 days: gill, muscle, eye and vertebrae lost significant amounts of copper during this period. The opportunity to shed copper during a short period when the poison supply to their tank failed, may have allowed fish exposed to 0.49 mg l⁻¹ to live 12 days longer than expected. The sensitivity of Noemacheilus to copper, cadmium and zinc was compared with that of rainbow trout. Salmo gairdneri.     

Characterization of copper bioreduction and biosorption by a highly copper resistant bacterium isolated from copper-contaminated vineyard soil

Copper is an essential but toxic heavy metal that negatively impacts living systems at high concentration. This study presents factors affecting copper bioremoval (bioreduction and biosorption) by a highly copper resistant monoculture of Pseudomonas sp. NA and copper bioremoval from soil. Seven bacteria resistant to high concentration of Cu(II) were isolated from enrichment cultures of vineyard soils and mining wastes. Culture parameters influencing copper bioreduction and biosorption by one monoculture isolate were studied. The isolate was identified by 16S rRNA gene sequence analysis as a Pseudomonas sp. NA (98% similarity to Pseudomonas putida, Pseudomonas plecoglossicida and other Pseudomonas sp.). The optimal temperature for growth was 30 °C and bioremoval of Cu(II) was maximal at 35 °C. Considerable growth of the isolate was observed between pH 5.0 and 8.0 with the highest growth and biosorption recorded at pH 6.0. Maximal bioreduction was observed at pH 5.0. Cu(II) bioremoval was directly proportional to Cu(II) concentration in media. Pseudomonas sp. NA removed more than 110 mg L^− 1 Cu(II) in water within 24 h through bioreduction and biosorption at initial concentration of 300 mg L^− 1. In cultures amended with 100 mg L^− 1, 20.7 mg L^− 1 of Cu(II) was biologically reduced and more than 23 mg L^− 1 of Cu(II) was biologically removed in 12 h. The isolate strongly promoted copper bioleaching in soil. Results indicate that Pseudomonas sp. NA has good potential as an agent for removing copper from water and soil.     

Impact of toxic cyanobacteria on gastropods and microcystin accumulation in a eutrophic lake (Grand-Lieu, France) with special reference to Physa (= Physella) acuta

Hepatotoxic microcystins (MCs) produced by cyanobacteria are known to accumulate in gastropods following grazing of toxic cyanobacteria and/or absorption of MCs dissolved in water, with adverse effects on life history traits demonstrated in the laboratory. In the field, such effects may vary depending on species, according to their relative sensitivity and ecology. The aims of this study were to i) establish how various intensities of MC-producing cyanobacteria proliferations alter the structure of gastropod community and ii) compare MC tissue concentration in gastropods in the field with those obtained in our previous laboratory experiments on the prosobranch Potamopyrgus antipodarum and the pulmonate Lymnaea stagnalis. We explored these questions through a one-year field study at three stations at Grand-Lieu Lake (France) affected by different intensities of cyanobacteria proliferations. A survey of the community structure and MC content of both cyanobacteria and gastropods was associated with a caging experiment involving P. antipodarum and L. stagnalis. In total, 2592 gastropods belonging to 7 prosobranch and 16 pulmonate species were collected. However, distribution among the stations was unequal with 62% vs 2% of gastropods sampled respectively at the stations with the lowest vs highest concentrations of MC. Irrespective of the station, pulmonates were always more diverse, more abundant and occurred at higher frequencies than prosobranchs. Only the pulmonate Physa acuta occurred at all stations, with abundance and MC tissue concentration (≤ 4.32 µg g DW^− 1) depending on the degrees of MC-producing cyanobacteria proliferations in the stations; therefore, P. acuta is proposed as a potential sentinel species. The caging experiment demonstrated a higher MC accumulation in L. stagnalis (≤ 0.36 µg g DW^− 1 for 71% of individuals) than in P. antipodarum (≤ 0.02 µg g DW^− 1 for 12%), corroborating previous laboratory observations. Results are discussed in terms of differential gastropod sensitivity and MC transfer through the food web.     

