Comparison of three microbial assay procedures for measuring toxicity of chemical compounds: ToxAlert10, CellSense and Biolog MT2 microplates

Methods for measuring toxicity or respiratory activity of microbial cultures can be used as tools for assessing the impact of chemicals or waste streams on biological wastewater treatment plants. Easy-to-use and highly standardised toxicity tests are gaining wide acceptance due to their capability to make an assessment of the overall composite toxicity of the discharge. In this study we evaluate the relative sensitivity and performance of three microbial assay procedures for measuring toxicity of nine commonly used organic compounds in pharmaceutical processes: ToxAlert^® (using Vibrio fischeri), CellSense biosensors (using activated sludge, Pseudomonas putida and V. fischeri sensors) and Biolog MT2 microplates (using industrial activated sludge). Results were statistically compared and characteristics of the three procedures are discussed in terms of their sensitivity, reproducibility, representativity and ease of execution. All tests were found to be easy to perform. ToxAlert^® gave the best quantitative results, best reproducibility and repeatability followed by Biolog MT2. The uncertainty in the estimates for CellSense was found to be large, leading to wide confidence intervals. Although ToxAlert^® gave the best quantitative results the Biolog MT2 microplates which used industrial activated sludge were found to be more representative of the microflora present in industrial biological treatment plants. The microplate respiration tests are recommended for screening large number of toxicants or wastewater samples. CellSense gave poor reproducibility, however, it is a method with potential since it uses whole cell biosensors which allow different species to be easily and quickly tested.     

A comparison study between a disposable electrochemical DNA biosensor and a Vibrio fischeri-based luminescent sensor for the detection of toxicants in water samples

In the present study, a comparison between a disposable electrochemical DNA biosensor and a Vibrio fischeri-based luminescent sensor for the detection of toxicants in water samples was made.In order to realize this study, a disposable electrochemical DNA biosensor has been reported. The DNA biosensor is assembled by immobilizing double stranded Calf Thymus DNA onto the surface of a disposable carbon screen-printed electrode. The oxidation signal of the guanine base, obtained by a square wave voltammetric scan, is used as analytical signal to detect the DNA damage; the presence of low molecular weight compounds with affinity for nucleic acids is measured by their effect on the guanine oxidation peak.Wastewater samples provided during First European Interlaboratory Exercise on water toxicity in the course of the project SWIFT-WFD were analyzed, and biosensor results were compared with a currently used toxicity test ToxAlert^®100 based on the bioluminescence inhibition of Vibrio fischeri. This test have been used because is rapid, easy handling and cost effectively responses for the toxicity assessment in real water samples.The results showed a promising correlation between two tests used for the detection of toxic compounds in water samples.     

Assessment of the acute toxicity of triclosan and methyl triclosan in wastewater based on the bioluminescence inhibition of Vibrio fischeri

In this work, the contributions of triclosan and its metabolite methyl triclosan to the overall acute toxicity of wastewater were studied using Vibrio fischeri. The protocol used in this paper involved various steps. First, the aquatic toxicities of triclosan and methyl triclosan were determined for standard substances, and the 50% effective concentrations (EC₅₀) were determined for these compounds. Second, the toxic responses to different mixtures of triclosan, methyl triclosan, and surfactants were studied in different water matrices, i.e., Milli-Q water, groundwater and wastewater, in order to evaluate (i) the antagonistic or synergistic effects, and (ii) the influence of the water matrices. Finally, chemical analysis was used in conjunction with the toxicity results in order to assess the aquatic toxicities of triclosan and its derivative in wastewaters. In this study, the toxicities of 45 real samples corresponding to the influents and effluents from eight wastewater treatment works (WWTW) were analyzed. Thirty-one samples were from a wastewater treatment plant (WWTP) equipped with two pilot-scale membrane bioreactors (MBR), and the influent and the effluent samples after various treatments were characterized via different chromatographic approaches, including solid-phase extraction (SPE), liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS), and SPE coupled to gas chromatography–mass spectrometry (GC–MS). The toxicity was determined by measuring the bioluminescence inhibition of Vibrio fischeri. In order to complete the study and to extrapolate the results to different WWTPs, the toxicity to V. fischeri of samples from seven more plants was analyzed, as were their triclosan and methyl triclosan concentrations. Good agreement was established between the overall toxicity values and concentrations of the biocides, indicating that triclosan is one of the major toxic organic pollutants currently found in domestic wastewaters.     

