Bioanalytical effect-balance model to determine the bioavailability of organic contaminants in sediments affected by black and natural carbon.

In sediments several binding phases dictate the fate and bioavailability of organic contaminants. Black carbon (BC) has a high sorptive capacity for organic contaminants and can limit their bioavailability, while the fraction bound to organic carbon (OC) is considered to be readily desorbable and bioavailable. We investigated the bioavailability and mixture toxicity of sediment-associated contaminants by combining different extraction techniques with in vitro bioanalytical tools. Sediments from a harbour with high fraction of BC, and sediments from remote, agricultural and urban areas with lower BC were treated with exhaustive solvent extraction, Tenax extraction and passive sampling to estimate total, bioaccessible and bioavailable fractions, respectively. The extracts were characterized with cell-based bioassays that measure dioxin-like activity (AhR-CAFLUX) and the adaptive stress response to oxidative stress (AREc32). Resulting bioanalytical equivalents, which are effect-scaled concentrations, were applied in an effect-balance model, consistent with a mass balance-partitioning model for single chemicals. Sediments containing BC had most of the bioactivity associated to the BC fraction, while the OC fraction played a role for sediments with lower BC. As effect-based sediment-water distribution ratios demonstrated, most of the bioactivity in the AhR-CAFLUX was attributable to hydrophobic chemicals while more hydrophilic chemicals activated AREc32, even though bioanalytical equivalents in the aqueous phase remained negligible. This approach can be used to understand the fate and effects of mixtures of diverse organic contaminants in sediments that would not be possible if single chemicals were targeted by chemical analysis; and make informed risk-based decisions concerning the management of contaminated sediments.

Bioanalytical evidence that chemicals in tattoo ink can induce adaptive stress responses.

Tattooing is becoming increasingly popular, particularly amongst young people. However, tattoo inks contain a complex mixture of chemical impurities that may pose a long-term risk for human health. As a first step towards the risk assessment of these complex mixtures we propose to assess the toxicological hazard potential of tattoo ink chemicals with cell-based bioassays. Targeted modes of toxic action and cellular endpoints included cytotoxicity, genotoxicity and adaptive stress response pathways. The studied tattoo inks, which were extracted with hexane as a proxy for the bioavailable fraction, caused effects in all bioassays, with the red and yellow tattoo inks having the greatest response, particularly inducing genotoxicity and oxidative stress response endpoints. Chemical analysis revealed the presence of polycyclic aromatic hydrocarbons in the tested black tattoo ink at concentrations twice the recommended level. The detected polycyclic aromatic hydrocarbons only explained 0.06% of the oxidative stress response of the black tattoo ink, thus the majority of the effect was caused by unidentified components. The study indicates that currently available tattoo inks contain components that induce adaptive stress response pathways, but to evaluate the risk to human health further work is required to understand the toxicokinetics of tattoo ink chemicals in the body.

In vitro bioassays to evaluate complex chemical mixtures in recycled water.

With burgeoning population and diminishing availability of freshwater resources, the world continues to expand the use of alternative water resources for drinking, and the quality of these sources has been a great concern for the public as well as public health professionals. In vitro bioassays are increasingly being used to enable rapid, relatively inexpensive toxicity screening that can be used in conjunction with analytical chemistry data to evaluate water quality and the effectiveness of water treatment. In this study, a comprehensive bioassay battery consisting of 36 bioassays covering 18 biological endpoints was applied to screen the bioactivity of waters of varying qualities with parallel treatments. Samples include wastewater effluent, ultraviolet light (UV) and/or ozone advanced oxidation processed (AOP) recycled water, and infiltrated recycled groundwater. Based on assay sensitivity and detection frequency in the samples, several endpoints were highlighted in the battery, including assays for genotoxicity, mutagenicity, estrogenic activity, glucocorticoid activity, arylhydrocarbon receptor activity, oxidative stress response, and cytotoxicity. Attenuation of bioactivity was found to be dependent on the treatment process and bioassay endpoint. For instance, ozone technology significantly removed oxidative stress activity, while UV based technologies were most efficient for the attenuation of glucocorticoid activity. Chlorination partially attenuated genotoxicity and greatly decreased herbicidal activity, while groundwater infiltration efficiently attenuated most of the evaluated bioactivity with the exception of genotoxicity. In some cases, bioactivity (e.g., mutagenicity, genotoxicity, and arylhydrocarbon receptor) increased following water treatment, indicating that transformation products of water treatment may be a concern. Furthermore, several types of bioassays with the same endpoint were compared in this study, which could help guide the selection of optimized methods in future studies. Overall, this research indicates that a battery of bioassays can be used to support decision-making on the application of advanced water treatment processes for removal of bioactivity.

