Biodegradation of chemicals tested in mixtures and individually: mixture effects on biodegradation kinetics and microbial composition.

Biodegradation in the aquatic environment occurs in the presence of many chemicals, while standard simulation biodegradation tests are conducted with single chemicals. This study aimed to investigate the effect of the presence of additional chemicals on (1) biodegradation kinetics of individual chemicals and (2) the microbial composition in test systems. Parallel mixture and single substance experiments were conducted for 9 chemicals (phenethyl benzoate, oxacycloheptadec-10-en-2-one, α-ionone, methyl 2-naphthyl ether, decan-5-olide, octan-2-one, 2'-acetonaphthanone, methyl N-methylanthranilate, (+)-menthone) using inoculum from a Danish stream. Biotic and abiotic test systems were incubated at 12 °C for 1-30 days. Primary biodegradation kinetics were then determined from biotic/abiotic peak area ratios using SPME GC/MS analysis. The effect of the mixture on biodegradation varied with test chemical and was more pronounced for chemicals with lag-phases above 14 days: two chemicals degraded in the mixture but not when tested alone (i.e., positive mixture effect), and two degraded when tested alone but not in the mixture (i.e., negative mixture effect). Microbial composition (16S rRNA gene amplicon sequencing) was highly affected by 14 days incubation and the presence of the mixture (significant carbon source), but less by single chemicals (low carbon source). Growth on chemical mixtures resulted in consistent proliferation of Pseudomonas and Malikia, while specific chemicals increased the abundance of putative degraders belonging to Novosphingobium and Zoogloea. The chemical and microbiological results support (1) that simulation biodegradation kinetics should be determined in mixtures at low environmentally relevant concentrations and (2) that degradation times beyond some weeks are associated with more uncertainty.

Biodegradation Kinetics of Fragrances, Plasticizers, UV Filters, and PAHs in a Mixture─Changing Test Concentrations over 5 Orders of Magnitude.

Biodegradation of organic chemicals emitted to the environment is carried out by mixed microbial communities growing on multiple natural and xenobiotic substrates at low concentrations. This study aims to (1) perform simulation type biodegradation tests at a wide range of mixture concentrations, (2) determine the concentration effect on the biodegradation kinetics of individual chemicals, and (3) link the mixture concentration and degradation to microbial community dynamics. Two hundred ninety-four parallel test systems were prepared using wastewater treatment plant effluent as inoculum and passive dosing to add a mixture of 19 chemicals at 6 initial concentration levels (ng/L to mg/L). After 1-30 days of incubation at 12 °C, abiotic and biotic test systems were analyzed using arrow solid phase microextraction and GC-MS/MS. Biodegradation kinetics at the highest test concentrations were delayed for several test substances but enhanced for the reference chemical naphthalene. Test concentration thus shifted the order in which chemicals were degraded. 16S rRNA gene amplicon sequencing indicated that the highest test concentration (17 mg C/L added) supported the growth of the genera Acidovorax, Novosphingobium, and Hydrogenophaga, whereas no such effect was observed at lower concentrations. The chemical and microbial results confirm that too high mixture concentrations should be avoided when aiming at determining environmentally relevant biodegradation data.

Passive Dosing of Petroleum and Essential Oil UVCBs-Whole Mixture Toxicity Testing at Controlled Exposure.

Petroleum products and essential oils are produced and used in large amounts and are categorized as "Substances of Unknown or Variable composition, Complex reaction products or Biological materials (UVCBs)." These UVCBs are notorious difficult-to-test substances, since they are complex mixtures of hydrophobic and volatile compounds. This study introduces two passive dosing (PD) approaches for whole UVCB toxicity testing: (1) headspace PD applies the UVCB and purified lipid oil as a donor to control exposure via the headspace and (2) silicone rod PD applies UVCB-loaded silicone rods to control exposure via an aqueous test medium and headspace. Headspace gas chromatography-mass spectrometry measurements were used to cross-validate the approaches at the saturation level and to confirm exposure and maintain mixture composition at varying donor concentration levels. Both approaches were applied to whole-mixture toxicity tests of petroleum and essential oil UVCBs with daphnia and algae. Finally, the observed toxicity was linked to concentrations in the donor and in lipid membranes at equilibrium with the donors. Dose-response curves were similar across the dosing approaches and tested species for petroleum products but differed by an order of magnitude between essential oils and PD systems. All observed toxic effects were consistent with baseline toxicity, and no excess mixture toxicity was observed.

