There is currently a lack of ecotoxicity tests adapted to earthworm species of higher ecological relevance and whose endpoints could be directly related to their ecological role in the soil. We propose a new and relatively simple ecotoxicity test based on the estimation of cast production (CP) by Lumbricus terrestris under laboratory conditions. CP was found to be linearly correlated to earthworm biomass and to be greatly influenced by soil water content. Azinphos-methyl had no effect on CP at all the concentrations tested. Significant decreases were observed at the normal application rate for other pesticides with (imidacloprid, carbaryl, methomyl) or without (ethyl-parathion and chlorpyrifos-ethyl) a clear concentration–effect response. For the highest concentration tested, reduction in CP varied between 35 and 67%. CP is straightforward and rapidly measured and ecologically meaningful. We thus believe it to be of great use as an endpoint in ecotoxicity testing. Cast production of Lumbricus terrestris is affected by pesticides under laboratory conditions.

The acute toxicity of CuO and ZnO nanoparticles in artificial freshwater (AFW) and in natural waters to crustaceans Daphnia magna and Thamnocephalus platyurus and protozoan Tetrahymena thermophila was compared. The L(E)C50 values of nanoCuO for both crustaceans in natural water ranged from 90 to 224 mg Cu/l and were about 10-fold lower than L(E)C50 values of bulk CuO. In all test media, the L(E)C50 values for both bulk and nanoZnO (1.1–16 mg Zn/l) were considerably lower than those of nanoCuO. The natural waters remarkably (up to 140-fold) decreased the toxicity of nanoCuO (but not that of nanoZnO) to crustaceans depending mainly on the concentration of dissolved organic carbon (DOC). The toxicity of both nanoCuO and nanoZnO was mostly due to the solubilised ions as determined by specific metal-sensing bacteria. Natural waters remarkably reduced the toxicity of nanoCuO but not nanoZnO.

Chiral pesticides comprise a new and important class of environmental pollutants nowadays. With the development of industry, more and more chiral pesticides will be introduced into the market. But their enantioselective ecotoxicology is not clear. Currently used synthetic pyrethroids, organophosphates, acylanilides, phenoxypropanoic acids and imidazolinones often behave enantioselectively in agriculture use and they always pose unpredictable enantioselective ecological risks on non-target organisms or human. It is necessary to explore the enantioselective toxicology and ecological fate of these chiral pesticides in environmental risk assessment. The enantioselective toxicology and the fate of these currently widely used pesticides have been discussed in this review article.

A Pb-mine site situated on acidic soil, but comprising of Ca-enriched islands around derelict buildings was used to study the spatial pattern of genetic diversity in Lumbricus rubellus. Two distinct genetic lineages (‘A’ and ‘B’), differentiated at both the mitochondrial (mtDNA COII) and nuclear level (AFLPs) were revealed with a mean inter-lineage mtDNA sequence divergence of approximately 13%, indicative of a cryptic species complex. AFLP analysis indicates that lineage A individuals within one central ‘ecological island’ site are uniquely clustered, with little genetic overlap with lineage A individuals at the two peripheral sites. FTIR microspectroscopy of Pb-sequestering chloragocytes revealed different phosphate profiles in residents of adjacent acidic and calcareous islands. Bioinformatics found over-representation of Ca pathway genes in ESTPb libraries. Subsequent sequencing of a Ca-transport gene, SERCA, revealed mutations in the protein's cytosolic domain. We recommend the mandatory genotyping of all individuals prior to field-based ecotoxicological assays, particularly those using discriminating genomic technologies. Landscapes punctuated by Pb-polluted islands have engendered local genetic differentiation in resident earthworms.

Cultures of the flagellate Isochrysis galbana were used to carry out the ecotoxicological evaluation of four PAHs [(naphthalene, phenanthrene, pyrene (Pyr) and fluoranthene (Flu)] by monitoring growth rate and the fluorescence variables F ₀, F m, F v and F v/F m, determined with a fast repetition rate fluorometer. The results presented in this investigation showed that F v was a suitable endpoint in acute ecotoxicological tests with marine phytoplankton. The derived effective concentrations followed the known narcotic mechanism of toxicity and showed sensitivity levels comparable to marine invertebrate embryo-larval bioassays. Pyr and Flu showed the lowest EC₁₀, which ranged between 168-279 and 189-697 nM, respectively.

