From Bioavailability Science to Regulation of Organic Chemicals.

The bioavailability of organic chemicals in soil and sediment is an important area of scientific investigation for environmental scientists, although this area of study remains only partially recognized by regulators and industries working in the environmental sector. Regulators have recently started to consider bioavailability within retrospective risk assessment frameworks for organic chemicals; by doing so, realistic decision-making with regard to polluted environments can be achieved, rather than relying on the traditional approach of using total-extractable concentrations. However, implementation remains difficult because scientific developments on bioavailability are not always translated into ready-to-use approaches for regulators. Similarly, bioavailability remains largely unexplored within prospective regulatory frameworks that address the approval and regulation of organic chemicals. This article discusses bioavailability concepts and methods, as well as possible pathways for the implementation of bioavailability into risk assessment and regulation; in addition, this article offers a simple, pragmatic and justifiable approach for use within retrospective and prospective risk assessment.

Use of factorial design for the multivariate optimization of polypropylene membranes for the cleanup of environmental samples using the accelerated membrane-assisted cleanup approach.

Accelerated membrane-assisted cleanup (AMAC) is a recently developed method to purify extracts from matrix rich samples such as fish tissue and sediments. In this study, we tested the applicability of cast polypropylene (CPP) membranes in AMAC and evaluated the optimized dialysis procedure for the cleanup of extracts of fish tissue. Design of experiments was used to optimize the factors temperature, solvents and static time of dialysis. Main factors influencing dialysis procedure were solvents and temperature as well as the number of cycles. For the CPP membrane the optimal parameters were a temperature of 55 °C, a solvent mixture of n-hexane:acetone (90:10, v:v), a static time of dialysis of 6 min and 20 dialysis cycles. Comparing to the LDPE membrane this was a reduction of dialysis time from 160 to 120 min, but a higher solvent use of 150 ml per sample. However, compared to LDPE membranes CPP exhibited a lower retention of fish tissue matrix and thus reduced cleanup efficiency. Compound specific structural descriptors such as the molecular weight, the van der Waals volume and a shape factor were calculated to explain differences in diffusivity of the different model compounds. We concluded that the permeation of the molecules was related to molecular shapes and the availability of free solvent cavities in the membranes.

Polar compounds dominate in vitro effects of sediment extracts.

Sediment extracts from three polluted sites of the river Elbe basin were fractionated using a novel online fractionation procedure. Resulting fractions were screened for mutagenic, aryl hydrocarbon receptor (AhR)-mediated, transthyretin (TTR)-binding, and estrogenic activities and their potency to inhibit gap junctional intercellular communication (GJIC) to compare toxicity patterns and identify priority fractions. Additionally, more than 200 compounds and compound classes were identified using GC-MS/MS, LC-MS/MS, and HPLC-DAD methods. For all investigated end points, major activities were found in polar fractions, which are defined here as fractions containing dominantly compounds with at least one polar functional group. Nonpolar PAH fractions contributed to mutagenic and AhR-mediated activities while inhibition of GJIC and estrogenic and TTR-binding activities were exclusively observed in the polar fractions. Known mutagens in polar fractions included nitro- and dinitro-PAHs, azaarenes, and keto-PAHs, while parent and monomethylated PAHs such as benzo[a]pyrene and benzofluoranthenes were identified in nonpolar fractions. Additionally, for one sample, high AhR-mediated activities were determined in one fraction characterized by PCDD/Fs, PCBs, and PCNs. Estrone, 17ß-estradiol, 9H-benz[de]anthracen-7-one, and 4-nonylphenol were identified as possible estrogenic and TTR-binding compounds. Thus, not only nonpolar compounds such as PAHs, PCBs, and PCDD/Fs but also the less characterized and investigated more polar substances should be considered as potent mutagenic, estrogenic, AhR-inducing, TTR-binding, and GJIC-inhibiting components for future studies.

Active bio-monitoring of contamination in aquatic systems--an in situ translocation experiment applying the PICT concept.