Which chemicals drive biological effects in wastewater and recycled water?

Removal of organic micropollutants from wastewater during secondary treatment followed by reverse osmosis and UV disinfection was evaluated by a combination of four in-vitro cell-based bioassays and chemical analysis of 299 organic compounds. Concentrations detected in recycled water were below the Australian Guidelines for Water Recycling. Thus the detected chemicals were considered not to pose any health risk. The detected pesticides in the wastewater treatment plant effluent and partially advanced treated water explained all observed effects on photosynthesis inhibition. In contrast, mixture toxicity experiments with designed mixtures containing all detected chemicals at their measured concentrations demonstrated that the known chemicals explained less than 3% of the observed cytotoxicity and less than 1% of the oxidative stress response. Pesticides followed by pharmaceuticals and personal care products dominated the observed mixture effects. The detected chemicals were not related to the observed genotoxicity. The large proportion of unknown toxicity calls for effect monitoring complementary to chemical monitoring.

Bioanalytical and chemical evaluation of disinfection by-products in swimming pool water.

Pool water disinfection is vital to prevent microbial pathogens. However, potentially hazardous disinfection by-products (DBP) are formed from the reaction between disinfectants and organic/inorganic precursors. The aim of this study was to evaluate the presence of DBPs in various swimming pool types in Brisbane, Australia, including outdoor, indoor and baby pools, and the dynamics after a complete water renewal. Chemical analysis of 36 regulated and commonly found DBPs and total adsorbable organic halogens as well as in vitro bioassays targeting cytotoxicity, oxidative stress and genotoxicity were used to evaluate swimming pool water quality. Dichloroacetic acid and trichloroacetic acid dominated in the pool water samples with higher levels (up to 2600 µg/L) than the health guideline values set by the Australian Drinking Water Guidelines (100 µg/L). Chlorinated DBPs occurred at higher concentrations compared to tap water, while brominated DBPs decreased gradually with increasing pool water age. Biological effects were expressed as chloroacetic acid equivalent concentrations and compared to predicted effects from chemical analysis and biological characterisation of haloacetic acids. The quantified haloacetic acids explained 35-118% of the absorbable organic halogens but less than 4% of the observed non-specific toxicity (cytotoxicity), and less than 1% of the observed oxidative stress response and genotoxicity. While the DBP concentrations in Australian pools found in this study are not likely to cause any adverse health effect, they are higher than in other countries and could be reduced by better hygiene of pool users, such as thorough showering prior to entering the pool and avoiding urination during swimming.

Realistic environmental mixtures of micropollutants in surface, drinking, and recycled water: herbicides dominate the mixture toxicity toward algae.

Mixture toxicity studies with herbicides have focused on a few priority components that are most likely to cause environmental impacts, and experimental mixtures were often designed as equipotent mixtures; however, real-world mixtures are made up of chemicals with different modes of toxic action at arbitrary concentration ratios. The toxicological significance of environmentally realistic mixtures has only been scarcely studied. Few studies have simultaneously compared the mixture effect of water samples with designed reference mixtures comprised of the ratios of analytically detected concentrations in toxicity tests. In the present study, the authors address the effect of herbicides and other chemicals on inhibition of photosynthesis and algal growth rate. The authors tested water samples including secondary treated wastewater effluent, recycled water, drinking water, and storm water in the combined algae assay. The detected chemicals were mixed in the concentration ratios detected, and the biological effects of the water samples were compared with the designed mixtures of individual detected chemicals to quantify the fraction of effect caused by unknown chemicals. The results showed that herbicides dominated the algal toxicity in these environmentally realistic mixtures, and the contribution by the non-herbicides was negligible. A 2-stage model, which used concentration addition within the groups of herbicides and non-herbicides followed by the model of independent action to predict the mixture effect of the two groups, could predict the experimental mixture toxicity effectively, but the concentration addition model for herbicides was robust and sufficient for complex mixtures. Therefore, the authors used the bioanalytical equivalency concept to derive effect-based trigger values for algal toxicity for monitoring water quality in recycled and surface water. All water samples tested would be compliant with the proposed trigger values associated with the appropriate guidelines.