European ring exercise on water toxicity using different bioluminescence inhibition tests based on Vibrio fischeri, in support to the implementation of the water framework directive

An inter-laboratory comparison exercise was conducted under the European Union funded project entitled: Screening Methods for Water Data Information in Support of the Implementation of the Water Framework Directive (SWIFT-WFD) and coordinated by the Consejo Superior de Investigaciones Científicas (CSIC), in order to evaluate the reproducibility of different toxicity tests based on the bioluminescence inhibition of Vibrio fischeri, for the rapid water toxicity assessment.For the first time, this type of exercise has been organized in Europe, and using different tests based on the same principle. In this exercise, 10 laboratories from 8 countries (Austria, Cyprus, Germany, Greece, Italy, Portugal, Romania, and Spain) took place, and a total number of 360 samples were distributed.During the exercise, six series of six samples were analyzed along 5 months. Every batch of samples was composed by three real samples and three standard solutions. The real samples were: a raw influent and the effluent of a wastewater treatment plant (WWTP), and a sample from a first settlement of the WWTP spiked with a mixture of toxicant standards.A final number of 330 (91.7%) samples was analyzed, 3300 values in duplicate were collected, and the results for each sample were expressed as the 50% effective concentration (EC₅₀) values calculated through five points of dilution inhibition curves, after 5 and 15 min of incubation times.A statistical study was initiated using 660 results. The mean values, standard deviations (σ), variances (σ²), and upper and lower warning limits (UWL and LWL) were obtained, using the EC₅₀ values calculated with the result from the participating laboratories.The main objectives of this toxicity ring study were to evaluate the repeatability (r) and reproducibility (R) when different laboratories conduct the test, the influence of complex matrix samples, the variability between different tests based on the same principle, and to determine the rate at which participating laboratories successfully completed tests initiated.In this exercise, the 3.93% toxicity values were outliers according with the Z-score values and the Dixon test. The samples with the greater number of outliers were those with the smallest variability coefficient, corresponding to the greater and the smaller toxicity level.No relation was found through the cluster analysis, between the final results and the different commercial devices involved. Testing by multiple commercial devices did not appear to reduce the precision of the results, and the variability coefficient for the exercise was nearby to the average value for past editions carried out at national level, where the different participants used the same commercial device.Stability of samples was also followed during the exercise. While statistical significance differences were not found for the greater part of samples, for the sample from the WWTP influent, a significant decrease of the toxicity value was found along this study. Nevertheless, this was a type of sample with a high toxicity level during all the exercise.On the other hand, in order to obtain the chemical characterization of real samples, those were analyzed by chromatographic techniques, using different sequential solid phase extraction (SSPE) procedures, followed by liquid chromatography coupled with mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS). Good agreement was found between the chemical analysis results and the toxicity level of the samples.     

Immunoenzyme assay of nonylphenol: study of selectivity and detection of alkylphenolic non-ionic surfactants in water samples

Immunoenzyme assay (ELISA) is proposed and characterized for determination of alkylphenol ethoxylates, a primary class of manufactured non-ionic surfactants. The assay is based on the obtained polyclonal antibodies against nonylphenol (NP), the main stable intermediate of the decomposition of nonylphenol ethoxylates. A mixture of non-modified branched isomers of NP was applied as hapten coupled to protein carriers by Mannich reaction with the use of formaldehyde. The proposed ELISA format is based on immobilized NP-(soybean trypsin inhibitor) conjugate as a competitor of antigen molecules contained in the tested sample for binding with specific antibodies indirectly labeled via an anti-species immunoperoxidase conjugate. The developed ELISA allows to reveal NP with the limit of detection about 10 ng ml⁻¹ and NP-related compounds such as octylphenol, alkylphenoletoxylates, alkylphenolcarboxylates and their halogenated derivatives. The ELISA was applied for assaying polluted water samples, namely influents and effluents from different wastewater treatment plants (WWTP) and tap water. ELISA and chromatographic data demonstrate good correlation (r = 0.94), while ELISA gives higher values. Due to endocrine disrupting and other toxic activities of some metabolites of alkylphenolic non-ionic surfactants, the developed assay may be effectively used in ecological monitoring and sanitary control.     

