Approach for detecting mutagenicity of biodegraded and ozonated pharmaceuticals, metabolites and transformation products from a drinking water perspective.

Many pharmaceuticals and related metabolites are not efficiently removed in sewage treatment plants and enter into surface water. There, they might be subject of drinking water abstraction and treatment by ozonation. In this study, a systematic approach for producing and effect-based testing of transformation products (TPs) during the drinking water ozonation process is proposed. For this, two pharmaceutical parent substances, three metabolites and one environmental degradation product were investigated with respect to their biodegradability and fate during drinking water ozonation. The Ames test (TA98, TA100) was used for the identification of mutagenic activity present in the solutions after testing inherent biodegradability and/or after ozonation of the samples. Suspicious results were complemented with the umu test. Due to the low substrate concentration required for ozonation, all ozonated samples were concentrated via solid phase extraction (SPE) before performing the Ames test. With the exception of piracetam, all substances were only incompletely biodegradable, suggesting the formation of stable TPs. Metformin, piracetam and guanylurea could not be removed completely by the ozonation process. We received some evidence that technical TPs are formed by ozonation of metformin and piracetam, whereas all tested metabolites were not detectable by analytical means after ozonation. In the case of guanylurea, one ozonation TP was identified by LC/MS. None of the experiments showed an increase of mutagenic effects in the Ames test. However, the SPE concentration procedure might lead to false-positive results due to the generation of mutagenic artefacts or might lead to false-negative results by missing adequate recovery efficiency. Thus, these investigations should always be accompanied by process blank controls that are carried out along the whole ozonation and SPE procedure. The study presented here is a first attempt to investigate the significance of transformation products by a systematic approach. However, the adequacy and sensitivity of the methodology need to be further investigated. The approach of combining biodegradation and ozonation with effect-based assays is a promising tool for the early detection of potential hazards from TPs as drinking water contaminants. It can support the strategy for the evaluation of substances and metabolites in drinking water. A multitude of possible factors which influence the results have to be carefully considered, among them the selectivity and sensibility of the mutagenicity test applied, the extraction method for concentrating the relevant compounds and the biocompatibility of the solvent. Therefore, the results have to be carefully interpreted, and possible false-negative and false-positive results should be considered.

Whole effluent assessment of industrial wastewater for determination of bat compliance: Part 1: Paper manufacturing industry.

BACKGROUND, AIM AND SCOPE: The applicability of the Whole Effluent Assessment concept for the proof of compliance with the "best available techniques" has been analysed with paper mill wastewater from Germany by considering its persistency (P), potentially bio-accumulative substances (B) and toxicity (T). MATERIALS AND METHODS: Twenty wastewater samples from 13 paper mills using different types of cellulose fibres as raw materials have been tested in DIN or ISO standardised bioassays: the algae, daphnia, luminescent bacteria, duckweed (Lemna), fish-egg and umu tests with lowest ineffective dilution (LID) as test result. The potentially bio-accumulative substances (PBS) were determined by solid-phase microextraction and referred to the reference compound 2,3-dimethylnaphthalene. Usually, a primary chemical-physical treatment of the wastewater was followed by a single or multi-stage biological treatment. One indirectly discharged wastewater sample was pre-treated biologically in the Zahn-Wellens test before determining its ecotoxicity. RESULTS: No toxicity or genotoxicity at all was detected in the acute daphnia and fish egg as well as the umu assay. In the luminescent bacteria test, moderate toxicity (up to LIDlb=6) was observed. Wastewater of four paper mills demonstrated elevated or high algae toxicity (up to LIDA=128), which was in line with the results of the Lemna test, which mostly was less sensitive than the algae test (up to LIDDW=8). One indirectly discharged wastewater sample was biodegraded in the Zahn-Wellens test by 96% and was not toxic after this treatment. Low levels of PBS have been detected (median 3.27 mmol L(-1)). The colouration of the wastewater samples in the visible band did not correlate with algae toxicity and thus is not considered as its primary origin. Further analysis with a partial wastewater stream from thermomechanically produced groundwood pulp (TMP) revealed no algae or luminescent bacteria toxicity after pre-treatment of the sample in the Zahn-Wellens test (chemical oxygen demand elimination 85% in 7 days). Thus, the algae toxicity of the respective paper mill cannot be explained with the TMP partial stream; presumably other raw materials such as biocides might be the source of algae toxicity. DISCUSSION: Comparative data from wastewater surveillance of authorities confirmed the range of ecotoxicity observed in the study. Wastewater from paper mills generally has no or a moderate ecotoxicity (median LID 1 and 2) while the maximum LID values, especially for the algae and daphnia tests, are considerably elevated (LIDA up to 128, LIDD up to 48). CONCLUSIONS: Wastewater from paper mills generally is low to moderately ecotoxic to aquatic organisms in acute toxicity tests. Some samples show effects in the chronic algae growth inhibition test which cannot be explained exclusively with colouration of the samples. The origin of elevated algae ecotoxicity could not be determined. In the algae test, often flat dose-response relationships and growth promotion at higher dilution factors have been observed, indicating that several effects are overlapping. RECOMMENDATIONS AND PERSPECTIVES: At least one bioassay should be included in routine wastewater control of paper mills because the paper manufacturing industry is among the most water consuming. Although the algae test was the most sensitive test, it might not be the most appropriate test because of the complex relationship of colouration and inhibition and the smooth dose-effect relationship or even promotion of algae growth often observed. The Lemna test would be a suitable method which also detects inhibitors of photosynthesis and is not disturbed by wastewater colouration.