The environmental risk assessment of toxicants is often derived from chemical monitoring, based on single species tests performed in the laboratory. However, to provide ecologically relevant information, community approaches are required. The aim of this study was to causally link prometryn exposure to community-level effects in complex field situations and to identify response times of adaptation to pollution and recovery from pollution. For this reason sensitivity shifts in communities were detected and related to structural changes within the periphyton community. Furthermore, it was intended to illustrate the possibility of a combined approach of community translocation and sensitivity assessment for active monitoring of polluted sites. Periphyton was grown at a reference (R) and at a polluted (P) site of the river Elbe basin for 26 days, was subsequently transferred from the polluted site to the reference site and vice versa. Sensitivity of communities to prometryn was determined according to the pollution-induced community tolerance (PICT)-concept in short-term tests by measuring photosynthesis inhibition and was related to structural changes in algal class and diatom species composition. Exposure to prometryn was determined using polar organic integrative samplers (POCIS), giving time-weighted average concentrations. Environmental concentrations of prometryn were significantly higher at the polluted site compared to the reference site. Communities grown at the polluted site showed a higher tolerance to prometryn in comparison to the reference site. 17 Days after the translocation to the reference site, EC(50) decreased 2-fold compared to the non-translocated P-community of the same age. By contrast, EC(50) of the community grown at the reference site was 5 times higher after 17 days exposure at the polluted site. Furthermore, P-R communities were less sensitive to prometryn (higher EC(50)) than R-P communities, 24 days after translocation. These changes in sensitivity to prometryn were consistent with changes in species composition and clearly indicate that the exposure history of communities is defining the time-response of recovery and adaptation. In conclusion, the PICT-concept is shown to be a suitable tool for analysis of recovery and adaptation processes of communities under natural conditions. Therefore, it improves the link between cause and effect in field situations. In situ translocation studies provide an ecological relevant assessment of pesticide effects under field conditions and could be used as a diagnostic tool in active monitoring for decision-making frameworks as used in the implementation of the European Water Framework Directive (WFD).

Effect directed analysis of riverine sediments--the usefulness of Potamopyrgus antipodarum for in vivo effect confirmation of endocrine disruption.

In vivo tests are not commonly used in effect directed analysis (EDA) approaches. In the present study, a novel methodology was developed whereby Potamopyrgus antipodarum, which is known to be sensitive to endocrine disrupting compounds, was used as test organism. Field sediments from a polluted site in the north of Belgium were extracted and fractionated using three coupled and automatically switched normal-phase HPLC columns. Part of the fractions were spiked to artificial sediments and tested in a sediment contact test with P. antipodarum. The other part was used for an in vitro effect confirmation with the ER-LUC and anti-AR CALUX assays. Two of the six tested fractions stimulated the reproduction of the snails, while two others inhibited the reproduction. The fractions that caused an increase in reproduction also showed an increased estrogenic potency in the ER-LUC assay. Chemical analysis revealed that one of the most prominent compounds in those fractions was bisphenol-A, which has already been reported to have a stimulating effect on the reproduction of P. antipodarum by other authors. Due to the fact that previous studies have shown that this snail is also present in the field at this certain site, it was possible to directly link the results with effects that were observed in the field. This study indicates that effect directed analyses, supported by in vivo biotests, are very useful tools in order to identify the compounds that cause adverse effects on organisms or even population level.

Characterizing field sediments from three European river basins with special emphasis on endocrine effects - A recommendation for Potamopyrgus antipodarum as test organism.

The assessment of endocrine disrupting potentials of field sediments has until now been mostly limited to classical chemical analysis, in vitro assays and in vivo bioassays performed with vertebrates. There is an urgent need for easy, cheap and reproducible invertebrate tests which may be applied in certain monitoring activities. Since the mudsnail Potamopyrgus antipodarum is known to be tolerant to natural stressors, but also sensitive to endocrine disrupting chemicals, it is very likely that this organism could be suitable for the assessment of endocrine effects of e.g. field sediments. Within this study the endocrine potential of sediments in three European river basins was assessed. The yeast estrogen screen (YES) and a sediment contact test with P. antipodarum were performed. Furthermore, analyses of physico-chemical properties and concentrations of heavy metals, PAHs, organotins, natural steroids and alkylphenols were done. In the sediment contact test, the reproduction of the snail was promoted by a part of the sediments. This phenomenon could not be explained by their physico-chemical properties. However, at some of those sites a high estrogenic activity was detected in the YES, leading to the assumption that endocrine disrupting compounds could be responsible for those effects. This assumption could be confirmed to some extent with partially high concentrations of xeno-estrogens (e.g. nonylphenol) at the certain sites. Our study demonstrates the applicability of the test with P. antipodarum for a variety of sediments and once again points out the need of suitable in vivo biotests for the risk assessment of field sediments.

Identification of a phytotoxic photo-transformation product of diclofenac using effect-directed analysis.

