Sulfamethoxazole is Metabolized and Mineralized at Extremely Low Concentrations.

The presence of organic micropollutants in water and sediments motivates investigation of their biotransformation at environmentally low concentrations, usually in the range of µg L-1. Many are biotransformed by cometabolic mechanisms; however, there is scarce information concerning their direct metabolization in this concentration range. Threshold concentrations for microbial assimilation have been reported in both pure and mixed cultures from different origins. The literature suggests a range value for bacterial growth of 1-100 µg L-1 for isolated aerobic heterotrophs in the presence of a single substrate. We aimed to investigate, as a model case, the threshold level for sulfamethoxazole (SMX) metabolization in pure cultures of Microbacterium strain BR1. Previous research with this strain has covered the milligram L-1 range. In this study, acclimated cultures were exposed to concentrations from 0.1 to 25 µg L-1 of 14C-labeled SMX, and the 14C-CO2 produced was trapped and quantified over 24 h. Interestingly, SMX removal was rapid, with 98% removed within 2 h. In contrast, mineralization was slower, with a consistent percentage of 60.0 ± 0.7% found at all concentrations. Mineralization rates increased with rising concentrations. Therefore, this study shows that bacteria are capable of the direct metabolization of organic micropollutants at extremely low concentrations (sub µg L-1).

Extrapolation of cytotoxic masked effects in planar in vitro assays.

The masking of specific effects in in vitro assays by cytotoxicity is a commonly known phenomenon. This may result in a partial or complete loss of effect signals. For common in vitro assays, approaches for identifying and quantifying cytotoxic masking are partly available. However, a quantification of cytotoxicity-affected signals is not possible. As an alternative, planar bioassays that combine high-performance thin layer chromatography with in vitro assays, such as the planar yeast estrogen screen (p-YES), might allow for a quantification of cytotoxically affected signals. Affected signals form a typical ring structure with a supressed or completely lacking centre that results in a double peak chromatogram. This study investigates whether these double peaks can be used for fitting a peak function to extrapolate the theoretical, unaffected signals. The precision of the modelling was evaluated for four individual peak functions, using 42 ideal, undistorted peaks from estrogenic model compounds in the p-YES. Modelled ED50-values from bisphenol A (BPA) experiments with cytotoxically disturbed signals were 13 times higher than for the apparent data without compensation for cytotoxicity (320 ± 63 ng versus 24 ± 17 ng). This finding has a high relevance for the modelling of mixture effects according to concentration addition that requires unaffected, complete dose-response relationships. Finally, we applied the approach to results of a p-YES assay on leachate samples of an elastomer material used in water engineering. In summary, the fitting approach enables the quantitative evaluation of cytotoxically affected signals in planar in vitro assays and also has applications for other fields of chemical analysis like distorted chromatography signals.

Fermented biochar has a markedly different effect on fate of pesticides in soil than compost, straw, and a mixed biochar-product.

Current knowledge about how biochars affect the fate of pesticides in soil is based on studies that used pure biochars. After finding that an additional biological post-pyrolysis treatment, such as co-composting or lactic fermentation, is required for biochars for superior performance in temperate arable soils, a knowledge gap formed of how such further processed biochar products would affect the fate of pesticides in soil. This study compared the effects of a novel fermented biochar alone or mixed with biogas residues on the fate of two pesticides, 4-chloro-2-methylphenoxyacetic acid (MCPA) and metalaxyl-M, in a temperate arable soil to the traditional organic amendments wheat straw and compost. The fate of 14C-labeled MCPA was markedly affected in different ways. Fermented biochar effectively reduced the water-extractability and mineralization due to adsorption that was comparable to adsorption strengths reported for pure biochars. However, this effect was weak for the biochar mixed with biogas residues. Straw reduced water-extractable amounts due to increased biodegradation and formation of likely biogenic non-extractable residues of MCPA. In contrast, compost decelerated mineralization and increased the water solubility of the MCPA residues due to released dissolved organic matter. The amendments' effects were minor regarding 14C-metalaxyl-M, except for the fermented biochar which again reduced water-extractability and delayed degradation due to adsorption. Thus, the effects of the organic amendments differed for the two pesticide compounds with only the fermented biochar's effect being similar for both. However, this effect was no longer present in the mixed product containing 20% biochar. Our findings clearly show that biologically treated biochar-containing products can affect the fate of pesticides in soil very differently, also when compared to traditional organic amendments. Such impacts and their desirable and undesirable ecotoxicological implications need to be considered before the large-scale application of biochars to temperate arable soils.

