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Bioassay-directed chemical analysis of River Elbe surface water including large volume extractions and high performance fractionation
Abstract
A bioassay-directed fractionation and identification (toxicity identification evaluation procedure) was performed on extracts of 10 1 River Elbe water samples. The experimental method included a SDB-1 solid phase extraction followed by RP-HPLC fractionation and subfractionation. Chemical analysis by GC-MS as well as acute toxicity testing using a luminescent bacteria assay were conducted in the respective fractions. Many substances were identified, among which were pesticides and pharmaceuticals, but many compounds remained unknown.
... As the concentration of chemicals in environmental waters is typically in the nanogram per litre to microgram per litre range, sample preparation prior to bioanalysis is required, with solidphase extraction (SPE) commonly applied to enrich water samples. [6][7][8][9][10] As bioassays are increasingly applied to cleaner matrices, such as surface water and drinking water, samples oen need to be enriched up to 100 times to detect an effect. 11 For practical purposes, the extracts are diluted in the bioassays, hence the initial enrichment of the water sample by SPE is typically 1000 to 2000 fold. ...
... f recovery;i ¼ C i;extract ðwater þ mixÞ À C water i;extract ðwaterÞ C i;nominal (6) Reverse recovery modelling was also applied to existing iceberg modelling studies from the literature. [19][20][21][22] The predicted loss of effect by SPE was calculated using eqn (7). ...
... Predicted loss of effect after SPE ¼ 1 À BEQ chem;extract BEQ chem;modelled 100% recovery (7) 4. Results and discussion ...
In vitro bioassays are increasingly used for water quality monitoring. Surface water samples often need to be enriched to observe an effect and solid-phase extraction (SPE) is commonly applied for this purpose. The applied methods are typically optimised for the recovery of target chemicals and not for effect recovery for bioassays. A review of the few studies that have evaluated SPE recovery for bioassays showed a lack of experimentally determined recoveries. Therefore, we systematically measured effect recovery of a mixture of 579 organic chemicals covering a wide range of physicochemical properties that were spiked into a pristine water sample and extracted using large volume solid-phase extraction (LVSPE). Assays indicative of activation of xenobiotic metabolism, hormone receptor-mediated effects and adaptive stress responses were applied, with non-specific effects determined through cytotoxicity measurements. Overall, effect recovery was found to be similar to chemical recovery for the majority of bioassays and LVSPE blanks had no effect. Multi-layer SPE exhibited greater recovery of spiked chemicals compared to LVSPE, but the blanks triggered cytotoxicity at high enrichment. Chemical recovery data together with single chemical effect data was used to retrospectively estimate with reverse recovery modelling that there was typically less than 30% effect loss expected due to reduced SPE recovery in published surface water monitoring studies. The combination of targeted experiments and mixture modelling clearly shows the utility of SPE as a sample preparation method for surface water samples, but also emphasizes the need for adequate controls when extraction methods are adapted from chemical analysis workflows.
... While EDAs in the late 1980s and early 1990s were predominantly based on assays with invertebrates such as Daphnia magna, Daphnia pulex, Ceriodaphnia dubia and the fish Pimephales promelas [57], in recent years acute bioluminescence inhibition of the bacteria Vibrio fischeri has become the predominant biotest [4,22,64,70,91,92,93]. This biotest is highly reproducible, rapid, easy to handle, does not consume too much sample, and may be advanced to a real high-throughput instrument by using microtiter plates [6,22] or direct combination with TLC [70,71]. ...
... This may be demonstrated on the basis of a recent study going for toxicity identification in river water with Vibrio fischeri [93]. River waters are believed to contain natural and anthropogenic compounds from the whole range of logK OW values. ...
... This is typically the case for specifically acting contaminants for example in wastewater effluents [15] or environments close to toxicant sources [30]. Increasing dilution with the distance from the sources and increasing shares of solely nonspecifically acting compounds in the mixture reduce the chances of success [93]. Thus, I feel, it is indicative to carefully pre-identify hot spots of pollution and toxicity by biological screening methods or on the basis of known sources and to focus EDA on these sites rather than to use EDA for screening purposes. ...
Wastewater effluents, groundwater, surface water, sediments, soils and air particulate matter are often contaminated by a multitude of chemicals. Since often no a priori knowledge of relevant toxicants exists, chemical analysis alone is not an appropriate tool for hazard assessment. Instead, a linkage of effect data and hazardous compounds is required. For that purpose, effect-directed analysis (EDA) was developed, which is based on a combination of biotesting, fractionation procedures and chemical analytical methods. Since a controversial discussion about the prospects of success in relation to the expense exists, the current methodological state of EDA for organic toxicants in complex mixtures and important results are reviewed in this paper with the aim of establishing criteria for the successful use of this promising tool. While EDA is a powerful tool to identify specifically acting individual toxicants close to the source of emission, it is inappropriate for screening purposes and often may fail in remote areas where the concentrations of specific toxicants are too low relative to the nonspecific toxicity of the whole mixture of natural and anthropogenic compounds. The biological tools have to be carefully selected with respect to their ability to detect specific effects and their significance in hazard assessment. Sophisticated chemical tools are required to identify individual toxicants in mixtures of thousands of compounds, which are typical for contaminated environments.
... Effects-directed analysis (EDA) is a technique utilized to isolate and identify biologically active chemicals such as endocrine-active chemicals (EACs) from complex mixtures. The technique has been applied for EAC isolation from waters and sediments (Reineke et al. 2002;Jenkins et al. 2003;Furuichi et al. 2004;Beck et al. 2006;Fetter et al. 2014;Jonker et al. 2015;Hashmi et al. 2018Hashmi et al. , 2020Sonavane et al. 2018;Zwart et al. 2018). Not only can EDA guide the identification of the responsible EACs, but it can also help rule out candidate EACs not responsible for the observed biological activity or effect(s). ...
... In addition, EDA is sometimes not possible due to the low concentrations of EACs often found in environmental waters (analytical chemistry detector limitations), and/or bioassay detection limitations. Flow diagrams for EDA exist in the literature that follow a common pattern of sample collection, extraction, crude fractionation, and fine fractionation (with bioassay assessment of extracts/fractions at each step; Reineke et al. 2002;Furuichi et al. 2004;Brennan et al. 2013;Fetter et al. 2014;Pochiraju et al. 2020). Using these flow diagrams as a framework, a conceptual EDA could provide investigators with a quick, inexpensive assessment of the elements required to successfully implement an EDA. ...
Effects‐directed analysis (EDA) is an important tool for identifying unknown bioactive components in a complex mixture. EDA of endocrine‐active chemicals (EACs) from water sources has promising regulatory implications but also unique logistical challenges. Here, we propose a conceptual EDA (framework) based on a critical review of EDA literature and concentrations of common EACs in waste and surface waters. Required water volumes for identification of EACs under this EDA framework were estimated based upon bioassay performance (in vitro and in vivo bioassays), limits of quantification by mass spectrometry [MS], and EAC water concentrations. Sample volume for EDA across the EACs showed high variation in the bioassay detectors with genistein, bisphenol A, and androstenedione requiring very high sample volumes and ethinylestradiol and 17☐‐trenbolone requiring low sample volumes. Sample volume based on the MS detector was far less variable across the EACs. The EDA framework equation was rearranged to calculate detector ‘thresholds’, and these thresholds were compared to the literature EAC water concentrations to evaluate the feasibility of the EDA framework. In the majority of instances, feasibility of the EDA was limited by the bioassay, not MS detection. Mixed model analysis showed the volumes required for a successful EDA were affected by the potentially responsible EAC, detection methods, and the water source type with detection method having the greatest effect on EDA of estrogens and androgens. The EDA framework, equation, and model presented here provides a valuable tool for designing a successful EDA. This article is protected by copyright. All rights reserved.
... ation. Then, the collected fractions are used for the toxicity tests [12][13][14]. These methods can also be modified to couple HPLC online to a biological test system [15]. ...
... An interlaboratory comparison with different commercial test kits has shown that the bioluminescence inhibition assay with Aliivibrio fischeri is highly reliable [26]. In addition to the investigation of sediments and wastewater [29][30][31], the assay was used in the analysis of surface and groundwater [13,32]. ...
... In a monitoring context, the data evaluations and investigations suggested in tier 1 and 2 will help to decide if EDA (tier 3) should be applied to unravel unexplained toxicity. The application of EDA is showing promise as an efficient tool for unraveling toxicity at a specific site and in a specific environmental compartment when: 1) effects of concern are exhibited by the sample or an extract thereof; 2) these effects cannot be explained by target analysis of chemicals known to cause this effect (in general or at the specific site); and 3) there are indications that the observed effect is due to specific or reactive toxicity (for explanation see chapter 4) rather than to baseline toxicity, which is typically caused by large numbers of contributing chemicals, resulting in a distribution of toxicity over many or all fractions (Reineke et al., 2002). ...
... EDA studies with bioassays addressing predominantly non-specific effects, for example measured via bioluminescence inhibition of Vibrio fischeri (Reineke et al., 2002) or other non-specific cytotoxic endpoints based on cell viability quantified by cell counting, or, by higher throughput indicator dyes (Borenfreund and Puerner, 1985;Mosmann, 1983) are not very helpful to identify individual chemicals in EDA since all chemicals contribute to the non-specific toxic burden according to their lipophilicity. Large effects, caused by one or several chemicals, would have to be the result of high concentrations of fairly hydrophobic chemicals in an otherwise weakly contaminated sample. ...
Aquatic environments are often contaminated with complex mixtures of chemicals that may pose a risk to ecosystems and human health. This contamination cannot be addressed with target analysis alone but tools are required to reduce this complexity and identify those chemicals that might cause adverse effects. Effect-directed analysis (EDA) is designed to meet this challenge and faces increasing interest in water and sediment quality monitoring. Thus, the present paper summarizes current experience with the EDA approach and the tools required, and provides practical advice on their application. The paper highlights the need for proper problem formulation and gives general advice for study design. As the EDA approach is directed by toxicity, basic principles for the selection of bioassays are given as well as a comprehensive compilation of appropriate assays, including their strengths and weaknesses. A specific focus is given to strategies for sampling, extraction and bioassay dosing since they strongly impact prioritization of toxicants in EDA. Reduction of sample complexity mainly relies on fractionation procedures, which are discussed in this paper, including quality assurance and quality control. Automated combinations of fractionation, biotesting and chemical analysis using so-called hyphenated tools can enhance the throughput and might reduce the risk of artifacts in laboratory work. The key to determining the chemical structures causing effects is analytical toxicant identification. The latest approaches, tools, software and databases for target-, suspect and non-target screening as well as unknown identification are discussed together with analytical and toxicological confirmation approaches. A better understanding of optimal use and combination of EDA tools will help to design efficient and successful toxicant identification studies in the context of quality monitoring in multiply stressed environments.
... The available literature data confirm that such tests are frequently used (e.g., particularly Daphtoxkit F magna, Daphtoxkit F pulex and Algaltoxkit F microbiotests [21,22]). Microtox and LumisTOX systems, based on the use of bacteria Vibrio fischeri, are also quite common [1,23,24]. ...
... However, it was not possible to conclude that those specific compounds caused the toxicity because a significant number of pollutants still remained unidentified, even after the thorough chemical analyses of the samples. The authors explained the lack of a relationship between the results from chemical analyses and the toxicity measurements using the microbiotests by the antagonistic and synergistic effects that occurred [23]. Integrated tools have also been used successfully for estimating the pollution level of protected waters (e.g., in the Albufera Natural Park, Spain). ...
