Acute and chronic toxicity of nickel on freshwater and marine tropical aquatic organisms.

Water quality guidelines and ecological risk assessment of chemical substances like nickel (Ni) in tropical regions such as South East Asia and Melanesia are often based on temperate information as a result of fewer Ni ecotoxicity data available for tropical species. This leaves an unknown margin of uncertainty in the risk assessment in the tropics. In order to fill this data gap, this study was designed to conduct standard toxicity tests on Ni with two freshwater species (acute tests) and three marine species (acute and chronic tests) originated from tropical Hong Kong. All tests were carried out using measured concentrations of Ni with control mortality below 15%. The median lethal concentrations (LC50s) were determined as 2520 (95% confidence interval: 2210, 2860) and 426 (351, 515) µg Ni L-1 for the freshwater gastropods Pomacea lineata (48 h) and Sulcospira hainanensis (96 h), respectively, while 96 h LC50s of 4300 (3610, 5090), 18,200 (6470, 51,200), 62,400 (56,800, 68,500), and 71,700 (68,200, 75,400) µg Ni L-1 were derived for the marine copepod Tigriopus japonicus, the gastropod Monodonta labio, juvenile and adult of the marine fish Oryzias melastigma, respectively. The chronic effect concentration of 10% (EC10) based on the intrinsic rate of increase of the population of T. japonicus was 29 (12, 69) µg Ni L-1. In terms of growth inhibition, the chronic EC10 for M. labio was 34 (17, 67) µg Ni L-1. The results also indicated that T. japonicus in maturation stage (LC10: 484 (349, 919) µg Ni L-1) was less sensitive than its nauplii stage (LC10: 44 (27, 72) µg Ni L-1). This study represents an important addition of high-quality toxicity data to the tropical Ni toxicity database which can be used for future ecological risk assessment of Ni and derivation of its water quality guidelines in tropical regions.

The use of immobilised metal affinity chromatography (IMAC) to compare expression of copper-binding proteins in control and copper-exposed marine microalgae.

Toxicity of metals to aquatic organisms is dependent on both external factors, such as exposure concentration and water quality parameters, and intracellular processes including specific metal-binding sites and detoxification. Current models used to predict copper toxicity in microalgae do not adequately consider these intracellular processes. This study compared the copper-binding proteins from four species of marine microalgae, Dunaliella tertiolecta, Tetraselmis sp., Phaedactylum tricornutum and Ceratoneis closterium, in controls (no added copper) and following a 72-h exposure to copper (sufficient to inhibit growth by approximately 50%). Cells were lysed by sonication, which was optimised to obtain 54-94% cell rupture for the different algae. Cell lysates were processed by immobilised metal affinity chromatography (IMAC) using Cu(2+) as the bound metal (i.e. Cu-IMAC). Bound proteins were subsequently analysed by SDS-PAGE, comparing proteins recovered from algae that were exposed to copper versus untreated control cells. Individual proteins for which copper exposure resulted in changes to proteins present were excised from gels and further analysed by nano LC ESI-MS/MS; proteins were identified using the Mascot database. Proteins identified in this way included heat-shock proteins, rubisco, α- and ß-tubulins and ATP synthase (ß subunit). The results established that Cu-IMAC is a useful approach to identify proteins involved in copper binding in algae. This study identified several proteins that may play an active role in responses to copper toxicity in marine microalgae.

Markers of intestinal inflammation, not bacterial burden, correlate with clinical outcomes in Clostridium difficile infection.

BACKGROUND: Clostridium difficile is a leading hospital-acquired infection. Many patients remain symptomatic for several days on appropriate antibiotic therapy. To assess the contribution of ongoing infection vs persistent inflammation, we examined the correlation between fecal cytokine levels, fecal C. difficile burden, and disease outcomes in C. difficile infection (CDI). METHODS: We conducted a prospective cohort study in Barnes Jewish Hospital between June 2011 and May 2012 of hospitalized adults with CDI. We determined fecal interleukin 8 (IL-8) and lactoferrin protein concentrations by enzyme immunoassay. We used real-time polymerase chain reaction (PCR) to measure relative fecal IL-8 and CXCL-5 RNA transcript abundances, and quantitative PCR to enumerate C. difficile burden. RESULTS: Of 120 study subjects, 101 (84%) were started on metronidazole, and 33 of those (33%) were subsequently given vancomycin. Sixty-two (52%) patients had diarrhea persistent for 5 or more days after starting CDI therapy. Initial fecal CXCL-5 messenger RNA (mRNA), IL-8 mRNA, and IL-8 protein correlated with persistent diarrhea and use of vancomycin. Time to diarrhea resolution was longer in patients with elevated fecal cytokines at diagnosis. Fecal cytokines were more sensitive than clinical severity scores in identifying patients at risk of treatment failure. Clostridium difficile burden did not correlate with any measure of illness or outcome at any point, and decreased equally with metronidazole and vancomycin. CONCLUSIONS: Persistent diarrhea in CDI correlates with intestinal inflammation and not fecal pathogen burden. These findings suggest that modulation of host response, rather than adjustments to antimicrobial regimens, might be a more effective approach to patients with unremitting disease.

