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.

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.

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°).

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.

Differential immunomodulatory responses to nine polycyclic aromatic hydrocarbons applied by passive dosing.

Studying the effects of hydrophobic chemicals using in vitro cell based methods is hindered by the difficulty in bringing and keeping these chemicals in solution. Their effective concentrations are often lower than their nominal concentrations. Passive dosing is one approach that provides defined and stable dissolved concentrations during in vitro testing, and was applied to control and maintain freely dissolved concentrations of polycyclic aromatic hydrocarbons (PAHs) at levels up to their aqueous solubility limit. The immunomodulatory effects of 9 different PAHs at aqueous solubility on human bronchial epithelial cells were determined by analysing the cytokine promoter expression of 4 different inflammatory cytokines using stably transfected recombinant A549 cell lines. Diverse immunomodulatory responses were found with the highest induction observed for the most hydrophobic PAHs chrysene, benzo(a)antracene and benzo(a)pyrene. Cytokine promoter expression was then studied in dose response experiments with acenaphthene, phenanthrene and benzo(a)anthracene. The strongest induction was observed for benzo(a)anthracene. Cell viability analysis was performed and showed that none of the PAHs induced cytotoxicity at any of the concentrations tested. Overall, this study shows that (1) immunomodulatory effects of PAHs can be studied in vitro at controlled freely dissolved concentrations, (2) the most hydrophobic PAHs were the strongest inducers and (3) induction was often higher at lower exposure levels and decreased then with concentration despite the apparent absence of cytotoxicity.

PAH toxicity at aqueous solubility in the fish embryo test with Danio rerio using passive dosing.

As part of the risk assessment process within REACh, prior to manufacturing and distribution of chemical substances their (eco)toxicological impacts have to be investigated. The fish embryo toxicity test (FET) with the zebrafish Danio rerio has gained a high significance as an in vitro alternative to animal testing in (eco)toxicology. However, for hydrophobic organic chemicals it remains a technical challenge to ensure constant freely dissolved concentration at the maximum exposure level during such biotests. Passive dosing with PDMS silicone was thus applied to control the freely dissolved concentration of ten PAHs at their saturation level in the FET. The experiments gave repeatable results, with the toxicity of the PAHs generally increasing with the maximum chemical activities of the PAHs. HPLC analysis confirmed constant exposure at the saturation level. In additional experiments, fish embryos without direct contact to the silicone surface showed similar mortalities as those exposed with direct contact to the silicone. Silicone oil overlaying the water phase as a novel passive dosing phase had no observable effects on the development of the fish embryos until hatching. This study provides further data to support the close relationship between the chemical activity and the toxicity of hydrophobic organic compounds. Passive dosing from PDMS silicone enabled reliable toxicity testing of (highly) hydrophobic substances at aqueous solubility, providing a practical way to control toxicity exactly at the maximum exposure level. This approach is therefore expected to be useful as a cost-effective initial screening of hydrophobic chemicals for potential adverse effects to freshwater vertebrates.

Impact of soil amendments and the plant rhizosphere on PAH behaviour in soil.

Carbonaceous amendments reduce PAH dissolved concentrations (Cfree), limiting their uptake and toxicity. A soil contaminated with PAHs was mixed with activated carbon (AC), charcoal or compost and planted with radish (Raphanus sativus L.), and Cfree, chemical activities and diffusive uptake of the PAHs measured over 2 months. For AC, Cfree and diffusive uptake were decreased by up to 94% compared to the unamended soil within one week. In addition, the sum chemical activity of the PAHs remained below the threshold for baseline toxicity. In contrast, charcoal and compost only led to modest reductions in Cfree and diffusive uptake, with sum chemical activities that could potentially result in baseline toxicity being observed. Furthermore, both Cfree and diffusive uptake were lower in the planted compared to unplanted soils. Therefore, only AC successfully reduced PAH acute toxicity in the soil, but plant-promoted microbial degradation may also play an important role in PAH attenuation.

Impact of activated carbon, biochar and compost on the desorption and mineralization of phenanthrene in soil.

Sorption of PAHs to carbonaceous soil amendments reduces their dissolved concentrations, limiting toxicity but also potentially biodegradation. Therefore, the maximum abiotic desorption of freshly sorbed phenanthrene (≤5 mg kg(-1)) was measured in three soils amended with activated carbon (AC), biochar or compost. Total amounts of phenanthrene desorbed were similar between the different soils, but the amendment type had a large influence. Complete desorption was observed in the unamended and compost amended soils, but this reduced for biochar (41% desorbed) and AC (8% desorbed). Cumulative amounts mineralized were 28% for the unamended control, 19% for compost, 13% for biochar and 4% for AC. Therefore, the effects of the amendments in soil in reducing desorption were also reflected in the extents of mineralization. Modeling was used to analyze key processes, indicating that for the AC and charcoal treatments bacterial activity did not limit mineralization, but rather desorption into the dissolved phase.