Determining the Temperature Dependency of Biodegradation Kinetics for 34 Hydrocarbons while Avoiding Chemical and Microbial Confounding Factors.

Biodegradation kinetics data are keystone for evaluating the environmental persistence and risk of chemicals. Biodegradation kinetics depend highly on the prevailing temperature, which influences microbial community structures, metabolic rates, and chemical availability. There is a lack of high-quality comparative biodegradation kinetics data that are determined at different test temperatures but with the same microbial inoculum and chemical availability. The present study was designed to determine the effect of test temperature on the biodegradation kinetics of hydrocarbons while avoiding confounding factors. We used inocula from a Northern river (2.7 °C) and a Central European river (12.5 °C). Aqueous stock solutions containing 45 individual hydrocarbons were generated by passive dosing and added to river water containing the native microorganisms. Compound-specific biodegradation kinetics were then determined at 2.7, 12, and 20 °C based on substrate depletion. Main findings comprise the following: (1) Degradation half-times (DegT50) of 34 test chemicals were determined at different test temperatures and were largely consistent with the Arrhenius equation (activation energy, 65.4 kJ/mol). (2) Differences in biodegradation kinetics between tested isomers were rather limited. (3) The recent lowering of standard test temperature from 20 to 12 °C results typically in a doubling of DegT50 values and can lead to a stricter persistency assessment.

Accelerated Passive Dosing of Hydrophobic Complex Mixtures-Controlling the Level and Composition in Aquatic Tests.

Petroleum products and essential oils are complex mixtures of hydrophobic and volatile chemicals and are categorized as substances of unknown or variable composition, complex reaction products, or biological materials (UVCBs). In aquatic testing and research of such mixtures, it is challenging to establish initial concentrations without the addition of cosolvents, to maintain constant concentrations during the test, and to keep a constant mixture composition in dilution series and throughout test duration. Passive dosing was here designed to meet these challenges by maximizing the surface area (Adonor/Vmedium = 3.8 cm2/mL) and volume (Vdonor/Vmedium > 0.1 L/L) of the passive dosing donor in order to ensure rapid mass transfer and avoid donor depletion for all mixture constituents. Cracked gas oil, cedarwood Virginia oil, and lavender oil served as model mixtures. This study advances the field by (i) showing accelerated passive dosing kinetics for 68 cracked gas oil constituents with typical equilibration times of 5-10 min and for 21 cederwood Virginia oil constituents with typical equilibration times < 1 h, (ii) demonstrating how to control mixture concentration and composition in aquatic tests, and (iii) discussing the fundamental differences between solvent spiking, water-accommodated fractions, and passive dosing.

Time-Resolved Freely Dissolved Concentrations of Semivolatile and Hydrophobic Test Chemicals in In Vitro Assays-Measuring High Losses and Crossover by Headspace Solid-Phase Microextraction.