Interlaboratory study of the bioluminescence inhibition tests for rapid wastewater toxicity assessment

Several toxicity procedures are currently being used for the wastewater toxicity assessment. We have undertaken an interlaboratory comparison of the use of different bioluminescence inhibition toxicity tests based on Vibrio fischeri, in order to evaluate their reproducibility for the rapid wastewater toxicity assessment. Twenty-two laboratories took part in this study organized by the Institut Català de Tecnologia (ICT) and the Consejo Superior de Investigaciones Cientificas (CSIC). During the exercise, six series of six samples were analyzed along 5 months. Every batch of samples was composed by three real samples and three standard solutions. The real samples were: an untreated effluent of a paper industry, a sample from a first settlement of a wastewater treatment plant (WWTP) and the final effluent of the WWTP. The goals of the interlaboratory study were to evaluate the repeatability (r) and reproducibility (R) when different laboratories conduct the test, the influence of different matrix samples, the variability between different tests based on the same principle: the bioluminescence inhibition of V. fischeri, but involving different commercial devices and to determine the rate at which participating laboratories successfully completed tests initiated. The maximum number of outlier values was corresponding to a non-treated effluent from a paper industry. This also was the most complex and toxic sample analyzed. An increase on the non-convergent values obtained for the participants was observed at higher matrix complexity and at lower toxicity level. In comparison with other editions of this interlaboratory study the matrixes of real samples analyzed were more complex, nevertheless the final variability coefficient for the exercise was nearby to the average value for the past editions. Due to the high complexity of some samples involved in this intercalibration the stability of real samples were also followed during the test. On the other hand, no relation was found between final results and the different devices, as show the cluster analysis.     

Profiling of the reactive oxygen species-related ecotoxicity of CuO, ZnO, TiO2, silver and fullerene nanoparticles using a set of recombinant luminescent Escherichia coli strains: differentiating the impact of particles and solubilised metals

We propose a novel combination of high-throughput luminescent bacterial tests for the evaluation of the reactive oxygen species (ROS)-generating potential of engineered nanoparticles (eNPs) and the role of solubilised metal ions in this process. The set of tests consists of differently engineered recombinant Escherichia coli strains: (1) a new sensor strain, which bioluminescence is induced by superoxide anions; (2) six recombinant E. coli strains (superoxide dismutase (sod) single, double and triple mutants and a respective wild-type strain), transformed with luxCDABE genes responding to toxic compounds by decreasing their luminescence; and (3) three strains in which bioluminescence is specifically induced by bioavailable metals (Cu, Zn and Ag). The applicability of this battery of tests in profiling oxidative potential of eNPs was evaluated on nTiO₂, nCuO, nZnO and nAg (25, 30, 70 and <100 nm, respectively) NPs and fullerenes. As controls for the size or solubility, the bulk formulations (bTiO₂, bCuO and bZnO) and soluble salts (ZnSO₄, CuSO₄ and AgNO₃) were also analysed. Bacterial toxicity tests showed that nCuO was four-fold more toxic, and nAg was 15-fold more toxic to triple sod mutant than to wild type (2-h EC₅₀ values were 8.1 and 2.0 mg Cu l⁻¹, respectively, and 46 and 3.1 mg Ag l⁻¹, respectively). Formation of ROS by nCuO and nAg was proved by superoxide anion-inducible strain. The metal sensor bacteria showed that the ROS formation by CuO NPs was caused by solubilised Cu ions, but in case of nAg, particles also had an effect. nZnO was remarkably more toxic to sod triple mutant than to wild type strain (2-h EC₅₀ were 4.5 and 54 mg Zn l⁻¹, respectively). Fullerenes inhibited the bioluminescence of sod triple mutant at 3,882 mg l⁻¹ but had no effect on the wild-type strain even at 20,800 mg l⁻¹. Nano and bTiO₂ showed some effect on viability of bacteria only at high concentrations (>4,000 mg l⁻¹) although nTiO₂ (but not bTiO₂) induced the bioluminescence of the superoxide anion sensing bacteria starting from 100 mg l⁻¹. Thus, our innovative combined approach is expected to provide more consistent and informative data concerning the general toxicity, ROS-production potential and also solubilisation of metals in the case of metallic NPs.     