JRODOS customization for Pakistan and assessment of the consequences of a hypothetical nuclear accident.

Since the Chernobyl disaster in 1986, decision support systems and modelling tools have been utilized in response to nuclear and radiological emergencies. The java-based real-time online decision support system (JRODOS) is a decision support tool that can be utilised in response to an emergency in managing off-site radiological consequences. This article documents the customization and use of JRODOS for Pakistan. JRODOS was tailored to the local Pakistan conditions, and a case study of a theoretical nuclear power plant accident was used to assess JRODOS's feasibility as a decision support tool. A worst-case probabilistic accident scenario was used to identify zones and areas where urgent protective actions, early protective actions and food restriction and other response actions could be required. The areas and distances identified for the implementation of protective and response actions for such a hypothetical accident were found to be in agreement with the emergency planning zones and distances suggested by the International Atomic Energy Agency (IAEA). Additionally, the implications of meteorological and source term input parameters on predicting the radiation doses to members of the public were investigated. It was identified that the output of such tools strongly depends on the availability and accuracy of the input parameters, such as radioactive release and meteorological data. Limitations and uncertainties associated with these tools need to be considered in deciding on protective and other response actions in response to a nuclear accident. As established by the IAEA, protective and other response actions need to be applied on a graded approach, taking into account the protection strategy and uncertainties and limitations in the available information and criteria, based on the conditions at the facility and off-site.

A look down the drain: Identification of dissolved and particle bound organic pollutants in urban runoff waters and sediments.

Urban runoff contains a range of organic micropollutants which, if not removed during wastewater treatment, pose a risk to aquatic environments. These mixtures are complex and often site-specific. Street drains provide an ideal sampling point given they collect the runoff from local and defined catchments. In this study, runoff was collected and sampled in five street drains located in a medium sized town in Germany. A specially constructed trap was used to collect the particulate and total water fractions of the runoff. In addition, passive samplers were deployed to determine the freely dissolved concentrations of selected compounds in the runoff. In sum, 187 polar organic micropollutants could be quantified using LC-HRMS. Thirty of these could only be detected by the use of passive samplers. Traffic derived pollutants such as corrosion inhibitors, rubber- and plastic additives, but also pollutants of industrial origin were strongly represented with sum median concentrations of 100 µg/kg dry weight (DW) in the sediment and 400 ng/L in the water fraction. Several of these substances are of concern due to their environmental persistence and mobility. Perfluorinated compounds and pesticides occurred at lower levels of several µg/kg DW sediment or ng/L water. A number of substances including pharmaceuticals, sweeteners and stimulants indicated domestic wastewater influences. Furthermore, a total of 62 parent and alkylated PAHs were quantified by GC-MS and contributed 30-70% to the sum concentrations of the micropollutants. Non-EPA PAHs dominated the carcinogenic PAH toxicity. The increased PAH alkylation indices (0.7-0.9) showed these primarily came from combustion sources. The runoff particles were additionally microscopically characterized, and correlations were found between the rubber particle counts and the PAH alkylation-index as well as the levels of 2-(methylthio)benzothiazole, a marker compound for tire leaching.

Effects of the antidepressant mirtazapine on the swimming behaviour and gene expression rate of Danio rerio embryos - Is the sedating effect seen in humans also evident for fish?

