Combined effect of copper, cadmium, and lead upon Cucumis sativus growth and bioaccumulation

Cucumis sativus (cucumber) was tested to assess an ecotoxicity in soils contaminated by the heavy metals copper (Cu), cadmium (Cd), and lead (Pb) separately and in combinations. The toxicity endpoint was plant growth, which was measured as shoot and root lengths after 5 day exposure. Sum of toxic unit (TU) at 50% inhibition for the mixture (EC50mix) was calculated from the dose (TU-based)-response relationships by the Trimmed Spearman-Karber method. Binary metal combinations of Cu+Cd, Cu+Pb, and Cd+Pb produced all three types of interactions; concentration additive (EC50mix=1TU), synergistic (EC50mix<1TU), and antagonistic (EC50mix>1TU) responses. Ternary combination of Cu+Cd+Pb produced an antagonistic response for the growth of Cucumis sativus. Bioaccumulations of Cu, Cd, and Pb were observed in Cucumis sativus and the bioaccumulation of one metal was influenced by the presence of other metals in metal mixtures. In general, antagonistic and/or synergistic responses reflected bioaccumulation patterns in some binary combinations, but the patterns in mixtures were not always consistent with toxicity data. This study indicated that TU approach appears to be a good model to estimate the combined effect of metals in plant systems, and mixture toxicity may be closely-related to the bioaccumulation pattern within plants. Combined effects of mixtures have to be taken into account to ecological risk assessment.     

The combined toxic effects of nonpolar narcotic chemicals to Pseudokirchneriella subcapitata

This paper presents the toxicity data of 10 nonpolar narcotic chemicals on Pseudokirchneriella subcapitata (green algae) assessed by a new algal toxicity testing technique conducted under air-tight environment. Based on DO production, median effective concentration (EC50) varies from 1.73 mg/L (1-octanol) to 8,040 mg/L (2-propanol). The endpoint of algal growth rate reveals similar sensitivity as that from DO production. Compared to literature data, Pseudokirchneriella subcapitata and Nitrosomonas are apparently more sensitive to nonpolar narcotics than other organisms such as minnow, daphnia, and Tetrahymena pyriformis. Furthermore, good correlations between toxic effects observed from Pseudokirchneriella subcapitata and other aquatic organisms were found. Hence, algal toxicity test can be considered as a surrogate test for estimating the toxicity of nonpolar chemicals to fathead minnow, Microtox, activated sludge, Daphina magna, and Tetrahymena pyriformis. The combined effects of 13 binary mixtures of nonpolar chemicals were investigated using both additive-index method and isobologram analysis. Overall speaking, the joint actions between these chemicals are strictly additive. Model analyses indicate that these compounds act on identical reaction sites or receptors, which verify that these chemicals are of the same toxicity mechanism (narcosis).     

Toxicity of four antifouling biocides and their mixtures on the brine shrimp Artemia salina

Zinc pyrithione (ZPT), Copper pyrihione (CPT), Chlorothalonil and Diuron are four of the most widely used as alternative to tributlytin (TBT) antifouling biocides in boat paints. As most previous laboratory bioassays for these biocides have been conducted solely based on acute tests with a single compound, information on the possible combined toxicity of these common biocides to marine organisms are limited. In this study, the toxicity of binary (in several proportions), ternary and quaternary mixtures were evaluated using the brine shrimp Artemia salina as test organism. Mixture toxicities were studied using the concentration addition model (isobolograms and toxic unit summation), and the mixture toxicity index (MTI). The ZPT-CPT combination had a strictly synergistic effect which requires attention because the coexistence of ZPT and CPT in the marine environment, due to transchelation of ZPT, may occur. The binary mixtures of Diuron with the metal pyrithiones exhibited various interactive effects (synergistic, antagonistic or additive) depending on concentration ratios, whereas all binary mixtures that contained Chlorothalonil exhibited antagonistic effects. The different types of combined effects subsequent to proportion variation of binary mixtures underline the importance of the combined toxicity characterization for various ratios of concentrations. The four ternary mixtures tested, also exhibited various interactive effects, and the quaternary mixture exhibited synergism. The models applied were in agreement in most cases. The observed synergistic interactions underline the requirement to review water quality guidelines, which are likely underestimating the adverse combined effects of these chemicals.     