The pharmaceutical diclofenac (DCF) is released in considerably high amounts to the aquatic environment. Photo-transformation of DCF was reported as the main degradation pathway in surface waters and was found to produce metabolites with enhanced toxicity to the green algae Scenedesmus vacuolatus. We identified and subsequently confirmed 2-[2-(chlorophenyl)amino]benzaldehyde (CPAB) as a transformation product with enhanced toxicity using effect-directed analysis. The EC(50) of CPAB (4.8 mg/L) was a factor of 10 lower than that for DCF (48.1 mg/L), due to the higher hydrophobicity of CPAB (log K(ow) = 3.62) compared with DCF (log D(ow) = 2.04) at pH 7.0.

Effect-directed analysis of contaminated sediments with partition-based dosing using green algae cell multiplication inhibition.

Effect-directed analysis (EDA) has been frequently and successfully used to identify key toxicants in sediment extracts. However, by disregarding bioavailability this approach may lead to a biased prioritisation of fractions and toxicants with respect to hazards and risks. To overcome this problem the present EDA of sediment components from the Bílina river (Most Czech Republic), that inhibit growth of the green algae Scenedesmus vacuolatus, applies a novel partition-based dosing technique to prioritize and identify major toxic fractions and compounds in comparison to conventional solvent dosing. The novel dosing technique is based on partitioning from loaded silicone rods to the aqueous phase similar to partition processes that determine exposure in native sediment-water systems. In the present study the application of partition-based dosing had a big influence suggesting polar compounds such as triclosan as key toxicants while polycyclic aromatic hydrocarbon (PAH) fractions did not exhibit significant effects. In contrast, conventional dosing prioritized mainly PAHs in agreement with previous studies. For both approaches individual toxicants could be confirmed quantitatively based on the index of confirmation quality (ICQ), which compares the effect of fractions and artificial mixtures of identified and quantified toxicants over the full range of effect levels.

Effect-directed analysis of contaminated sediment from the wastewater canal in Pancevo industrial area, Serbia.

Wastewater canal (WWC) in Pancevo industrial area in Serbia, whose main environmental receptor is the River Danube, is a well known hot-spot of contamination. WWC sediments have been assessed by UNEP based on chemical target analysis. However, integrative biological data on exposure to hazardous compounds are only provided by the present study which aims at evaluating whether the monitored compounds sufficiently reflect potential hazards and to suggest additional compounds to include in monitoring and hazard assessment by applying effect-directed analysis (EDA) based on arylhydrocarbon receptor-mediated activity and cytotoxicity. Multistep NP-HPLC fractionation provided 18 fractions co-eluting with polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), polycyclic aromatic hydrocarbons (PAHs) and more polar compounds. PAHs fractions exhibited great potencies to induce ethoxyresorufin-o-deethylase (EROD) in H4IIE rat hepatoma cell line expressed as 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TCDD-EQ) (0.1-34.6 x 10(3) pg g(-1)dry weight). Chemical analysis of the most active fractions revealed great concentrations of PAHs (up to 292 x 10(2)ngg(-1) sediment equivalents (SEQ)), methylated PAHs (up to 900 x 10(2) ng g(-1) SEQ), and other alkyl-substituted PAHs. Only minor portions of biologically derived TCDD-EQs could be attributed to monitored PAHs with known relative potencies (REPs). We hypothesize that a major part of the activity is due to non-monitored alkylated and heterocyclic PAHs. Results of the cell cytotoxicity/proliferation assay on H4IIE cell line suggest the presence of sediment pollutants with pronounced potency to disturb cell growth.

Partitioning-based dosing: an approach to include bioavailability in the effect-directed analysis of contaminated sediment samples.

One of the major challenges of effect-directed identification of hazardous chemicals in sediments is the appropriate consideration of bioavailability for a hazard-based prioritization of fractions and toxicants. The equilibrium partitioning approach is frequently used to estimate the bioavailability of sediment contaminants. To simulate partitioning ofthe constituents of complex sediment contaminations in the sediment-water-biota system, a new dosing technique based on silicone elastomer stirrer bars (silicone rods) for use in a growth inhibition test with the green algae Scenedesmus vacuolatus was developed. The dosing technique has a high loading capacity and a fast achievement of equilibrium (5-50 min) for a broad range of model compounds. The subsequent delivery of compounds stored by the silicone during the bioassay compensates possible losses of compounds and keeps the concentrations constant. Furthermore, a dose-dependent response is achieved for single compounds and complex mixtures from sediment extracts. The extracts of three sediment samples from the Elbe River Basin were fractionated using a multistep HPLC method and investigated in the bioassay using conventional DMSO dosing and loaded silicone rods. The results of the toxicity test clearly show that the toxicity patterns are quite different for the two dosing techniques, whereas the differences between the sampling locations are less pronounced. We suggestthat partitioning-based dosing better reflects exposure in sediments and thus provides a more realistic identification of key toxicants.