In the last decade, mirtazapine has become an important antidepressant in clinical use and has also been found at many different environmental sampling sites. Several homologies between the zebrafish Danio rerio and humans, combined with a number of advantages for behavioural and gene expression research using zebrafish embryos, make their use for the analysis of mirtazapine appropriate. The sedative effect of mirtazapine in humans was also found for a specific concentration range in zebrafish embryos (1333.4 µg/L - 2666.9 µg/L). Specifically, 116 hpf old zebrafish embryos showed a reduced swimming distance when exposed to 1334.4 µg/L mirtazapine. Furthermore, changes at the gene regulatory level could be measured (1333.4 µg/L), in particular in the superordinate regulatory systems. For selected transporters of all regulatory systems, an up regulation of the genes by a factor of more than five times could be measured at the highest mirtazapine exposure concentration that was tested. Finally, studies on the protein levels demonstrated an increase in acetylcholinesterase activity for several exposure concentrations (83.3 µg/L and 666.7 µg/L). The physiological changes in zebrafish embryos caused by mirtazapine demonstrate the relevance of these types of studies in aquatic non-target organisms. Such neuroactive substances could pose a potential risk for aquatic organisms below the previously considered concentration threshold for morphological effects.

Passive Sampling and Dosing of Aquatic Organic Contaminant Mixtures for Ecotoxicological Analyses.

Toxicity results from exposure to mixtures of organic contaminants. Assessing this using ecotoxicity bioassays involves sampling of the environmental mixture and then introducing this into the test. The first step is accounting for the bioavailable levels of all mixture constituents. Passive sampling specifically targets these bioavailable fractions but the sampler-accumulated mixture varies with the compound and sampler properties as well as time. The second step involves reproducing and maintaining the sampled mixture constituents in the bioassay. Passive sampler extraction and spiking always leads to a skewed mixture profile in the test. Alternatively, the recovered passive samplers might be directly used in passive dosing mode. Here, the reproduced contaminant mixture depends on whether kinetic or equilibrium sampling applies. These concepts were tested for determining the combined toxicity of laboratory and field mixtures of aquatic contaminants in the Microtox and ER-Calux bioassays. Aqueous sample extraction and spiking, passive sampler extraction and spiking, and passive sampling and dosing were compared for first sampling and then introducing mixtures in toxicity bioassays. The analytical and toxicity results show that the correct way to first sample the bioavailable mixture profile, and then to reproduce and maintain this in the toxicity test, is by combining equilibrium passive sampling and dosing.

Comparing straw, compost, and biochar regarding their suitability as agricultural soil amendments to affect soil structure, nutrient leaching, microbial communities, and the fate of pesticides.

The emission of nutrients and pesticides from agricultural soils endangers natural habitats. Here, we review to which extent carbon-rich organic amendments help to retain nutrients and pesticides in agricultural soils and to reduce the contamination of surrounding areas and groundwater. We compare straw, compost, and biochar to see whether biochar outperforms the other two more traditional and cheaper materials. We present a list of criteria to evaluate the suitability of organic materials to be used as soil amendments and discuss differences in elemental compositions of straw, compost, and biochar to understand, how soil microorganisms utilize those materials. We review their effects on physical and chemical soil characteristics, soil microbial communities, as well as effects on the transformation and retention of nutrients and pesticides in detail. It becomes clear that for all three amendments their effects can vary greatly depending on numerous aspects, such as the type of soil, application rate, and production procedure of the organic material. Biochar is most effective in increasing the sorption capacity of soils but does not outperform straw and compost with regards to the other aspects investigated. Nevertheless, the possibility to design biochar properties makes it a very promising material. Finally, we provide critical comments about how to make studies about organic amendments more comparable (comprehensive provision of material properties), how to improve concepts of future work (meta-analysis, long-term field studies, use of deep-insight microbial DNA sequencing), and what needs to be further investigated (the link between structural and functional microbial parameters, the impact of biochar on pesticide efficiency).

Kinetic Passive Sampling: In Situ Calibration Using the Contaminant Mass Measured in Parallel Samplers with Different Thicknesses.

The use of single-phase passive samplers is a common method for sampling bioavailable concentrations of hydrophobic aquatic pollutants. Often such samplers are used in the kinetic stage, and in situ calibration is necessary. Most commonly, exchange kinetics are derived from the release rates of performance reference compounds (PRCs). In this study, a complementary calibration approach was developed, in which measuring the contaminant mass ratio (CMR) from two samplers with different thicknesses allows the dissolved concentrations to be determined. This new CMR calibration was tested (1) in a laboratory experiment with defined and constant concentrations and (2) in the field, at a storm water retention site. Silicone passive samplers with different thicknesses were used to sample a range of dissolved polycyclic aromatic hydrocarbons. In the laboratory study, the concentrations derived from the CMR calibration were compared with those from water extraction and passive dosing and differences below a factor 2 were found. In the field study, CMR-derived concentrations were compared to those from PRC calibration. Here, differences ranged by only a factor 1 to 3 between both methods. These findings indicate that the CMR calibration can be applied as a stand-alone or complementary calibration method for kinetic passive sampling.

