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.     

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.     

Strategies for the identification of non-polar toxicants in aqueous environmental samples using toxicity-based fractionation and gas chromatography-mass spectrometry.

Toxicity-based fractionation is a useful tool for the isolation and identification of non-polar organic compounds that are present at toxic concentrations in aqueous environmental samples. Methods for isolating such toxicants from the aqueous sample matrix and techniques for fractionating the compounds for the purpose of reducing the complexity of the sample matrix and thus facilitating identification are evaluated. Strategies for analyzing gas chromatographic-mass spectrometric data and confirming toxicant identification are presented. Studies that use toxicity-based fractionation for identifying the cause of toxicity in aqueous environmental samples such as municipal and industrial wastewater treatment plant effluents and ambient waters are discussed.     

Effects of gamma-ray treatment on wastewater toxicity from a rubber products factory

In order to reduce the toxicity of both raw wastewater and effluent from a rubber products factory, γ-ray treatment was applied at different dose levels. The γ-ray treatment did not completely removed the toxicity, suggesting that there were major toxicants other than destroyable organic compounds. Toxicity identification evaluation (TIE phase 1) was conducted to characterize major toxicants using Daphnia magna. The suspected toxicants in both raw wastewater and effluent were mostly filterable materials and EDTA chelatable metals and, to some degree, non-polar organic compounds. Anion-exchange removable compounds, most likely organics, were found only in raw wastewater. Metal analyses showed that zinc and copper concentrations were above levels causing toxicity to D. magna. After 20 kGy γ-ray treatment of raw wastewater, filtrations both at pH 3 and at the initial pH (pH 3.6) showed dramatic change (9 to 77% and 29 to 85%, respectively) in toxicity reduction, suggesting the formation of toxic filterable materials which are stable even at acidic conditions. Unlike raw wastewater, there was no significant change in TIE results after γ-ray treatment at 20 kGy for rubber effluent.     

Determination of the plasticizer N-butylbenzenesulfonamide and the pharmaceutical Ibuprofen in wastewater using solid phase microextraction (SPME)

A rapid, inexpensive and solvent-free method for the simultaneous determination of the polyamide plasticizer N-butylbenzenesulfonamide (NBBS) and the widely used pharmaceutical Ibuprofen by solid phase microextraction (SPME) combined with gas chromatography/mass spectrometry (GC/MSD) in wastewater samples was developed. Besides the optimized analytical conditions, results of investigations with varying analytical parameters are reported. Problems, which may occur during the analytical procedure (e.g. salt deposits, adsorption phenomena, carry-over), are discussed. For the determination of Ibuprofen, it is important to carry out the extraction under acidic conditions with sufficiently buffered samples; the GC/MSD system must be very clean and well maintained. SPME allows an extraction of Ibuprofen without derivatization of its carboxylic group. For quantification in complex matrices, the standard addition technique is necessary. Limit of detection and limit of determination are 0.1 μg/L for both analytes. NBBS and Ibuprofen were detected in several raw and treated wastewater samples from municipal wastewater treatment plants in the range from < 0.1 to 3.5 μg/L. Received: 13 March 1998 / Revised: 16 June 1998 / Accepted: 19 June 1998     

Immunochemical array for the identification of cross-reacting analytes

A method for the identification and quantification of cross-reacting analytes using competitive immunochemical assays is described. The method uses information both of antibodies with and without significant inhibition caused by a sample. Maximum concentrations of all possible analytes were estimated for all antibodies not showing a significant inhibition. These maximum concentrations could be used to exclude certain analytes from the further identification process. A minimum variance method was used for the identification of analytes from the data given by antibodies showing significant inhibition. All samples were measured in a parallel affinity sensor array (PASA). The PASA system allows the parallel performance of numerous individual immunochemical assays. Triazines were used as a model substance class. Samples containing either atrazine, terbuthylazine, simazine or deethylatrazine at different concentration levels were generated and analyzed in the PASA system. 11 out of 13 samples were correctly identified, 2 samples could not be identified without doubt, no wrong identification was observed. Samples of atrazine, terbuthylazine and simazine at a concentration level of 0.1 μg/L, the EU maximum admissible concentration for individual pesticides, and of deethylatrazine at 0.3 μg/L could be quantified. Received: 25 September 1998 / Revised: 30 November 1998 / Accepted: 5 December 1998     

Identification of toxic compounds in wastewater treatment plants during a field experiment