Intestinal inflammatory biomarkers and outcome in pediatric Clostridium difficile infections.

OBJECTIVES: To identify specific fecal biomarkers for symptomatic Clostridium difficile infection and predictors of poor outcomes. STUDY DESIGN: We enrolled 65 children with positive C difficile testing (cases) and 37 symptomatic controls. We also analyzed stool samples from colonized and non-colonized asymptomatic children. We performed enzyme immunoassays to determine fecal interleukin (IL)-8, lactoferrin, and phosphorylated-p38 protein concentrations, and quantitative polymerase chain reaction to determine IL-8 and chemokine ligand (CXCL)-5 RNA relative transcript abundances, and C difficile bacterial burden. RESULTS: Of 68 asymptomatic controls, 16 were colonized with C difficile. Phosphorylated-p38 was specific for C difficile infection but lacked sensitivity. Fecal cytokines were elevated in samples from symptomatic children, whether cases or controls. In children with C difficile infection, fecal CXCL-5 and IL-8 messenger RNA abundances at diagnosis correlated with persistent diarrhea after 5 days of C difficile infection therapy and with treatment with vancomycin. When children with concomitant viral gastroenteritis were excluded, these correlations persisted. Time-to-diarrhea resolution was significantly longer in patients with elevated fecal cytokines at diagnosis. A logistic regression model identified high CXCL-5 messenger RNA abundance as the only predictor of persistent diarrhea. Conversely, fecal C difficile bacterial burden was not different in symptomatic and asymptomatic children and did not correlate with any clinical outcome measure. CONCLUSIONS: Fecal inflammatory cytokines may be useful in distinguishing C difficile colonization from disease and identifying children with C difficile infection likely to have prolonged diarrhea.

Viral co-infections are common and are associated with higher bacterial burden in children with clostridium difficile infection.

Clostridium difficile infections in children are increasing. In this cohort study, we enrolled 62 children with diarrhea and C difficile. We performed polymerase chain reaction assays to detect viral agents of gastroenteritis and quantify C difficile burden. Fifteen (24%) children diagnosed as having C difficile infection had a concomitant viral co-infection. These patients tended to be younger and had a higher C difficile bacterial burden than children with no viral co-infections (median difference = 565,957 cfu/mL; P = 0.011), but were clinically indistinguishable. The contribution of viral co-infection to C difficile disease in children warrants future investigation.

Applicability of Chronic Multiple Linear Regression Models for Predicting Zinc Toxicity in Australian and New Zealand Freshwaters.

Bioavailability models, for example, multiple linear regressions (MLRs) of water quality parameters, are increasingly being used to develop bioavailability-based water quality criteria for metals. However, models developed for the Northern Hemisphere cannot be adopted for Australia and New Zealand without first validating them against local species and local water chemistry characteristics. We investigated the applicability of zinc chronic bioavailability models to predict toxicity in a range of uncontaminated natural waters in Australia and New Zealand. Water chemistry data were compiled to guide a selection of waters with different zinc toxicity-modifying factors. Predicted toxicities using several bioavailability models were compared with observed chronic toxicities for the green alga Raphidocelis subcapitata and the native cladocerans Ceriodaphnia cf. dubia and Daphnia thomsoni. The most sensitive species to zinc in five New Zealand freshwaters was R. subcapitata (72-h growth rate), with toxicity ameliorated by high dissolved organic carbon (DOC) or low pH, and hardness having a minimal influence. Zinc toxicity to D. thomsoni (reproduction) was ameliorated by both high DOC and hardness in these same waters. No single trophic level-specific effect concentration, 10% (EC10) MLR was the best predictor of chronic toxicity to the cladocerans, and MLRs based on EC10 values both over- and under-predicted zinc toxicity. The EC50 MLRs better predicted toxicities to both the Australian and New Zealand cladocerans to within a factor of 2 of the observed toxicities in most waters. These findings suggest that existing MLRs may be useful for normalizing local ecotoxicity data to derive water quality criteria for Australia and New Zealand. The final choice of models will depend on their predictive ability, level of protection, and ease of use. Environ Toxicol Chem 2023;42:2614-2629. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Sediment spiking and equilibration procedures to achieve partitioning of uranium similar to contamination in tropical wetlands near a mine site.