The fundamental goal underlying studies of the environment and environmental processes is to be able to evaluate the impact of various contaminants on live organisms, including humans. Until recently, data obtained by common chemical and physicochemical methods were used in environmental risk assessment. However, such data could be characterized as routine and limited to the determinations of chosen toxic substances expressed as generalized chemical indicators of environmental pollution.In recent years, there has been intense development of bioanalytical techniques that employ live organisms as indicators. Biotests detect the presence of toxic substances in the environment, and determine their toxicity in the samples analyzed by quantitatively estimating the harm that they cause to live organisms.Ecotoxicological tests are also employed as one of the tools in comprehensive assessment of the state of the environment. In this case, the environmental data and the information on the impact of pollutants on live organisms are obtained from environmental samples that have been analyzed with both chemical techniques and biotests.In this article, we present the data available on the application of integrated research for assessing the quality of the aquatic environment.
... In this study, bioassays for acute and chronic toxicity to Vibrio fischeri failed to identify toxic fractions. This goes in line with a previous study performing EDA aiming to identify toxicants in river water (Reineke et al. 2002). The initial toxicity of the whole sample observed using Vibrio fischeri was distributed over several fractions which all exerted low toxicity. ...
... The initial toxicity of the whole sample observed using Vibrio fischeri was distributed over several fractions which all exerted low toxicity. In all of these fractions some compounds could be chemically analysed, however no toxicants explaining the observed effects were identified (Reineke et al. 2002). Brack (2003) states in a review on EDA studies that the bioassay using acute toxicity to Vibrio fischeri often fails to identify specific toxicants in complex environmental samples. ...
The establishment of cause-effect relationships between observed
biological effects and environmental stressors is a key challenge in
ecotoxicology. This challenge comprises the identification of toxicants present
in biologically active environmental samples and the quantification of effect
contributions of these toxicants to the observed adverse effects. Furthermore,
the identification of milieu factors affecting toxicity of compounds and samples
is needed, in order to transfer results obtained in the laboratory to realistic
environmental situations. For the prediction of the influence of milieu factors
on toxicity of toxicants, models are needed, in order to assess the toxicity for
specific environmental situations quantitatively. This thesis addresses
different aspects of these challenges in four consecutive chapters.
The aim of the first part of the study was to investigate whether it is
possible to identify potentially relevant toxicants in samples with relatively low
levels of contamination using effect-directed analysis (EDA). For this purpose
compounds extracted from a marine sediment sample from the Swedish west
coast were separated into distinct fractions, using two preparative
chromatographic techniques. As detector for toxicity a biotest battery was
applied, consisting of one algal and two bacterial bioassays, comprising acute
and chronic endpoints. Chronic algal toxicity was a powerful tool to
discriminate between toxic and non-toxic fractions, while acute and chronic
bacterial toxicity tests failed to identify toxic fractions. Eight compounds were
identified as potentially relevant toxicants to algae by chemical analysis of
toxic fractions as inferred from the algal bioassay tests: anthracene,
fluoranthene, pyrene, benzo[a]anthracene, benzo[b]fluoranthene, benzo[a]pyrene,
benzo[k]fluoranthene and indeno[1,2,3-cd]pyrene.
After identification of compounds using EDA, a confirmation step is
commonly performed in order to verify whether the identified compounds can
be held responsible for the observed effects. The aim of the subsequent
chapter was to advance the methodology for the confirmation of cause-effect
relationships in cases where mixtures of compounds are identified in the
investigated samples. Tools for the assessment of mixture toxicity accounting
for unknown modes of action and heterogeneity of concentration-response
curves were adapted to be used in the confirmation step. Two samples were
investigated using effect-directed analysis. All identified compounds were
tested individually as pure compounds as well as in mixtures at ratios equal to
those found in the sediment extracts. The observed extract toxicity was then
compared with the expected combined effects calculated according to the
models of concentration addition and independent action as well as with the
observed toxicity of the synthetic mixture. Drawbacks of the established
approach using toxic unit summation were demonstrated. Instead, the Index
of Confirmation Quality was introduced, an easy to read representation
allowing a quantitative measure of confirmation over a range of different effect
levels.
The majority of compounds identified and confirmed as relevant toxicants
in chapter 2 and 3 were polycyclic aromatic hydrocarbons (PAHs). Toxicity of
PAHs was previously reported to depend on light conditions. The aim of
chapter 4 was to analyse the influence of simulated sun light on the toxicity of
the previously investigated samples. Additionally, toxicity of identified
toxicants and mixtures of these compounds was assessed under these
environmentally realistic light conditions. It was shown that the toxicity of the
samples, the PAH compounds and the created synthetic mixtures exert
photoenhanced toxicity. The combined effects of the mixtures can be
predicted using the model of concentration addition. The confirmation step in
EDA revealed that, that only a smaller part of the samples’ toxicity can be
attributed to the combined effects of the identified compounds under
simulated sun light revealed than under standard growth light.
In chapter 5 previously proposed models for the assessment of
photoinduced PAH toxicity were tested with regard to their applicability to the
prediction of photoinduced toxicity to the green algae Scenedesmus
vacuolatus qualitatively and quantitatively. For this purpose, 14 different PAH
compounds were tested under up to three different light conditions for their
effect on the algae reproduction. Illumination conditions comprised standard
algae growth light, simulated sun light aiming to mimic environmental light
conditions, and UV-filtered light, in order to minimise light influence on PAH
toxicity. Models proposed for the prediction of photoinduced toxicity were
modified, in order to account for different exposure conditions and toxic
endpoints used in the bioassay tests. The present results show that the gap
between the highest occupied molecular orbital (HOMO) and the lowest
unoccupied molecular orbital (LUMO) proposed as indication for
photoinduced toxicity to Daphnia magna (Mekenyan et al. 1994) can be used
as a qualitative indication of a potential photoinduced toxicity to the green
algae Scenedesmus vacuolatus. The impact of light conditions on PAH
toxicity can be quantified by a linear model which allows the estimation of the
ED50 of a compound from the amount of absorbed photons and an empirically
determined relative phototoxic efficacy value of the compound.
In conclusion, cause-effect relationships were established between identified
toxic compounds and observed effects in an environmental sample as well as
between the light conditions and the toxicity induced by environmental
samples. Furthermore, the cause-effect relationship between the light
absorbance and the algal toxicity of PAH compounds was quantitatively
modelled.
... Acute toxicity testing was conducted with the bioluminescent bacteria assay with Vibrio fischeri (NRRL-B-11177, Dr. Lange GmbH, Berlin, Germany) according to standard protocols (ISO 1998, Reineke et al. 2002. Results were expressed as percent of loss of luminescence (data range 0100%; level significantly above background 10%). ...
... Summarising the results of different working groups in this field, it becomes obvious that providing evidence for a direct link between contaminant and effect can be difficult ( Kosian et al. 1998, Reineke et al. 2002, even after sophisticated fractionation procedures and especially in the marine environment ( Carr et al. 2001, Burgess 2000. In the present study, the combination of chemical analysis with a variety of biological test systems resulted in the identification of ecotoxicologically-relevant fractions. ...
-Part I: Determination and identification of organic pollutants
Part II: Results of the biotest battery and development of a biotest index
-Preamble. This series of two papers presents the results of an interdisciplinary research project (ISIS) dealing with bioassay-directed
fractionation of marine sediment extracts. Part I presents the extraction and fractionation procedure as well as the results
of chemical analysis, including non-target analysis of sediments. Part II describes the results of the biotest battery in
relation to chemicals possibly causing parts of the observed effects. A biotest index is used to compare the toxicities of
the samples.
-AUTHORS / AFFILIATIONS
Ninja Reineke (3), Werner Wosniok (4), Dirk Danischewski (1), Heinrich Hühnerfuss (3), Angelika Kinder (5), Arne Sierts-Herrmann
(5), Norbert Theobald (2), Hans-Heinrich Vahl (6), Michael Vobach (1), Johannes Westendorf (6) and Hans Steinhart (5).
-(1) Federal Research Centre for Fisheries, Institute for Fishery Ecology, Palmaille 9, 22767 Hamburg, Germany
(2) Federal Maritime and Hydrographic Agency, Bernhard-Nochtstr. 78, 20359 Hamburg, Germany
(3) University of Hamburg, Institute for Organic Chemistry, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany
(4) University of Bremen, Institute of Statistics, Bibliothekstr. 1, 28334 Bremen, Germany
(5) University of Hamburg, Institute for Food Chemistry, Grindelallee 117, 20146 Hamburg, Germany
(6) University of Hamburg, University Hospital Hamburg-Eppendorf, Department for Toxicology, Vogt-Kölln-Str. 30, 22527 Hamburg,
Germany
(7) Eurofins Wiertz-Eggert-Jörissen, Stenzelring 14b, 21107 Hamburg, Germany
Goal, Scope and Background The ecological relevance of contaminants in mixtures is difficult to assess, because of possible interactions and due to
lacking toxicity data for many substances present in environmental samples. Marine sediment extracts, which contain a mixture
of environmental contaminants in low concentrations, were the object of this study. The extracts were investigated with a
set of different biotests in order to identify the compound or the substance class responsible for the toxicity. For this
goal, a combination of biotests, biotest-directed fractionation and chemical analysis has been applied. Further on, a strategy
for the development of a biotest index to describe the toxicity of the fractions without a prior ranking of the test results
is proposed. This article (Part II) focuses on the biological results of the approach.
Methods The toxicological potential of organic extracts of sediments from the North Sea and the Baltic Sea was analyzed in a bioassay-directed
fractionation procedure with a set of biotests: luciferase reporter gene assays on hormone receptor and Ah receptor, arabinose
resistance test, fish embryo test (Danio rerio), comet assay, acetylcholinesterase inhibition test, heat-shock protein 70
induction, oxidative stress and luminescence inhibition test (Vibrio fischeri). The test results provided the basis for the
calculation of a biotest index by factor analysis to compare the toxicity of the samples and fractions.
Results and Discussion Results of 11 biotests on different fractionation levels of the samples were described and discussed with regard to the occurrence
of contaminants and their toxic potentials. Polychlorinated biphenyls, polycyclic aromatic hydrocarbons, quinones, brominated
indoles and brominated phenols were in the focus of interest. A biotest index was constructed to compare the toxic responses
in the samples and to group the biotest results.
Conclusion The procedure presented in this study is well suited for bioassay-directed fractionation of marine sediment extracts. However,
in relatively low contaminated samples, high enrichment factors and sufficient fractionation is necessary to allow identification
of low concentrations of contaminants which is required to link effects and possible causes. In the present case, the relation
between substances and effects was difficult to uncover due to relatively low concentrations of pollutants compared to the
biogenic matrix and to the remaining complexity of the fractions. The results, with respect to the brominated phenols and
indoles in the samples, highlight the successful use of bioassay directed fractionation in the case of high concentrations
and high toxicity.
Recommendation and Outlook In general, it has been shown that a marine risk assessment requires focusing on the input of diffuse sources and taking
into account the fact of mixture toxicity. Effects resulting from biogenic substances will make the assessment of the influence
of anthropogenic substances even more difficult.
... Bioassays for acute and chronic toxicity to Vibrio fischeri failed to identify toxic fractions. This finding is in agreement with a previous study that used EDA to identify toxicants in river water (Reineke et al., 2002). The initial toxicity of the whole sample observed using Vibrio fischeri was distributed over several fractions all exhibiting low toxicity. ...
... The initial toxicity of the whole sample observed using Vibrio fischeri was distributed over several fractions all exhibiting low toxicity. In all of these fractions some compounds could be chemically analyzed; however, no toxicants were identified that explained the observed effects (Reineke et al., 2002). Brack (2003) stated in a review of EDA studies that the bioassay using acute toxicity to Vibrio fischeri often fails to identify specific toxicants in complex environmental samples. ...