Chemical analysis and toxicity bioassays were used in conjunction to determine the toxic compounds present in wastewater. This combined methodology was applied to wastewater samples collected at two wastewater treatment plants (WWTP) from the area of Barcelona (Spain), during a field experiment carried out from 1-4 April 2000. The efficiency of the WWTP was evaluated by collecting and analyzing samples at various stages of the water treatment process. The samples corresponded to the raw influent, from first settlement—before biological treatment—and from the effluent.Two bioluminescence inhibition assays: ToxAlert^®10 and ToxAlert^®100 from Merck both based on the bioluminescence inhibition of Vibrio fischeri (marine bacterial specie) were used. ToxAlert^®10 is a portable device with no temperature control and uses freeze-dried bacterial reagent and ToxAlert^®100 uses liquid-dried bacterial reagent and the incubation takes place at controlled temperature. Both tests showed similar results.Besides the toxicity studies, the wastewater samples were characterized by various analytical protocols involving the use of solid phase extraction (SPE) followed by liquid chromatography-mass spectrometry (LC-MS). Phenols, non-ionic surfactants, linear alkyl benzene sulphonates, benzene and naphthalene sulphonates and micro-pollutants with high endocrine-disrupting effects like estradiol and ethynyl estradiol were identified at the WWTP.The toxic responses obtained for the samples collected at WWTP were defined by the 50% effective concentration (EC₅₀), the Toxicity Units (TU) and the toxicity impact index (TII₅₀). The toxic effect at the different steps of the WWTP was attributed to the compounds identified and quantified by LC-MS like transformation products of nonylphenol polyethoxylate such as nonylphenol and nonylphenol carboxylate.     

Determination of arsenic species in human urine using HPLC with on-line photooxidation or microwave-assisted oxidation combined with flow-injection HG-AAS

An improved analytical procedure is presented for the separation and simultaneous determination of hydride-forming (toxic) and not hydride-forming (non-toxic) arsenic species in human urine. Separation was performed by cation-exchange chromatography using a new solid phase type based on the continuous bed chromatography (CBC) technology. This column permits by a factor of 4 higher flow rates than conventional columns resulting in a drastical reduction of retention times without any loss of resolution. Using this type of column, arsenobetaine (AsBet), arsenocholine (AsChol), and dimethylarsinic acid (DMA) were separated from the more toxic arsenic species arsenous acid (As(III)), arsenic acid (As(V)), and methylarsonic acid (MA) within only 4 min. The HPLC system was coupled via a flow injection system and either a UV or a microwave (MW) reactor to the HG-AAS instrument. UV photolysis and MW digestion were used to transform AsBet and AsChol to hydride-forming species and to make them accessible to HG-AAS. UV photolysis turned out to be more suitable for this application than MW digestion, because the latter technique led to peak broadening and poorer performance. The described procedure was applied to the determination of arsenic species in urine samples of non-occupationally exposed persons before and 12 h after seafood consumption. Detection limits were about 1 μg/L for each arsenic species. After consumption, the AsBet and DMA excretion increased by at least a factor of 150 for AsBet and by a factor of 6 for DMA, respectively, while the excretion of the other species did not increase significantly. This invalidates the use of total urinary arsenic as well as total hydride-forming arsenic as an indicator for exposure to inorganic arsenic. Received: 12 August 1998 / Revised: 30 October 1998 / Accepted: 24 November 1998     

In silico prediction of drug toxicity

It is essential, in order to minimise expensive drug failures due to toxicity being found in late development or even in clinical trials, to determine potential toxicity problems as early as possible. In view of the large libraries of compounds now being handled by combinatorial chemistry and high-throughput screening, identification of putative toxicity is advisable even before synthesis. Thus the use of predictive toxicology is called for. A number of in silico approaches to toxicity prediction are discussed. Quantitative structure-activity relationships (QSARs), relating mostly to specific chemical classes, have long been used for this purpose, and exist for a wide range of toxicity endpoints. However, QSARs also exist for the prediction of toxicity of very diverse libraries, although often such QSARs are of the classification type; that is, they predict simply whether or not a compound is toxic, and do not give an indication of the level of toxicity. Examples are given of all of these. A number of expert systems are available for toxicity prediction, most of them covering a range of toxicity endpoints. Those discussed include TOPKAT, CASE, DEREK, HazardExpert, OncoLogic and COMPACT. Comparative tests of the ability of these systems to predict carcinogenicity show that improvement is still needed. The consensus approach is recommended, whereby the results from several prediction systems are pooled. It is simply amazing that we can formulate any kind of QSAR. The (desired activity) is only the starting point. The truly formidable problem is that of toxicity, especially the difficult long-term toxicities resulting from chronic usage'. (Hansch & Leo [1])     

Trace analysis of herbicides in wastewaters by a dispersive liquid–liquid microextraction approach and liquid chromatography with quadrupole linear ion trap mass spectrometry: Evaluation of green parameters