In vitro assays are normally conducted in plastic multiwell plates open to exchange with the ambient air. The concentration of test substances freely available to cells is often not known, can change over time, and is difficult to measure in the small volumes in microplates. However, even a well-characterized toxicological response is of limited value if it cannot be linked to a well-defined exposure level. The aim of this study was to develop and apply an approach for determining time-resolved freely dissolved concentrations of semivolatile and hydrophobic organic chemicals (SVHOCs) in in vitro assays: (1) free fractions were measured by a new medium dilution method and (2) time-resolved loss curves were obtained by measurements of total concentrations in 96-well plates during incubations at 37 °C. Headspace solid-phase microextraction was used as an analytical technique for 24 model chemicals spanning 6 chemical groups and 4-5 orders of magnitude in Kow and Kaw. Free fractions were >30% for chemicals with log Kow < 3.5 and then decreased with increasing log Kow. Medium concentrations declined significantly (>50%) within 24 h of incubation for all 20 chemicals having log Kow > 4 or log Kaw > -3.5 in serum-free medium. Losses of chemicals were lower for medium containing 10% fetal bovine serum, most significantly for chemicals with log Kow > 4. High crossover to neighboring wells also was observed below log Kow of 4 and log Kaw of -3.5. Sealing the well plates had limited effect on the losses but clearly reduced crossover. The high losses and crossover of most tested chemicals question the suitability of multiwell plates for in vitro testing of SVHOCs and call for (1) test systems that minimize losses, (2) methods to control in vitro exposure, (3) analytical confirmation of exposure, and (4) exposure control and confirmation being included in good in vitro reporting standards.

Mixture Effects on Biodegradation Kinetics of Hydrocarbons in Surface Water: Increasing Concentrations Inhibited Degradation whereas Multiple Substrates Did Not.

Most biodegradation tests are conducted using single chemicals at high concentrations, although these chemicals are present in the environment as mixtures at low concentrations. A partitioning-based platform was recently developed for biodegradation testing of composed mixtures of hydrophobic chemicals at ng/L to µg/L concentrations. We used this platform to study the concentration and mixture effect on biodegradation kinetics. Biodegradation tests were conducted in 20 mL vials using environmental water samples as inocula. Passive dosing was applied (1) to vary initial test concentrations of individual test compounds and (2) to vary the number of mixture components between 1 and 16. Automated solid-phase microextraction coupled to gas chromatography-mass spectrometry was used to measure substrate depletion relative to abiotic controls. The number of mixture components had no or only a limited effect on the biodegradation half times for three compounds when tested at environmentally relevant concentrations. In contrast, longer lag phases and half lives were observed for single compounds when tested at higher concentrations that approached aqueous solubility. The obtained results support that simultaneous testing of multiple chemicals at low concentrations can accelerate the generation of biodegradation kinetic data, which are more environmentally relevant compared with data from tests conducted with single chemicals at much higher concentrations.

Biodegradation of Volatile Chemicals in Soil: Separating Volatilization and Degradation in an Improved Test Setup (OECD 307).

During environmental risk assessments of chemicals, higher-tier biodegradation tests in soil, sediment, and surface-water systems are required using OECD standards 307, 308, and 309 guidelines, respectively. These guidelines are not suitable for testing highly volatile chemicals, and a biometer closed-incubation setup is recommended for testing slightly volatile chemicals. In this setup, the degradation kinetics of highly volatile chemicals can largely be influenced by volatilization. Additionally, guidelines lack sufficient information on test-system geometry and guidance on how to measure and maintain aerobic conditions during the test. Our objectives were (1) to design a closed test setup for biodegradation tests in soil in which the maintaining and measuring of aerobic conditions was possible without the loss of volatile test chemicals and (2) to suggest data-treatment measures for evaluating the degradation kinetics of volatile test chemicals. With the new setup, full-scale OECD 307 tests were performed using the volatile 14C-labeled chemicals decane and tetralin. For both test chemicals, reproducible complete mass balances were observed, and the new setup ensured that the volatilization losses were kept below the mineralized fraction. Based on the obtained data, an extended model was developed that enabled consideration of the volatilization in the modeling of degradation kinetics.

Determining Biodegradation Kinetics of Hydrocarbons at Low Concentrations: Covering 5 and 9 Orders of Magnitude of Kow and Kaw.