Tetracycline antibiotics in hospital and municipal wastewaters: a pilot study in Portugal

This study investigated the occurrence of tetracyclines (TCs), namely minocycline (MIN), TC, and its epimer epitetracycline (ETC), and doxycycline (DC), in four hospital wastewater effluents and its fate in municipal wastewater treatment plants (WWTPs), in Coimbra, Portugal. Analytical determination was carried out by solid-phase extraction followed by liquid chromatography with fluorescence detection. A gradient system with a mobile phase containing oxalic acid 0.02 M and acetonitrile was used. After postcolumn derivatization with magnesium reagent, TCs were detected at λ _exc 386 nm and λ _em 500 nm. The proposed method allowed good sensitivity, accuracy, and precision. LOQs were 0.5 μg l⁻¹ for ETC and TC and 15 and 5 μg l⁻¹ for MIN and DC, respectively. The recovery values ranged between 66.4% and 117.1%, and intraday and interday repeatability was lower than 6.8%. The method was successfully used to determine the presence of the above-mentioned TCs in 24 wastewater composite samples obtained from hospital effluents and from influent and effluent of the WWTP located in Coimbra, Portugal. MIN and TC were found in 41.7% of the samples; ETC and DC were found in 25% and 8.3% of the samples, respectively. The levels found ranged from 6 to 531.7 μg l⁻¹ in hospital effluents, while its concentrations in WWTP ranged from 95.8 to 915.3 μg l⁻¹. A seasonal influence in the concentrations found has also been observed, the levels found in samples collected during spring being higher than those observed in samples collected during autumn; however, these are only preliminary results. The WWTP removal rate ranged between 89.5% and 100%.     

Determination of estrogens in water by HPLC-UV using cloud point extraction

A method based on cloud point extraction was developed to determine four kinds of estrogens: estriol (E3), estradiol (E2), estrone (E1), and progesterone (P) in water by high performance liquid chromatography separation and ultraviolet detection (HPLC-UV). The non-ionic surfactant Triton X-114 was chosen as extractant solvent. The parameters affecting extraction efficiency, such as concentrations of Triton X-114 and Na₂SO₄, equilibration temperature, equilibration time and centrifugation time were evaluated and optimized. Under the optimum conditions, preconcentration factors of 99 for E3, 73 for E2, 152 for E1 and 86 for P were obtained for 10 mL water sample. The detection of limitation was 0.23 ng mL⁻¹ for E3, 0.32 ng mL⁻¹ for E2, 0.25 ng mL⁻¹ for E1 and 5.0 ng mL⁻¹ for P. The proposed method was successfully applied to the determination of trace amount of estrogens in wastewater treatment plant (WWTP) effluent water and exposure water with 10 ng mL⁻¹ E2 for toxicological study in our lab. For the case of WWTP effluent water samples, no estrogen was found. The accuracy of the proposed method was tested by recovery measurements of spiked samples and good recoveries of 81.2-99.5% were obtained.     

Characterization of wastewater toxicity by means of a whole-cell bacterial biosensor, using Pseudomonas putida, in conjunction with chemical analysis

A new amperometric biosensor based on inhibition of Pseudomonas putida has been developed to assess the acute toxicity of wastewater. This system uses the biological component immobilized on disposable screen-printed electrodes. The responses for a selected group of polar organic standard substances were studied using Pseudomonas putida as biological component. The results have been compared with responses obtained using the same system and Escherichia coli as biological component and with the bioluminescence inhibition of Vibrio fisheri using ToxAlert 100. Different properties, e.g. the standard deviation (SD) of the data, the goodness of fit (R2) and the standard deviation (Syx) of the vertical distances of the points from the inhibition curve, the 50% effective concentration (EC50) and the toxicity units (TII50) of the standard substance, were calculated and compared. This biosensor was used to assess the acute toxicity of real wastewater samples collected at different wastewater treatment plants (WWTP). Finally, a sequential solid-phase extraction (SSPE) procedure followed by liquid chromatography-mass spectrometry (LC-MS) was used to determine the polar organic toxic substances present in the wastewater samples.     