The derivation of sediment quality guideline values (SQGVs) presents significant challenges. Arguably the most important challenge is to conduct toxicity tests using contaminated sediments with physico-chemistry that represents real-world scenarios. We used a novel metal spiking method for an experiment that ultimately aims to derive a uranium SQGV. Two pilot studies were conducted to inform the final spiking design, i.e. percolating a uranyl sulfate solution through natural wetland sediments. An initial pilot study that used extended mixing equilibration phases produced hardened sediments not representative of natural sediments. A subsequent percolation method produced sediment with similar texture to natural sediment and was used as the method for spiking the sediments. The range of total recoverable uranium (TR-U) concentrations achieved was 8-3200 mg/kg. This reflected the concentrations found in natural wetlands and water management ponds found on a uranium mine site and was above natural levels. Dilute-acid extractable uranium (AE-U) concentrations were >80% of total concentrations, indicating that much of the uranium in the spiked sediment was labile and potentially bioavailable. The portion of TR-U extractable as AE-U was similar at the start and end of the 4.5-month field-deployment. Porewater uranium (PW-U) analyses indicated that partition coefficients (Kd) were 2000-20,000 L/kg, and PW-U was greater in post- than pre-field-deployed samples when TR-U was ≤1500 mg/kg, indicating the binding became weaker during the field-deployment period. At higher spiked-U concentrations, the PW-U was lower post-field-deployment. Comparing the physico-chemical data of the spiked sediments with environmental monitoring data from sediments in the vicinity of a uranium mining operation indicated that they were representative of sediments contaminated by mining and that the U-spiked sediments had a clear U concentration gradient. This confirmed the suitability of the spiking procedure for preparing sediments that were suitable for deriving a SQGV for uranium.

Uptake and internalisation of copper by three marine microalgae: comparison of copper-sensitive and copper-tolerant species.

Although it has been well established that different species of marine algae have different sensitivities to metals, our understanding of the physiological and biochemical basis for these differences is limited. This study investigated copper adsorption and internalisation in three algal species with differing sensitivities to copper. The diatom Phaeodactylum tricornutum was particularly sensitive to copper, with a 72-h IC50 (concentration of copper to inhibit growth rate by 50%) of 8.0 microg Cu L(-1), compared to the green algae Tetraselmis sp. (72-h IC50 47 microg Cu L(-1)) and Dunaliella tertiolecta (72-h IC50 530 microg Cu L(-1)). At these IC50 concentrations, Tetraselmis sp. had much higher intracellular copper (1.97+/-0.01 x 10(-13)g Cu cell(-1)) than P. tricornutum (0.23+/-0.19 x 10(-13)g Cu cell(-1)) and D. tertiolecta (0.59+/-0.05 x 10(-13)g Cu cell(-1)), suggesting that Tetraselmis sp. effectively detoxifies copper within the cell. By contrast, at the same external copper concentration (50 microg L(-1)), D. tertiolecta appears to better exclude copper than Tetraselmis sp. by having a slower copper internalisation rate and lower internal copper concentrations at equivalent extracellular concentrations. The results suggest that the use of internal copper concentrations and net uptake rates alone cannot explain differences in species-sensitivity for different algal species. Model prediction of copper toxicity to marine biota and understanding fundamental differences in species-sensitivity will require, not just an understanding of water quality parameters and copper-cell binding, but also further knowledge of cellular detoxification mechanisms.

Growth-inhibiting effects of 12 antibacterial agents and their mixtures on the freshwater microalga Pseudokirchneriella subcapitata.