A partitioning-based experimental platform was developed and applied to determine primary biodegradation kinetics of 53 hydrocarbons at ng/L to µg/L concentrations covering C8-C20, 11 structural classes, and several orders of magnitude in hydrophobicity and volatility: (1) Passive dosing from a loaded silicone donor was used to set the concentration of each hydrocarbon in mixture stock solutions; (2) these solutions were combined with environmental water samples in gastight auto sampler vials for 1-100 days incubation, and (3) automated solid phase microextraction (SPME) coupled to GC-MS was applied directly on these test systems for measuring primary biodegradation relative to abiotic controls. First order biodegradation kinetics were obtained for 40 hydrocarbons in activated sludge filtrate, 18 in seawater, and 21 in lake water. Water phase half-lives in seawater and lake water were poorly related to hydrophobicity and volatility but were, with a few exceptions, within a factor of 10 or shorter than BioHCwin predictions. The most persistent hydrocarbons, 1,1,4,4,6-pentamethyldecalin, perhydropyrene, 1,2,3,6,7,8-hexahydropyrene, and 2,2,4,4,6,8,8-heptamethylnonane, showed limited or inconsistent degradation in all three environmental media. This biodegradation approach can cover a large chemical space at low substrate concentrations, which makes it highly suited for optimizing predictive models for environmental biodegradation.

Retrospective comparison of GnRH agonist trigger with HCG trigger in GnRH antagonist cycles in anticipated high-responders.

All IVF-ICSI cycles carried out between October 2009 and October 2012 using GnRH agonist (GnRHa) ovulation trigger (n = 62) followed by a single dose of HCG plus progesterone and oestradiol in the luteal phase because of anticipated ovarian hypertsimulation were retrospectively compared with historic control cycles using HCG trigger (n = 29) and standard luteal phase support. Women's mean age, body mass index, anti-Müllerian hormone, FSH, LH, starting and total stimulation dose, number of follicles, oocytes, embryos, fertilization, implantation, polycystic ovary syndrome, ICSI, live birth and ongoing pregnancy rates per embryo transfer were similar (GnRHa 40.7% versus HCG 35.0%). For each started cycle, GnRHa resulted in 11.4% higher (statistically non-significant) live birth and ongoing pregnancy rate (OR 1.73, CI 0.64 to 4.69), with a similar difference for double-embryo transfers (OR 1.62, CI 0.44 to 6.38) and less need for freezing all embryos (9.7% versus 27.6%; P = 0.04). Incidence of mild-to-moderate OHSS was 16.2% with GnRHa trigger and 31.0% with HCG trigger) and no severe OHSS in the former. The addition of single low-dose HCG in the luteal phase after GnRHa trigger for suspected high-responders reduced the incidence of OHSS with good clinical outcomes, compared with HCG trigger.

Velocity dependent passive sampling for monitoring of micropollutants in dynamic stormwater discharges.

Micropollutant monitoring in stormwater discharges is challenging because of the diversity of sources and thus large number of pollutants found in stormwater. This is further complicated by the dynamics in runoff flows and the large number of discharge points. Most passive samplers are nonideal for sampling such systems because they sample in a time-integrative manner. This paper reports test of a flow-through passive sampler, deployed in stormwater runoff at the outlet of a residential-industrial catchment. Momentum from the water velocity during runoff events created flow through the sampler resulting in velocity dependent sampling. This approach enables the integrative sampling of stormwater runoff during periods of weeks to months while weighting actual runoff events higher than no flow periods. Results were comparable to results from volume-proportional samples and results obtained from using a dynamic stormwater quality model (DSQM). The paper illustrates how velocity-dependent flow-through passive sampling may revolutionize the way stormwater discharges are monitored. It also opens the possibility to monitor a larger range of discharge sites over longer time periods instead of focusing on single sites and single events, and it shows how this may be combined with DSQMs to interpret results and estimate loads over extended time periods.

Model-based monitoring of stormwater runoff quality.

Monitoring of micropollutants (MP) in stormwater is essential to evaluate the impacts of stormwater on the receiving aquatic environment. The aim of this study was to investigate how different strategies for monitoring of stormwater quality (combining a model with field sampling) affect the information obtained about MP discharged from the monitored system. A dynamic stormwater quality model was calibrated using MP data collected by automatic volume-proportional sampling and passive sampling in a storm drainage system on the outskirts of Copenhagen (Denmark) and a 10-year rain series was used to find annual average (AA) and maximum event mean concentrations. Use of this model reduced the uncertainty of predicted AA concentrations compared to a simple stochastic method based solely on data. The predicted AA concentration, obtained by using passive sampler measurements (1 month installation) for calibration of the model, resulted in the same predicted level but with narrower model prediction bounds than by using volume-proportional samples for calibration. This shows that passive sampling allows for a better exploitation of the resources allocated for stormwater quality monitoring.