Paper-based chromatic toxicity bioassay by analysis of bacterial ferricyanide reduction

Water quality assessment requires a continuous and strict analysis of samples to guarantee compliance with established standards. Nowadays, the increasing number of pollutants and their synergistic effects lead to the development general toxicity bioassays capable to analyse water pollution as a whole. Current general toxicity methods, e.g. Microtox^®, rely on long operation protocols, the use of complex and expensive instrumentation and sample pre-treatment, which should be transported to the laboratory for analysis. These requirements delay sample analysis and hence, the response to avoid an environmental catastrophe. In an attempt to solve it, a fast (15 min) and low-cost toxicity bioassay based on the chromatic changes associated to bacterial ferricyanide reduction is here presented. E. coli cells (used as model bacteria) were stably trapped on low-cost paper matrices (cellulose-based paper discs, PDs) and remained viable for long times (1 month at −20 °C). Apart from bacterial carrier, paper matrices also acted as a fluidic element, allowing fluid management without the need of external pumps. Bioassay evaluation was performed using copper as model toxic agent. Chromatic changes associated to bacterial ferricyanide reduction were determined by three different transduction methods, i.e. (i) optical reflectometry (as reference method), (ii) image analysis and (iii) visual inspection. In all cases, bioassay results (in terms of half maximal effective concentrations, EC₅₀) were in agreement with already reported data, confirming the good performance of the bioassay. The validation of the bioassay was performed by analysis of real samples from natural sources, which were analysed and compared with a reference method (i.e. Microtox). Obtained results showed agreement for about 70% of toxic samples and 80% of non-toxic samples, which may validate the use of this simple and quick protocol in the determination of general toxicity. The minimum instrumentation requirements and the simplicity of the bioassay open the possibility of in-situ water toxicity assessment with a fast and low-cost protocol.     

Monitoring of di-(2-ethylhexyl)phthalate,nonylphenol, nonylphenol ethoxylates,and polychlorinated biphenyls in anaerobic and aerobic sewage sludge by gas chromatography–mass spectrometry

The occurrence of di-(2-ethyhexyl)phthalate (DEHP), nonylphenol and nonyphenol mono- and diethoxylates (NPEs) and seven polychlorinated biphenyl (PCB) congeners in different types of sludge samples is reported. The analysis of these compounds was carried out by sonication-assisted extraction and analytical determination by gas chromatography coupled with a mass spectrometry detector, following a previously described method. The applicability of the method was tested by monitoring the organic pollutants in primary, secondary, mixed, and digested-dehydrated sludge samples from two wastewater treatment plants (WWTPs) based on aerobic and on anaerobic biological stabilization. The occurrence of these compounds in sewage sludge and the influence of sludge stabilization process on the further farmland application of the sludge were evaluated. DEHP and NPEs were detected in all analysed sludge samples from both WWTPs at concentration levels in the range of 22.3–601?mg?kg^?1 and 136–2357?mg?kg^?1 dm (dry matter), respectively. PCBs were detected in all types of sludge analysed from the anaerobic WWTP but was not detected in any sludge sample from the aerobic WWTP. Concentration levels of the sum of the seven PCBs congeners were up to 1.5?mg?kg^?1 dm. The concentration of DEHP, sum of NPEs, and sum of the seven PCB congeners were higher than the limits fixed in the third draft of the future Sludge Directive for land application of sludge in the 67%, 100%, and 11% of samples from the anaerobic WWTP and in the 83%, 92%, and 0% of samples from the aerobic WWTP, respectively.     