The growth-inhibiting and binary joint effects of 12 antibacterial agents on the freshwater green alga Pseudokirchneriella subcapitata (Korschikov) Hindák were investigated over 72-h exposures. The toxicity values (the median inhibitory concentration value, in micromoles) in decreasing order of sensitivity were triclosan (0.0018)>triclocarban (0.054)>roxithromycin (0.056)>clarithromycin (0.062)>tylosin (0.20)>tetracycline (2.25)>chlortetracycline (3.49)>norfloxacin (5.64)>sulfamethoxazole (7.50)>ciprofloxacin (20.22)>sulfamethazine (31.26)>trimethoprim (137.78). Several of these antibacterial compounds would be toxic at the micrograms per liter concentrations reported in surface waters and sewage effluents. Simple additive effects were observed in binary mixtures of sulfonamides, and most tylosin, triclosan, or triclocarban combinations. Potentially synergistic effects were observed in binary mixtures of the same class, such as macrolides, tetracyclines, and fluoroquinolones, as well as in some combined drugs, such as trimethoprim and sulfonamides or tylosin and tetracyclines. Potentially antagonistic effects were only observed between tylosin and triclocarban, triclosan and norfloxacin, and triclocarban and norfloxacin. Although present at low concentrations in the aquatic environment, mixtures of these antibacterial agents can potentially affect algal growth in freshwater systems due to their combined action.

Toxicity of a secondary-treated sewage effluent to marine biota in Bass Strait, Australia: development of action trigger values for a toxicity monitoring program.

Melbourne Water's Eastern Treatment Plant (ETP) produces a secondary-treated sewage effluent which is chlorinated and discharged into Bass Strait at Boags Rocks, Victoria, Australia. Disappearance of the sensitive brown seaweed Hormosira banksii from rock platforms immediately adjacent to the shore-line discharge was identified in the early 1990s. Subsequently, Melbourne Water and CSIRO undertook an environmental impact assessment and review of land and marine effluent disposal options, which included ambient water quality monitoring, biological monitoring, bioaccumulation studies and toxicity testing of existing effluent to assess the nature and magnitude of the environmental effects. This paper presents data from the toxicity monitoring programs since 2001. Chronic toxicity testing using macroalgal germination and cell division (H. banksii), microalgal growth rate (Nitzschia closterium) and scallop larval development (Chlamys asperrima), confirmed that ammonia was the major cause of effluent toxicity. Results from this toxicity monitoring program were used to develop action trigger values for toxicity for each species, which were then used in a refined monitoring program in 2005-2007. An upgrade of the ETP is in progress to improve nitrification/denitrification in order to reduce ammonia concentrations and the toxicity of the effluent. Toxicity testing with a simulated upgraded effluent confirmed that ammonia concentrations and toxicity were reduced. Estimated "safe" dilutions of effluent, calculated using species sensitivity distributions, decreased from 1:140-300 for existing ETP effluent to 1:20 for nitrified effluent, further confirming that treatment improvements should reduce the impact on marine biota in the vicinity of the discharge.

Toxicity of nickel to tropical freshwater and sediment biota: A critical literature review and gap analysis.

More than two-thirds of the world's nickel (Ni) lateritic deposits are in tropical regions, and just less than half are within South East Asia and Melanesia (SEAM). With increasing Ni mining and processing in SEAM, environmental risk assessment tools are required to ensure sustainable development. Currently, there are no tropical-specific water or sediment quality guideline values for Ni, and the appropriateness of applying guideline values derived for temperate systems (e.g., Europe) to tropical ecosystems is unknown. Databases of Ni toxicity and toxicity tests for tropical freshwater and sediment species were compiled. Nickel toxicity data were ranked, using a quality assessment, identifying data to potentially use to derive tropical-specific Ni guideline values. There were no data for Ni toxicity in tropical freshwater sediments. For tropical freshwaters, of 163 Ni toxicity values for 40 different species, high-quality chronic data, based on measured Ni concentrations, were found for just 4 species (1 microalga, 2 macrophytes, and 1 cnidarian), all of which were relevant to SEAM. These data were insufficient to calculate tropical-specific guideline values for long-term aquatic ecosystem protection in tropical regions. For derivation of high-reliability tropical- or SEAM-specific water and sediment quality guideline values, additional research effort is required. Using gap analysis, we recommend how research gaps could be filled. Environ Toxicol Chem 2018;37:293-317. © 2017 SETAC.

Sensitivity of marine microalgae to copper: the effect of biotic factors on copper adsorption and toxicity.