Effect-directed analysis (EDA) has been reported to be a powerful tool for the identification of the responsible toxicants in contaminated, hazardous environmental samples. The aim of this study was to investigate whether it also is possible to use currently available EDA methodology to identify potentially relevant toxicants in samples that do not pose obvious problems. For this purpose, compounds extracted from a marine sediment sample from the west coast of Sweden were separated into distinct fractions, using two preparative chromatographic techniques. One algal bioassay using Scenedesmus vacuolatus and two bacterial bioassays using Vibrio fischeri were applied as detectors of toxicity, representing acute and chronic end points. Chronic algal toxicity was a powerful tool for discriminating between toxic and nontoxic fractions, whereas acute and chronic bacterial toxicity failed to identify toxic fractions. Eight compounds were identified as potentially relevant toxicants by chemical analysis of toxic fractions: anthracene, fluoranthene, pyrene, benzo[a]anthracene, benzo[b]fluoranthene, benzo[a]pyrene, benzo[k]fluoranthene, and indeno[1,2,3-cd]pyrene.
... Since unspecific effects such as lethality occur due to exposure to very broad range of compounds, fractionation typically results in the distribution of toxicity over many different fractions (Di Paolo et al. 2015). This phenomenon has been observed previously for other bioassays detecting mainly unspecific effects (Reineke et al. 2002). In contrast, specific effects such as AChE inhibition should be detectable by the AChE assay in a limited number of individually fractions. ...
Acetylcholinesterase (AChE) inhibitors are an important class of neuroactive chemicals that are often detected in aquatic and terrestrial environments. The correct functionality of the AChE enzyme is linked to many important physiological processes such as locomotion and respiration. Consequently, it is necessary to develop new analytical strategies to identify harmful AChE inhibitors in the environment. It has been shown that mixture effects and oxidative stress may jeopardize the application of in vivo assays for the identification of AChE inhibitors in the environment. To confirm that in vivo AChE assays can be successfully applied when dealing with complex mixtures, an extract from river water impacted by non-treated wastewater was bio-tested using the acute toxicity fish embryo test (FET) and AChE inhibition assay with zebrafish. The zebrafish FET showed high sensitivity for the extract (LC10 = relative extraction factor 2.8) and we observed a significant inhibition of the AChE (40%, p < 0.01) after 4-day exposure. Furthermore, the extract was chromatographically fractionated into a total of 26 fractions to dilute the mixture effect and separate compounds according to their physico-chemical properties. As expected, non-specific acute effects (i.e., mortality) disappeared or evenly spread among the fractions, while AChE inhibition was still detected in five fractions. Chemical analysis did not detect any known AChE inhibitors in these active fractions. These results confirm that the AChE assay with Danio rerio can be applied for the detection of neuroactive effects induced in complex environmental samples, but also, they highlight the need to increase analytical and identification techniques for the detection of neurotoxic substances.
... For the chemical fractionation, the extract from SPE underwent chromatographic separation using High-Performance Liquid Chromatography (HPLC, LC-2030, Prominence-i, Shimadzu, Japan) coupled with a Fractionation Collector (CF-1, Spectra/Chrom, Fisher Scientific). Plastic extracts (40 μL) were injected into a C18 column (4.6 mm I.D.×150 mm, Shiseido, Japan), according to Reineke et al. [44] with modifications. The plastic leachate samples were separated using an isocratic solvent mixture of 50% water and 50% methanol over 30 min. ...
In this study, fragments of polyethylene plastic bags were treated with simulated gastric juice of fish for 16 hr. Following solid-phase extraction, methanol eluents caused acute toxicity to embryos and larvae of Japanese medaka. Chromatographic fractions (polar to more non-polar with numbers increasing) of the extract were evaluated for toxicity and estrogenic activity using medaka and an estrogen receptor (ER) cell-line. Fractions 6 and 9 had the highest estrogenic effects with relative hydrophobic chemicals. The vtg expression in fraction 6 was 22-fold higher than control, and the ER cellular response in fraction 9 was 8.5-fold higher than controls. Following non-target screening (NTS), several novel phthalates and phenols were identified in the above two fractions. Fractions 1 and 2 appeared to be primarily responsible for the acute toxicity observed with the whole extract. The hatching rate decreased to 36% in fraction 2, and the hatching of medaka larvae from exposed embryos was even not observed following exposure to fraction 1. NTS of these fractions indicated 635 and 808 entities, respectively, most without toxicity information. These results indicate plastic leachates from gastric juices of fish are complex mixtures of many compounds that can have acute reproductive and sublethal endocrine impacts in fish.
... Tabella 9 -Nome chimico e struttura delle sostanza oggetto della ricerca. Per la determinazione delle metodiche analitiche necessarie all'individuazione delle sostanze scelte come indicatrici della ricerca, sono state condotte inizialmente ricerche bibliografiche, privilegiando i lavori inerenti la problematica e soprattutto quelli nei quali si ricercano le sostanze selezionate e la matrice indagata è acqua (Balcerzak 1997, Barbante, Cozzi et al. 1999, Bencs, Ravindra et al. 2003, Braza, Modamio et al. 2000, Colodner, Boyle et al. 1993, Farre, Ferrer et al. 2001, Galassi, Guzzella et al. 2004, Gòmez, Gòmez et al. 2000, Hall & Pelchat 1993, Hayen & Karst 2003, Jaworowicz, Filipowski et al. 1999, Kanitsar, Kollensperger et al. 2003, Koutsouba, Heberer et al. 2003, Krachler, Alimonti et al. 1998, Loffler & Ternes 2003, Lopez de Alda, Diaz-Cruz et al. 2003, Miao, Koenig et al. 2002, Niessen 1998, Ollers, Singer et al. 2001, Reineke, Bester et al. 2002, Rodriguez, Quintana et al. 2003, Sacher, Lange et al. 2001, Ternes 2001, Ternes, Bonerz et al. 2001, Weigel, Bester et al. 2001, Wissiack & Rosenberg 2002, Zuccato 2000. Infatti, essendo le sostanze ricercate di tipo farmaceutico, è disponibile un numero molto elevato di lavori relativi alla determinazione dei prodotti in matrici biologiche quali, sangue, plasma, urine, ecc. ...
L'uso di sostanze per uso farmaceutico, sia come disinfettanti o utilizzate a livello clinico per fini diagnostici sia per prodotti cosmetici o per l'igiene personale, è ampio e diffuso in tutti gli ambiti della vita quotidiana e lavorativa.Negli ultimi anni gli studi sulla contaminazione ambientale causata dai PPCPs (Pharmaceuticals and Personal Care Products) si sono essenzialmente occupati delle conseguenze derivanti dalla dispersione di sostanze antibiotiche o ormonali, in relazione rispettivamente al fenomeno dell'aumento della resistenza batterica ed all'interferenza sui sistemi endocrini. La ricerca descritta nel presente volume si colloca nell'ambito degli studi nazionali ed internazionali inerenti la problematica, proponendosi di offrire un contributo nell'ampliamento delle conoscenze su tali temi, ed ha come obiettivo la determinazione dell'eventuale presenza nelle acque del fiume Arno di residui di farmaci.
... Combining chemical and bioanalytical techniques to characterize water samples shows great potential for identifying pollutants of potential concern more coherently, as component-based effect assessment can be compared with effect observations in the same sample [55]. Current limitations mostly relate to the amount of sample required for biological analysis, which typically involves multiple bioassays and enriched samples [1]. ...
Abstract Environmental water quality monitoring aims to provide the data required for safeguarding the environment against adverse biological effects from multiple chemical contamination arising from anthropogenic diffuse emissions and point sources. Here, we integrate the experience of the international EU-funded project SOLUTIONS to shift the focus of water monitoring from a few legacy chemicals to complex chemical mixtures, and to identify relevant drivers of toxic effects. Monitoring serves a range of purposes, from control of chemical and ecological status compliance to safeguarding specific water uses, such as drinking water abstraction. Various water sampling techniques, chemical target, suspect and non-target analyses as well as an array of in vitro, in vivo and in situ bioanalytical methods were advanced to improve monitoring of water contamination. Major improvements for broader applicability include tailored sampling techniques, screening and identification techniques for a broader and more diverse set of chemicals, higher detection sensitivity, standardized protocols for chemical, toxicological, and ecological assessments combined with systematic evidence evaluation techniques. No single method or combination of methods is able to meet all divergent monitoring purposes. Current monitoring approaches tend to emphasize either targeted exposure or effect detection. Here, we argue that, irrespective of the specific purpose, assessment of monitoring results would benefit substantially from obtaining and linking information on the occurrence of both chemicals and potentially adverse biological effects. In this paper, we specify the information required to: (1) identify relevant contaminants, (2) assess the impact of contamination in aquatic ecosystems, or (3) quantify cause–effect relationships between contaminants and adverse effects. Specific strategies to link chemical and bioanalytical information are outlined for each of these distinct goals. These strategies have been developed and explored using case studies in the Danube and Rhine river basins as well as for rivers of the Iberian Peninsula. Current water quality assessment suffers from biases resulting from differences in approaches and associated uncertainty analyses. While exposure approaches tend to ignore data gaps (i.e., missing contaminants), effect-based approaches penalize data gaps with increased uncertainty factors. This integrated work suggests systematic ways to deal with mixture exposures and combined effects in a more balanced way, and thus provides guidance for future tailored environmental monitoring.
... Kaiser demonstrated the significant relationship between luminescent bacterial test data and acute toxicity data for many aquatic and land species, including fishes (Kaiser, 1998). The Water Framework Directive established by the European Union in 2000 requires an integrated approach for assessing water quality in a river basin, so the V. fischeri-based bioassay has been applied extensively for the monitoring or control of river water quality, particularly in Europe (Reineke et al., 2002;Zhang et al., 2012;Serpa et al., 2014). There are also a number of reports of its application in other aqueous environments, including industrial waste water (Reemtsma et al., 1999;Wang et al., 2002;Eilersen et al., 2004;Dries at al., 2014;Carbajo et al., 2015). ...
... It is an advantage of SPE to capture and stabilize the compounds on the sorbents when sampled (Hillebrand et al., 2013). Different approaches and devices for the sampling of large volumes of water have been developed since the 1970s (CIAgent, 2012;Coes et al., 2014;Dawson et al., 1976;de Lappe et al., 1983;Dean et al., 2009;Ehrhardt and Bums, 1990;Ellis et al., 2008;Gomez-Belinchon et al., 1988;Green et al., 1986;Hanke et al., 2012;Lakshmanan et al., 2010;McKenzie-Smith et al., 1994;Petrick et al., 1996;Reineke et al., 2002;Sarkar and Sen, 1989;SEASTAR INSTRUMENTS, 1984;Sturm et al., 1998;Suarez et al., 2006;Supowit et al., 2016;Thomas et al., 2001;Weigel et al., 2001;Yunker et al., 1989). Briefly, many of the devices were best suited for low water volumes (for analytical purposes), are not (anymore) commercially available or do not operate in a fully automated mode (see Supporting material for detailed information). ...
The implementation of targeted and nontargeted chemical screening analysis in combination with in vitro and organism-level bioassays is a prerequisite for a more holistic monitoring of water quality in the future. For chemical analysis, little or no sample enrichment is often sufficient, while bioanalysis often requires larger sample volumes at a certain enrichment factor for conducting comprehensive bioassays on different endpoints or further effect-directed analysis (EDA). To avoid logistic and technical issues related to the storage and transport of large volumes of water, sampling would benefit greatly from onsite extraction. This study presents a novel onsite large volume solid phase extraction (LVSPE) device tailored to fulfill the requirements for the successful effect-based and chemical screening of water resources and complies with available international standards for automated sampling devices. Laboratory recovery experiments using 251 organic compounds in the log D range from − 3.6 to 9.4 (at pH 7.0) spiked into pristine water resulted in acceptable recoveries and from 60 to 123% for 159 out of 251 substances. Within a European-wide demonstration program, the LVSPE was able to enrich compounds in concentration ranges over three orders of magnitude (1 ng L− 1 to 2400 ng L− 1). It was possible to discriminate responsive samples from samples with no or only low effects in a set of six different bioassays (i.e. acetylcholinesterase and algal growth inhibition, androgenicity, estrogenicity, fish embryo toxicity, glucocorticoid activity). The LVSPE thus proved applicable for onsite extraction of sufficient amounts of water to investigate water quality thoroughly by means of chemical analysis and effect-based tools without the common limitations due to small sample volumes.