The potential ecological hazard of metals in soils may be measured directly using a combination of chemical and biological techniques or estimated using appropriate ecological models. Terrestrial ecotoxicity testing has gained scientific credibility and growing regulatory interest; however, toxicity of metals has often been tested in freshly amended soils. Such an approach may lead to derivation of erroneous toxicity values (EC₅₀) and thresholds. In this study, the impact of metal amendments on soil ecotoxicity testing within a context of ion competition was investigated. Four coarse-textured soils were amended with copper (Cu) and nickel (Ni), incubated for 16 weeks and conditioned by a series of total pore water replacements. RhizonTM extracted pore water Cu, Ni, pH and dissolved organic carbon (DOC) concentrations were measured after each replacement. Changes in ecotoxicity of soil solutions were also monitored using a lux-based biosensor (Escherichia coli HB101 pUCD607) and linked to variations in soil solution metal and DOC concentrations, pH and selected characteristics of the experimental soils (exchangeable calcium (Ca) and magnesium (Mg)). Prior to conditioning of soils, strong proton competition produced relatively high EC₅₀ values (low toxicity) for both, Cu and Ni. The successive replacement of pore waters lead to a decline of labile pools of metals, DOC and alleviated the ecotoxicological protective effect of amendment impacted soil solution chemistry. Consequently, derived ecotoxicity values and toxicity thresholds were more reflective of genuine environmental conditions and the relationships observed more consistent with trends reported in historically contaminated soils.

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.

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 µS 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 LIDA = 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 (LIDA = 1,536) and luminescent bacteria (LIDlb = 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 (LIDA up to 3,072, LIDD up to 512 and LIDlb 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.

Background, aim, and scope The chemical substance 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) is a non-ionic surfactant used as an industrial defoaming agent and in various other applications. Its commercial name is Surynol 104® and the related ethoxylates are also available as Surfynol® 420, 440, 465 and 485 which are characterized by different grades of ethoxylation of TMDD at both hydroxyl functional groups. TMDD and its ethoxylates offer several advantages in waterborne industrial applications in coatings, inks, adhesives as well as in paper industries. TMDD and its ethoxylates can be expected to reach the aquatic environment due its widespread use and its physico-chemical properties. TMDD has previously been detected in several rivers of Germany with concentrations up to 2.5 µg/L. In the United States, TMDD was also detected in drinking water. However, detailed studies about its presence and distribution in the aquatic environment have not been carried out so far. The aim of the present study was the analysis of the spatial and temporal concentration variations of TMDD in the river Rhine at the Rheingütestation Worms (443.3 km). Moreover, the transported load in the Rhine was investigated during two entire days and 7 weeks between November 2007 and January 2008. Materials and methods The sampling was carried out at three different sampling points across the river. Sampling point MWL1 is located in the left part of the river, MWL2 in the middle part, and MWL4 in the right part. One more sampling site (MWL3) was run by the monitoring station until the end of 2006, but was put out of service due to financial constrains. The water at the left side of the river Rhine (MWL1) is influenced by sewage from a big chemical plant in Ludwigshafen and by the sewage water from this city. The water at the right side of the river Rhine (MWL4) is largely composed of the water inflow from river Neckar, discharging into Rhine 14.9 km upstream from the sampling point and of communal and industrial wastewater from the city Mannheim. The water from the middle of the river (MWL2) is largely composed of water from the upper Rhine. Water samples were collected in 1-L bottles by an automatic sampler. The water samples were concentrated by use of solid-phase extraction (SPE) using Bond Elut PPL cartridges and quantified by use of gas chromatography-mass spectrometry (GC-MS). The quantification was carried out with the internal standard method. Based on these results, concentration variations were determined for the day profiles and week profiles. The total number of analyzed samples was 219. Results The results of this study provide information on the temporal concentration variability of TMDD in river Rhine in a cross section at one particular sampling point (443.3 km). TMDD was detected in all analyzed water samples at high concentrations. The mean concentrations during the 2 days were 314 ng/L in MWL1, 246 ng/L in MWL2, and 286 ng/L in MWL4. The variation of concentrations was low in the day profiles. In the week profiles, a trend of increasing TMDD concentrations was detected particularly in January 2008, when TMDD concentrations reached values up to 1,330 ng/L in MWL1. The mean TMDD concentrations during the week profiles were 540 ng/L in MWL1, 484 ng/L in MWL2, and 576 ng/L in MWL4. The loads of TMDD were also determined and revealed to be comparable in all three sections of the river. The chemical plant located at the left side of the Rhine is not contributing additional TMDD to the river. The load of TMDD has been determined to be 62.8 kg/d on average during the entire period. By extrapolation of data obtained from seven week profiles the annual load was calculated to 23 t/a. Discussion The permanent high TMDD concentrations during the investigation period indicate an almost constant discharge of TMDD into the river. This observation argues for effluents of municipal wastewater treatment plants as the most likely source of TMDD in the river. Another possible source might be the degradation of ethoxylates of TMDD (Surfynol® series 400), in the WWTPs under formation of TMDD followed by discharge into the river. TMDD has to be considered as a high-production-volume (HPV) chemical based on the high concentrations found in this study. In the United States, TMDD is already in the list of HPV chemicals from the Environmental Protection Agency (EPA). However, the amount of TMDD production in Europe is unknown so far and also the biodegradation rates of TMDD in WWTPs have not been investigated. Conclusions TMDD was found in high concentrations during the entire sampling period in the Rhine river at the three sampling points. During the sampling period, TMDD concentrations remained constant in each part of the river. These results show that TMDD is uniformly distributed in the water collected at three sampling points located across the river. ‘Waves' of exceptionally high concentrations of TMDD could not be detected during the sampling period. These results indicate that the effluents of WWTPs have to be considered as the most important sources of TMDD in river Rhine. Recommendations and perspectives Based also on the occurrence of TMDD in different surface waters of Germany with concentrations up to 2,500 ng/L and its presence in drinking water in the USA, more detailed investigations regarding its sources and distribution in the aquatic environment are required. Moreover, the knowledge with respect to its ecotoxicity and its biodegradation pathway is scarce and has to be gained in more detail. Further research is necessary to investigate the rate of elimination of TMDD in municipal and industrial wastewater treatment plants in order to clarify the degradation rate of TMDD and to determine to which extent effluents of WWTPs contribute to the input of TMDD into surface waters. Supplementary studies are needed to clarify whether the ethoxylates of TMDD (known as Surfynol 400® series) are hydrolyzed in the aquatic environment resulting in formation of TMDD similar to the well known cleavage of nonylphenol ethoxylates into nonylphenols. The stability of TMDD under anaerobic conditions in groundwater is also unknown and should be studied.