Quantitative evaluation of polyethersulfone and polytetrafluoroethylene membrane sorption in a polar organic chemical integrative sampler (POCIS).

The lag effect in the polar organic chemical integrative sampler (POCIS) equipped with a polyethersulfone (PES) membrane (POCIS-PES) is a potential limitation for its application in water environments. In this study, a POCIS with a poly(tetrafluoroethylene) (PTFE) membrane (POCIS-PTFE) was investigated for circumventing membrane sorption in order to provide more reliable concentration measurements of organic contaminants. Sampler characteristics such as sampling rates (RS) and sampler-water partition coefficients (KSW) were similar for POCIS-PES and POCIS-PTFE, indicating that partitioning into Oasis HLB as the receiving phase dominates the overall partitioning from the aqueous phase to the POCIS. Membrane sorption was quantified in both laboratory and field experiments. Although POCIS-PTFE showed minor membrane sorption, the PTFE membranes were not robust enough to prevent changes in the sorption of the pollutants to the inner Oasis HLB sorbent due to biofouling. This was reflected in significant ionization effects in the electrospray ionization (ESI) source during the LC-MS/MS analysis. Despite clear differences in the ionization effects, the two POCISs types provided similar time-weighted average (CTWA) concentrations after a two-week passive sampling campaign in surface water and the outflow of a wastewater treatment plant. This study contributes to a more detailed understanding of POCIS application by providing a quantitative evaluation of membrane sorption and its associated effects in the laboratory and field.

Do you smell the danger? Effects of three commonly used pesticides on the olfactory-mediated antipredator response of zebrafish (Danio rerio).

Fish are warned about the presence of predators via an alarm cue released from the skin of injured conspecifics. The detection of this odor inherently initiates an antipredator response, which increases the chance of survival for the individual. In the present study, we assessed the effect of three commonly used pesticides on the antipredator response of zebrafish (Danio rerio). For this, we analyzed the behavioral response of zebrafish to a conspecific skin extract following 24 h of exposure to the respective contaminants. Results demonstrate that fish exposed to 20 µg/L of the organophosphate insecticide chlorpyrifos significantly reduced bottom-dwelling and freezing behavior, suggesting an impairment of the antipredator response. For the urea-herbicide linuron and the pyrethroid insecticide permethrin, no statistically significant effects could be detected. However, linuron-exposed fish appeared to respond in an altered manner to the skin extract; some individuals failed to perform the inherent behaviors such as erratic movements and instead merely increased their velocity. Furthermore, we determined whether zebrafish would avoid the pesticides in a choice maze. While fish avoided permethrin, they behaved indifferently to chlorpyrifos and linuron. The study demonstrates that pesticides may alter the olfactory-mediated antipredator response of zebrafish in distinct ways, revealing that particularly fish exposed to chlorpyrifos may be more prone to predation.

Bioavailability of estrogenic compounds from sediment in the context of flood events evaluated by passive sampling.

Studies worldwide have demonstrated through in vitro bioassays and chemical analysis that endocrine-disrupting chemicals (EDCs) can accumulate in river sediments. However, remobilization of sediment-bound EDCs due to bioturbation or re-suspension during flood events remains poorly understood. The aim of this study was to evaluate the bioavailability of EDCs, more specifically estrogenic compounds (EC), from sediment under turbulent conditions using a passive sampling approach. Sediment was sampled along the Luppe River, Germany, previously described as a "hotspot" for ECs. The concentration of target ECs and estrogenic activity were investigated using chemical analysis (LC MS/MS) in addition to a novel screening tool (planar Yeast Estrogen Screen; p-YES) that utilizes high performance thin-layer chromatography plates in combination with an in vitro bioassay (YES). Estrone (50%, E1) and nonylphenol (35%, NP) accounted for the majority of estrogenic activity reported of up to 20 ±â€¯2.4 µg E2 equivalents per kg dry weight in the Luppe sediments. Two types of passive samplers (polar organic chemical integrative sampler (POCIS) and Chemcatcher) were used to investigate the bioavailability of ECs from suspended sediment under laboratory conditions. NP, E1, E2 and ethynylestradiol (EE2) were remobilized from Luppe sediment when subjected to turbulent conditions, such as in a flood event, and were readily bioavailable at ecotoxicologically relevant concentrations (NP 18 µg/L, E1 14 ng/L, E2 0.2 ng/L, EE2 0.5 ng/L).