Benchmarking organic micropollutants in wastewater, recycled water and drinking water with in vitro bioassays.

Thousands of organic micropollutants and their transformation products occur in water. Although often present at low concentrations, individual compounds contribute to mixture effects. Cell-based bioassays that target health-relevant biological endpoints may therefore complement chemical analysis for water quality assessment. The objective of this study was to evaluate cell-based bioassays for their suitability to benchmark water quality and to assess efficacy of water treatment processes. The selected bioassays cover relevant steps in the toxicity pathways including induction of xenobiotic metabolism, specific and reactive modes of toxic action, activation of adaptive stress response pathways and system responses. Twenty laboratories applied 103 unique in vitro bioassays to a common set of 10 water samples collected in Australia, including wastewater treatment plant effluent, two types of recycled water (reverse osmosis and ozonation/activated carbon filtration), stormwater, surface water, and drinking water. Sixty-five bioassays (63%) showed positive results in at least one sample, typically in wastewater treatment plant effluent, and only five (5%) were positive in the control (ultrapure water). Each water type had a characteristic bioanalytical profile with particular groups of toxicity pathways either consistently responsive or not responsive across test systems. The most responsive health-relevant endpoints were related to xenobiotic metabolism (pregnane X and aryl hydrocarbon receptors), hormone-mediated modes of action (mainly related to the estrogen, glucocorticoid, and antiandrogen activities), reactive modes of action (genotoxicity) and adaptive stress response pathway (oxidative stress response). This study has demonstrated that selected cell-based bioassays are suitable to benchmark water quality and it is recommended to use a purpose-tailored panel of bioassays for routine monitoring.

Toxicity characterization of urban stormwater with bioanalytical tools.

Stormwater harvesting has become an attractive alternative strategy to address the rising demand for urban water supply due to limited water sources and population growth. Nevertheless, urban stormwater is also a major source of surface water pollution. Runoff from different urban catchments with source contributions from anthropogenic activities and various land uses causes variable contaminant profiles, thus posing a challenging task for environmental monitoring and risk assessment. A thorough understanding of raw stormwater quality is essential to develop appropriate treatment facilities for potential indirect potable reuse of stormwater. While some of the key chemical components have previously been characterized, only scarce data are available on stormwater toxicity. We benchmarked stormwater samples from urban, residential and industrial sites across various Australian capital cities against samples from the entire water cycle, from sewage to drinking water. Six biological endpoints, targeting groups of chemicals with modes of toxic action of particular relevance for human and environmental health, were investigated: non-specific toxicity (Microtox and combined algae test), the specific modes of action of phytotoxicity (combined algae test), dioxin-like activity (AhR-CAFLUX), and estrogenicity (E-SCREEN), as well as reactive toxicity encompassing genotoxicity (umuC) and oxidative stress (AREc32). Non-specific toxicity was highly variable across sites. The baseline toxicity equivalent concentrations of the most polluted samples were similar to secondary treated effluent from wastewater treatment plants. Phytotoxicity results correlated well with the measured herbicide concentrations at all sites. High estrogenicity was found in two sampling events and could be related to sewage overflow. Genotoxicity, dioxin-like activity, and oxidative stress response were evident in only three of the samples where the stormwater drain was beside a heavy traffic road, confirming that road runoff is the potential source of contaminants, while the bioanalytical equivalent concentrations (BEQ) of these samples were similar to those of raw sewage. This study demonstrates the benefit of bioanalytical tools for screening-level stormwater quality assessment, forming the basis for the evaluation of future stormwater treatment and reuse schemes.