Passive dosing of polycyclic aromatic hydrocarbon (PAH) mixtures to terrestrial springtails: linking mixture toxicity to chemical activities, equilibrium lipid concentrations, and toxic units.

A 7-day mixture toxicity experiment with the terrestrial springtail Folsomia candida was conducted, and the effects were linked to three different mixture exposure parameters. Passive dosing from silicone was applied to tightly control exposure levels and compositions of 12 mixture treatments, containing the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene, and pyrene. Springtail lethality was then linked to sum chemical activities (∑a), sum equilibrium lipid concentrations (∑C(lipid eq.)), and sum toxic units (∑TU). In each case, the effects of all 12 mixture treatments could be fitted to one sigmoidal exposure-response relationship. The effective lethal chemical activity (La50) of 0.027 was well within the expected range for baseline toxicity of 0.01-0.1. Linking the effects to the lipid-based exposure parameter yielded an effective lethal concentration (LC(lipid eq 50)) of 133 mmol kg(-1) lipid in good correspondence with the lethal membrane burden for baseline toxicity (40-160 mmol kg(-1) lipid). Finally, the effective lethal toxic unit (LTU50) of 1.20 was rather close to the expected value of 1. Altogether, passive dosing provided tightly controlled mixture exposure in terms of both level and composition, while ∑a, ∑C(lipid eq.), and ∑TU allowed baseline toxicity to be linked to mixture exposure.

Baseline toxic mixtures of non-toxic chemicals: "solubility addition" increases exposure for solid hydrophobic chemicals.

This study addresses the question whether hydrophobic organic chemicals exerting no toxicity at their solubility limit (saturation) can form a toxic mixture. Spiking methods generally do not allow testing exactly at saturation without introducing microcrystals. Passive dosing was thus applied to test the acute toxicity of several high melting point PAHs and their mixtures at the respective saturation levels to aquatic and terrestrial invertebrates. With the aquatic Daphnia magna, anthracene, chrysene, and benzo(a)pyrene resulted in no or limited acute toxicity (0-20%), whereas binary and tertiary mixtures of these resulted in significant acute toxicity (70-88%). Toxicity of PAHs and their mixtures could be fitted with one (sum) chemical activity-response curve in accordance with a similar mode of toxic action (i.e., concentration addition). The effective chemical activity (Ea-50) of 0.029 and the effective concentration on a lipid basis (EC(lipid, eq.)-50) of 95.7 mM were well within the range for baseline toxicity. Similar mixtures showed less toxicity to the terrestrial Folsomia candida due to steady-state body-burdens being below equilibrium partitioning levels. The results of the present study raise questions about the focus of risk assessment schemes and toxicity testing guidelines on individual substances, since apparently non-toxic chemicals might become toxic in a mixture.

Comparing the desorption and biodegradation of low concentrations of phenanthrene sorbed to activated carbon, biochar and compost.

Carbonaceous soil amendments are applied to contaminated soils and sediments to strongly sorb hydrophobic organic contaminants (HOCs) and reduce their freely dissolved concentrations. This limits biouptake and toxicity, but also biodegradation. To investigate whether HOCs sorbed to such amendments can be degraded at all, the desorption and biodegradation of low concentrations of (14)C-labelled phenanthrene (≤5 µg L(-1)) freshly sorbed to suspensions of the pure soil amendments activated carbon (AC), biochar (charcoal) and compost were compared. Firstly, the maximum abiotic desorption of phenanthrene from soil amendment suspensions in water, minimal salts medium (MSM) or tryptic soy broth (TSB) into a dominating silicone sink were measured. Highest fractions remained sorbed to AC (84±2.3%, 87±4.1%, and 53±1.2% for water, MSM and TSB, respectively), followed by charcoal (35±2.2%, 32±1.7%, and 12±0.3%, respectively) and compost (1.3±0.21%, similar for all media). Secondly, the mineralization of phenanthrene sorbed to AC, charcoal and compost by Sphingomonas sp. 10-1 (DSM 12247) was determined. In contrast to the amounts desorbed, phenanthrene mineralization was similar for all the soil amendments at about 56±11% of the initially applied radioactivity. Furthermore, HPLC analyses showed only minor amounts (<5%) of residual phenanthrene remaining in the suspensions, indicating almost complete biodegradation. Fitting the data to a coupled desorption and biodegradation model revealed that desorption did not limit biodegradation for any of the amendments, and that degradation could proceed due to the high numbers of bacteria and/or the production of biosurfactants or biofilms. Therefore, reduced desorption of phenanthrene from AC or charcoal did not inhibit its biodegradation, which implies that under the experimental conditions these amendments can reduce freely dissolved concentration without hindering biodegradation. In contrast, phenanthrene sorbed to compost was fully desorbed and biodegraded.