Bioactivation of Quinolines in a Recombinant Estrogen Receptor Transactivation Assay Is Catalyzed by N-Methyltransferases.

Hydroxylation of polyaromatic compounds through cytochromes P450 (CYPs) is known to result in potentially estrogenic transformation products. Recently, there has been an increasing awareness of the importance of alternative pathways such as aldehyde oxidases (AOX) or N-methyltransferases (NMT) in bioactivation of small molecules, particularly N-heterocycles. Therefore, this study investigated the biotransformation and activity of methylated quinolines, a class of environmentally relevant N-heterocycles that are no native ligands of the estrogen receptor (ER), in the estrogen-responsive cell line ERα CALUX. We found that this widely used cell line overexpresses AOXs and NMTs while having low expression of CYP enzymes. Exposure of ERα CALUX cells to quinolines resulted in estrogenic effects, which could be mitigated using an inhibitor of AOX/NMTs. No such mitigation occurred after coexposure to a CYP1A inhibitor. A number of N-methylated but no hydroxylated transformation products were detected using liquid chromatography-mass spectrometry, which indicated that biotransformations to estrogenic metabolites were likely catalyzed by NMTs. Compared to the natural ER ligand 17ß-estradiol, the products formed during the metabolization of quinolines were weak to moderate agonists of the human ERα. Our findings have potential implications for the risk assessment of these compounds and indicate that care must be taken when using in vitro estrogenicity assays, for example, ERα CALUX, for the characterization of N-heterocycles or environmental samples that may contain them.

Directed OmniChange Evolution Converts P450 BM3 into an Alkyltrimethylammonium Hydroxylase.

Cetyl-trimethylammonium bromide (CTAB) is a widely used cationic surfactant that is biodegradable in nature. CTAB biodegradation requires hydroxylation in the first step, which is rate-limiting and crucial for solubility in water. In this study, the OmniChange multi-site mutagenesis method was applied to reengineer the P450 BM3 substrate specificity towards the hydroxylation of CTAB by simultaneous mutagenesis of four previously reported positions (R47, Y51, F87, and L188). 1740 clones from the P450 BM3 OmniChange library were screened with the NADPH depletion assay. A total of 696 clones were rescreened with the NADPH depletion and an Ampliflu™ Red/ horseradish peroxidase based H2 O2 detection assay. Several improved P450 BM3 variants were identified and finally four were kinetically characterized with respect to CTAB hydroxylation, based on both performance and coupling efficiency. Based on NADPH consumption, the P450 BM3 variant P3A8 (R47E/Y51M/F87V/L188E) displayed an initial activity (64.9±4.8 s-1 , 13.5-fold increased activity compared with wild-type P450 BM3), which nearly matches the specific activity for its natural fatty acid substrate (palmitic acid (32-122 s-1 )). Variant P3A8 showed high coupling efficiency (92.5 %), whereas wild-type P450 BM3 displayed a low coupling efficiency (0.5 %). HPLC-MS/MS detection confirmed that P3A8 and P2E7 (R47D/Y51L/F87V/L188A) form 13 and 35 times more 2-hydroxylated CTAB than P450 BM3. In addition, di-hydroxylated CTAB products were detected for all four investigated P450 BM3 variants (up to a yield of 77 %; P3A8). Di-hydroxylated quaternary amines are highly interesting bolaform surfactants with a high hydrophilicity (surface contact angle: θ=16.7°).