Microalgae are sensitive indicators of environmental change and, as the basis of most freshwater and marine ecosystems, are widely used in the assessment of risk and development of environmental regulations for metals. However, interspecies differences in sensitivity to metals are not well understood. The relationship between metal-algal cell binding and copper sensitivity of marine microalgae was investigated using a series of 72-h growth-rate inhibition bioassays and short-term (1-h) uptake studies. A range of marine algae from different taxonomic groups were screened to determine whether copper adsorption to the cell membrane was influenced by biotic factors, such as the ultrastructure of cell walls and cell size. Minutocellus polymorphus was the most sensitive species to copper and Dunaliella tertiolecta the least sensitive, with 72-h IC50 values (concentration to inhibit growth-rate by 50%) of 0.6 and 530 microg Cu/L, respectively. Copper solution-cell partition coefficients at equilibrium (K(d)) were calculated for six species of algae on a per cell and surface area basis. The largest and smallest cells had the lowest and highest K(d) values, respectively (on a surface area basis), with a general (non-linear) trend of decreasing K(d) with increasing cell surface area (p=0.026), however, no relationship was found between K(d) and copper sensitivity, nor cell size and copper sensitivity. Interspecies differences in copper sensitivity were not related to cell size, cell wall type, taxonomic group or K(d) values. The differences in sensitivity may be due to differences in uptake rates across the plasma membrane, in internal binding mechanisms and/or detoxification mechanisms between the different microalgal species.

Spectators or participants: How can SETAC become more engaged in international climate change research programs?

Environmental toxicologists and chemists have been crucial to evaluating the chemical fate and toxicological effects of environmental contaminants, including chlorinated pesticides, before and after Rachel Carson's publication of Silent Spring in 1962. Like chlorinated pesticides previously, global climate change is widely considered to be one of the most important environmental challenges of our time. Over the past 30 yr, climate scientists and modelers have shown that greenhouse gases such as CO2 and CH4 cause radiative forcing (climate forcing) and lead to increased global temperatures. Despite significant climate change research efforts worldwide, the climate science community has overlooked potential problems associated with chemical contaminants, in particular how climate change could magnify the ecological consequences of their use and disposal. It is conceivable that the impacts of legacy or new chemical contaminants on wildlife and humans may be exacerbated when climate changes, especially if global temperatures rise as predicted. This lack of attention to chemical contaminants represents an opportunity for environmental toxicologists and chemists to become part of the global research program, and our objective is to highlight the importance of and ways for that to occur. Environ Toxicol Chem 2017;36:1971-1977. © 2017 SETAC.

A review of nickel toxicity to marine and estuarine tropical biota with particular reference to the South East Asian and Melanesian region.

The South East Asian Melanesian (SEAM) region contains the world's largest deposits of nickel lateritic ores. Environmental impacts may occur if mining operations are not adequately managed. Effects data for tropical ecosystems are required to assess risks of contaminant exposure and to derive water quality guidelines (WQG) to manage these risks. Currently, risk assessment tools and WQGs for the tropics are limited due to the sparse research on how contaminants impact tropical biota. As part of a larger project to develop appropriate risk assessment tools to ensure sustainable nickel production in SEAM, nickel effects data were required. The aim of this review was to compile data on the effects of nickel on tropical marine, estuarine, pelagic and benthic species, with a particular focus on SEAM. There were limited high quality chronic nickel toxicity data for tropical marine species, and even fewer for those relevant to SEAM. Of the data available, the most sensitive SEAM species to nickel were a sea urchin, copepod and anemone. There is a significant lack of high quality chronic data for several ecologically important taxonomic groups including cnidarians, molluscs, crustaceans, echinoderms, macroalgae and fish. No high quality chronic nickel toxicity data were available for estuarine waters or marine and estuarine sediments. The very sparse toxicity data for tropical species limits our ability to conduct robust ecological risk assessment and may require additional data generation or read-across from similar species in other databases (e.g. temperate) to fill data gaps. Recommendations on testing priorities to fill these data gaps are presented.

Toxicity, biotransformation, and mode of action of arsenic in two freshwater microalgae (Chlorella sp. and Monoraphidium arcuatum).