Technical guidance on biodegradation testing of difficult substances and mixtures in surface water.

The aim of this article is to address critical challenges in the OECD 309 "Aerobic mineralization in surface water - simulation biodegradation test" for volatile chemicals, highly hydrophobic chemicals, mixtures or UVCBs (unknown or variable composition, complex reaction products or biological materials). Several modifications are presented to address technical challenges (minimize and account for losses), make testing more environmentally relevant (lower concentrations) and generate data for multiple substances (more and better aligned data):•Minimizing and accounting for test substance losses: Aqueous solutions are handled using gas tight syringes, tests are conducted in gas tight vials, and automated analysis is performed directly on unopened test vials. Abiotic losses are accounted for via concentration ratios between test systems and abiotic controls that are incubated and measured in parallel.•Testing at low environmentally relevant concentrations: Substances are tested at low concentrations to avoid toxicity and solubility artefacts and analyzed using a sensitive analytical method. Substances are added without co-solvent (using passive dosing) or with a minimum of co-solvent (using microvolume spiking).•Testing of multiple chemicals in mixtures combined with constituent specific analysis: Primary biodegradation kinetics of chemicals are determined in tests of multi-constituent mixtures or UVCBs using constituent specific analysis.

Closed aerobic biodegradation kinetics test with activated sludge and low concentration chemical mixtures.

The activated sludge process at wastewater treatment plants is important to prevent discharge of organic pollutants to the environment. Determination of biodegradation kinetics in activated sludge is challenging for mixtures that cover a diverse range of structures. The aims of this study were to (1) design a closed aerobic biodegradation batch test with activated sludge and (2) develop a sample preparation procedure that is compatible with LC-MS and Solid Phase Microextraction (SPME) coupled to GC-MS. A headspace:sludge ratio of 4:1 was sufficient to ensure aerobic conditions in activated sludge for 7 days at co-solvent concentrations <0.01%. Ethanol was added to sub-samples (50%) to stop biodegradation, extract sorbed chemicals and allow storage at -18 °C without ice formation. The ethanol extracted the chemicals from the sludge before filtration (0.2 µm). The filtrate was diluted in ultrapure water to <12% ethanol before analysis by SPME GC-MS/MS and was suitable for direct injection on LC-MS/MS. Biodegradation was distinguished from sorption through abiotic controls using autoclaved poisoned sludge. Linalool, naphthalene, α-isomethylionone, phenanthrene, citronellol, drometrizole, 2-ethylhexyl 4-methoxycinnamate, dicyclohexyl phthalate, BP-1, BP-3, methyl-, ethyl-, propylparaben, alkyl sulfates and isethionates degraded within 48 h in activated sludge, while musk ketone, tonalide and 1,3,5-trichlorobenzene did not. A 10 times reduction of sludge density did not markedly affect the microbial diversity but slowed biodegradation kinetics (partly explained by theory). This study demonstrated a 'cold' alternative to an OECD 314b test and how biodegradation kinetics can be determined for mixtures of diverse chemicals in closed batch tests with activated sludge.

Linking biodegradation kinetics, microbial composition and test temperature - Testing 40 petroleum hydrocarbons using inocula collected in winter and summer.