A modified method for the analysis of organics in industrial wastewater as directed by their toxicity to Vibrio fischeri

A method for the identification of toxic compounds in industrial wastewater is presented, consisting of sequential solid phase extraction (SPE), fractionation by HPLC and GC-MS for compound identification. All analytical steps are accompanied by an automated detection of the aquatic toxicity by luminescence inhibition of Vibrio fischeri, which helps to reduce the large number of samples and subsamples that have to be processed by exluding those without toxic effects. The advantages of this procedure in comparison to previous methods of toxicity directed water analysis are discussed. The procedure was successfully applied to various samples of tannery wastewater, showing that benzothiazoles account for the major toxicity of tanyard wastewater. For very polar wastewater constituents, such as in beamhouse wastewaters, the use of LC-MS/MS for the final compound identification is suggested.     

A Voltammetric Electronic Tongue for the Quantitative Analysis of Quality Parameters in Wastewater

The use of a voltammetric electronic tongue for the quantitative analysis of quality parameters in influent wastewater from a wastewater treatment plant (WWTP) that treats domestic and industrial wastewater is proposed. The electronic voltammetric tongue consists of a set of four noble electrodes (iridium, rhodium, platinum and gold) housed inside a stainless steel cylinder. These noble metals have high durability and are low maintenance‐demanding, as required for developing future automated equipment. A pulse voltammetry study was conducted in 35 wastewater samples to determine ammonia (NH₄⁺‐N), nitrates (NO₃⁻‐N), total phosphate (tot‐P), soluble chemical oxygen demand (CODs) and conductivity. These parameters were also determined in these samples by routine analytical methods in the WWTP laboratory. A partial least squares (PLS) analysis was run to obtain a model to predict each parameter. Twenty‐five samples were included in the calibration set and 10 in the validation set. Calibration and validation sets were selected randomly, except for the extreme values of each parameter, which were included in the calibration set. Variable selection was performed on the voltammetric data using Genetic Algorithms in the calibration data set for each parameter. The electronic tongue showed good predictive power to determine the concentrations of NH₄⁺‐N, NO₃⁻‐N and tot‐P and CODs.     

Physicochemical characterization of ion-exchange membranes in water-methanol mixtures

A complete physicochemical characterization of two ion-exchange membranes—CM2 and Nafion^®117—used in electrodialysis and in direct methanol fuel cells (DMFC) has been carried out. For each membrane, in different methanol-water mixtures—0%, 20%, 40%, 60%, 80% and 100%—and at different temperatures (25.0; 40.0 et 55.0 °C), we have measured the variations of the geometrical dimensions, the proton electrical conductivity, the swelling rate and the amount of methanol in the membrane. The FTIR analysis of Nafion^®117 was performed at different methanol contents of the external solution.The results show that the CM2 membrane presents the best geometrical stability, and the lowest conductivity at any methanol content. At high methanol contents, Nafion^®117 is 10 times more conductive than the CM2 membrane. It was found that the methanol is absorbed more by Nafion^®117, and its effect is more noticeable on the microstructure of this membrane, under standard conditions. The high methanol permeability of these membranes, particularly of the Nafion^®117, induces bad cell efficiencies and lifetimes.     

A modified method for the analysis of organics in industrial wastewater as directed by their toxicity to Vibrio fischeri

A method for the identification of toxic compounds in industrial wastewater is presented, consisting of sequential solid phase extraction (SPE), fractionation by HPLC and GC-MS for compound identification. All analytical steps are accompanied by an automated detection of the aquatic toxicity by luminescence inhibition of Vibrio fischeri, which helps to reduce the large number of samples and subsamples that have to be processed by exluding those without toxic effects. The advantages of this procedure in comparison to previous methods of toxicity directed water analysis are discussed. The procedure was successfully applied to various samples of tannery wastewater, showing that benzothiazoles account for the major toxicity of tanyard wastewater. For very polar wastewater constituents, such as in beamhouse wastewaters, the use of LC-MS/MS for the final compound identification is suggested. Received: 25 September 1998 / Revised: 20 November 1998 / Accepted: 26 November 1998     

Determination of fluoroquinolone antibiotics in hospital and municipal wastewaters in Coimbra by liquid chromatography with a monolithic column and fluorescence detection