... Due to the large numbers of toxicity tests to be carried out simultaneously, rapid tests are generally used for acute toxicity evaluation in these procedures [8,3]. While bacterial and invertebrate organisms [9,10] are mostly used for rapid screening tests, the use of alga inhibition test would be particularly important to protect the aquatic environment in a trophic-level strategy. In previous studies, Selenastrum capricornutum growth inhibition, which represents a short-term method for estimating the chronic toxicity [11], proved effective to assess the risk associated to the organic micropollutants in contaminated sites of Po River basin (Northern Italy) [12][13][14]. ...
The risk associated to the organic micropollutants was evaluated in surface waters from a contaminated river site, downstream from a highly antropized area in Northern Italy. The sensitivity of a chronic toxicity test toward organics was evaluated in a TRE approach by comparing the toxic effects of the concentrated organic-extract with the toxicity detected in the original mixture. The river water collected during a one-year sampling was treated by SPE extraction with Lichrolut resin and EDTA addition, while Selenastrum capricornutum growth inhibition test was used as ecotoxicological model. While the results were suggesting the organic micropollutants as primary toxicants in the riverwater, the relationship found between the Toxicity Reduction, as evaluated by the resin-EDTA treatment, and the toxicity of the organic mixture, as actually measured in the organic-extracts, was further supporting the predictive capacity of the adopted procedure to monitor the organic pollution in surface waters.
... Therefore, a procedure is necessary for providing toxicity data, as well as identification of the compounds causing the effects [2]. An integrated approach based on a parallel application of bioassays and chemical analysis is the most promising tool for the assessment of environmental pollution [2][3][4][5]. ...
Lake Turawskie, an artificial reservoir on the Mała Panew River, was selected for a preliminary project financed by the Province Environment Protection Fund in Opole (Poland). The aim of this project was to assess the ecological state of this lake, and testing aqueous extracts from bottom sediments for toxic effects was one of the approaches. The toxicity of aqueous extracts of sediments was assessed applying the measurements of bioluminescence inhibition of Vibrio fischeri bacteria. In addition, analyses of organic compounds in sediment extracts obtained by aqueous and subsequent dichloromethane extraction were performed. The chromatograms from coupled gas chromatography -mass spectrometry (GC/MS) indicated a very complex composition of the examined dichloromethane extracts. The GC/MS non target screening analyses were conducted on a set of selected samples as an attempt to identify chemical substances responsible for the observed toxicity effects. However, the differences in sediment toxicity were not reflected in the results of the GC/MS analyses and it was not possible to correlate sediment toxicity with specific organic compounds. Lake sediments contain thousands of substances of natural and anthropogenic origin. Taking into account that an unknown number of them are toxic, the presence of some compounds may have a negative influence on an aquatic ecosystem. Therefore, it is very important to obtain reliable information about the toxicity of the lake's sediments.
... To detect and identify the substances responsible for the observed estrogenic activities in environmental samples and trace their sources, the bioassay combined with gas (or liquid) chromatography-mass spectrometry was proved to be an effectively method (GS-MS or LC-MS) (Reineke et al., 2002;Matthiessen et al., 2006;Jonkers et al., 2010). Of the bioassays, in vivo experiments for investigating estrogenic effects are usually expensive and time-consuming, while in vitro bioassays are relatively rapid, inexpensive, and easy to perform to give first evidence of the occurrence of the total estrogenic contaminants in environmental samples (Campbell et al., 2006). ...
Studies on estrogenic disrupting compounds (EDCs) occurrence and identification of main responsible compounds in river water discharged into the sea are of significance. In the present research, we screened estrogenic activities of 10 river water samples from 3 main rivers discharged into Bohai Sea in Tianjin using a recombinant two-hybrid yeast assay and chemical analysis by gas chromatography-mass spectrometry. All sample extracts induced significant estrogenic activity, with 17beta-estradiol equivalents (EEQ) of raw water ranging from 5.72 to 59.06 ng/L. Six most concerned EDCs in the river water samples including estrone, 17beta-estradiol, 17alpha-ethinylestradiol, estriol, diethylstilbestrol and estradiol valerate were determined, with their concentrations up to 50.70, 31.40, 24.40, 37.20, 2.56, and 8.47 ng/L, respectively. Through causality analysis by comparing the EEQ values of yeast assay and chemical analysis, 17alpha-ethinylestradiol and 17beta-estradiol were identified as the main contributors to the estrogenic effects of the river samples, accounting for the whole estrogenic activities (62.99% to 185.66%), and estrogen antagonistic compounds might presented in the heavy polluted water samples. The proposed approach using both chemical analysis and bioassay could be used for identification and evaluation of the estrogenic activity of EDCs in river water.
... Nevertheless, the effects of flow conditions on the ecotoxicity of raw and treated wastewater have not been investigated in detail so far. Commonly, effect-directed analysis[EDA]has been used as a powerful tool for the investigation of potentially toxic xenobiotics in complex matrices such as wastewater[8][9][10][11][12]. This approach integrates biotesting with physicochemical fractionation procedures and chemical analysis. ...
Background
Flow conditions in the sewer systems are particularly important for the chemical and toxicological characteristics of raw and treated wastewater. Nevertheless, this topic has not been thoroughly investigated to date. In this study, composite wastewater samples were taken daily from the influent and effluent of a municipal sewage treatment plant. Polarity-based fractionation of the samples was carried out through sequential solid phase extractions. Biological testing of single and recombinant fractions was performed using bioluminescence inhibition assay according to DIN EN ISO 11348-2. Selected compounds (pharmaceuticals and polycyclic aromatic hydrocarbons) were also included in the chemical analysis by liquid chromatography coupled with tandem mass spectrometry and gas chromatography coupled with mass spectrometry. By analyzing different flow conditions, this study clarifies how these fractions contribute to the total toxicity of organic substances in wastewater. Additionally, it demonstrates the extent to which the potentially hazardous effects of the fractions can be reduced at the examined sewage treatment plant.
Results
Summarizing, medium to highly polar organic compounds were particularly relevant for the total toxicity of organic xenobiotics. For rising wastewater flow under wet weather conditions, we observed a significant decrease in the overall toxicity of the organic pollutants and specifically in the toxic effects of the moderately polar fraction 2.
Conclusions
The results provide the starting point for an important risk assessment regarding the occurrence and behavior of potentially toxic xenobiotics by differentiated polarity in municipal wastewater for varying flow conditions.
... This information is necessary to the removal of pollutants or for environmental remediation (Lijana and Per-Anders, 2007). Combined in vitro bioassays and chemical analysis tools have, therefore, now been A c c e p t e d M a n u s c r i p t 4 / 25 recognized as effective methods for screening estrogenic chemicals and for environmental risk assessments (Reineke et al., 2002; Matthiessen et al., 2006). Chemical analytical methods, using gas (or liquid) chromatography-mass spectrometry (GS-MS or LC-MS) combined with solid phase extraction (SPE) for determining concentrations of EDCs in water, have proved to be very useful complementary methods associated with bioassays (Kasprzyk-Hordern et al., 2008; Jonkers et al., 2010). ...
Detection of estrogenic disrupting compounds (EDCs) in drinking waters around China has led to rising concerns about health risks associated with these compounds. There is, however, a paucity of studies on the occurrence and identification of the main compounds responsible for this pollution in the source waters. To fill this void, we screened estrogenic activities of 23 source water samples from six main river systems in China, using a recombinant two-hybrid yeast assay. All sample extracts induced significant estrogenic activity, with E2 equivalents (EEQ) of raw water ranging from 0.08 to 2.40 ng/L. Additionally, 16 samples were selected for chemical analysis by gas chromatography-mass spectrometry. The EDCs of most concern, including estrone (E1), 17beta-estradiol (E2), 17alpha-ethinylestradiol (EE2), estriol (E3), diethylstilbestrol (DES), estradiol valerate (EV), 4-t-octylphenol (4-t-OP), 4-nonylphenols (4-NP) and bisphenol A (BPA), were determined at concentrations of up to 2.98, 1.07, 2.67, 4.37, 2.52, 1.96, 89.52, 280.19 and 710.65 ng/L, respectively. Causality analysis, involving comparison of EEQ values from yeast assay and chemical analysis identified E2, EE2 and 4-NP as the main responsible compounds, accounting for the whole estrogenic activities (39.74% to 96.68%). The proposed approach using both chemical analysis and yeast assay could be used for the identification and evaluation of EDCs in source waters of China.
... Bioassays provide data about the effect without pinpointing the substances and the potential source, so a tool is needed to provide toxicity data as well to identify the compounds causing the effects. An integrated approach based on parallel application of bioassays and chemical analysis is the most powerful way of assessing environmental pollution [9,10]. ...
The present study deals with the assessment of the quality of sediments using chemometric interpretation of monitoring data from physicochemical parameters and toxicity tests. The dataset comprised results from lake-sediment samples. All 260 samples were analyzed for 44 physicochemical parameters, as well as for acute and chronic toxicity by respective biological tests. The monitoring results were considered both separately (chemical and toxicity data) and simultaneously in order to identify pollution sources in the region and to detect correlation between chemical and toxicity tests.The chemometric methods applied were cluster analysis and principal components analysis. Our effort to establish some simple relation between the two different types of parameters measured (chemical and ecotoxicity) did not give any hint of serious correlation between them. Obviously, sediment assessment needs to be done in two separate analytical tracks – chemical (as until now) and ecotoxicological (which uses biological tests to obtain independent information about acute or chronic toxicity).
... Overall, there is no one species or endpoint that is consistently the most sensitive for all species or all chemicals in all soils, and differences in bioavailability among compounds may confound comparison of test results (Clark et al., 1993). Therefore, bioassays can provide additional information, with acceptable reproducibility (Ritz et al., 2006) on herbicide uptake and translocation (Horowitz, 1980;Reineke et al., 2002). ...
... Conventionally, when dealing with bioaffinity assessment of mixtures, they are first fractionated [1], the collected fractions are evaporated, and microplate bioassays are used to detect the bioactive fractions, eventually in parallel with compound identification by mass spectrometry (MS) and/or nuclear magnetic resonance (NMR) spectroscopy [2][3][4][5][6][7]. The general workflow of such a process is depicted in Fig. 1. ...
In the screening of complex mixtures, for example combinatorial libraries, natural extracts, and metabolic incubations, different approaches are used for integrated bioaffinity screening. Four major strategies can be used for screening of bioactive mixtures for protein targets—pre-column and post-column off-line, at-line, and on-line strategies. The focus of this review is on recent developments in post-column on-line screening, and the role of mass spectrometry (MS) in these systems. On-line screening systems integrate separation sciences, mass spectrometry, and biochemical methodology, enabling screening for active compounds in complex mixtures. There are three main variants of on-line MS based bioassays: the mass spectrometer is used for ligand identification only; the mass spectrometer is used for both ligand identification and bioassay readout; or MS detection is conducted in parallel with at-line microfractionation with off-line bioaffinity analysis. On the basis of the different fields of application of on-line screening, the principles are explained and their usefulness in the different fields of drug research is critically evaluated. Furthermore, off-line screening is discussed briefly with the on-line and at-line approaches.