Bioanalytical and chemical assessment of the disinfection by-product formation potential: role of organic matter.

Disinfection by-products (DBP) formed from natural organic matter and disinfectants like chlorine and chloramine may cause adverse health effects. Here, we evaluate how the quantity and quality of natural organic matter and other precursors influence the formation of DBPs during chlorination and chloramination using a comprehensive approach including chemical analysis of regulated and emerging DBPs, total organic halogen quantification, organic matter characterisation and bioanalytical tools. In vitro bioassays allow us to assess the hazard potential of DBPs early in the chain of cellular events, when the DBPs react with their molecular target(s) and activate stress response and defence mechanisms. Given the reactive properties of known DBPs, a suite of bioassays targeting reactive modes of toxic action including genotoxicity and sensitive early warning endpoints such as protein damage and oxidative stress were evaluated in addition to cytotoxicity. Coagulated surface water was collected from three different drinking water treatment plants, along with reverse osmosis permeate from a desalination plant, and DBP formation potential was assessed after chlorination and chloramination. While effects were low or below the limit of detection before disinfection, the observed effects and DBP levels increased after disinfection and were generally higher after chlorination than after chloramination, indicating that chlorination forms higher concentrations of DBPs or more potent DBPs in the studied waters. Bacterial cytotoxicity, assessed using the bioluminescence inhibition assay, and induction of the oxidative stress response were the most sensitive endpoints, followed by genotoxicity. Source waters with higher dissolved organic carbon levels induced increased DBP formation and caused greater effects in the endpoints related to DNA damage repair, glutathione conjugation/protein damage and the Nrf2 oxidative stress response pathway after disinfection. Fractionation studies indicated that all molecular weight fractions of organic carbon contributed to the DBP formation potential, with the humic rich fractions forming the greatest amount of DBPs, while the low molecular weight fractions formed more brominated DBPs due to the high bromide to organic carbon ratio. The presence of higher bromide concentrations also led to a higher fraction of brominated DBPs as well as proportionally higher effects. This study demonstrates how a suite of analytical and bioanalytical tools can be used to effectively characterise the precursors and formation potential of DBPs.

Mixture effects of organic micropollutants present in water: towards the development of effect-based water quality trigger values for baseline toxicity.

In this study we propose for the first time an approach for the tentative derivation of effect-based water quality trigger values for an apical endpoint, the cytotoxicity measured by the bioluminescence inhibition in Vibrio fischeri. The trigger values were derived for the Australian Drinking Water Guideline and the Australian Guideline for Water Recycling as examples, but the algorithm can be adapted to any other set of guideline values. In the first step, a Quantitative Structure-Activity Relationship (QSAR) describing the 50% effect concentrations, EC50, was established using chemicals known to act according to the nonspecific mode of action of baseline toxicity. This QSAR described the effect of most of the chemicals in these guidelines satisfactorily, with the exception of antibiotics, which were more potent than predicted by the baseline toxicity QSAR. The mixture effect of 10-56 guideline chemicals mixed at various fixed concentration ratios (equipotent mixture ratios and ratios of the guideline values) was adequately described by concentration addition model of mixture toxicity. Ten water samples were then analysed and 5-64 regulated chemicals were detected (from a target list of over 200 chemicals). These detected chemicals were mixed in the ratios of concentrations detected and their mixture effect was predicted by concentration addition. Comparing the effect of these designed mixtures with the effect of the water samples, it became evident that less than 1% of effect could be explained by known chemicals, making it imperative to derive effect-based trigger values. The effect-based water quality trigger value, EBT-EC50, was calculated from the mixture effect concentration predicted for concentration-additive mixture effects of all chemicals in a given guideline divided by the sum of the guideline concentrations for individual components, and dividing by an extrapolation factor that accounts for the number of chemicals contained in the guidelines and for model uncertainties. While this concept was established using the example of Australian recycled water, it can be easily adapted to any other set of water quality guidelines for organic micropollutants. The cytotoxicity based trigger value cannot be used in isolation, it must be applied in conjunction with effect-based trigger values targeting critical specific modes of action such as estrogenicity or photosynthesis inhibition.

Most oxidative stress response in water samples comes from unknown chemicals: the need for effect-based water quality trigger values.