Analysis of dissolved microcystins in surface water samples from Kovada Lake, Turkey

Dissolved (extracellular) microcystin (MC) concentrations were determined at 3 sampling stations on Lake Kovada, Turkey. The dominant species of cyanobacteria found in August and September of 2006 were Microcystis aeruginosa, Synechococcus sp., Phormidium limosum, Phormidium formosa and Planktothrix limnetica. MC concentrations in water were measured by ELISA and MC variants were examined by HPLC-PDA. Quantitative analysis by HPLC indicated that five MC variants (MC-LR, -RR, -LA, -LW, -LF) were identified in water samples from Kovada Lake. The maximum concentration of dissolved MC-LW was 98.9 µg l^− 1 in October. MC-LR was only detected in May at a concentration of 0.5 µg l^− 1. The cross reactivity of the antibody (MC10E7) to variants such as MC-LA MC-LW & MC-LF was low. Hence the results determined by ELISA were lower than those determined by HPLC in September and October samples due to differences in the specificity of the antibody to MC variants. Total extraceullar MCs was quantified by ELISA and ranged from 0.73 to 48.5 µg MC-LR equivalents l^− 1, which in some cases exceeded the WHO provisional Guideline Value for MC-LR in drinking water. This study confirms that the lakes of Turkey should be monitored for toxic cyanobacteria and for MCs to avoid or reduce the potential exposure of people to these health hazards.     

Simultaneous determination of microcystin contaminations in various vertebrates (fish, turtle, duck and water bird) from a large eutrophic Chinese lake, Lake Taihu, with toxic Microcystis blooms

This is the first to conduct simultaneous determination of microcystin (MC) contaminations in multi-groups of vertebrates (fish, turtle, duck and water bird) from Lake Taihu with Microcystis blooms. MCs (-RR, -YR, -LR) in Microcystis scum was 328 μg g^− 1 DW. MCs reached 235 μg g^− 1 DW in intestinal contents of phytoplanktivorous silver carp, but never exceeded 0.1 μg g^− 1 DW in intestinal contents of other animals. The highest MC content in liver of fish was in Carassius auratus (150 ng g^− 1 DW), followed by silver carp and Culter ilishaeformis, whereas the lowest was in common carp (3 ng g^− 1 DW). In livers of turtle, duck and water bird, MC content ranged from 18 to 30 ng g^− 1 DW. High MC level was found in the gonad, egg yolk and egg white of Nycticorax nycticorax and Anas platyrhynchos, suggesting the potential effect of MCs on water bird and duck embryos. High MC contents were identified for the first time in the spleens of N. nycticorax and A. platyrhynchos (6.850 and 9.462 ng g^− 1 DW, respectively), indicating a different organotropism of MCs in birds. Lakes with deaths of turtles or water birds in the literatures had a considerably higher MC content in both cyanobacteria and wildlife than Lake Taihu, indicating that toxicity of cyanobacteria may determine accumulation level of MCs and consequently fates of aquatic wildlife.     

Whole lake selective withdrawal experiment to control harmful cyanobacteria in an urban impoundment

Different environmental conditions support optimal growth by Aphanizomenon and Microcystis in Ford Lake, Michigan, USA, based on weekly species biovolume and water chemistry measurements from June through October 2005-2007. Experimental withdrawal of hypolimnetic water through the outlet dam was conducted in 2006, with 2005 and 2007 acting as control years, to test theory regarding management of nuisance and toxic cyanobacteria. The dynamics of Aphanizomenon and Microcystis blooms in Ford Lake appear to be driven largely by NO₃⁻ concentrations, with higher levels shifting the advantage to Microcystis (P < 0.0001). Aphanizomenon was most successful with a mean TN:TP ratio (mol:mol) of 48.3:1, whereas Microcystis thrived with a mean ratio of 70.1:1. Withdrawal of hypolimnetic water successfully destabilized the water column and led to higher levels of NO₃⁻ and the near elimination of the Aphanizomenon bloom in 2006 (P < 0.0001). Selective withdrawal did not reduce Microcystis biovolume or microcystin toxicity. Microcystis biovolume and NO₃⁻ levels were positively correlated with microcystin toxin (P = 0.01) and jointly accounted for 30.5% of the variability in the data. Selective withdrawal may be a viable management option for improving water quality under certain circumstances. To fully address the problem of nuisance and toxic algal blooms in Ford Lake, however, an integrated approach is required that targets cyanobacteria biovolume dynamics as well as conditions suited for toxin production.     