Combined toxicity effects of MTBE and pesticides measured with Vibrio fischeri and Daphnia magna bioassays

Methyl-tert-butyl ether (MTBE), a fuel oxygenate that is added to gasoline, commonly contaminates aquatic systems, many of which are already contaminated with pesticides. The toxic effects (EC(50) value) of several pure pesticides (Diuron, Linuron, Dichlofluanid, Sea nine, Irgarol and tributyltin (TBT)) were measured and compared with the EC(50) value of the pesticide mixed with MTBE, using the Vibrio fischeri and Daphnia magna acute toxicity assays. The interaction between chemicals was evaluated in terms of the effects of mixing on the EC(50) value (i.e. the concentration (mg/L) of a compound or mixture that is required to produce a 50% change in a toxic response parameter) and the time required to generate the toxic response. Presence of MTBE enhanced the EC(50) value of several pesticides (Diuron, Dichlofluanid, TBT and Linuron) and/or the toxic response manifested more rapidly than with pure pesticides. Toxicity enhancements were quite substantial in many cases. For example, the presence of MTBE increased the toxicity of Diuron by more than 50% when tested with the V. fischeri assay (5, 15 and 30 min exposure). Also, the toxic response manifested itself within 5 min whereas without the MTBE the same response arose in 30 min. Presence of MTBE increased the toxicity of Dichlofluanid by 30% when measured with the D. magna assay. Toxicities of only two pesticides (Sea nine and Irgarol) were not raised by the presence of MTBE.     

An analysis of the combined effects of organic toxicants

This paper presents a basic database for the joint actions of 44 binary mixtures of various organic toxicants on Escherichia coli. The multiple toxicity behaviors observed from the E. coli organisms were analyzed and compared with previous works based on the Microtox tests. The two kinds of tests produced quite different responses, in terms of the joint action mode and the sum of toxic units, to various organic mixtures. However, detailed analyses with the considerations of the chemical's mechanisms of toxicity and the slope of toxicant's dose-response curve have revealed several general criteria for the prediction of combined effects of organic toxicants. First, for both reactive and non-reactive toxicants, either additive or less than additive (antagonistic) joint actions will be observed for chemicals of the same mechanism of toxicity. Second, the mixture of reactive toxicants with different mechanisms is the only category of organic mixtures associated with frequent observations of synergism. Third, greater-than-additive (synergistic) effects are inherently associated with toxicants having flat dose-response curves. Less than additive effects are, however, mainly related to a chemical's display steep dose-response curves. Model analyses indicate that the observed synergistic effects are due to response addition or response multiplication joint actions. Hence, most of the synergistic joint actions are non-interactive in nature and are governed by the dose-response relationships of individual toxicants.     

Hormesis in mixtures -- can it be predicted?

Binary mixture studies are well established for mixtures of pollutants, pesticides, or allelochemicals and sound statistical methods are available to evaluate the results in relation to reference models. The majority of mixture studies are conducted to investigate the effect of one compound on the inhibitory action of another. However, since stimulatory responses to low concentrations of chemicals are gaining increased attention and improved statistical models are available to describe this phenomenon of hormesis, scientists are challenged by the question of what will happen in the low concentration range when all or some of the chemicals in a mixture induce hormesis? Can the mixture effects still be predicted and can the size and concentration range of hormesis be predicted? The present study focused on binary mixtures with one or both compounds inducing hormesis and evaluated six data sets of root length of Lactuca sativa L. and areal growth of Lemna minor L., where substantial and reproducible hormetic responses to allelochemicals and herbicides have been found. Results showed that the concentration giving maximal growth stimulatory effects (M) and the concentration where the hormetic effect had vanished (LDS) could be predicted by the most-used reference model of concentration addition (CA), if the growth inhibitory concentrations (EC50) followed CA. In cases of deviations from CA at EC50, the maximum concentration M and the LDS concentration followed the same deviation patterns, which were described by curved isobole models. Thus, low concentration mixture effects as well as the concentration range of hormesis can be predicted applying available statistical models, if both mixture partners induce hormesis. Using monotonic concentration-response models instead of biphasic concentration-response models for the prediction of joint effects, thus ignoring hormesis, slightly overestimated the deviation from CA at EC20 and EC50, but did not alter the general conclusion of the mixture study in terms of deviation from the reference model. Mixture effects on the maximum stimulatory response were tested against the hypothesis of a linear change with mixture ratio by constructing 95% prediction intervals based on the single concentration-response curves. Four out of the six data sets evaluated followed the model of linear interpolation reasonably well, which suggested that the size of the hormetic growth stimulation can be roughly predicted in mixtures from knowledge of the concentration-response relationships of the individual chemicals.     