An analytical method for determining phenylureas (monuron, isoproturon, diuron, linuron and neburon) and propanil herbicides in wastewater has been developed and validated, and the most significant parameters were compared with the corresponding ones found in the literature, thus showing the method performance. The method involves pre‐concentration by a simple, rapid, sensitive and low environmental toxicity temperature‐controlled ionic liquid dispersive liquid–liquid microextraction procedure. The herbicides were identified and determined by liquid chromatography with a hybrid triple quadrupole linear ion trap mass spectrometer. Data acquisition in selected‐reaction monitoring mode allowed the simultaneous identification and quantification of the analytes using two transitions. The information dependent acquisition scan was performed to carry out the identification of those analytes whose second transition was present at low intensity, also providing extra confirmation for the other analytes. Limits of quantification were in the range 1.0–5.0 ng/L. Good recoveries (95–103%) were obtained for the extraction of the target analytes in wastewater samples. The methodology developed was applied to analyze effluent wastewater samples from a wastewater treatment plant located in an agricultural zone of Almería (Spain) and the results indicated the presence of diuron at mean concentration levels of 73.5 ng/L.     

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.     

Toxicological evaluation of exposed SPMD membranes

This article is focused on bioassay evaluation of exposed SPMDs. Toxicity testing of SPMD extract is a suitable complementary parameter to chemical analysis. Vtox as an integrative parameter describing toxic properties of extract from membrane and thus a level of contamination of assessed ambient is presented here. Two examples of practical use of Vtox is demonstrated: a course of toxicity throw wastewater treatment plant with industrial inlets and increase of toxicity in surface water body caused by leakages of POPs from industrial plant.     

Electrochemical oxidation of p-hydroxybenzoic and protocathecuic acids at a dimensional stable anode (DSA) in the presence of NaCl

This work is part of a wider research programme on innovative technologies for industrial wastewater treatment. Results from electrolyses at DSA commercial anodes of synthetic solutions with composition analogous to that of agro-industrial wastes are presented. The results obtained indicate that the rate of degradation of phenolic compounds is high, provided that chloride ions are present in solution. Oxidation of phenolic compounds is faster than that of biodegradable substances, such as sugars or amminoacids. Moreover, investigation on the trend of toxicity during the treatment, seems to exclude that toxic intermediates persist in solution when phenolic compounds are removed. Experiments on olive oil mill wastewater (OMW) samples show that the results obtained from synthetic solutions are extensible to real wastewater. When phenolic compounds are completely removed, the toxicity of the solution is very low; the initial dark colour of the solution, due to the brown pigment which characterises OMW, is nearly completely disappeared.     

Development and validation of a rapid and wide-scope qualitative screening method for detection and identification of organic pollutants in natural water and wastewater by gas chromatography time-of-flight mass spectrometry

In this work, a multiclass screening method for organic contaminants in natural and wastewater has been developed and validated for qualitative purposes, i.e. to ensure the reliable and sensitive identification of compounds detected in samples at a certain level of concentration. The screening is based on the use of GC-TOF MS, and the sample procedure involves solid phase extraction with C(18) cartridges. Around 150 organic contaminants from different chemical families were investigated, including PAHs, octyl/nonyl phenols, PCBs, PBDEs and a notable number of pesticides, such as insecticides (organochlorines, organophosphorus, carbamates and pyrethroids), herbicides (triazines and chloroacetanilides), fungicides and several relevant metabolites. Surface water, ground water and effluent wastewater were spiked with all target analytes at three concentration levels (0.02, 0.1 and 1 μg/L). Influent wastewater and raw leachate from a municipal solid waste treatment plant were spiked at two levels (0.1 and 1 μg/L). Up to five m/z ions were evaluated for every compound. The identification criterion was the presence of, at least, two m/z ions at the expected retention time, measured at their accurate mass, and the accomplishment of the Q/q(i) intensity ratio within specified tolerances. The vast majority of compounds investigated were correctly identified in the samples spiked at 1 μg/L. When analyte concentration was lowered down to 0.1 μg/L the identification was more problematic, especially in complex-matrix samples like influent wastewater. On the contrary, many contaminants could be properly identified at the lowest level 0.02 μg/L in cleaner matrices. The procedure was applied to the screening of water samples of different origin and matrix composition and allowed the detection of several target contaminants. A highly reliable identification could be carried out thanks to the sensitive full-spectrum acquisition at accurate mass, the high selectivity reached with the use of narrow-mass window extracted ion chromatograms, the low mass errors observed in the positive detections and the Q/q ratio accomplishment.     