A comparison of equilibrium and kinetic passive sampling for the monitoring of aquatic organic contaminants in German rivers.

The performances of an equilibrium and a kinetic passive sampler for monitoring a range of organic contaminants (Log KOW from -0.03 to 6.26) were evaluated in the effluent of a wastewater treatment plant, the receiving river Saar as well as the river Mosel in Germany. The polar organic chemical integrative sampler (POCIS) and a new mixed polymer sampler (MPS) were selected as kinetic and equilibrium passive samplers, respectively. Concentrations were described in terms of a time-weighted average concentration (CTWA) from the POCIS measurements and as an equilibrium concentration from the MPS (CEquil-MPS) and POCIS membrane (CEquil-PES) analyses. Twenty-seven compounds could be detected, including eight priority substances of the EU Water Framework Directive. Both sampler types detected a similar range of compounds in the low ng/L to µg/L range, with a high proportion of pharmaceuticals being detected at all sampling sites. To account for uncertainty in the POCIS sampling rates, a range in CTWA was estimated by applying low and high sampling rates. For the compounds that were detected in the POCIS this range was within a factor of 3.5. Interestingly, the MPS extracts showed lower ionisation artefacts than the POCIS extracts during the LC-MS/MS analysis. Finally, total water concentrations (CTotal) were estimated from the dissolved concentrations, literature organic carbon partition coefficients (KOC) and the total organic carbon levels measured in the rivers. For the compounds in this study, negligible differences between CTotal and the passive sampler-derived dissolved concentrations were found with a maximum difference of 15% for diclofenac. Overall, this study demonstrated that the parallel application of kinetic and equilibrium passive samplers can improve the description of water quality.

Comparison of the sampling rates and partitioning behaviour of polar and non-polar contaminants in the polar organic chemical integrative sampler and a monophasic mixed polymer sampler for application as an equilibrium passive sampler.

In this work, Oasis HLB® beads were embedded in a silicone matrix to make a single phase passive sampler with a higher affinity for polar and ionisable compounds than silicone alone. The applicability of this mixed polymer sampler (MPS) was investigated for 34 aquatic contaminants (log KOW -0.03 to 6.26) in batch experiments. The influence of flow was investigated by comparing uptake under static and stirred conditions. The sampler characteristics of the MPS was assessed in terms of sampling rates (RS) and sampler-water partition coefficients (KSW), and these were compared to those of the polar organic chemical integrative sampler (POCIS) as a reference kinetic passive sampler. The MPS was characterized as an equilibrium sampler for both polar and non-polar compounds, with faster uptake rates and a shorter time to reach equilibrium than the POCIS. Water flow rate impacted sampling rates by up to a factor of 12 when comparing static and stirred conditions. In addition, the relative accumulation of compounds in the polyethersulfone (PES) membranes versus the inner Oasis HLB sorbent was compared for the POCIS, and ranged from <1% to 83% depending on the analyte properties. This is indicative of a potentially significant lag-phase for less polar compounds within POCIS. The findings of this study can be used to quantitatively describe the partitioning and kinetic behaviour of MPS and POCIS for a range of aquatic organic contaminants for application in field sampling.

Equilibrium partitioning of organic compounds to OASIS HLB® as a function of compound concentration, pH, temperature and salinity.

Oasis hydrophilic lipophilic balance® (Oasis HLB) is commonly employed in solid phase extraction (SPE) of environmental contaminants and within polar organic chemical integrative passive samplers (POCIS). In this study batch experiments were carried out to evaluate the relative affinity of a range of relevant organic pollutants to Oasis HLB in aqueous systems. The influence of sorbate concentration, temperature, pH, and salinity on the equilibrium sorption was investigated. Equilibrium partition ratios (KD) of 28 compounds were determined, ranging over three orders of magnitude from 1.16 × 103 L/kg (atenolol) to 1.07 × 106 L/kg (isoproturon). The Freundlich model was able to describe the equilibrium partitioning to Oasis HLB, and an analysis of the thermodynamic parameters revealed the spontaneous and exothermic nature of the partitioning process. Ionic strength had only a minor effect on the partitioning, whereas pH had a considerable effect but only for ionizable compounds. The results show that apolar interactions between the Oasis HLB and analyte mainly determine the equilibrium partitioning. These research findings can be used to optimize the application of SPE and POCIS for analyses of environmental contaminants even in complex mixtures.