Evaluation of toxic and interactive toxic effects of three agrochemicals and copper using a battery of microbiotests

Three commonly used test organisms of different trophic levels (Vibrio fischeri, Pseudokirchneriella subcapitata and Daphnia magna) were exposed to selected agrochemicals (fosthiazate, metalaxyl-M, imidacloprid) and copper, in single doses or in binary mixtures. The toxicity of each single compound varied up to two orders of magnitude, depending on the test species examined. V. fischeri was the most sensitive test organism regarding fosthiazate and metalaxyl-M, indicating an IC₅₀ value of 0.20 mg/L (0.17-0.25 mg/L) and 0.88 mg/L (0.35-1.57 mg/L), respectively. Imidacloprid was the least toxic compound, indicating an EC₅₀ value on D. magna of 64.6 mg/L (43.3-122.5 mg/L) and an IC₅₀ value on V. fischeri of 226 mg/L (159-322 mg/L), while for imidacloprid at a concentration of 1000 mg/L the effect on P. subcapitata was lower than 50%. Copper was the most toxic compound towards all test organisms exhibiting the highest toxic effect on P. subcapitata, with an IC₅₀ value of 0.05 mg/L (0.003-0.008 mg/L). The toxic effects of the binary mixtures have been compared to the theoretically expected effect, resulting from a simple mathematical model based on the theory of probabilities. The independent action model was used in order to predict the theoretically expected effect. The interactive effects were mostly antagonistic or additive, while in few cases (interactive effects of metalaxyl-M and copper on V. fischeri) a synergistic mode of action was observed for some concentration combinations. Experiments showed that interactive effects of chemicals may vary depending on the test species used as well as on the chemicals and their respective concentrations. Although most of the concentrations of chemicals tested in this study are higher than the ones usually found in natural environment, the evaluation of their interactive toxic effects using a battery of bioassays may comprise a useful tool for the estimation of the environmental hazard of chemicals.     

A Daphnia magna feeding bioassay as a cost effective and ecological relevant sublethal toxicity test for Environmental Risk Assessment of toxic effluents

Environmental Risk Assessment of chemical products and effluents within EC countries require the use of cost effective standardized toxicity tests that in most cases are restricted to acute responses to high doses. Thus, subtle ecological effects are underestimated. Here we propose a short-term one day Daphnia magna feeding inhibition test as a cost effective and ecological relevant sublethal bioassay. The sensitivity and reliability of the proposed bioassay was tested in the laboratory against standardized bacteria, algae growth, D. magna and fish acute toxicity test by using 16 chemical mixture x water type combinations that included four different water types fortified with four complex chemical mixtures. Water types included ASTM hard water and three selected effluents diluted 1/10 in water to mimic worse field situations that many overexploited arid river ecosystems suffer during summer months when effluents are discharged into them with little dilution. The results obtained denoted a greater sensitivity of the proposed feeding bioassay in 51 out of 65 tests performed with an average sensitivity 50 fold greater than that of the standardized tests. The greater differences were obtained for mixtures that included narcotic chemicals and the lowest differences for those containing pesticides. Furthermore, feeding responses to the studied contaminant mixtures behaved differently to increasing TOC content than those based on bioluminescent bacteria and algae. Increasing TOC coming from sewage treated effluents decrease toxicity to the latter bioassays but increased those of D. magna feeding bioassays. These results empathize the need to include additional bioassays to monitor more accurately and realistically the toxicity of effluents or surface waters dominated by effluent discharges, a quite common situation in America and Mediterranean arid regions.     