The induction of adaptive stress response pathways is an early and sensitive indicator of the presence of chemical and non-chemical stressors in cells. An important stress response is the Nrf-2 mediated oxidative stress response pathway where electrophilic chemicals or chemicals that cause the formation of reactive oxygen species initiate the production of antioxidants and metabolic detoxification enzymes. The AREc32 cell line is sensitive to chemicals inducing oxidative stress and has been previously applied for water quality monitoring of organic micropollutants and disinfection byproducts. Here we propose an algorithm for the derivation of effect-based water quality trigger values for this end point that is based on the combined effects of mixtures of regulated chemicals. Mixture experiments agreed with predictions by the mixture toxicity concept of concentration addition. The responses in the AREc32 and the concentrations of 269 individual chemicals were quantified in nine environmental samples, ranging from treated effluent, recycled water, stormwater to drinking water. The effects of the detected chemicals could explain less than 0.1% of the observed induction of the oxidative stress response in the sample, affirming the need to use effect-based trigger values that account for all chemicals present.

Water quality assessment using the AREc32 reporter gene assay indicative of the oxidative stress response pathway.

The reporter gene assay AREc32 is based on the induction of the Nrf2 mediated oxidative stress response pathway in the human breast cancer cell line MCF7, where eight copies of the antioxidant response element (ARE) are linked to a reporter gene encoding for luciferase. The Nrf2-ARE pathway is responsive to many chemicals that cause oxidative stress, among them a large number of pesticides and skin irritants. We adopted and validated the AREc32 bioassay for water quality testing. tert-Butylhydroquinone served as the positive control, phenol as the negative control and other reactive chemicals were assessed for their specificity. An environmentally relevant reference chemical, benzo(a)pyrene was the most potent inducer of all tested chemicals. The concentration causing an induction ratio (IR) of 1.5 (EC(IR1.5)) was chosen as the effect benchmark value. The assay was applied to 21 water samples ranging from sewage to drinking water, including secondary treatment and various tertiary treatment options (ozonation, biologically activated carbon filtration, membrane filtration, reverse osmosis, advanced oxidation, chlorination, chloramination). The samples were enriched by solid phase extraction. In most samples the oxidative stress response was far more sensitive than cytotoxicity. The primary and secondary treated effluent exceeded the effect threshold IR 1.5 at a relative enrichment factor (REF) of 1, i.e., the native samples were active. All tertiary treated samples were less potent and their EC(IR1.5) lay between REF 1 and 10. The Nrf2 pathway was induced at a REF of approximately 10 for surface waters and drinking water, and above this enrichment cytotoxicity took over in most samples and quenched the induction. The blank (ultrapure water run through the sample enrichment process) was cytotoxic at an REF of 100, which is the limit of concentrations range that can be evaluated. Treatment typically decreased both the cytotoxicity and oxidative stress response apart from drinking water treatment where chlorination caused an increase in oxidative stress response, presumably due to the formation of disinfection by-products. This study demonstrates the relevance and applicability of the oxidative stress response pathway for water quality monitoring.

Bioanalytical assessment of the formation of disinfection byproducts in a drinking water treatment plant.

Disinfection of drinking water is the most successful measure to reduce water-borne diseases and protect health. However, disinfection byproducts (DBPs) formed from the reaction of disinfectants such as chlorine and monochloramine with organic matter may cause bladder cancer and other adverse health effects. In this study the formation of DBPs through a full-scale water treatment plant serving a metropolitan area in Australia was assessed using in vitro bioanalytical tools, as well as through quantification of halogen-specific adsorbable organic halogens (AOXs), characterization of organic matter, and analytical quantification of selected regulated and emerging DBPs. The water treatment train consisted of coagulation, sand filtration, chlorination, addition of lime and fluoride, storage, and chloramination. Nonspecific toxicity peaked midway through the treatment train after the chlorination and storage steps. The dissolved organic matter concentration decreased after the coagulation step and then essentially remained constant during the treatment train. Concentrations of AOXs increased upon initial chlorination and continued to increase through the plant, probably due to increased chlorine contact time. Most of the quantified DBPs followed a trend similar to that of AOXs, with maximum concentrations observed in the final treated water after chloramination. The mostly chlorinated and brominated DBPs formed during treatment also caused reactive toxicity to increase after chlorination. Both genotoxicity with and without metabolic activation and the induction of the oxidative stress response pathway showed the same pattern as the nonspecific toxicity, with a maximum activity midway through the treatment train. Although measured effects cannot be directly translated to adverse health outcomes, this study demonstrates the applicability of bioanalytical tools to investigate DBP formation in a drinking water treatment plant, despite bioassays and sample preparation not yet being optimized for volatile DBPs. As such, the bioassays are useful as monitoring tools as they provide sensitive responses even at low DBP levels.