Earthy odor compounds production and loss in three cyanobacterial cultures

Geosmin and 2-methylisoborneol (MIB) related odor events caused by cyanobacteria have been a very common problem to water supply. This paper investigated the effects of temperature (18 and 25 °C) and light intensity (10 and 100 μmol photons m⁻² s⁻¹) on the production behaviors of earthy odor compounds by three odorous cyanobacteria, i.e., the geosmin-producing planktonic Anabaena circinalis (Ana 318), geosmin-producing benthic Phormidium amoenum (Pho 012) and MIB-producing benthic Phormidium sp. (Pho 689). At the same time, the effects of biodegradation and volatilization on the fates of the released odor compounds in water were also evaluated. The combination of high temperature (25 °C) and light intensity (100 μmol photons m⁻² s⁻¹) favored the growth of the three cyanobacteria and the production of chl-a and odor compounds. However, higher chl-a and odor yields (average odor compounds per cell) were achieved for the two benthic cyanobacteria at the temperature of 18 °C. Most of geosmin was included within the cells for Ana 318 (95-99%) and Pho 012 (85-60%), while only 20-40% MIB was bound to the cells for Pho 689. The half-life times of MIB and geosmin due to volatilization varied between 18.8 and 35.4 days, while 8 out of 10 samples exhibited a half-life time (t_1/2) for geosmin biodegradation shorter than 1 day (0.38-15.0 h), showing that biodegradation could affect the fate of geosmin significantly in aquatic environments. In comparison, biodegradation of MIB was much slower (t_1/2: 122-2166 h). Denaturing gradient gel electrophoresis (DGGE) analysis showed that Pseudomonas- and Sphingomonas-like bacteria coexisted with cyanobacteria in the cultures, and may have played an important role in geosmin/MIB biodegradation. The result of this study will be helpful for better understanding and managing the earthy odor problems caused by cyanobacteria in water supply.     

Selective suppression of harmful cyanobacteria in an entire lake with hydrogen peroxide

Although harmful cyanobacteria form a major threat to water quality, few methods exist for the rapid suppression of cyanobacterial blooms. Since laboratory studies indicated that cyanobacteria are more sensitive to hydrogen peroxide (H₂O₂) than eukaryotic phytoplankton, we tested the application of H₂O₂ in natural waters. First, we exposed water samples from a recreational lake dominated by the toxic cyanobacterium Planktothrix agardhii to dilute H₂O₂. This reduced the photosynthetic vitality by more than 70% within a few hours. Next, we installed experimental enclosures in the lake, which revealed that H₂O₂ selectively killed the cyanobacteria without major impacts on eukaryotic phytoplankton, zooplankton, or macrofauna. Based on these tests, we introduced 2 mg L⁻¹ (60 μM) of H₂O₂ homogeneously into the entire water volume of the lake with a special dispersal device, called the water harrow. The cyanobacterial population as well as the microcystin concentration collapsed by 99% within a few days. Eukaryotic phytoplankton (including green algae, cryptophytes, chrysophytes and diatoms), zooplankton and macrofauna remained largely unaffected. Following the treatment, cyanobacterial abundances remained low for 7 weeks. Based on these results, we propose the use of dilute H₂O₂ for the selective elimination of harmful cyanobacteria from recreational lakes and drinking water reservoirs, especially when immediate action is urgent and/or cyanobacterial control by reduction of eutrophication is currently not feasible. A key advantage of this method is that the added H₂O₂ degrades to water and oxygen within a few days, and thus leaves no long-term chemical traces in the environment.     

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.     

Production of Shiga-like toxins in viable but nonculturable Escherichia coli O157:H7

Escherichia coli O157:H7, a causative agent of hemolytic uremic syndrome, can enter into a viable but nonculturable (VBNC) state when under stress. To date, it is unknown whether VBNC cells produce Shiga-like toxins (Stx). To address this question, we confirmed the expression of the stx1 and stx2 genes and the production of Stx in VBNC E. coli O157:H7 cells. To quantitatively assess the production of Stx in VBNC cells, we developed a Vero-cell microplate cytotoxicity assay based on the correspondence of the cytotoxicity of VBNC cells on Vero cells to the number of inoculated VBNC cells. Using this method, we found that all VBNC cells induced by river water, PBS buffer, deionized water, or chloraminated water retained the ability to produce Stx, and that they had differing levels of Stx. Both aged (19-month-old) VBNC cells induced by river water and fresh VBNC cells induced by chloraminated water showed very low half maximal inhibitory concentration (IC₅₀; 6.6 × 10⁴ and 7.1 × 10⁴ respectively), corresponding to higher levels of toxins produced than VBNC cells induced by deionized water and PBS buffer. VBNC cells originating from different isolates may vary in Stx production, and the VBNC cells from bovine isolates produced higher levels of Stx than those from clinical isolates. These results demonstrate a potential health risk of VBNC E. coli O157:H7 in environmental water and the importance of monitoring VBNC E. coli O157:H7.     