Many factors affect the biodegradation kinetics of chemicals in test systems and the environment. Empirical knowledge is needed on how much test temperature, inoculum, test substances and co-substrates influence the biodegradation kinetics and microbial composition in the test. Water was sampled from the Gudenaa river in winter (2.7 °C) and summer (17 °C) (microbial inoculum) and combined with an aqueous stock solution of >40 petroleum hydrocarbons prepared by passive dosing. This resulted in low-concentration test systems that were incubated for 30 days at 2.7, 12 and 20 °C. Primary biodegradation kinetics, based on substrate depletion relative to abiotic controls, were determined with automated Solid Phase Microextraction coupled to GC/MS. Biodegradation kinetics were remarkably similar for summer and winter inocula when tested at the same temperature, except when cooling summer inoculum to 2.7 °C which delayed degradation relative to winter inoculum. Amplicon sequencing was applied to determine shifts in the microbial composition between season and during incubations: (1) the microbial composition of summer and winter inocula were remarkably similar, (2) the incubation and the incubation temperature had both a clear impact on the microbial composition and (3) the effect of adding >40 petroleum hydrocarbons at low test concentrations was limited but resulted in some proliferation of the known petroleum hydrocarbon degraders Nevskia and Sulfuritalea. Overall, biodegradation kinetics and its temperature dependency were very similar for winter and summer inoculum, whereas the microbial composition was more affected by incubation and test temperature compared to the addition of test chemicals at low concentrations.

Inter-laboratory comparison of water solubility methods applied to difficult-to-test substances.

Water solubility is perhaps the single most important physical-chemical property determining the environmental fate and effects of organic compounds. Its determination is particularly challenging for compounds with extremely low solubility, frequently referred to as "difficult-to-test" substances and having solubility's generally less than 0.1 mg/L. The existing regulatory water solubility test for these compounds is the column elution method. Its applicability, however, is limited, to non-volatile solid or crystalline hydrophobic organic compounds. There currently exists no test guideline for measuring the water solubility of very hydrophobic liquid, and potentially volatile, difficult-to-test compounds. This paper describes a "slow-stir" water solubility methodology along with results of a ring trial across five laboratories evaluating the method's performance. The slow-stir method was applied to n-hexylcyclohexane, a volatile, liquid hydrophobic hydrocarbon. In order to benchmark the inter-laboratory variability associated with the proposed slow-stir method, the five laboratories separately determined the solubility of dodecahydrotriphenylene, a hydrophobic solid compound using the existing column elution guideline. Results across the participating laboratories indicated comparable reproducibility with relative standard deviations (RSD) of 20% or less reported for each test compound - solubility method pair. The inter-laboratory RSD was 16% for n-hexylcyclohexane (mean 14 µg/L, n = 5) using the slow-stir method. For dodecahydrotriphenylene, the inter-laboratory RSD was 20% (mean 2.6 µg/L, n = 4) using the existing column elution method. This study outlines approaches that should be followed and the experimental parameters that have been deemed important for an expanded ring trial of the slow-stir water solubility method.

Biodegradation of an essential oil UVCB - Whole substance testing and constituent specific analytics yield biodegradation kinetics of mixture constituents.

Testing and assessing the persistency, bioaccumulative and toxic properties of UVCBs (substances of Unknown or Variable composition, Complex reaction products or Biological materials) pose major technical and analytical challenges. The main aim of this study was to combine whole substance biodegradation testing with constituent specific analytics for determining primary biodegradation kinetics of the main UVCB constituents. An additional aim was to link the primary biodegradation kinetics of the main constituents to the bioaccumulation potential and baseline toxicity potential of the UVCB. Two closed biodegradation experiments were conducted using similar test systems but different analyses. The model substance, cedarwood Virginia oil, was tested at a low concentration and wastewater treatment plant effluent served as inoculum. We used microvolume solvent spiking for a quantitative mass transfer of the UVCB, while avoiding that co-solvent degradation would lead to anaerobic conditions. The biodegradation of UVCB constituents was determined with automated solid-phase microextraction coupled to GC-MS/MS using targeted analysis for main constituents and non-targeted analysis for minor constituents and non-polar degradation products. Primary biodegradation kinetics of main constituents, accounting for 73% w/w of the mixture, were successfully determined with degradation rate constants ranging from 0.09 to 0.25 d-1. Minor constituents were also degraded and non-polar degradation products were not observed. Finally, the bioaccumulation potential and baseline toxicity potential of the mixture at test start were calculated and both parameters decreased then substantially. The strength of the new approach is the possibility of biodegradation testing of a whole UVCB at low concentration while generating constituent specific biodegradation kinetics.