Effect-directed analysis of sediment-associated algal toxicants at selected hot spots in the river Elbe basin with a special focus on bioaccessibility.

Benthic algae are the dominant primary producers in many rivers and may be at risk of being affected by sediment-associated toxicants. Toxicity as well as bioaccessibility should be considered for the identification of major toxicants affecting benthic algae. Thus, effect-directed analysis (EDA) combining biotesting, fractionation, and chemical analysis was combined with both exhaustive and bioaccessibility-directed extraction to identify the major toxicants in sediments at three industrial sites in the Elbe River basin (Bitterfeld, Most, and Prelouc). Exhaustive accelerated solvent extraction was used together with bioaccessibility-directed extraction with TENAX in aqueous suspension. Growth inhibition of the green alga Scenedesmus vacuolatus identified polycyclic aromatic compounds as important algal toxicants at all three sites, while in Bitterfeld and Prelouc additional toxicants, such as N-phenyl-2-naphthylamine and 7H-benzo[de]anthracen-7-one, played an important role. Confirmation of the identified compounds was based on the index of confirmation quality (ICQ) comparing the effect of an artificial mixture to that of the original sample. The results suggest a successful confirmation of identified toxicants for most fractions with ICQ values between 0.5 and 2. The bioaccessibility of toxicants decreased in the order Prelouc > Bitterfeld > Most as determined by the investigation of TENAX samples. Similar total concentrations of toxicants in Prelouc and Most sediment result in bioaccessible concentrations in Prelouc that exceed those in Most by a factor of 10 to 20. The extraction with TENAX with a subsequent EDA proved to be a powerful tool for prioritizing contaminants and contaminated sites with respect to the hazard of sediment-associated toxicants to green algae.

Polycyclic aromatic hydrocarbon (PAH) contamination of surface sediments and oysters from the inter-tidal areas of Dar es Salaam, Tanzania.

Surface sediment and oyster samples from the inter-tidal areas of Dar es Salaam were analyzed for 23 polycyclic aromatic hydrocarbons (PAHs) including the 16 compounds prioritized by US-EPA using GC/MS. The total concentration of PAHs in the sediment ranged from 78 to 25,000 ng/g dry weight, while oyster concentrations ranged from 170 to 650 ng/g dry weight. Hazards due to sediment contamination were assessed using Equilibrium Partitioning Sediment Benchmarks and Threshold Effect Levels. Diagnostic indices and principle component analysis were used to identify possible sources. Interestingly, no correlation between sediment and oyster concentrations at the same sites was found. This is supported by completely different contamination patterns, suggesting different sources for both matrices. Hazard assessment revealed possible effects at six out of eight sites on the benthic communities and oyster populations. The contribution of PAH intake via oyster consumption to carcinogenic risks in humans seems to be low.

Direct and indirect effects of pollutants on algae and algivorous ciliates in an aquatic indoor microcosm.

An aquatic indoor microcosm was used to study effects of the pesticides parathion-methyl and prometryn on phototrophic flagellates (Cryptomonas sp.) and predatory ciliates (Urotricha furcata). Parathion-methyl caused effects to flagellates and ciliates at the range of low mg L(-1), regardless of whether the organisms were exposed separately or combined in the multi-species test system. Prometryn caused effects on the flagellates at low microg L(-1) concentrations, resulting in a NOEC of 6.9 microg L(-1) in the single-species test and a NOEC of 15.2 microg L(-1) in the multi-species microcosm. For ciliates the NOEC decreased by factor 145 in the multi-species test compared to the NOEC of 2.2 mg L(-1) in the single-species test when exposed to prometryn. The lower NOEC for ciliates exposed to prometryn in the microcosm was most likely caused by an indirect effect due to reduced availability of flagellates as food. The measurement of nutrient concentrations in the test media and organisms facilitated the modelling of effects. The presented aquatic indoor microcosm is considered as a tool which could be standardised and applied as an instrument to provide data for higher tier risk assessment.

Automated fractionation procedure for polycyclic aromatic compounds in sediment extracts on three coupled normal-phase high-performance liquid chromatography columns.