The dosing determines mutagenicity of hydrophobic compounds in the Ames II assay with metabolic transformation: passive dosing versus solvent spiking.

The Ames II bacterial mutagenicity assay is a new version of the standard Ames test for screening chemicals for genotoxic activity. However, the use of plastic micro-titer plates has drawbacks in the case of testing hydrophobic mutagens, since sorptive and other losses make it difficult to control and define the exposure concentrations, and they reduce availability for bacterial uptake or to the S9 enzymes. With passive dosing, a biocompatible polymer such as silicone is loaded with the test compound and acts as a partitioning source. It compensates for any losses and results in stable freely dissolved concentrations. Passive dosing using silicone O-rings was applied in the Ames II assay to measure PAH mutagenicity in strains TA98 and TAMix - a mixture of six different bacterial strains detecting six different base-pair substitutions - after metabolic activation by S9. Initially, 10 PAHs were tested with passive dosing from saturated O-rings, aiming at levels in the test medium close to aqueous solubility. Fluoranthene, pyrene and benzo(a)pyrene were mutagenic in both TA98 and TAMix, whereas benz(a)anthracene was mutagenic in TA98 only. The concentration-dependent mutagenic activity of benzo(a)pyrene was then compared for passive dosing and solvent spiking. With spiking, nominal concentrations greatly exceeded aqueous solubility before mutagenicity was observed, due to sorptive losses and limiting dissolution kinetics. In contrast, the passive dosing concentration-response curves were more reproducible, and shifted towards lower concentrations by several orders of magnitude. This study raises fundamental questions about how to introduce hydrophobic test substances in the Ames II assay with biotransformation, since the measured mutagenicity not only depends on the compound potency but also on its supply, sorption and consumption during the assay.

Dynamic passive dosing for studying the biotransformation of hydrophobic organic chemicals: microbial degradation as an example.

Biotransformation plays a key role in hydrophobic organic compound (HOC) fate, and understanding kinetics as a function of (bio)availability is critical for elucidating persistence, accumulation, and toxicity. Biotransformation mainly occurs in an aqueous environment, posing technical challenges for producing kinetic data because of low HOC solubilities and sorptive losses. To overcome these, a new experimental approach based on passive dosing is presented. This avoids using cosolvent for introducing the HOC substrate, buffers substrate depletion so biotransformation is measured within a narrow and defined dissolved concentration range, and enables high compound turnover even at low concentrations to simplify end point measurement. As a case study, the biodegradation kinetics of two model HOCs by the bacterium Sphingomonas paucimobilis EPA505 were measured at defined dissolved concentrations ranging over 4 orders of magnitude, from 0.017 to 658 µg L(-1) for phenanthrene and from 0.006 to 90.0 µg L(-1) for fluoranthene. Both compounds had similar mineralization fluxes, and these increased by 2 orders of magnitude with increasing dissolved concentrations. First-order mineralization rate constants were also similar for both PAHs, but decreased by around 2 orders of magnitude with increasing dissolved concentrations. Dynamic passive dosing is a useful tool for measuring biotransformation kinetics at realistically low and defined dissolved HOC concentrations.

Measuring binding and speciation of hydrophobic organic chemicals at controlled freely dissolved concentrations and without phase separation.

The binding and speciation of hydrophobic organic chemicals (HOCs) in aqueous solutions were determined by controlling chemical activity and measuring total concentrations. Passive dosing was applied to control chemical activities of HOCs in aqueous solutions by equilibrium partitioning from a poly(dimethylsiloxane) polymer preloaded with the chemicals. The HOC concentrations in the equilibrated solutions [C(solution(eq))] and water [C(water(eq))] were then measured. Free fractions of the HOCs were determined as C(water(eq))/C(solution(eq)), whereas enhanced capacities (E) of the solutions for HOCs were determined as C(solution(eq))/C(water(eq)). A mixture of polycyclic aromatic hydrocarbons served as model analytes, while humic acid, sodium dodecyl sulfate, hydroxypropyl-ß-cyclodextrin, and NaCl served as model medium constituents. The enhanced capacities were plotted versus the concentrations of medium constituents, and simple linear regression provided precise partition ratios, salting out constants, and critical micelle concentrations. These parameters were generally in good agreement with published values obtained by solid phase microextraction and fluorescence quenching. The very good precision was indicated by the low relative standard errors for the partition ratios of 0.5-8%, equivalent to 0.002-0.03 log unit. This passive dosing approach allows binding and speciation of HOCs to be studied without any phase separation steps or mass balance assumptions.