Whole effluent assessment of industrial wastewater for determination of BAT compliance. Part 2: metal surface treatment industry.

BACKGROUND, AIM AND SCOPE: Toxicity testing has become a suitable tool for wastewater evaluation included in several reference documents on best available techniques of the Integrated Pollution Prevention and Control (IPPC) Directive. The IPPC Directive requires that for direct dischargers as well as for indirect dischargers, the same best available techniques should be applied. Within the study, the whole effluent assessment approach of OSPAR has been applied for determining persistent toxicity of indirectly discharged wastewater from the metal surface treatment industry. MATERIALS AND METHODS: Twenty wastewater samples from the printed circuit board and electroplating industries which indirectly discharged their wastewater to municipal wastewater treatment plants (WWTP) have been considered in the study. In all factories, the wastewater partial flows were separated in collecting tanks and physicochemically treated in-house. For assessing the behaviour of the wastewater samples in WWTPs, all samples were biologically pretreated for 7 days in the Zahn-Wellens test before ecotoxicity testing. Thus, persistent toxicity could be discriminated from non-persistent toxicity caused, e.g. by ammonium or readily biodegradable compounds. The fish egg test with Danio rerio, the Daphnia magna acute toxicity test, the algae test with Desmodesmus subspicatus, the Vibrio fischeri assay and the plant growth test with Lemna minor have been applied. All tests have been carried out according to well-established DIN or ISO standards and the lowest ineffective dilution (LID) concept. Additionally, genotoxicity was tested in the umu assay. The potential bioaccumulating substances (PBS) were determined by solid-phase micro-extraction and referred to the reference compound 2,3-dimethylnaphthalene. RESULTS: The chemical oxygen demand (COD) and total organic carbon (TOC) values of the effluents were in the range of 30-2,850 mg L(-1) (COD) and 2-614 mg L(-1) (TOC). With respect to the metal concentrations, all samples were not heavily polluted. The maximum conductivity of the samples was 43,700 microS cm(-1) and indicates that salts might contribute to the overall toxicity. Half of the wastewater samples proved to be biologically well treatable in the Zahn-Wellens test with COD elimination above 80%, whilst the others were insufficiently biodegraded (COD elimination 28-74%). After the pretreatment in the Zahn-Wellens test, wastewater samples from four (out of ten) companies were extremely ecotoxic especially to algae (maximum LID(A) = 16,384). Three wastewater samples were genotoxic in the umu test. Applying the rules for salt correction of test results as allowed in the German Wastewater Ordinance, only a small part of toxicity could be attributed to salts. Considering the PBS, wastewater from the metal surface treatment industry exhibited very low levels of PBS. In one factory, the origin of ecotoxicity has been attributed to the organosulphide dimethyldithiocarbamate (DMDTC) used as a water treatment chemical for metal precipitation. The assumption based on rough calculation of input of the organosulphide into the wastewater was confirmed in practice by testing its ecotoxicity at the corresponding dilution ratio after pretreatment in the Zahn-Wellens test. Whilst the COD elimination of DMDTC was only 32% in 7 days, the pretreated sample exhibited a high ecotoxicity to algae (LID(A) = 1,536) and luminescent bacteria (LID(lb) = 256). DISCUSSION: Comparative data from wastewater surveillance by authorities (data from 1993 to 2007) confirmed the range of ecotoxicity observed in the study. Whilst wastewater from the metal surface treatment industry usually did not exhibit ecotoxicity (median LID 1-2), the maximum LID values reported for the algae, daphnia and luminescent bacteria tests were very high (LID(A) up to 3,072, LID(D) up to 512 and LID(lb) up to 2,048). DMDTC was found to be one important source of ecotoxicity in galvanic wastewater. DMDTC is added in surplus, and according to the supplier, the amount in excess should be detoxified with ferric chloride or iron sulphate. The operator of one electroplating company had not envisaged a separate treatment of the organosulphide wastewater but was assuming that excess organosulphide would be bound by other heavy metals in the sewer. DMDTC degrades via hydrolysis to carbon disulfide (which is also toxic to animals and aquatic organisms), carbonyl sulphide, hydrogen sulphide and dimethylamine, but forms complexes with metals which stabilise the compound with respect to transformation. Although no impact on the WWTP is expected, the question arises whether the organosulphide is completely degraded during the passage of the WWTP. CONCLUSIONS AND RECOMMENDATIONS: The results show that the organic load of wastewater from the electroplating industry has been underestimated by focussing on inorganic parameters such heavy metals, sulphide, cyanide, etc. Bioassays are a suitable tool for assessing the ecotoxicological relevance of these complex organic mixtures. The proof of biodegradability of the organic load (and its toxicity) can be provided by the Zahn-Wellens test. The environmental safety of water treatment chemicals should be better considered. The combination of the Zahn-Wellens test followed by the performance of ecotoxicity tests turned out to be a cost-efficient suitable instrument for the evaluation of indirect dischargers and considers the requirements of the IPPC Directive.