Polychlorinated biphenyls, naphthalenes, dibenzo-p-dioxins and furans, as well as polyaromatic hydrocarbons (PAHs), hydroxy-, keto- and nitro-PAHs and sulphur, oxygen and nitrogen heterocycles represent major groups of toxicants in contaminated sediments. The identification of toxic substances in effect-directed analysis is often based on a group-specific fractionation of these polycyclic aromatic compounds (PACs). Several fractionation steps using different methods, solvent exchange and concentration steps are generally necessary to achieve this aim increasing the risk of losses and artefact formation. In order to avoid these disadvantages and to enhance the throughput, an automated on-line fractionation method was developed using coupled and automatically connected columns, including cyanopropyl- and nitrophenylpropyl-bonded silica and porous graphitised carbon stationary phases. Exploiting the potential of each column, compounds are separated mainly according to their polarity, number of aromatic carbons and planarity. Excellent group-specific resolution, high reproducibility and good recoveries suggest that this method is suitable for the fractionation of a multitude of major sediment contaminants in one HPLC-run.

How to confirm identified toxicants in effect-directed analysis.

Due to the production and use of a multitude of chemicals in modern society, waters, sediments, soils and biota may be contaminated with numerous known and unknown chemicals that may cause adverse effects on ecosystems and human health. Effect-directed analysis (EDA), combining biotesting, fractionation and chemical analysis, helps to identify hazardous compounds in complex environmental mixtures. Confirmation of tentatively identified toxicants will help to avoid artefacts and to establish reliable cause-effect relationships. A tiered approach to confirmation is suggested in the present paper. The first tier focuses on the analytical confirmation of tentatively identified structures. If straightforward confirmation with neat standards for GC-MS or LC-MS is not available, it is suggested that a lines-of-evidence approach is used that combines spectral library information with computer-based structure generation and prediction of retention behaviour in different chromatographic systems using quantitative structure-retention relationships (QSRR). In the second tier, the identified toxicants need to be confirmed as being the cause of the measured effects. Candidate components of toxic fractions may be selected based, for example, on structural alerts. Quantitative effect confirmation is based on joint effect models. Joint effect prediction on the basis of full concentration-response plots and careful selection of the appropriate model are suggested as a means to improve confirmation quality. Confirmation according to the Toxicity Identification Evaluation (TIE) concept of the US EPA and novel tools of hazard identification help to confirm the relevance of identified compounds to populations and communities under realistic exposure conditions. Promising tools include bioavailability-directed extraction and dosing techniques, biomarker approaches and the concept of pollution-induced community tolerance (PICT). [figure: see text]

Determination of 10 particle-associated multiclass polar and semi-polar pesticides from small streams using accelerated solvent extraction.

A new analytical method using accelerated solvent extraction was developed for the determination of 10 particle-associated polar and semipolar pesticides. In addition, six deuterated analogues of the target compounds were evaluated as internal standards. The method yielded acceptable accuracy (73-103% recovery) and precision (<25% relative standard deviation) for eight compounds. Using size exclusion chromatography (SEC) as cleanup step resulted in higher recoveries compared to solid phase extraction (SPE) cleanup. Deuterated standards with 10 or more deuterium atoms performed well as internal standards concerning similar recovery and correlation with the target analytes. The method was employed to extract particle-associated pesticides from 16 streams located in an area with intense agriculture in France. Acetochlor, pirimicarb, tebuconazole, fenpropidin, alpha-endosulfan and chlorfenvinphos were detected at concentrations up to 1 mg kg(-1) dry weight. A comparison with aquatic toxicity data indicated potential risk to the benthic fauna exposed to these concentrations of pirimicarb, alpha-endosulfan and chlorfenvinphos. We suggest that the method presented here be used for the extraction and quantitation of particle-associated polar pesticides.

Development and validation of a method for determination of trace levels of alkylphenols and bisphenol A in atmospheric samples.

A method has been developed and validated in order to assess the occurrence of the alkylphenols tert-octylphenol and the isomers of technical nonylphenol as well as bisphenol A in gasphase and aerosol samples of a remote area. Gasphase samples were adsorbed to XAD2 resin, aerosol samples were taken on glass fiber filters. After ultrasonic extraction, clean-up by column chromatography and silylation of the analytes, ten nonylphenol peaks were quantified separately using a GC-MSD-SIM method. The absolute limits of detection and determination are in the range of a few pg per compound, which is a prerequisite for the quantification of the analytes in relatively unpolluted air. The precision of the whole analytical method is in the range of 1-17% and the recoveries range from 57% to 80%. Problems were encountered during method development due to the tendency of the analytes to sorb to glass surfaces. Silanisation of glassware was crucial to achieve acceptable recoveries. The widespread use of the analytes in plastic resins resulted in sample contamination. For this reason a careful choice of sampling material was necessary. Measured concentrations in gasphase samples (lower nanogram per m3 range) and aerosol samples (upper picogram per m3 range) are one to three orders of magnitude below already published concentrations.