In vitro bioassay for reactive toxicity towards proteins implemented for water quality monitoring.

Reactive organic chemicals comprise a large number of compounds with a variety of reactive moieties. While most assays for reactive toxicity focus on DNA damage, reactivity towards proteins can also lead to irreparable damage, but reactivity towards proteins is typically not included in any test battery for water quality assessment. Glutathione (GSH) is a small tripeptide whose cysteine moiety can serve as a model for nucleophilic sites on proteins. GSH is also an important indicator of detoxification processes and the redox status of cells and due to its protective role, depletion of GSH ultimately leads to adverse effects. A bioassay based on genetically modified Escherichia coli strains was used to quantify the specific reactivity towards the protein-like biological nucelophile GSH. The significance of GSH for detoxification was assessed by comparing the growth inhibition induced by reference chemicals or water samples in a GSH-deficient strain to its fully functional parent strain. The GSH deficient strain showed the same sensitivity as the GSH proficient strain to non-reactive and DNA damaging chemicals, but was more sensitive to chemicals that attack cysteine in proteins. The difference in effect concentrations for 50% inhibition of growth assessed as biomass increase (EC(50)) between the two strains indicates the relevance of GSH conjugation as a detoxification step as well as direct reactivity with cysteine-containing proteins. Seven reference compounds serving as positive and negative controls were investigated. The E. coli strain that lacks GSH was four times more sensitive towards the positive control Sea-Nine, while negative controls benzo[a]pyrene, 2-aminoanthracene, phenol, t-butylhydroquinone, methyl methane sulfonate and 4-nitroquinoline oxide showed equal effect concentrations in both strains. Water samples collected across an indirect potable reuse scheme representing the complete water cycle from sewage to drinking water in South East Queensland, Australia were used to evaluate the applicability of the E. coli assay for reactive toxicity in water samples. While the EC(50) values of the GSH+ strain showed similar trends as in other biological endpoints over the various treatment chains, the specific response indicative of protein damage was only observed in samples that had undergone chlorination as a disinfection process. High natural organic matter or other matrix components disturbed the bioassay so much that we recommend it for future routine testing only in tertiary treated water or drinking water.

Combining passive sampling and toxicity testing for evaluation of mixtures of polar organic chemicals in sewage treatment plant effluent.

Effluent from sewage treatment plants has been associated with a range of pollutant effects. Depending on the influent composition and treatment processes the effluent may contain a myriad of different chemicals which makes monitoring very complex. In this study we aimed to monitor relatively polar organic pollutant mixtures using a combination of passive sampling techniques and a set of biochemistry based assays covering acute bacterial toxicity (Microtox), phytotoxicity (Max-I-PAM assay) and genotoxicity (umuC assay). The study showed that all of the assays were able to detect effects in the samples and allowed a comparison of the two plants as well as a comparison between the two sampling periods. Distinct improvements in water quality were observed in one of the plants as result of an upgrade to a UV disinfection system, which improved from 24x sample enrichment required to induce a 50% response in the Microtox assay to 84x, from 30x sample enrichment to induce a 50% reduction in photosynthetic yield to 125x, and the genotoxicity observed in the first sampling period was eliminated. Thus we propose that biochemical assay techniques in combination with time integrated passive sampling can substantially contribute to the monitoring of polar organic toxicants in STP effluents.

The ichthyotoxic alga Chattonella marina induces Na+, K+ -ATPase, and CFTR proteins expression in fish gill chloride cells in vivo.