Toxicity assessment of common xenobiotic compounds on municipal activated sludge: comparison between respirometry and Microtox

The toxicity of four xenobiotic compounds 3,5-dichlorophenol, formaldehyde, 4-nitrophenol and dichloromethane, representative of industrial wastewater contaminants was evaluated by a simple respirometric procedure set up on the basis of OECD Method 209 and by the Microtox bioassay. Very good reproducibility was observed for both methods, the variation coefficients being in the range of 2-10% for the respirometric procedure and 6-15% for Microtox, values that can be considered very good for a biological method. Comparison of EC(50) data obtained with the two methods shows that in both cases 3,5-dichlorophenol is more toxic than other compounds investigated and dichloromethane has a very low toxicity value. Intermediate EC(50) values were found for the two other chemicals, formaldehyde and 4-nitrophenol. Moreover, the Microtox EC(50) values are generally lower (except for dichloromethane) than the respirometric ones: these differences could be explained by the fact that the Microtox method uses a pure culture of marine species and, therefore, should not necessarily be expected to behave like a community of activated sludge bacteria. In conclusion, both methods can be usefully applied for toxicity detection in wastewater treatment plants but it is advisable to take into account that Microtox is more sensitive than respirometry in estimating the acute toxicity effect on the biomass operating in the plant.     

Assessment of ammonia toxicity in tests with the microalga,Nephroselmis pyriformis,Chlorophyta

Previous studies of an industrial effluent have indicated toxic effects by the ammonium/water system in tests with the unicellular green alga, Nephroselmis pyriformis. This investigation was undertaken to determine the toxicity of ammonia to this alga, and to identify the dominant toxicant in an industrial process effluent. The algal standard test was modified to improve the pH control, i.e. by testing at shorter exposure time and in the presence of a buffering agent, 3 mM HEPES. Ammonia was found to be the predominant toxic form in the dissociating ammonium/water system and the specific toxicity of ammonia (EC50, 24 h exposure) was 2.34 microM (32.8 microg ammonia nitrogen/L). Due to the dissociation, the toxicity is strongly pH dependent. Joint toxicity with additive effects of ammonia and ammonium ions was indicated, but the toxicity of ammonium ions was almost a factor 100 less (EC50, 24 h exposure, 224 microM or 3140 microg ammonium-nitrogen/L). According to the results the EC50 for effect on growth rate of N. pyriformis in seawater of pH 8.0 is 71 microM total ammonium. Hence, this alga appears to be more sensitive to ammonia than other studied marine plankton algae. Ammonia was found to be the dominant toxicant in the industrial effluent, using N. pyriformis as test organism.     

Novel methods for integrated risk assessment of cumulative stressors — Results from the NoMiracle project

This special issue covers the main results of the European Sixth Framework Integrated Research project NoMiracle (Novel Methods for Risk Assessment of Cumulative stressors in Europe). New tools to analyse, characterise and quantify the combined risks to health or the environment from multiple stressors are presented or reviewed. Examples of cumulative stressors are mixtures of chemicals alone or in combination with biological or physical environmental factors such as pathogens and climate extremes. Among the main findings, the scientific work points at the importance of time in dealing with toxicity, and in particular the toxicity of chemical mixtures, the natures of the uncertainties associated with risk assessment and the value of visualisation in identifying and quantifying the most relevant risks. A major conclusion of the project is that researchers and regulators should focus on the receptor rather than on the single stressor or combination of agents. There is also a need for more efforts on mechanistic understanding and for a mechanism-based framework for interpreting mixture/multiple stressor effects.The new tools are available on the internet (http://nomiracle.jrc.ec.europa.eu).     

Toxicity of metals and metal mixtures: analysis of concentration and time dependence for zinc and copper

The toxic effects of heavy metals, zinc and copper, in unary and binary solutions were studied using the Microtox acute toxicity test which relies upon the attenuation of light intensity emitted by Vibrio fischeri. The toxic effect Gamma (ratio of the light intensity lost at time t to the light intensity remaining at time t) of zinc could be related to its concentration [X] by a two-parameter equation Gamma=a(1-exp(-b[X])), where parameter a was a function of time and b equal to 0.88L/mg. The toxic effect of zinc asymptotically approached a maximum with respect of to concentration at all times. The toxic effect of copper was fundamentally different from that of zinc, and increased exponentially with concentration without any limiting maximum value. It could also be described by a two-parameter equation, however, the equation had the form Gamma=aexp(b[X]), where parameter a was a constant and b a function of time. The different functional dependencies (of the toxic effect on the metal concentration) of zinc and copper indicate that different toxicity/inhibition mechanisms were possibly responsible for the attenuation of light intensity for the two metals. The toxic effects of binary mixtures were substantially higher than those expected on the basis of additivity of individual metals. No simple correlations were obtained that could relate the toxic effect of binary mixture to those of individual metals. A better understanding of metal-microbe interactions is needed for achieving predictive capability for toxic effect of mixtures.     