Defining and Controlling Exposure During In Vitro Toxicity Testing and the Potential of Passive Dosing.

Toxicity testing using in vitro bioassays is assuming an increasingly important role. Nevertheless, several issues remain with regard to their proper application, which mainly relate to the proper definition and control of the test chemical(s) concentrations to which the cells or tissues are exposed. This has fundamental implications for understanding the underlying relationship between the in vitro exposure regime and response, and leads to uncertainty in the resulting bioassay data. This chapter covers the definition and control of exposure of hydrophobic organic chemicals (HOCs) in in vitro bioassays aimed at measuring their toxicity. A review of the fate of HOCs in typical in vitro set-ups is followed by a discussion of how to define the test exposure. Currently applied approaches for introducing HOCs into in vitro bioassays are then related to these different definitions of test exposure. Finally, passive dosing as one possible approach for giving defined and constant dissolved concentrations of HOCs in in vitro toxicity tests is introduced, using examples taken from the literature, and how this might be better integrated into high throughput in vitro toxicity testing is discussed.

Passive dosing versus solvent spiking for controlling and maintaining hydrophobic organic compound exposure in the Microtox® assay.

Microbial toxicity bioassays such as the Microtox® test are ubiquitously applied to measure the toxicity of chemicals and environmental samples. In many ways their operation is conducive to the testing of organic chemicals. They are of short duration, use glass cuvettes and take place at reduced temperatures in medium lacking sorbing components. All of these are expected to reduce sorptive and volatile losses, but particularly for hydrophobic organics the role of such losses in determining the bioassay response remains unclear. This study determined the response of the Microtox® test when using solvent spiking compared to passive dosing for introducing the model hydrophobic compounds acenaphthene, phenanthrene, fluoranthene and benzo(a)pyrene. Compared to solvent spiking, the apparent sensitivity of the Microtox® test with passive dosing was 3.4 and 12.4 times higher for acenaphthene and phenanthrene, respectively. Furthermore, fluoranthene only gave a consistent response with passive dosing. Benzo(a)pyrene did not result in a response with either spiking or passive dosing even at aqueous solubility. Such differences in the apparent sensitivity of the Microtox® test can be traced back to the precise definition of the dissolved exposure concentrations and the buffering of losses with passive dosing. This highlights the importance of exposure control even in simple and short-term microbial bioassays such as the Microtox® test.

Transfer and effects of 1,2,3,5,7-pentachloronaphthalene in an experimental food chain.

Polychlorinated naphthalenes are environmentally relevant compounds that are measured in biota at concentrations in the µg/kg lipid range. Despite their widespread occurrence, literature data on the accumulation and effects of these compounds in aquatic ecosystems are sparsely available. The goal of this study was to gain insights into the biomagnification and effects of 1,2,3,5,7-pentachloronaphthalene (PeCN52) in an experimental food chain consisting of benthic worms and juvenile rainbow trout. Worms were contaminated with PeCN52 by passive dosing from polydimethylsiloxane silicone. The contaminated worms were then used to feed the juvenile rainbow trout at 0.12, 0.25 or 0.50 µg/g fish wet weight/day, and the resulting internal whole-body concentrations of the individual fish were linked to biological responses. A possible involvement of the cellular detoxification system was explored by measuring PeCN52-induced expression of the phase I biotransformation enzyme gene cyp1a1 and the ABC transporter gene abcb1a. At the end of the 28-day study, biomagnification factors were similar for all dietary intake levels with values between 0.5 and 0.7 kg lipid(fish)/kg lipid(worm). The average uptake efficiency of 60% indicated that a high amount of PeCN52 was transferred from the worms to the fish. Internal concentrations of up to 175 mg/kg fish lipid in the highest treatment level did not result in effects on survival, behavior, or growth of the juvenile trout, but were associated with the induction of phase I metabolism which was evident from the significant up-regulation of cyp1a1 expression in the liver. In contrast, no changes were seen in abcb1a transcript levels.