Schematic view of an on-line bioaffinity analysis or HRS setup with MS based bioassay detection
... The overall chemical analysis of fractions as directed by the outcome of the ecotoxicity tests is today often called effects-directed analysis (EDA). Such fractionations have been applied for tracking harmful components from a wide variety of environmental samples, such as sediments (Carr et al., 2001), effluents (Burkhard and Jenson, 1993; Yang et al., 1999; Mount and Hockett, 2000; Grung et al., 2007), groundwater (Gustavson et al., 2000) and surface water (Reineke et al., 2002). The methods are somewhat case-and country-specific, but the mostly applied and most formal concept is the Toxicity Identification Evaluation (TIE) published by the U.S. Environmental Protection Agency (U.S. EPA) in the 1980's. ...
Screening of wastewater effluents from municipal and industrial wastewater treatment plants with biotests showed that the treated wastewater effluents possess only minor acute toxic properties towards whole organisms (e.g. bacteria, algae, daphnia), if any. In vitro tests (sub-mitochondrial membranes and fish hepatocytes) were generally more susceptible to the effluents. Most of the effluents indicated the presence of hormonally active compounds, as the production of vitellogenin, an egg yolk precursor protein, was induced in fish hepatocytes exposed to wastewater. In addition, indications of slight genotoxic potential was found in one effluent concentrate with a recombinant bacteria test. Reverse electron transport (RET) of mitochondrial membranes was used as a model test to conduct effluent assessment followed by toxicant characterisations and identifications. Using a modified U.S. EPA Toxicity Identification Evaluation Phase I scheme and additional case-specific methods, the main compound in a pulp and paper mill effluent causing RET inhibition was characterised to be an organic, relatively hydrophilic high molecular weight (HMW) compound. The toxicant could be verified as HMW lignin by structural analyses using nuclear magnetic resonance. In the confirmation step commercial and in-house extracted lignin products were used. The possible toxicity related structures were characterised by statistical analysis of the chemical breakdown structures of laboratory-scale pulping and bleaching effluents and the toxicities of these effluents. Finally, the biological degradation of the identified toxicant and other wastewater constituents was evaluated using bioassays in combination with chemical analyses. Biological methods have not been used routinely in establishing effluent discharge limits in Finland. However, the biological effects observed in this study could not have been predicted using only routine physical and chemical effluent monitoring parameters. Therefore chemical parameters cannot be considered to be sufficient in controlling effluent discharges especially in case of unknown, possibly bioaccumulative, compounds that may be present in small concentrations and may cause chronic effects. Suomessa jätevedet johdetaan jätevedenpuhdistamoille ennen kuin ne päästetään vastaanottavaan vesistöön. Kattavasta jätevedenkäsittelystä huolimatta nämä jätevedet sisältävät monia aineita, joista osa voi olla haitallisia ympäristölle. Jätevesipäästöjen ominaisuuksia tarkkaillaan ja niille asetetaan kemiallisiin ja fysikaalisiin tekijöihin perustuvia lupaehtoja. Lisäksi eräissä maissa lainsäädäntö ja ohjeistukset edellyttävät biologisten menetelmien käyttöä. Suomessa biologisia menetelmiä ei ole käytetty rutiininomaisesti lupaehtoja laadittaessa. Jätevesien arviointiin soveltuvia biologisia menetelmiä ovat ekotoksikologiset testit. Niitä käytetään arvioimaan vaikutuksia esimerkiksi organismien kasvuun, lisääntymiseen tai käyttäytymiseen. Perinteisesti ekotoksikologiset vaikutustutkimukset ovat perustuneet kokoeliöihin, esim. kaloihin, mutta nykyään suuntaus on ollut kohti taloudellisesti ja eettisesti toteuttamiskelpoisempia testimenetelmiä. Pienen mittakaavan testit ovat mielenkiintoinen vaihtoehto perinteisille menetelmille. Väitöstyön tarkoituksena oli soveltaa useita pienen mittakaavan testejä jätevesien arviointiin ja edelleen kehittää menetelmiä haitta-aineiden tunnistamiseen. Tarkoituksena oli tuottaa työkaluja jätevesien tai muiden monikomponenttisten vesinäytteiden arviointiin biologisilla menetelmillä sekä haitallisten aineiden ja rakenteiden tunnistamiseen ja karakterisointiin. Kunnallisten ja teollisuuslaitosten jätevesien tutkiminen vahvisti sen, että suomalaiset jätevedenpuhdistamot ovat tehokkaita poistamaan aineita, jotka saattaisivat aiheuttaa akuutteja vaikutuksia kokonaisissa eliöissä. Kuitenkin havaittiin viitteitä hormonivaikutuksista ja perimämyrkyllisyydestä. Tutkimuksen myöhemmässä vaiheessa jäljitettiin myrkyllisyyttä aiheuttavia komponentteja puunjalostusteollisuuden jätevedestä. Ligniini tunnistettiin pääasialliseksi myrkyllisyyden aiheuttajaksi käytettäessä mitokondriaalisilla membraaneilla tapahtuvaan elektroninsiirtoon perustuvaa in vitro -testiä (RET-testi). Kemiallisia rakenteita, jotka voisivat selittää ligniinin myrkyllisyyttä, tutkittiin yhdistämällä kemiallisia, biologisia ja tilastollisia menetelmiä. Lisäksi tutkittiin biohajotuksen vaikutusta jätevesissä esiintyvien orgaanisten yhdisteiden (ligniini ja nonyylifenolietoksylaatit) myrkyllisyyteen.
... However, the most often applied fractionation scheme involves the reversed phase (RP) HPLC fractionation, used directly to fractionate bulk extracts or for fine fractionation of SPE fractions. Linear gradients of methanol and water [9,10,11,12] or acetonitrile and water [13], respectively were preferably used to split the components in relation to their Kow range. ...
An overview of Toxicity Identification and Evaluation (TIE) procedures, used for the effect-based analysis of endocrine disrupting compounds (EDCs) in environmental samples, is presented. Future trends in advanced chemical analysis of EDCs and some emerging contaminants are outlined. The review also gives an overview of concentration levels found in environmental samples and discusses the correlation of calculated estrogenicity (based on chemical analysis of target EDCs) with that measured by various bioassays.
... Acute toxicity testing was conducted with a bioluminescence inhibition assay using the marine bacterium V. fischeri (NRRL-B- Reineke et al. 2002). Tests of the sediment and water extracts as well as brominated phenols and indoles (Table 3) were performed in duplicate in 2% NaCl solution containing 0.5% DMSO as the solvent for sediment and water extracts. ...
This work presents results from analytical as well as ecotoxicologic investigations of sediment and water samples from the North and Baltic Seas. A bioassay-directed procedure was used to investigate cause-effect relationships between observed effects in acute laboratory bioassays (luminescent bacteria assay with Vibrio fischeri and embryo test with Danio rerio) and analyte concentrations in extracted samples. Brominated phenols and indoles-including 4-bromophenol; 2,4-dibromophenol; 4- and 6-bromoindole; 3,4-, 4,6-, and 3,6-dibromoindole; and tribrominated compounds-were identified in partly remarkable concentrations (up to 40,000 ng g(-1) total organic carbon TOC for 4-bromophenol) in North Sea sediment samples and water samples (913 ng L(-1) 3,6-dibromoindole) from the German Bight. The toxicity of some of the identified brominated substances was low, with median effect concentration levels (EC(50)) ranging from 0.08 to 21.7 mg/L for V. fischeri and 4.3 to 46.3 mg/L for D. rerio. Comparison of the concentrations of analytes with ECs showed a toxicity contribution of brominated phenols and indoles to overall toxicity of the fraction. In the case of one water sample from the German Bight, brominated phenols and indoles accounted for the observed toxicity. Brominated phenols and indoles, which are assumed to be of biogenic origin, have rarely been discussed so far in the context of ecotoxicologic effects in marine ecosystems.
This paper presents the results of projects dealing with the assessment of the threats to the environment posed by old, unsealed municipal waste landfills, industrial landfills and polluted lake sediments. Acute and chronic toxicity studies were conducted on ground water samples collected from beneath municipal waste landfills, samples of waste from a post-metallurgical dumping site and sediment samples taken from the retention reservoir bottom. Simultaneously typical chemical analyses were conducted within the range required by the proper Decree of the Minister of the Environment of Poland (2002), as well as analyses of samples for organic chemicals content, the presence of which in the environment is not regulated by law. The results of studies presented here show new areas of possible application of biotests, where ecotoxicological results can efficiently support quality assessment of solid waste and further decisions about its management including their disposal in the environment.
The Urban Water Security Research Alliance (UWSRA) is a $50 million partnership over five years between the Queensland Government, CSIRO's Water for a Healthy Country Flagship, Griffith University and The University of Queensland. The Alliance has been formed to address South East Queensland's emerging urban water issues with a focus on water security and recycling. The program will bring new research capacity to South East Queensland tailored to tackling existing and anticipated future issues to inform the implementation of the Water Strategy.
A Toxicity Reduction Evaluation (TRE) conducted on various water treatments was successful in optimizing the acute toxicity of the final effluent. In particular, we found that the acute toxicity of the sample water treated by a water treatment process should not be correlated with the concentrations of the parent compounds still remaining in the water. Therefore, it was concluded that an acute toxicity test based on a bioassay must be performed in order to evaluate the efficiency of the water treatment process for various effluents containing environmental pollutants. In addition, the treatability and the resultant toxicity was evaluated as a simple scoring system. The scoring system ranked the treated water in three classes of toxicity with the calculation of several factors for the concerned toxicity. Using an impact evaluation based on the score of the characteristics, different water treatment processes could be directly compared. The new methodology presented here is specifically effective in listing possible unit operations for a water treatment process based on the impact, and thereby enables targeted and cost-effective water treatment.
The present study deals with the assessment of the quality of natural waters by the use of chemometric interpretation of monitoring data from physicochemical parameters and toxicity tests. The data set consists from data originating from the Turawa reservoir (surface water, sediment core water and groundwater). All 154 samples were analysed for 33 physicochemical parameters as well as for acute and chronic toxicity by respective biotests. The monitoring results were considered both separately (chemical and toxicity data) and simultaneous in order to identify pollution sources in the region and to detect correlation between chemical and toxicity tests. The chemometric methods applied were cluster and principal components analysis. The identified latent factors responsible for the data structure resemble the impact of sources like marine influence, water hardness, turbidity, nutrition etc. It is quite interesting to detect that there is no direct high correlation between chemical and toxicity data, which is an important indication that the water quality has to be assessed by both groups of parameters. Still, some indications for the formation of acute and chronic toxicity by some chemical parameters are found and discussed.
The advantages of combined approaches for the evaluation of drinking-water quality, such as effect-directed analysis (EDA) consisting of biological and chemical analytical methods are illustrated in this work by comparing a classical and EDA approach applied to samples from China and Germany. The China samples contain polycyclic aromatic hydrocarbons, alkylanilines, chloroanilines, chloronitrobenzenes, chlorobenzothiazoles, chloroalkylphosphates, as well as pesticides ((4-chloro-2-methylphenoxy)ethanoic acid, metolachlor, atrazine, and the insect repellent N,N-diethyl-3-toluamide) in concentrations between 1 and 1000 µg L−1. The toxicity profiles of two River Elbe samples and one sewage-treatment-plant sample, as determined by the luminescent bacteria test, selectively reflect the contamination background: the first profile only possesses two out of eight fractions well above the significance level of 10% inhibition, in the second profile, all but one fraction lie well above this level, exhibiting also a much higher total toxicity in each fraction as compared with the latter sample. In the third example, all fractions are far above the significance level and show a much higher total toxicity, but also the profile shifts towards less polar fractions.