Passive dosing to determine the speciation of hydrophobic organic chemicals in aqueous samples.

A new analytical approach to determine the speciation of hydrophobic organic analytes is presented. The freely dissolved concentration in a sample is controlled by passive dosing from silicone (poly(dimethylsiloxane)), and the total sample concentration at equilibrium is measured. The free fraction is determined as the ratio between measured concentrations in pure water and sample. (14)C-labeled fluoranthene served as model analyte, and total sample concentrations were easily measured by liquid scintillation counting. The method was applied to surface water, stormwater runoff, and wastewater. In the untreated wastewater, 61% of the fluoranthene was bound to suspended solids, 28% was associated to dissolved organic matter, and 11% was freely dissolved, while in treated wastewater, the speciation was 16% bound to suspended solids, 4% bound to dissolved organic matter, and 80% freely dissolved. The free fraction in roof runoff (85%) and surface water (91%) was markedly higher than in runoff from paved areas, which ranged from 27 to 36%. A log K(DOC) value of 5.26 was determined for Aldrich humic acid, which agrees well with reported values obtained by fluorescence quenching and solid phase microextraction (SPME). This analytical approach combines simplicity with high precision, and it does not require any phase separation steps.

Biodegradation testing of volatile hydrophobic chemicals in water-sediment systems - Experimental developments and challenges.

Degradation data are crucial for the persistence assessment of chemicals and they are generated using standard OECD guidelines. The OECD 308 describes a simulation biodegradation test of chemicals in water-sediment systems. This guideline is not applicable for testing highly volatile chemicals and recommends a closed biometer test setup for testing slightly volatile chemicals. However, proper details on system geometries, construction and monitoring of aerobic conditions are not provided. The choice of system geometry and sediment:water ratio influences the partitioning of test chemicals between different compartments (water, sediment and headspace) and can therefore affect their degradation. The guideline recommends the addition of test chemical via aqueous solutions, which however is not possible for hydrophobic volatile chemicals due to their volatilization losses and low solubility. Thus, the use of a co-solvent is necessary for the application of such chemicals but its effects in a closed setup has not been studied. We recently developed an improved closed test setup for testing volatile chemicals in soil. The objective was to adapt this improved test setup to conduct OECD 308 tests using 14C labelled chemicals with different volatilities. Using the adapted test setup it was possible to obtain a complete mass balance even for n-decane and tetralin having the highest Henry's constants of the tested chemicals. However, the use of co-solvent affected the oxygen levels, which in turn affected microbial activity and likely also the degradation of test chemicals. Therefore, the adapted test setup needs further developments for the testing of volatile hydrophobic chemicals.

Biodegradation kinetics testing of two hydrophobic UVCBs - potential for substrate toxicity supports testing at low concentrations.

The biodegradation kinetics of UVCB substances (unknown or variable composition, complex reaction products or biological materials) should be determined below the solubility limit to avoid experimental artefacts by the non-dissolved mixture. Recently, we reported delayed biodegradation kinetics of single petroleum hydrocarbons even at concentrations just below the solubility limit and attributed this to toxicity. The present study aimed to determine the concentration effect on biodegradation kinetics for constituents in two UVCBs, using surface water from a rural stream as the inoculum. Parallel biodegradation tests of diesel and lavender oil were conducted at concentrations just below the solubility limit and two orders of magnitude lower. The biodegradation kinetics of diesel oil constituents were generally similar at the two concentrations, which coincided with the stimulation of bacterial productivity (growth) at both concentrations, determined by [3H]leucine incorporation. By contrast, the biodegradation of lavender oil constituents was significantly delayed or even halted at the high test concentration. This was consistent with lavender oil stimulating bacterial growth at low concentration but inhibiting it at high concentration. The delayed biodegradation kinetics of lavender oil constituents at high concentration was best explained by mixture toxicity near the solubility limit. Consequently, biodegradation testing of hydrophobic UVCBs should be conducted at low, environmentally relevant concentrations ensuring that mixture toxicity does not affect the biodegradation kinetics.