The toxicity of As(V) and As(III) to two axenic tropical freshwater microalgae, Chlorella sp. and Monoraphidium arcuatum, was determined using 72-h growth rate-inhibition bioassays. Both organisms were tolerant to As(III) (72-h concentration to cause 50% inhibition of growth rate [IC50], of 25 and 15 mg As[III]/L, respectively). Chlorella sp. also was tolerant to As(V) with no effect on growth rate over 72 h at concentrations up to 0.8 mg/L (72-h IC50 of 25 mg As[V]/L). Monoraphidium arcuatum was more sensitive to As(V) (72-h IC50 of 0.25 mg As[V]/L). An increase in phosphate in the growth medium (0.15-1.5 mg PO4(3-)/L) decreased toxicity, i.e., the 72-h IC50 value for M. arcuatum increased from 0.25 mg As(V)/L to 4.5 mg As(V)/L, while extracellular As and intracellular As decreased, indicating competition between arsenate and phosphate for cellular uptake. Both microalgae reduced As(V) to As(III) in the cell, with further biological transformation to methylated species (monomethyl arsonic acid and dimethyl arsinic acid) and phosphate arsenoriboside. Less than 0.01% of added As(V) was incorporated into algal cells, suggesting that bioaccumulation and subsequent methylation was not the primary mode of detoxification. When exposed to As(V), both species reduced As(V) to As(III); however, only M. arcuatum excreted As(III) into solution. Intracellular arsenic reduction may be coupled to thiol oxidation in both species. Arsenic toxicity most likely was due to arsenite accumulation in the cell, when the ability to excrete and/or methylate arsenite was overwhelmed at high arsenic concentrations. Arsenite may bind to intracellular thiols, such as glutathione, potentially disrupting the ratio of reduced to oxidized glutathione and, consequently, inhibiting cell division.

Applications of flow cytometry to ecotoxicity testing using microalgae.

Flow cytometry is a rapid method for the quantitative measurement of light scattering and fluorescent properties of cells. Although this technique has been widely applied to biomedical and environmental studies, its potential as a tool in ecotoxicological studies has not yet been fully exploited. This article describes the application of flow cytometry to the development of bioassays with marine and freshwater algae for assessing the bioavailability of contaminants in waters and sediments.

The effect of water hardness on the toxicity of uranium to a tropical freshwater alga Chlorella sp.

Uranium (U) derived from mining activities is of potential ecotoxicological concern to freshwater biota in tropical northern Australia. Few data are available on the effects of water hardness (Ca and/or Mg), which is elevated in U mine wastewaters, on the toxicity and bioavailability of U to freshwater biota, particularly algae. This study determined the effect of water hardness (8, 40, 100 and 400 mg CaCO(3) x l(-1), added as calcium (Ca) and magnesium (Mg) sulphate) on the toxicity (72 h growth rate inhibition) of U to the unicellular green alga, Chlorella sp., in synthetic freshwater, at constant pH (7.0) and alkalinity (8 mg CaCO(3) x l(-1)), similar in chemical composition to sandy coastal streams in tropical northern Australia. A 50-fold increase in water hardness resulted in a 5-fold decrease (P0.05) differences in the predicted speciation (% distribution) of U amongst the four water hardness levels. The reduction in U toxicity with increasing water hardness was most likely due to competition between U and Ca and/or Mg for binding sites on the algal cell surface. The minimum detectable effect concentrations of U were approximately 3 and 24 times higher (at 8 and 400 mg CaCO(3)x l(-1) hardness, respectively) than the national interim U guideline value (0.5 micro g x l(-1)) for protecting aquatic ecosystems. Overall, the results reinforce the need for a more flexible U guideline based on a hardness-dependent algorithm, which may allow environmental managers to relax the national guideline for U on a site-specific basis.

Development of a whole-sediment toxicity test using a benthic marine microalga.