IntroductionProfiling of Noncovalent Protein–Protein and Protein–Ligand Interactions Based on Mass Spectrometry Flow AnalysisIntegration of Flow Analysis and High-Performance Liquid Chromatography for the Bioaffinity Screening of MixturesConclusions
The topic of endocrine disruption in the aquatic environment is a clear example of a problem-driven research area. Field observations
of endocrine abnormalities in wild life have prompted the growth of scientific attention and concern about the topic. Multiple
studies have reported the presence of endocrine disrupting activities in various compartments of the aquatic environment,
without, at the time, knowing the cause of the observations. The application of effect-directed analysis (EDA) has shown to
be a valuable approach in investigating the nature of the compounds responsible for endocrine disrupting activities in environmental
samples. Various research groups have applied EDA approaches and thereby successfully identified compounds responsible for
endocrine disrupting effects. The research field of endocrine disruption is thus one of the research areas that has extensively
experienced the benefits of EDA. This chapter describes the issue of endocrine disruption in the aquatic environment and discusses
examples of the application of EDA for the identification of responsible compounds.
Keywords17a-ethynylestradiol-Androgen-CALUX-Estradiol-Feminisation-Glucocorticoid-Hormone-Progesterone-Thyroid-hormone-Yeast Oestrogen Screen
Comprehensive GC/MS analysis was applied to both the mobile liquid phase (seepage water) and the immobile solid matter of discrete layers derived from a waste deposit landfill. The vertical distribution of organic compounds supports information on the transport, transfer and transformation processes with depth and, consequently, with time.Numerous low molecular weight organic contaminants of natural and xenobiotic origin were identified and partially quantified. Several were selected to act as molecular indicators for different processes. Interpretation of their occurrence and concentration profiles (considering possible waste sources) and their molecular properties allowed us to (i) differentiate immobile and mobile fractions, (ii) reveal restrictions in the vertical transport by transfer processes between particulate and water phase, (iii) identify dynamic accumulations of individual contaminants and (iv) estimate approximate residence times. In addition, intensive degradation processes were pointed out for the natural fraction of the organic matter by way of determination of specific transformation products. Besides the transformation of natural components, transformation of numerous xenobiotics was recognised. In particular, with respect to an important group of contaminants, the phthalate-based plasticisers, a detailed view of (i) the influence of transfer and transport phenomena on transformation processes as well as (ii) the consecutive appearance of different degradation steps in both seepage water and solid waste was pointed out. The information provides a valuable base for the prediction of the long term behaviour of organic contaminants in waste deposit landfills.
The present work is devoted to the study of the decontamination of chars obtained in the co-pyrolysis of plastics, biomass and tyre wastes. The chars were extracted with several organic solvents of different polarities either individually or in sequence. The ability of each selected extractant to remove toxic pollutants was evaluated by comparing the extraction yields and by characterizing the crude extracts with a combination of chemical analysis and toxicity bioassays. Also, the mineral composition of the treated and non-treated chars was assessed. The results obtained in this study indicate that hexane is the more efficient extraction solvent to be used in the organic decontamination of chars obtained in the co-pyrolysis of plastics, tyres and biomass. A sequential extraction with solvents of increasing polarity can provide a better decontamination of the raw pyrolysis char than any individual extraction. The compounds removed from the char during the decontamination process are mainly aliphatic hydrocarbons and aromatic hydrocarbons, therefore a material that may be upgraded to be used as a fuel and/or as raw material for the organic chemical industry.
Identification of unknown compounds remains one of the biggest challenges for the assignment of adverse effects of sediment contamination and other complex environmental mixtures to responsible toxicants by effect-directed analysis (EDA). The identification depends on information gained from biotesting, chromatographic separation, and mass spectrometric detection. Thus, a methodology is provided for non-target identification of partial polar mutagenic polyaromatic compounds in sediment extracts by using polymeric reversed-phase HPLC column, high-resolution mass spectrometry and PubChem database. After visualization and processing the chromatogram constituents by using deconvolution software, the unambiguous elemental compositions generated were used as input in PubChem database to find a possible identity for the suspected species. The retrieved structures from the database search were refined by characterized chromatographic and mass spectrometric classifiers based on 55 model compounds comprising eight different classes representing mutagenic substructures. The applicability of the method was demonstrated by positive and tentative identification of constituents of mutagenic sediment fractions similar to selected model compounds.
The Nackdong River is the longest river in South Korea and passes through major cities that have several industrial complexes, including chemical, electric, and petrochemical complexes, and municipal characteristics such as apartment complexes. Along the river, the Gumi region has an electric industrial complex and an apartment complex that may be possible point sources of xenoestrogens such as phenolic compounds. To identify the causative chemicals for estrogenic activity in the river water of this region, bioassay-directed chemical analysis was performed. All samples from six sampling sites (an upstream point: SI; hot spot points: S2-1, S2-2, and S2-3: and downstream points: S3, and S4) showed estrogenic activity in the E-screen assay, with bio-EEQs (17 beta-E(2)-equivalent quantities) ranging from 25.35-677.15 pg/L Samples from S2-2, the sampling point downstream of the junction of stream water, and domestic and industrial wastewater, contained the highest estrogenic activity. Since the bio-EEQ of the organic acid fraction (F2) of the S2-2 sample had the highest activity (823.25 pg-EEQ/L) and F2 may contain phenolic compounds, GC-MS analyses for phenolic xenoestrogens were conducted with the organic acid fractions of the river water samples. Six estrogenic phenolic chemicals. 4-NP, BPA, 4-t-OP, 4-t-BP, 4-n-OP, and 4-n-HTP, were detected, with the highest concentrations (I-EEQ) found in S2-2 (231.80 pg/L). Among these phenolic chemicals, 4-NP was the most potent estrogen (bio-EEF; 8.12 x 10(-5)) and acted as a full agonist. Furthermore, 4-NP was present at levels (2.0 mu g/L in S2-2) that can induce VTG induction in fish (>1 mu g/L). In addition, we confirmed that river water (S2-2) significantly increased serum VTG levels in crucian carp (Carassius auratus) in a fish exposure experiment under laboratory conditions. Therefore, phenolic xenoestrogens, especially 4-NP, may be the main causative compounds responsible for the estrogenic effect on the Nackdong River.
In order to determine the potential toxicities of organic pollutants in the river water of Chongqing City (China), chemicals were extracted from surface water of the Yangtze River and Jialing River between August 2004 and January 2005. Gas chromatography/mass spectrometry (GC/MS) analysis showed that the main compounds detected were polycyclic aromatic hydrocarbons (PAH) and phthalate acid esters (PAE). The ethoxyresorufin O-deethylase (EROD) test showed that the toxic equivalency (TEQ) values of the samples ranged from 0.9 to 13.3 x 10(-4) pg 2,3,7,8-TCDD/L river water. Incubation of H4IIE cells with organic extracts produced a time-dependent induction of cytochrome P-450 1A1 (CYP1A1) mRNA expression as determined by (1) reverse-transcription polymerase chain reaction (RT-PCR), (2) positive binding to aryl hydrocarbon receptor (AhR), and (3) activation of xenobiotic response element (XRE) by electrophoretic mobility shift assay (EMSA). Data indicated that organic extracts from the river water of Chongqing City induced CYP1A1 activity in hepatocytes in vitro. A possible mechanism underlying toxicity might involve the AhR signal pathway, but further studies are necessary.
Due to the requirements of the EU Water Framework Directive and other legal regulations (e.g., national laws like the German Federal Soil Protection Act), but also due to the implementation of the new EU chemicals management system REACH, environmental monitoring will gain increasing importance for the surveillance of environmental quality as well as for the assessment of chemicals. Against this background, the Work Group on 'Environmental Monitoring' of the Division of 'Environmental Chemistry and Ecotoxicology' within the German Chemical Society has compiled a position paper on substance-related environmental monitoring.
Core elements of this position paper are the definitions of important terms like monitoring, exposure monitoring, effect monitoring, and integrated monitoring. Moreover, temporal and spatial aspects (monitoring of spatial distributions, trend monitoring, and retrospective monitoring) and their applications are discussed. The main focus of this position paper is the coverage of aspects which have to be observed for the preparation and implementation of a monitoring program. Essential is the clear specification of the targets of the monitoring which determine the development of the monitoring concept and its realization, e.g., if environmental media (compartments) or organisms are most appropriate as samples for the aim of the study. Of course, also the properties of the target substances are important (e.g., lipophilicity/bioaccumulation as pre-requisite for an exposure monitoring with organisms). Finally, the monitoring phases of sample planning, sampling, storage and transport of samples, selection of analytical methods, quality assurance measures as well as reporting are discussed.
An important issue for the future is to link the quantification of chemicals in environmental compartments (exposure and pollution monitoring) more closely to the study of biological effects (effect monitoring) than has been the case up to now. Furthermore, by inclusion of a spatial differentiation, a comprehensive evaluation of the state of an ecosystem can be obtained and the relevance of the results improved. Practical examples of monitoring studies which illustrate various aspects covered in this position paper will be presented in a series of publications by members of the Work Group in the following issues of this journal.
A new concept based on hyphenation of biotests, for biological selection, and chemical analysis is introduced for water analysis. Biomolecular recognition components such as receptors, enzymes, and nucleic acids integrated in biological reaction chains are used for binding and selective enrichment of known and unknown biologically active substances in water samples; this is followed by identification and quantitation. The coupling of biomolecular recognition and binding to chemical analysis can be achieved either in discrete analytical steps, e.g. binding and elution of bioactive ligands from affinity columns followed by chemical analysis, or by methods capable of monitoring the binding of the ligand and simultaneous verification of its identity. This analytical strategy, denoted bioresponse-linked instrumental analysis (BLIA), enables detection of potential biological effects and identification of the analyte causing these effects. Several examples are presented.
The current challenge to ecotoxicology is to develop tools that allow rapid and cost-efficient detection of those environmental chemicals or their combinations that are responsible for sublethal, chronic toxic effects in exposed organisms. Bioanalytical tools may meet these challenges, particularly if they are mechanism-based. Technically, bioanalytical tools allow rapid and cost-efficient analysis of environmental matrices. Mechanism-based, bioanalytical tools, however, do not only indicate that certain chemicals are there, but-and this is the major advantage of mechanism-based bioanalytical tools (MBBTs)-they indicate that chemicals with a specific mode of toxic action or a specific toxic potential are there. In this way MBBTs bridge exposure and effect assessment and help in a faster identification of the causative agent(s). Several principles of MBBTs, including immunoassays, enzyme inhibition assays, receptor assays and gene induction assays are briefly discussed and their application in processes such as bioassay-directed fractionation is illustrated. The focus of this manuscript is the analytical power of MBBTs in exposure and effect assessment. MBBTs have, however, a much broader potential and can support research on other challenges in ecotoxicology such as mixture effects or multiple effects caused by single pollutants or by various stresses simultaneously.
Complex mixtures of toxic substances occurring in surface waters are difficult to characterise by chemical analyses because each compound occurs at a very low concentration and requires a specific analytical method to be identified. Ecotoxicological tests on water extracts can be used as a screening tool to evaluate quickly and simply the overall quality of a water body with regard to micropollutant contamination. In this work, a pre-concentration procedure based on solid-phase extraction (SPE), suitable for both biological testing and analytical determination, is proposed. The extraction procedure is an improved version of a methodology used to evaluate the toxicity of organic micropollutants occurring in surface waters. It offers the advantage of using disposable commercial cartridges, which are easier to manage than the columns prepared with macromolecular resins. Water extracts from two representative Italian rivers, characterised by a different gradient of potential contamination and prepared according to the new concentration techniques, are used. The acute toxicity of the water extracts is tested on Daphnia magna and the bioluminescence inhibition in Vibrio fischeri. Criteria based on the concentration factor (CF) are proposed for assessing the hazard to aquatic life due to the exposure to toxic substances in surface waters. The aim of hazard ranking is to focus analytical efforts towards those samples that show the highest toxic potential.