Background, aim and scope For reliable environmental risk assessment of pollutants, knowledge on the effects at different levels of biological organisation is needed. During the early days of biomarker research in environmental studies approximately two decades ago, biochemical biomarkers were considered as the most promising tool for such purposes. Among these, three enzymes have often been studied: catalase (CAT), glutathione S-transferase (GST) and cholinesterase (ChE). However, despite their intensive research, their measurements in invertebrates have not been commonly applied in environmental risk assessment (ERA) or for regulatory purposes. Main features In the present review, we summarise our past experiences in biochemical biomarker research in two crustacean species: water flea Daphnia magna and terrestrial isopod Porcellio scaber. This is to orientate their use and to provide recommendations for the use of novel biomarkers in environmental studies, such as proteomic or genomic responses. Results and discussion We assessed the intrinsic properties of biochemical biomarkers CAT, GST and ChE in the D. magna and the isopod P. scaber. It was found that they are not in agreement with the expectations that were previously given for their use in environmental studies. To advance their use in environmental risk assessment, we suggest that based on their properties, their role should be more specifically defined. ERA includes several distinct steps, among them hazard identification, effect assessment and finally risk characterisation, each of which requires a different type of toxicity data. We recommend that the use of biochemical markers is most appropriate for hazard identification because this is a procedure whose purpose is to characterise the potential hazard of the substance in question and is more flexible in terms of using different tools. Furthermore, our results imply that biochemical markers are not always more sensitive than whole-organism responses, as was anticipated. Their sensitivity depends on the mode of action, duration of exposure and test species. Therefore, we suggest that combining both a battery of biomarkers from different levels of biological organisation and an array of biomarkers within a single level could identify hazard adequately. Conclusions The lesson learnt from biochemical biomarkers in environmental studies utilizing crustacean model species is that, for successful application of each group of biomarkers, their intrinsic properties are needed to be known before an (eco)toxicity study is designed. We suggest that a substantial body of experience obtained with biochemical biomarkers should be exploited to new emerging biomarkers in environmental studies in order to facilitate their application. Recommendations and perspectives The future of biomarkers lies in a combination of traditional biochemical and new-generation biomarkers. The latter are not only a potential replacement for existing biomarkers but will also provide new knowledge which might encourage renewed research and development of traditional biomarkers. For research purposes, complete ecotoxicity information should include contributions from molecular fingerprint of an organism, as well as whole organism, population and ecosystem responses. Still, the type of biomarkers used for routine purposes will depend on their reproducibility, their ease of use, robustness, affordability of the methodology and the type of chemicals, organisms and ecosystem of interest.