The management of undesirable cyanobacteria blooms in channel catfish ponds using a constructed wetland: Contribution to the control of off-flavor occurrences

An exploratory study on the management of undesirable cyanobacteria blooms with respect to off-flavor problems using an integrated vertical-flow constructed wetland (CW) was performed at a small commercial-scale channel catfish farm from 2004 to 2007. The results of the three-year experiment indicated that water treatment by the CW could reduce the possibility of dominance by undesirable cyanobacteria species that often cause off-flavor problems. A detailed investigation in 2007, showed that the concentrations of geosmin, MIB (2-methylisoborneol), and β-cyclocitral in the water of the recirculating pond (4.3 ng L⁻¹, U.D. (undetected) and 0.2 ng L⁻¹, respectively) treated by the CW were significantly lower than those in the control pond (152.6 ng L⁻¹, 63.3 ng L⁻¹ and 254.8 ng L⁻¹, respectively). In addition, the relationships among the cyanobacteria species, the off-flavor compounds and ten environmental variables were explored by canonical correspondence analysis (CCA). The results showed that Oscillatoria sp., Oscillatoria kawamurae and Microcystis aeruginosa were the main sources of off-flavor compounds in the catfish ponds. The successful manipulation of undesirable cyanobacteria species potentially resulted in lower concentrations of odorous compounds in the water of the recirculating pond. An investigation of the concentrations of geosmin and MIB in catfish fillets showed that the levels of odorous compounds were below the OTC (odor threshold concentration) values in the recirculating pond but were above the OTC values from July to October in the control pond. Water recycling by the CW could potentially be one of the best management practices to control off-flavor occurrences in aquaculture.     

Controlling eutrophication by combined bloom precipitation and sediment phosphorus inactivation

The hypothesis that the combination of the flocculent polyaluminium chloride (PAC) with the lanthanum-modified bentonite Phoslock^® (Flock & Lock) could sink effectively a water bloom of cyanobacteria and could shift a turbid, cyanobacteria infested lake to a clear water lake was tested in a controlled laboratory experiment and a whole lake experiment. In the laboratory, a relatively low dose of the flocculent PAC (2.2 and 4.4 mg Al l⁻¹) was insufficient to sediment positively buoyant cyanobacteria (Microcystis aeruginosa). Similarly, the lanthanum modified clay (dosed at 390 mg l⁻¹) was insufficient to sediment the positively buoyant cyanobacteria. However, the combination of PAC and Phoslock^® effectively sedimented cyanobacteria flocks. Likewise, a combined treatment of 2 tons PAC and 18 tons Phoslock^® in Lake Rauwbraken in April 2008 effectively sedimented a developing cyanobacteria bloom of Aphanizomenon flos-aquae. The average chlorophyll-a concentration in the two years prior to this Flock & Lock treatment was 19.5 (±36.5) μg l⁻¹, while it was as low as 3.7 (±4.5) μg l⁻¹ in the years following the treatment. The combined treatment effectively reduced the amount of total phosphorus (TP) in the water column from on average 169 (±126) μg P l⁻¹ before the application to 14 (±15) μg P l⁻¹ after the treatment. Based on mean summer chlorophyll-a and TP concentrations, the lake was shifted from a eutrophic/hypertrophic state to an oligo/mesotrophic state. From directly after treatment in April 2008 until and including 2013, Lake Rauwbraken remained in an oligo-mesotrophic clear water state with TP reduced to less than 10% of the pre-treatment. This result shows that eutrophication in relatively small, isolated, stratifying lakes can be restored by targeting both water column and sediment P using a combination of flocculent and solid phase P-sorbent.     

Feeding characteristics of a golden alga (Poterioochromonas sp.) grazing on toxic cyanobacterium Microcystis aeruginosa

Microcystis aeruginosa has quickly risen in infamy as one of the most universal and toxic bloom-forming cyanobacteria. Here we presented a species of golden alga (Poterioochromonas sp. strain ZX1), which can feed on toxic M. aeruginosa without any adverse effects from the cyanotoxins. Using flow cytometry, the ingestion and maximal digestion rates were estimated to be 0.2∼1.2 and 0.2 M. aeruginosa cells (ZX1 cell)⁻¹ h⁻¹, respectively. M. aeruginosa in densities below 10⁷ cells mL⁻¹ could be grazed down by ZX1, but no significant decrease was observed when the initial density was 3.2 × 10⁷ cells mL⁻¹. ZX1 grazing was a little influenced by the light intensity (0.5∼2500 lx) and initial pH of the medium (pH = 5.0∼9.5). ZX1 could not survive in continuous darkness for longer than 10 days. The pH value was adjusted to 8 by ZX1 while to 10 by M. aeruginosa. This study may shed light on understanding the ecological interactions between M. aeruginosa and mixotrophic Poterioochromonas sp. in aquatic ecosystems.     