Toxicity of three phenolic compounds and their mixtures on the gram-positive bacteria Bacillus subtilis in the aquatic environment

Although phenolic compounds are intensively studied for their toxic effects on the environment, the toxicity of catechol, resorcinol and hydroquinone mixtures are still not well understood because most previous bioassays are conducted solely using single compound based on acute tests. In this work, the adverse effect of individual phenolic compounds (catechol, resorcinol and hydroquinone) and the interactive effect of the binary and tertiary mixtures on Bacillus subtilis (B. subtilis) using microcalorimetric method were examined. The toxicity of individual phenolic compounds follows the order catechol > resorcinol > hydroquinone with their respective half inhibitory concentration as 437, 728 and 934 µg mL⁻¹. The power-time curve of B. subtilis growth obtained by microcalorimetry is in complete agreement with the change in turbidity of B. subtilis against time, demonstrating that microcalorimetric method agrees well with the routine microbiological method. The toxicity data obtained from phenolic compound mixtures show that catechol and hydroquinone mixture possess synergistic effect while the other mixtures display additive joint actions. Furthermore, the concentration addition (CA) and independent action (IA) models were employed to predict the toxicities of the phenolic compounds. The experimental results of microcalorimetry show no significant difference on the toxicity of the phenolic compound mixtures from that predicted by CA. However, IA prediction underestimated the mixture effects in all the experiments.     

Testing for the toxicity of chemicals with Tetrahymena pyriformis

A test was developed using Tetrahymena pyriformis in order to determine the toxicity of various chemicals. Pre-cultured T. pyriformis was exposed for 24 h at 30 degrees C to various concentrations of chemicals, and the number of T. pyriformis surviving were then counted. The concentration of the chemical, at which the proliferation of T. pyriformis was restricted to one-half of the blank test (EC50), was determined. The method, applied to 57 chemicals, demonstrated that it could be used to detect the chemicals at low concentrations rapidly and with ease. The EC50 values showed a good relationship with 48 h LC50 values for Himedaka (Orizias latipes), and could be explained on the basis of the partition coefficient between water and n-octanol.     

Use of partition coefficients to predict mixture toxicity

By using the C(18)-Empore disks/water partition coefficient (K(MD)) to describe the toxicity of 50 mixed halogenated benzenes to Photobacterium phosphoreum, an approach is proposed in this study. Application of the approach to the 15 other related mixtures prove the predictive capability of this K(MD)-based approach, due to the consistency between the predicted toxicity and the observed ones with r(2)=0.929, SE=0.104, F=169.513 at P<0.001. Further analysis of this approach finds that, for the mixtures, although the toxicity is highly correlated with their hydrophobicity, this correlation is free from the range difference of the hydrophobicity, the ratio or the number of the individual chemicals. These analysis results suggest that this K(MD)-based approach is able to predict the toxicity of mixture pollutants in wastewater.     

A comparison of microbial bioassays for the detection of aquatic toxicants

The toxicity of 35 test chemicals was analyzed using two microbial bioassay systems. The commercially available Microtox Toxicity Analyzer System™ and the two-organism procedure of Tchan were used to determine the concentration of test chemicals resulting in a 50% reduction in response (EC₅₀). Both of the tests employed a luminescent bacterium while the procedure of Tchan also utilized an alga. Results from the two microbial tests were compared with available data obtained with fish toxicity bioassays and with each other. The Microtox™ procedure was somewhat more sensitive than the Tchan bioassay in detecting most of the test chemicals and fish bioassays were generally more sensitive than either of the microbial tests. As a notable exception, photosynthesis-inhibiting herbicides were detected at remarkably lower concentrations with the procedure of Tchan than any of the other bioassays. Potential applications for these tests are discussed.     