Our previous studies demonstrated that the ichthyotoxic Chattonella marina stimulated proliferation of branchial chloride cell (CC) and induced osmotic distress akin to hyperactive elimination of ions in fish (Rhabdosargus sarba). To ascertain the in vivo effects of C. marina on key CC ion transporters, the localization and expression of Na(+), K(+)-ATPase (NKA) and cystic fibrosis transmembrane conductance regulator (CFTR) proteins in response to C. marina exposure were investigated, using a quantitative immunocytochemical approach. The polarized distributions of NKA (alpha subunit) and CFTR proteins in branchial CCs of R. sarba remained unchanged under C. marina exposure. However, significant inductions of these two ion-transporters were detected in CCs of fish after 6h exposure. By real-time PCR, no significant changes in gill NKA and CFTR mRNA expressions were detected, suggesting a post-transcriptional pathway is likely involved in regulating the ion transporters abundance. This study is the first to demonstrate the in vivo effects of harmful algal toxin on NKA and CFTR protein expressions in gill transepithelial cells. Taken together, an augmentation of branchial CCs together with hyper-stimulation of NKA and CFTR in CCs attribute to the rapid development of osmotic distress in C. marina susceptible fish.

Hydrogen peroxide is not the cause of fish kills associated with Chattonella marina: cytological and physiological evidence.

Chattonella marina, a harmful algal bloom (HAB) causative species, was used to study the mortality, physiology, and pathology of a marine stenohaline fish, goldlined seabream exposed to the toxic alga. The median lethal time (LT50) was 3 h upon exposure to 8000 cells/ml of C. marina. Significant induction of filamental chloride cells (CCs) [i.e. increases in CC fractional area and in the volume density of CCs], concomitant with significant reduction of blood osmolality, were found in C. marina treated fish. To verify whether the toxicity of C. marina was mediated through oxidative stress, a hydrogen peroxide exposure experiment was carried out and the toxicity as well as cytological and physiological changes were compared with the C. marina treatment. Hydrogen peroxide at a concentration of 500 microM H2O2, (i.e. 25 times higher than that produced by 8000 cells/ml of C. marina (20 microM H2O2)) was unable to induce similar CC alterations and osmoregulatory impairment in fish as observed in the C. marina treatment. Non-specific membrane damage such as severe loss of microvilli projections on the CC apical opening and rupture of epithelial membranes in the lamellae were observed. The LT50 was 6 h, two times longer than that with 8000 cells/ml of C. marina. Based on the cytological and physiological evidence and toxicity data, the mechanism by which C. marina kills fish appears to be very different from that caused by H2O2/ROS. Osmoregulatory distress is the major cause of fish death upon exposure to C. marina.

Osmotic distress: a probable cause of fish kills on exposure to a subbloom concentration of the toxic alga Chattonella marina.

Mortality, changes in blood osmolality, and pO2 in the goldlined seabream (Rhabdosargus sarba) on exposure to a subbloom concentration (2,000 cells/ml) of a toxic red tide alga, Chattonella marina, were investigated and related to quantitative ultrastructural alterations of the gill. The median lethal time (LT50) was 6 h. Significant induction of filamental chloride cells (CCs) (increases in CC density, apical opening area, fractional area, volume densities of CCs, and mitochondria within CCs), concomitant with a significant reduction in blood osmolality, was found within 3 h of exposure to C. marina. Further reduction in blood osmolality (67%) and a drastic decline of pO2 (70%) were detected in moribund fish after 6 h. Fish were also subjected to severe salinity stress (abrupt transfer to 0 and 60% salinities), and the same parameters were measured. Our quantitative ultrastructural and physiological data suggest that fish exposed for 6 h to C. marina (2,000 cells/ml) suffered similar but more severe osmotic distress as compared to that induced by abrupt transfer to 60% hypersaline water. Results of the salinity stress experiment also showed that suffocation was not a secondary response induced by osmotic impairment in the moribund fish. Osmoregulatory failure in conjunction with suffocation may be the cause of death following exposure to C. marina. The findings of this study provide evidence that C. marina, even in concentrations below visible blooms, can pose a significant threat to marine fish.