Municipal and industrial effluents were screened with a battery of biotests and with a modified toxicity identification evaluation Phase I procedure. The acute toxicities of the effluent samples were low and the submitochondrial reverse electron-transport (RET) test was the most sensitive toxicity test. Estrogenic effects were found in almost all effluent samples, and genotoxicity was detected in one concentrated effluent sample. The fractionation methods we used proved to be especially effective at tracking toxicity caused by metals and organic contaminants, with the RET test being particularly suited to evaluating pH-dependent toxicity. The used solid-phase extraction columns with both hydrophilic and hydrophobic binding properties turned out to be suitable for removing or reducing organic toxicity-causing substances from the effluent samples. The results of this study show that the use of only conventional acute toxicity tests for effluent assessment will not be sufficient-the genotoxic, hormonal, and even bioaccumulative potential of the effluents and effluent fractions should be evaluated as well.
A new toxicity identification evaluation method for water exhibiting toxicity has been suggested by evaluating toxicity reduction resulting from adsorption and ion-exchange treatments. Adsorption using various adsorbents such as activated carbon, chitosan and zeolite, and ion-exchange using cationic and anionic ion-exchange resins were performed. In addition, toxicity was evaluated by a cell survival test using human liver cancer origin cells (HepG2), and the dose response data was applied to treatment characteristics. The amount of toxicity adsorbed by the various adsorbents was defined based on adsorption isotherm. Parameters of the toxicity adsorption isotherm provided information that allowed us to identify the toxicity-controlling chemicals in environmental water containing various chemicals. The method is promising for water quality management because it can be used to identify toxicity-controlling chemicals among various environmental pollutants.
Tris(chloro-isopropyl)phosphate (TCPP) was identified by GC-MS by comparing mass spectra and retention times to original standards. The concentrations in wastewater of a sewage treatment plant’s influent and effluent were analysed (520 ng l−1 and 380 ng l−1, respectively (mean values). The concentrations of TCPP in the wastewater inflow exhibited a high variability. The elimination of this compound in the sewage treatment plant also exhibits a high variability but is low. Additionally the concentrations in sewage sludge of the same plant were determined (mean value 5100 ng g−1 dry weight; 1700 ng g−1 wet weight, respectively). For a comparison sludge samples from twenty other plants were analysed. In these samples concentrations ranging from 1000–20000 ng g−1
(dry weight) were determined. Thus sorption to sludge does occur to some extent.
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 method has been developed that allows the solid-phase extraction of microorganic compounds from large volumes of water (10 l) for non-target analysis of filtered seawater. The filtration–extraction system is operated with glass fibre filter candles and the polymeric styrene–divinylbenzene sorbent SDB-1 at flow-rates as high as 500 ml/min. Recovery studies carried out for a couple of model substances covering a wide range of polarity and chemical classes revealed a good performance of the method. Especially for polar compounds (log KOW 3.3–0.7) quantitative recovery was achieved. Limits of detection were between 0.1 and 0.7 ng/l in the full scan mode of the MS. The suitability of the method for the analysis of marine water samples is demonstrated by the non-target screening of water from the German Bight for the presence of organic contaminants. In the course of this screening a large variety of substances was identified including pesticides, industrial chemicals and pharmaceuticals. For some of the identified compounds their occurrence in marine ecosystems has not been reported before, such as dichloropyridines, carbamazepine, propyphenazone and caffeine.
In this study a part of the organic compounds present in Rhine water was isolated by XAD-resins and fractionated. Isolates as well as fractions were tested for mutagenicity and toxicity.The highest mutagenic effects in the Ames test were observed with Salmonella typhimurium strain TA98 in the pH 7 isolate. Comparison of past data showed that mutagenicity remained the same in the period 1980–1990. The water samples had to be concentrated at least 25 times by XAD to induce short-term mortality in waterfleas (Daphnia magna), which indicates a substantial improvement in comparison with pollution during the seventies. Chronic toxicity was observed in Daphnia magna after lower levels of XAD-concentration. Extrapolation of these results to field cladocerans is discussed.Most mutagenicity was recovered in the moderately hydrophilic diethylether, ethylacetate and ethanol fractions, but toxicity was almost exclusively located in the lipophilic cyclohexane fraction. However, assuming concentration addition to be dominant in mixtures, the major part (more than 89%) of the toxicity in the cyclohexane fraction could not be attributed to the GC-MS-identified compounds, for which EC50 values were obtained from databases. Several probable causes for this discrepancy are discussed. However, the major contribution lacking is expected to be from identified compounds for which no information was found in the databases or from compounds that could not be identified by GC-MS.It is concluded that the emission reduction along the Rhine should continue, with a more important role for toxicological assays.Our study did not cover metals, very hydrophilic or very lipophilic compounds.
The acute toxicity of narcotic organic chemical mixtures is related to the total molar concentration within organisms. In this study, the use of polyacrylate SPME fibers is investigated for the purpose of biomimetic extractions, a procedure used to simulate bioconcentration and to estimate total concentrations in aquatic organisms. Experimental SPME-water partition coefficients correlate well with octanol-water and membrane-water partition coefficients, indicating that these passive sampling devices provide a good surrogate for lipid partitioning. On the basis of these relationships, the total internal concentration resulting from exposure to an aqueous sample can be estimated from the total moles of chemicals that sorb to the fiber. The aquatic toxicity of the sample is then predicted by comparison to critical internal concentrations that elicit adverse effects. This procedure offers a number of practical advantages over previous biomimetic extraction techniques (e.g., Empore disk) due to faster sorption kinetics, smaller sample volumes, and applicability to volatile compounds.
Within this study all three monochloronitrobenzenes were identified and quantified in water of the German Eight of the North Sea. The concentrations ranged from < 0.02 to 8.9 ng/L. The latter high value was found in the estuary of the River Elbe, though. Additionally, 2,5-dichloronitrobenzene and 3,4-dichloronitrobenzene were identified and quantified. The concentrations of these compounds ranged from < 0.05 to 7.7 ng/L. The major path of input for the chloronitrobenzenes to this marine area is the River Elbe. A method for quantification is described.
Organic extracts of coastal sediments collected off-shore from Barcelona were submitted to a three-level bioassay-directed chemical fractionation, including gel permeation chromatography (GPC) and normal-phase (NP) and reversed-phase (RP) liquid chromatography (LC). The chemical characterization, directed by the Salmonella microsome mutagenicity assay (TA98+S9, TA98NR+S9, and TA98/1,8DNP+S9), was carried out by capillary GC (CGC) coupled to selective detection systems and by CGC-MS techniques. The mutagenic activity recovered among the GPC fractions was concentrated (78-98%) in the second fraction (GPC-2), which was further fractionated by NP-LC. The mutagenicity among the 35 NP-LC-collected fractions exhibited a three-modal distribution, the highest level being recovered in the intermediate-polarity fractions (10-15%), which were further fractionated by RP-LC. Detailed chemical analysis of the mutagenic fractions led to the identification of 140 aromatic compounds, 57 of them classified as mutagenic or belonging to mutagenic chemical classes, among them, 1-nitropyrene, 6-nitrochrysene, and 6-nitrobenzo[a]pyrene were positively identified for the first time in a coastal sediment.
A partial toxicity identification evaluation (TIE) was conducted with a publicly owned treatment works (POTW) effluent that received no secondary treatment. The effluent was consistently acutely toxic to fathead minnow (Pimephales promelas) and the cladoceran Ceriodaphnia dubia. Initial Phase I toxicity characterization and Phase II identification work with Ceriodaphnia dubia suggested that surfactants were responsible for a significant portion of the toxicity of the effluent. Subsequent experiments concerning the behavior of various mixtures of surfactants in Phase I and Phase II TIE procedures provided additional evidence that these compounds were important determinants of toxicity of the POTW effluent. Finally, measurement of two general classes of surfactants in the test effluent (anionic, nonionic) indicated that there were sufficient concentrations of these compounds to be of toxicological concern. The identification of surfactants as potentially important toxicants in a primary effluent is not particularly surprising in light of their concentrations in untreated or inadequately treated wastewater.
Recent studies conducted at our laboratory have shown that sediments contaminated with complex mixtures of polycyclic aromatic hydrocarbons (PAHs) can exhibit enhanced toxicity (lethality) to a variety of aquatic species when the samples are tested under ultraviolet (UV) light designed to mimic the wavelengths present in sunlight. However, because these contaminated sediments can contain literally thousands of chemicals, it is difficult to use conventional analytical techniques to identify those compounds responsible for photo-induced toxicity. The purpose of this study was to adapt existing toxicity identification evaluation methods to attempt to identify those compounds contributing to the phototoxicity observed in our sediment samples. Pore water obtained from sediments collected near an oil refinery discharge was toxic to Lumbriculus variegatus following exposure to UV light, while organisms exposed to the same pore water, but without subsequent UV treatment, showed no toxic effect. Solid-phase extraction disks and high-performance liquid chromatography were used, in conjunction with toxicity tests with L. variegatus, to extract and fractionate phototoxic chemicals from the pore water. Phototoxic fractions analyzed by gas chromatography–mass spectrometry revealed the presence of a number of aliphatic hydrocarbons, substituted PAHs, and PAHs containing heteroatoms. Chemicals were screened for their phototoxic potential based on empirical data and predictive models. A refined list of PAHs was then evaluated on the basis of their phototoxic potency as defined by a recently developed quantitative structure–activity relationship model and estimates of their bioaccumulation potential. Based on the model predictions of potency and bioaccumulation, nine likely phototoxic chemicals were identified.
Pesticides are applied to the rice fields in the Sacramento Valley to prevent the growth of plants, algae and insects that reduce rice yields. Following the pesticide application, field water is released into agricultural drains that in turn discharge into the Sacramento River and delta. Rice irrigation is the largest single use of irrigation water in the Sacramento Valley, and because the irrigation water (or rice return) flows are the primary source of drain effluent during the spring and summer (up to 33% of the total flow), these discharges can significantly affect drain water quality and resident aquatic organisms. Acute and chronic toxicity to freshwater organisms (Ceriodaphnia dubia) was observed in the drain water during the period that coincides with the initial draining of the fields in 1986, 1987 and 1988. In 1988, a toxicity identification evaluation (TIE) was conducted using Ceriodaphnia dubia in an effort to identify the cause of toxicity. Both methyl parathion and carbofuran were identified as possible toxicants. Mixture tests and chronic toxicity tests indicated that the concentrations of methyl parathion and carbofuran in the water sample account for the toxicity observed in Ceriodaphnia dubia.
A styrene-divinylbenzene (St-DVB) sorbent with a specific surface area of ca. 1000 m2 g−1 packed in a disposable cartridge was investigated and compared with C18 silicas and other St-DVB polymers such as those entrapped in Empore disks and the LC-grade PRP-1 or PLRP-S with specific surface areas arpund 5000 m3g−1. A much higher retention was obtained for polar pesticides and acidic pesticides were also retained in their ionic form. The recoveries obtained on percolating 500 ml of drinking water samples at pH 7 spiked with a mixture of seventeen acidic, neutral and basic pesticides through a cartridge packed with 200 mg of SDB-1 sorbent (1060 m2 g−1) were above 80% for each analyte. Another important result was that humic and fulvic acids were not co-extracted and co-eluted on perculating a sample adjusted to pH 7, so no clean-up was necessary. Application to the analysis of a 500-ml river Seine water sample spiked at 0.1 μg l−1 showed that detection limits in this range can be obtained without any additional clean-up step.
Recently, a new procedure was developed to estimate total body residues (TBRs) in biota after exposure to complex mixtures of organic chemicals in water. The procedure is based on a simulation of bioconcentration using a hydrophobic phase and on the measurement of total molar concentrations on this hydrophobic phase via vapor pressure osmometry and gas chromatography–mass spectrometry. In this paper, the results of the application of this procedure to effluents and surface water are presented. Estimated TBRs (TBRests) give information on the potential total bioaccumulation of complex mixtures. Moreover, using these estimated total body burdens, baseline toxicity effects can be predicted, including the contributions of chemicals with specific modes of action to the overall baseline toxicity. The advantage of the parameter TBRest is that it determines total molar concentrations of organic chemicals, including those chemicals that are usually not measured because they cannot be identified or because their concentrations are below the detection limits of individual compounds.