An acute whole-sediment toxicity test with a benthic marine microalga was developed and optimized using flow cytometry to distinguish algae (based on their chlorophyll a autofluorescence) from sediment particles. Of seven benthic marine algae screened, the diatom Entomoneis cf punctulata was most suitable because of its tolerance of a wide range of water and sediment physicochemical parameters, including salinity, pH, ammonia, and sulfide. A whole-sediment and water-only toxicity test based on inhibition of esterase activity in this species was developed. Enzyme activity rather than growth was used as the test endpoint, as nutrient release from sediments has previously been found to stimulate algal growth, potentially masking contaminant toxicity. The sensitivity of the bioassay to a range of metals (copper, zinc, cadmium, lead, arsenic, manganese) and phenol in water-only exposures was compared to the standard 72-h growth rate inhibition test. The esterase enzyme inhibition test was sensitive to copper, with a 3-h inhibitory concentration to cause a 50% (IC50) reduction in a fluorescein diacetate fluorescence value of 97 +/- 39 microg Cu/L. A concentration-dependent response was also observed in the presence of sediment particles (copper tailings), with and without dilution, using a control clean sediment. The primary route of exposure to copper was via pore water rather than by direct contact with tailings particles. This is the first whole-sediment bioassay developed with a benthic alga suitable for sediment quality assessment in marine/estuarine systems, and its advantages and limitations are discussed.

Development of multispecies algal bioassays using flow cytometry.

Multispecies algal bioassays, suitable for assessing copper toxicity, were developed with three marine (Micromonas pusilla, Phaeodactylum tricornutum, and Heterocapsa niei) and three freshwater (Microcystis aeruginosa, Pseudokirchneriella subcapitata, and Trachelomonas sp.) microalgae. Flow cytometry was used to separate and count algal signals based on pigment fluorescence and cell size. Species were mixed together on the basis of equivalent surface areas to avoid the confounding effect on toxicity of increased biomass for metal binding. Under control conditions (no added copper), M. pusilla growth was inhibited in the presence of the other marine microalgae compared to single-species tests, while the opposite was true (i.e., growth stimulation) for M. aeruginosa and P. subcapitata in freshwater mixtures. Competition for nutrients, including CO2, and algal exudate production may account for these effects. Interactions between microalgal species also had a significant effect on copper toxicity to some species. In freshwater multispecies bioassays, the toxicity of copper to Trachelomonas sp. was greater in the presence of other species, with copper concentrations required to inhibit growth (cell division) rate by 50% (72-h [IC50]) decreasing from 9.8 to 2.8 microg Cu/L in single- and multispecies bioassays, respectively. In contrast, in marine multispecies bioassays, copper toxicity to the marine diatom P. tricornutum was reduced compared to single-species bioassays, with an increase in the 72-h IC50 value from 13 to 24 microg Cu/L. This reduction in copper toxicity was not explained by differences in the copper complexing capacity in solution (as a result of exudate production) because labile copper, measured by anodic stripping voltammetry, was similar for P. tricornutum alone and in the mixture. These results demonstrate that single-species bioassays may over- or underestimate metal toxicity in natural waters.

Toxicity of metal mixtures to a tropical freshwater alga (Chlorella sp): the effect of interactions between copper, cadmium, and zinc on metal cell binding and uptake.

The individual and combined effects of copper, cadmium, and zinc on the cell division rate of the tropical freshwater alga Chlorella sp. were determined over 48 to 72 h. Metal mixtures were prepared based on multiples of their single-metal median effective concentration (EC50) values, i.e., toxic units (TU) using a triangular mixture design with five toxicant levels (0, 0.75, 1.0, 1.25, and 1.5 TU). Single-metal EC50 values after a 72-h exposure were 0.11, 0.85, and 1.4 microM for copper, cadmium, and zinc, respectively. Significant interactions were observed for all metal combinations after 48 and 72 h. An equitoxic mixture of Cu + Cd was more than concentration additive (synergistic) to the growth of Chlorella sp., while combinations of Cu + Zn, Cd + Zn, and Cu + Cd + Zn were all less than concentration additive or were antagonistic. To determine the effect of each metal on the uptake of the other, extracellular (membrane-bound) and intracellular metal concentrations, both alone and in mixtures, were compared. The increased growth inhibition observed for mixtures of Cu + Cd was due to higher concentrations of cell-bound and intracellular copper in the presence of cadmium compared with copper alone (i.e., cadmium-enhanced copper uptake). In contrast, both extra- and intracellular cadmium concentrations were reduced in the presence of copper. In mixtures of Cu + Zn, copper also inhibited the binding and cellular uptake of zinc, which resulted in decreased toxicity. Zinc had no appreciable effect on the uptake of copper by Chlorella sp. Our results suggest that all three metals share some common uptake and transport sites on Chlorella cells and that copper out competes both cadmium and zinc for cell binding. Determination of metal cell distribution coefficients (K(d)) confirmed that K(d) values for cadmium and zinc in single-metal exposures decreased in the presence of copper.