Group separation of ortho-PCBs, coplanar non-ortho-PCBs and PCDDs/PCDFs using an alumina column as an one-step clean-up procedure

 A novel, simple inexpensive and rapid clean-up procedure is presented for the separation and quantitative determination of polychlorinated biphenyls (ortho-PCBs), coplanar non-ortho-PCBs (PCB No. 77, 126, 169) and polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) in environmental samples. This clean-up procedure is the first method separating ortho-PCBs, non-ortho-PCBs and PCDD/PCDFs in one step with a single activated alumina column. Firstly, the ortho-PCBs are eluted from the activated alumina with a non-polar solvent. The non-ortho-PCBs are isolated in the second fraction with a more polar solvent-mixture, and finally the PCDD/PCDFs are collected in a polar fraction. This clean-up procedure was used to determine the congener-specific concentrations of ortho-PCBs, non-ortho-PCBs 77, 126 and 169 and PCDD/PCDFs in two different seepage waters and in one sewage sludge by HRGC and HRMS. The toxic equivalent quantities (TEQs) of the individual PCDD/PCDF-congeners and some toxic coplanar PCB-congeners were estimated. Received: 26 June 1996/Revised: 11 September 1996/Accepted: 14 September 1996     

Synthesis and antibacterial activity of trinems bearing nitrogen derivatives at C(4)

Optimization of the antibacterial activity of 4-amino trinem, obtained through chemical modification of the basicity of the amino group at position 4, has led to the identification of a very interesting compound characterized by a broad spectrum of activity includingPseudomonas aeruginosa.Medicines Research Centre, GlaxoWellcome S.p. A Via A. Fleming 4, 37100 Verona, Italy. Published in Khimiya Geterotsiklicheskikh Soedinenii, No. 11, pp. 1553–1559, November, 1998.     

Automated continuous monitoring of drug dissolution process using a flow injection on-line dialysis sampling – solvent extraction separation spectrophotometric system

Inductively coupled plasma optical emission spectrometry (ICP-OES) was used for the element-specific determination of water-soluble organosilicon species separated by high-performance liquid chromatography (HPLC). Leachates from different waste deposit sites were investigated. The polydimethylsiloxane (PDMS) degradation product dimethylsilanediol (DMSD) could be detected in almost all samples in the low mg L^–1 range, and it was furthermore possible to determine trimethylsilanol (TMSOL) in some leachate samples in the μg L^–1 range. TMSOL was additionally analyzed by a method established before (LT-GC/ICP-OES). This study proved the occurrence of silanol compounds in leachates from locations were silicone material is deposited. Received: 10 July 1998 / Revised: 2 November 1998 / Accepted: 5 November 1998     

Determination of Cd, Cu, Pb and Sb in environmental samples by ICP-AES using polyaniline for separation

The anion exchange properties of polyaniline for Cd, Cu, Pb and Sb in potassium iodide were studied. The analytes converted into anionic complexes by KI (0.03–0.96 mol/L) in HCl were adsorbed on polyaniline and eluted with HNO₃. The optimum conditions for adsorption and elution were determined. Quantitative recoveries were obtained for Cd, Cu and Pb, whereas, the recoveries for Sb were about 75%. This separation procedure was used with subsequent ICP-AES determination for Cd, Cu, and Pb in NIST-coal fly ash (1633b) and a sea plant with an R.S.D of 5% (n = 5). Received: 13 November 1997 / Revised: 2 February 1998 / Accepted: 7 February 1998     

Whole cell immobilised biosensors for toxicity assessment of a wastewater treatment plant treating phenolics-containing waste

Wastewater treatment plants dealing with industrial wastes are often susceptible to overload of toxic influent that can partially or completely destroy treatment for extended periods. An obvious candidate for monitoring toxicity in such wastewater systems is bioluminescent bacteria. However, the natural bioluminescent bacteria can be particularly sensitive to some industrial wastes and therefore their response to normal operational conditions does not reflect the status of the microbial community responsible for treatment. Moreover, the salt dependence of the marine bioluminescent bacteria, and the temperature sensitivity of some strains, further complicate their use. Here we describe the construction of whole cell genetically modified bioluminescent biosensors and their immobilisation for use in monitoring the toxicity of a complex industrial wastewater containing phenolic materials. A hand-held luminometer was designed for laboratory or field use, and the immobilisation system designed with several things in mind: the geometry of the instrument; the need for containment of GM bacteria; the maximisation of the bioavailability of the wastewater to the biosensor. The performance of a candidate GM sensor was compared with Vibrio fischeri in liquid culture and after immobilisation in thin films of poly(vinyl alcohol) (PVA) cryogels. The biosensors were tested against pure phenol and 3-chlorophenol as a reference toxic chemical known to be much more toxic to bacteria than phenol. The biosensors were then tested with the phenolics-containing industrial wastewater. The immobilisation system proved to operate predictably with pure toxicants, and was able to discriminate toxicity of various zones within the wastewater treatment plant.