A method is described for the investigation of non-volatile organochlorine compounds in water.
Using a specially developed water sampler, samples can be taken from down to a depth of 2000 m. The sample is extracted in the sampler with hexane and after a column chromatographic preliminary cleansing, investigated each time on a nonpolar and a medium polar capillary column by temperature programmed gas chromatography.
The result of a network measurement survey in the North Sea is given. The distribution patterns which resulted are different with regard to the substances involved.
In the case of α- and γ-HCH, one can recognize a large scale structure of the concentration corresponding with the crucial pollution positions and rates of the water exchange in the North Sea.
For HCB, one can recognize a crucial position off the Federal German and Netherlands coasts apparently caused by the influx of the rivers Rhein, Weser, and Elbe.
Some selected PCB isomers do not permit the certain recognition of a structured distribution in water in the same way as those substances the concentrations of which are only slightly above the level of detection.
A non-target screening by gas chromatography-mass spectrometry has been carried out on water extracts of the river Elbe in order to obtain an overview of organic compounds being dissolved or bound to suspended matter in the Elbe. Samples of 1001 volume were taken at the freshwater border at Stade and extracted by liquid-liquid extraction with pentane. Before GC-MS analysis, the water extracts were fractionated into 15 subsamples by HPLC on an SiO2 column. A sensitivity of ca. 50 to 250 pg/l was reached for the GC-MC analysis. Several hundreds of natural and anthropogenic compounds could be identified or at least grouped into a compound class. The presence of a number of compounds could be verified from earlier investigations, in addition a great number of anthropogenic compounds were described which have previously not been reported for the river Elbe.
GC/MS non target screening has been applied to water samples taken during 1992–1994 from the Elbe river and its tributaries Mulde, Saale, Weie Elster, Schwarze Elster, and Havel. Based on full scan electron impact mass spectra and supplemented by extensive use of chemical ionisation and high resolution data as well as by synthetic reference compounds, several new classes of compounds, whose possible environmental effects are yet unknown at present, have been identified. Tetrachlorinated bis-(propyl)ethers are new among the most prominent contaminants throughout the Elbe river. The confluence with the Mulde river adds a variety of compounds, related to the chemistry of chloro- and nitroaromatics, azo dyes, benzanilides, carbamates, thiophosphates, and pesticides. The combined load of the Weie Elster and Saale rivers carries oligoformals, oxathiamacrocycles, and dichloro- and trichloro-bis-(propyl)ethers, whereas chloropropylphosphates are introduced via the Schwarze Elster. The majority of these compounds, originating from sources at the tributaries, are still present at the mouth of the Elbe river. In addition to specific industrial emissions, a variety of more generally observed organic compounds like long chain aliphatics, sterols, phenylalkanes, and plasticizers as well as ubiquitous environmental trace pollutants like polycyclic aromatic hydrocarbons, chlorobenzenes, and hexachlorocyclohexane isomers have been encountered throughout the Elbe river drainage system.
This report describes the isolation and identification of organic toxicants present in marine surface waters collected from industrially impacted areas around the UK in 1997. The characterization process utilized Toxicity Identification Evaluation (TIE) procedures that have been adapted for marine samples and a small volume bioassay using the marine copepod Tisbe battagliai. The dissolved organic content of bulk water samples was isolated using a layered solid phase extraction system to provide sample concentrates which were tested for acute toxicity. Where acute toxicity was demonstrated, the extracts were fractionated using reverse phase HPLC and tested further. Each fraction showing toxicity was then analysed by gas chromatography-mass spectrometry. The results of this investigation demonstrate that surface water concentrates collected from several impacted estuaries show acute toxicity. Organic extracts from surface waters may be concentrated using simple chromatographic techniques to induce toxicity in test species. Fractionation showed that the majority of this toxicity is associated with low to medium polarity contaminants. Compounds that were identified by gas chromatography–mass spectrometry as the possible cause of toxic effect include alkylphenols, alkyl substituted naphthalenes, alkyl-substituted fluorenes and dimethyl benzoquinone, however, the identity of certain toxic compounds remains unknown. It is concluded that the technique is a valuable tool in identifying compounds that may be potentially harmful to the aquatic environment.
Water samples of the River Elbe near Hamburg were analyzed for 145 organic chemical compounds. In part I results of the investigations concerning the following groups of compounds are presented (57 individual compounds): volatile chlorinated hydrocarbons, chloroalkylethers (haloethers), chlorobenzenes, nitrobenzenes, chloronitrobenzenes, and chloroanilines. Highest concentrations were found for the chlorinated bispropylethers and 1,7-dichloro-3,5-dioxaheptane (haloethers). Other important compounds were nitrobenzene, nitrotoluenes, and chloronitrobenzenes. The results were assessed on the basis of German surface water quality criteria.
Organic extracts of airborne particulate matter, collected in the city of Barcelona, were subjected to three-level, bioassay-directed, chemical fractionation, including gel permeation chromatography (GPC) and normal-phase (NP) and reversed-phase (RP) liquid chromatography (LC). The chemical characterization, directed by the Salmonella microsome mutagenicity assay (TA98, TA98NR and TA98/1,8DNP6 +/- S9), was carried out by capillary GC (CGC) coupled to selective detection systems, and by GC-MS techniques. The results obtained with the nitroreductase deficient strains show the important contribution of nitroaromatic compounds. Detailed chemical analysis of the mutagenic fractions led to the identification of 82 aromatic compounds and revealed the large contribution of chemical classes that are more polar than polycyclic aromatic hydrocarbons such as aromatic ketones, quinones and aldehydes.
This study evaluates the applicability of three bacterial short-term genotoxicity test systems to aquatic suspended particulate matter of the Elbe river. This material was sampled in sedimentation vessels after deposition periods of one month. It was extracted in a Soxhlet apparatus with toluene and methanol. Aqueous elutriates were prepared additionally. A solid phase method was developed that enables to incubate bacteria in contact with the particulate material. The test battery consists of two mutagenicity assays (the Ames-test and the Ara-test) and an SOS induction assay (the umu-test). Both mutagenicity assays came to nearly the same assessment of the samples of particulate matter of the Elbe. The quantitative response, however, was higher in the Ara-test. The particulate river material generally induced lower genotoxic potencies in the umu-test than in the mutagenicity assays. This lead to a completely different outcome of the umu-test; 29 out of 35 mutagenic samples were not SOS inducing. No quantitative or rank correlations between the concentrations of anthropogenic contaminants (PAHs, chlorinated hydrocarbons and metals) and the observed effects could be established on a 90% confidence limit. However, there is obvious correspondence between more contaminated regions of the river system and mutagenic effects (Ames- and Ara-test) in the samples from this region, as well as correspondence between low contaminated regions and the absence of mutagenicity. For this reason, the mutagenicity assays appear more favourable to describe the anthropogenic contamination with genotoxins in complex mixtures than the umu-test. The authors recommend the Ara-test for a first genotoxicity screening of complex environmental mixtures. This forward mutagenicity assay is advantageous due to higher effects and lower costs compared to the Ames-test. The development and use of a solid phase version of Ames- and Ara-test revealed the occurrence of a major part of particle-bound mutagens. The hydrophobic nature of these mutagens was also confirmed by the gradually decreasing effects with decreasing lipophilicity of the solvents. The results suggest that the solid phase test and the use of extracts complement each other in detecting mutagens of different lipophilicity. Both versions should be used in order to include a broad variety of compounds.
Bioassay-directed identification of toxicants in an acetonic extract of a sediment of the riverine Spittelwasser in the industrial region of Bitterfeld (Germany) was conducted. For this purpose, a combination of chromatographical fractionation, chemical analysis, and a biotest battery including Vibrio fischeri (inhibition of bioluminescence), Daphnia magna (immobilization), and Scenedesmus vacuolatus (inhibition of cell multiplication) was applied. Major toxicants identified and confirmed were methyl parathion (D. magna), prometryn, N-phenyl-beta-naphthalene amine, PAHs (S. vacuolatus), and tributyltin (all biotests). Toxicity to V. fischeri was dominated by elemental sulfur. Results indicate high toxicant loads in the sediment about 7 years after closedown of a majority of chemical production sites at Bitterfeld. Comparison of potential exposure and toxicity data indicate a severe hazard potential to aquatic organisms due to organic toxicants. The results illustrate the potency of a biotest battery for identification of toxicants in contaminated sediment within the frame of toxicity identification procedures.http://link.springer-ny. com/link/service/journals/00244/bibs/37n2p164.html
An efficient and novel method for the identification of toxic compounds in industrial wastewater was developed. In the first step, the samples collected were tested for toxicity using the recently developed ToxAlert 10 system based upon luminescence inhibition of freeze-dried Vibrio fischeri. In the second step, sequential solid-phase extraction (SSPE) and liquid chromatography/mass spectrometry (LC/MS) for compound identification were employed to isolate and identify compounds in the waters. Average recoveries ranging from 72 to 95% were obtained using the SSPE methodology for ubiquitous analytes such as poly(ethylene glycol)s, nonylphenol and alcohol polyethoxylates, phenols, linear alkylbenzenesulfonates, and benzene- and naphthalenesulfonates. In the third step, all the extracts obtained after SSPE followed by LC/MS identification were tested again with the ToxAlert system. The procedure was applied to influent and effluent samples of a sewage treatment plant (STP) and to a raw tannery effluent that constitutes the main type of influent in the receiving waters of the STP. This method has shown that, despite the complexity of the untreated tannery wastewaters with an average total organic carbon (TOC) value of 1960 mg of C/L, the biological treatment of the STP eliminates compounds that inhibit luminiscence of V. fischeri. In the final step, the chemical toxicity of the tentatively identified chemicals was tested to identify the toxicants in the waters. Comparison of the toxicities of the sample, the extracts, and individual components has shown that diverse classes of pollutants were responsible for toxicity, as all fractions of toxic samples gave significant bioluminescence inhibition values. Toxicity of the two intermediate-polarity SSPE fractions was attributed to alcohol ethoxylates, nonylphenol ethoxylates, bis(2-ethylhexyl)phthalate, and linear alkylbenzenesulfonates. In the most nonpolar and most polar fractions, identification of the compounds responsible for toxicity was unclear. By the toxicity-based fractionation, followed by LC/MS methodology, it was feasible to identify between 1.4 and 7.5% of the TOC, thus expanding the number of toxicants identified in these complex wastewaters as compared to those identified by conventional gas chromatography/mass spectrometric (GC/MS) methods. When artificial water samples were reconstituted using similar concentrations of the chemicals detected in the wastewaters, nonsynergetic toxicity effects were observed for all analytes with the exception of 2,6-naphthalenedisulfonate (2,6-NPS), which promoted the bioluminescence inhibition. The toxicity-directed identification was successful for the STP's samples and showed 1400 times higher toxicity for the raw tannery wastewaters as compared to the mixed industrial and domestic wastewaters by applying the Weibull model.
Several toxicity-based procedures have been proposed for waste water risk assessment but the toxic agents could never be identified in these very complex mixtures. A procedure was adopted using disposable solid-phase extraction cartridges to extract organic chemicals and preparative HPLC to fractionate them in relation of their hydrophobicity. Acute toxicity of whole samples and their fractions was measured on Daphnia magna, using a commercially available biokit. The procedure was applied to leachate from an industrial landfill and a textile effluent. In both cases the toxic effects due to xenobiotics were highest in the most hydrophobic HPLC fraction. The compounds responsible for the observed toxicity were identified and quantified by GC-MS. Reconstructed mixtures were analysed to assess their fitting with GC profiles and tested for toxicity to compare the responses of individual chemicals and mixtures.
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