Determination of copper in water by means of chalcocite copper ion-selective electrode

The possibility of determination of copper in natural waters was tested by direct potentiometry with chalcocite copper sensitive ion selective electrode. This can be carried out for the copper concentration down to 6 μg l⁻¹ when the standard additions procedure is used. The electrode characteristics was investigated and as a medium was proposed a TFB solution containing Tris, potassium fluoride and potassium nitrate. However at the extreme low concentrations of copper the water samples after addition of TFB should be heated to boiling. In these conditions the accuracy of determination for the average of five measurements is not worse than 25%, and precision is of the order of 20%.     

Speciation and rate of loss of copper from lakewater with implications to toxicity

The rates of disappearance of total dissolved copper in six prairie “pothole” hard water lakes treated with copper were measured. The first-order rate constants ranged from 1.01 to 0.11 day⁻¹, and the half-times from 1 to 7 days. Concentrations of 12 inorganic copper species and the humic acid-copper complex were calculated for the pH range of each lake. Less than 0.5% of total dissolved copper was calculated to be present at any time as free cupric ion. The total dissolved concentration of added copper was reduced with time probably through precipitation of tenorite (in some cases malachite) and by adsorption on solids.The effectiveness of copper as an algicide in these lakes at the relatively low concentration of added copper is explained in terms of a “total toxic copper” concept, which includes the species Cu(OH)₂⁰ and CuOH⁺ in addition to cupric ion, the generally recognized toxic species.     

Effect of copper on nitrification in activated sludge

The concentration of free copper in activated sludge with copper added is strongly influenced by pH. For example, at pH 6.5 with 9.13 × 10⁻⁵ mol Cu l⁻¹, the free copper concentration is 4.0 × 10⁻⁷ mol l⁻¹ (pCu = 6.4) and at pH 8.4 this concentration is 10⁻⁸ mol l⁻¹ (pCu = 8.0). In both cases the activated sludge concentration is 0.7 g MLSS l⁻¹. The free copper concentration is also affected by the concentration of mixed liquor suspended solids (MLSS).In batch experiments with constant pH, the effect of copper on the nitrification rate was not regulated by total copper concentration but by copper/sludge ratio or by free copper concentrations. Experiments at different pH showed a linear correlation between nitrification capacity and free copper concentration, suggesting that the pH effect on nitrification below 8.3 is in fact a copper effect.Activated sludge with copper did not become acclimatized to the copper in a period of three days. Addition of nitrilotriacetic acid (NTA) within one day did cancel the copper inhibition.The results were compared with the effect of copper on acetate removal by heterotrophic micro-organisms. The nitrifiers proved to be no more susceptible to copper than heterotrophic micro-organisms.     

Parameters predictive of Legionella contamination in hot water systems: association with trace elements and heterotrophic plate counts

The contamination of hot water samples with Legionella spp. was studied in relation to temperature, total hardness, trace element concentrations (iron, zinc, manganese, and copper) and heterotrophic plate counts (HPC) at both 22 and 37 °C. Factor analysis and receiver operating characteristic (ROC) curves were used to establish the cut-off of water parameters as predictors for Legionella contamination. Legionella spp. was isolated in 194 out of 408 samples (47.5%), with Legionella pneumophila being the most common (92.8%). After multiple logistic regression analysis, the risk for legionellae colonisation was positively associated with Mn levels >6 μg l⁻¹, HPC at 22 °C >27 CFU l⁻¹, and negatively with temperature >55 °C and Cu levels >50 μg l⁻¹. Multiple regression analysis revealed that Legionella spp. counts were positively associated with Mn, HPC at 37 °C and Zn and negatively associated with temperature. Only 1 out of the 97 samples (1%) having a Mn concentration, an HPC at 22 °C and an HPC at 37 °C below the respective median values exhibited a Legionella spp. concentration exceeding 10⁴ CFU l⁻¹vs. 41 out of the 89 samples (46.1%) with the three parameters above the medians. Our results show a qualitative and quantitative relationship between Legionella spp., the Mn concentration and heterotrophic plate counts in hot water samples from different buildings, suggesting that these parameters should be included in a water safety plan. The role of manganese in biofilm formation and its possible involvement in the mechanisms favouring Legionella survival and growth in water niches should be investigated further.