Assessing chemical toxicity with the bioluminescent photobacterium (Vibrio fischeri): a comparison of three commercial systems

The inhibition of light emitted by the bioluminescent bacterium, Vibrio fischeri, is the basis for several toxicity bioassays. The inhibitory effects of 81 chemicals, after 5 min contact time, were studied at eight concentrations using reagents from three commercial assay systems (ToxAlert 10, Microtox and LUMIStox). Solubility in water was the limiting factor in determining the selection of chemicals for study. The effective nominal concentrations (EC) resulting in 20, 50 and 80% inhibition were determined using Ln dose/Ln gamma plots and the results obtained for each system were compared by linear regression. The chemical concentrations producing 10-90% inhibition extended over 9 orders of magnitude and ranged from a minimum of 0.001 ppm to a maximum of 1,000,000 ppm. The toxicity of many chemicals was apparently related to their pH in solution and at high chemical concentrations, to osmotic imbalance. The fact that the same operator tested the same solutions simultaneously on three different systems reduced sources of error and variability and improved the consistency and reliability of the results. Only five compounds gave EC 50s that varied more than three-fold between assays. These data provide comparisons of toxicity that have not been previously available and demonstrate that, when used under standardised conditions, these bioluminescence-based toxicity assays produce very similar results.     

Synergy and other ineffective mixture risk definitions

A substantial effort has been spent over the past few decades to label toxicologic interaction outcomes as synergistic, antagonistic, or additive. Although useful in influencing the emotions of the public and the press, these labels have contributed fairly little to our understanding of joint toxic action. Part of the difficulty is that their underlying toxicological concepts are only defined for two chemical mixtures, while most environmental and occupational exposures are to mixtures of many more chemicals. Furthermore, the mathematical characterizations of synergism and antagonism are inextricably linked to the prevailing definition of 'no interaction,' instead of some intrinsic toxicological property. For example, the US EPA has selected dose addition as the no-interaction definition for mixture risk assessment, so that synergism would represent toxic effects that exceed those predicted from dose addition. For now, labels such as synergism are useful to regulatory agencies, both for qualitative indications of public health risk as well as numerical decision tools for mixture risk characterization. Efforts to quantify interaction designations for use in risk assessment formulas, however, are highly simplified and carry large uncertainties. Several research directions, such as pharmacokinetic measurements and models, and toxicogenomics, should promote significant improvements by providing multi-component data that will allow biologically based mathematical models of joint toxicity to replace these pairwise interaction labels in mixture risk assessment procedures.     

Bioanalytical tools for the evaluation of organic micropollutants during sewage treatment, water recycling and drinking water generation

A bioanalytical test battery was used for monitoring organic micropollutants across an indirect potable reuse scheme testing sites across the complete water cycle from sewage to drinking water to assess the efficacy of different treatment barriers. The indirect potable reuse scheme consists of seven treatment barriers: (1) source control, (2) wastewater treatment plant, (3) microfiltration, (4) reverse osmosis, (5) advanced oxidation, (6) natural environment in a reservoir and (7) drinking water treatment plant. Bioanalytical results provide complementary information to chemical analysis on the sum of micropollutants acting together in mixtures. Six endpoints targeting the groups of chemicals with modes of toxic action of particular relevance for human and environmental health were included in the evaluation: genotoxicity, estrogenicity (endocrine disruption), neurotoxicity, phytotoxicity, dioxin-like activity and non-specific cell toxicity. The toxicity of water samples was expressed as toxic equivalent concentrations (TEQ), a measure that translates the effect of the mixtures of unknown and potentially unidentified chemicals in a water sample to the effect that a known reference compound would cause. For each bioassay a different representative reference compound was selected. In this study, the TEQ concept was applied for the first time to the umuC test indicative of genotoxicity using 4-nitroquinoline as the reference compound for direct genotoxicity and benzo[a]pyrene for genotoxicity after metabolic activation.The TEQ were observed to decrease across the seven treatment barriers in all six selected bioassays. Each bioassay showed a differentiated picture representative for a different group of chemicals and their mixture effect. The TEQ of the samples across the seven barriers were in the same order of magnitude as seen during previous individual studies in wastewater and advanced water treatment plants and reservoirs. For the first time a benchmarking was performed that allows direct comparison of different treatment technologies and covers several orders of magnitude of TEQ from highly contaminated sewage to drinking water with TEQ close or below the limit of detection. Detection limits of the bioassays were decreased in comparison to earlier studies by optimizing sample preparation and test protocols, and were comparable to or lower than the quantification limits of the routine chemical analysis, which allowed monitoring of the presence and removal of micropollutants post Barrier 2 and in drinking water. The results obtained by bioanalytical tools were reproducible, robust and consistent with previous studies assessing the effectiveness of the wastewater and advanced water treatment plants. The results of this study indicate that bioanalytical results expressed as TEQ are useful to assess removal efficiency of micropollutants throughout all treatment steps of water recycling.