The main goal of this work was determination of residues of the antibiotics ofloxacin (OFLO), norfloxacin (NOR), ciprofloxacin (CIPRO), and enrofloxacin (ENRO) in wastewater samples. The samples, after acidification to pH 4.5 and addition of EDTA, were extracted on an anion-exchange cartridge in tandem with an Oasis HLB cartridge. The LC–FD method, developed in previous studies, was based on application of a monolithic C₁₈ column. The limit of quantification (LOQ) of the method was 250 ng L⁻¹ for OFLO, 25 ng L⁻¹ for NOR and CIPRO, and 50 ng L⁻¹ for ENRO. Mean recovery ranged between 75 and 121% for OFLO, NOR, CIPRO, and ENRO. A total of 14 wastewater samples were analyzed; these were collected from four hospitals and from influent and effluent from a wastewater-treatment plant in Coimbra, Portugal, during spring and autumn. CIPRO was present in all the samples, NOR was detected second most often, followed by OFLO. ENRO was found at concentrations under the LOQ in five hospital samples, and the highest level was found in influent from the WWTP.     

Novel coatings for stir bar sorptive extraction to determine pharmaceuticals and personal care products in environmental waters by liquid chromatography and tandem mass spectrometry

Two new commercially available polar coatings for stir bar sorptive extraction (SBSE), consisting of polyacrylate (PA) with a proportion of polyethyleneglycol (PEG) (Acrylate Twister^®) and PEG modified silicone (EG Silicone Twister^®), were evaluated and compared with the classic coating based on polydimethylsiloxane (PDMS Twister^®) for the extraction of a group of pharmaceuticals and personal care products (PPCPs) from wastewater samples.     

Application of ring study: water toxicity determinations by bioluminescence assay with Vibrio fischeri

Acceptance of toxicity bioassays as effective analytical tools in environmental areas needs guarantees of standardization but also validation. Ten European laboratories took part in an inter-laboratory study using different commercial devices based on bioluminescence inhibition of bacteria Vibrio fischeri. Reproducibility and stability by short toxicity endpoints, effective concentration that gives 10%, 50% and 80% of inhibition (EC₁₀, EC₅₀ and EC₈₀) is evaluated. Parametric and non-parametric statistic is applied and performance of participant laboratories is addressed by z-scores calculated by non-parametric statistic. z-Score classification was based on harmonised protocol for proficiency testing of analytical laboratories (satisfactory |z| ≤ 2; questionable 2 < |z| ≤ 3; unsatisfactory |z| > 3). Tested samples were phenol, 3,5-dichlorophenol and influent wastewater. Based on z-score classification, more than 70% of the laboratories showed a satisfactory performance for phenol, 3,5-dichlorophenol and influent wastewater (86%, 90% and 70%, respectively). Reproducibility and stability was observed in toxicant references and in wastewater samples. EC₈₀ determination appears to be more robust that EC₁₀ and EC₅₀. EC determinations can be considered favorable at 5 and 15 min of exposition, in particular for EC₈₀. The use of different commercial devices can not be considered an additional source of variation.     

One-step Bioluminescence ATPase Assay for the Evaluation of Neurotoxic Effects of Metal Ions

Summary. Membrane-bound ATPases, such as Na,K-ATPase and nucleotide triphospho-diphosphohydrolase (NTPDase), being one of the first targets of a toxic action are generally considered as good markers for estimating toxicity. A bioluminescence assay was applied for fast and sensitive evaluation of heavy metals effect on the rat brain synaptosomal membrane ATPase activity. The assay consists of ATP-consuming reaction catalyzed by synaptic plasma membrane ATPases coupled to the luminescent firefly luciferase reaction, which consumes residual ATP after the course of ATPase reaction. The bioluminescence ATPase assay was applied to study the effect of heavy and transitional metals (Cu²⁺, Pb²⁺, Cd²⁺, Hg²⁺) on rat brain ATPase activity after assay optimization. All metals applied inhibited synaptic membrane ATPase activity in a concentration dependent manner. The IC ₅₀ values (Hg²⁺ < Cu²⁺ < Cd²⁺ < Pb²⁺) obtained with the bioluminescence assay were highly correlated with those obtained by the spectrophotometric method. The fast bioluminescence ATPase assay with small sample and substrate requirements could be adjusted for high-throughput environmental and pharmacological screening.