A comparative assessment of the CLP calculation method and in vivo testing for the classification of plant protection products.

In Europe, animal testing for the purpose of regulatory plant protection product (PPP) assessment should be undertaken only as a last resort. Nevertheless, there is a need to improve the acceptance of alternative methods, which has been slow due to a lack of data regarding the predictivity of in vivo effects. The CLP calculation method is an alternative method based on the concentration addition of all adverse substances in a mixture. It is often applied as a conservative approach for the estimation of toxicodynamic interactions. However, PPPs consist of pesticides and co-formulants, which in combination can also exhibit altered toxicokinetic properties. Our analysis revealed that oral and inhalation toxicity was underestimated for approximately 45% of the in vivo classified products by the CLP calculation method as compared to in vivo testing. With regard to skin and eye irritation, the CLP calculation method underestimated the irritating potential in 22% and 6% of PPPs, respectively. Based on specific concentration limits, skin sensitisation was underestimated in 34% of PPPs. Similar false negative rates have been reported for PPP in vitro testing. Hence, we suggest the development of an integrated assessment strategy, weighing all available information and considering relevant parameters influencing predictivity and uncertainty.

The impact of chemosensitisation on bioaccumulation and sediment toxicity.

Cellular multixenobiotic resistance (MXR) transport proteins enhance the efflux of numerous organic pollutants. However, MXR proteins may be blocked or saturated by xenobiotic compounds, acting as inhibitors - also called chemosensitisers. Although effective on a cellular level, the environmental relevance of chemosensitisers has not been conclusively demonstrated. Since sediments are an important source of bioaccumulating compounds in aquatic ecosystems, sediments and sediment-associated hydrophobic pollutants were investigated for their potential to increase exposure and toxicity in the presence of chemosensitisation. In this study, we address this issue by (1) comparing the net uptake of 17 hydrophobic environmental pollutants by zebrafish (Danio rerio) embryos in the presence and absence of the model chemosensitiser verapamil and (2) investigating the impact of verapamil on the dose-dependent effect on zebrafish embryos exposed to polluted sediment extracts. None of the 17 pollutants showed a reproducible increase in bioaccumulation upon chemosensitisation with verapamil. Instead, internal concentrations were subject to intra-species variation by a factor of approximately two. However, a significant increase in toxicity was observed upon embryo co-exposure to verapamil for one of three sediment extracts. In contrast, another sediment extract exhibited less toxicity when combined with verapamil. In general, the results indicate only a minor impact of verapamil on the uptake of moderately hydrophobic chemicals in zebrafish embryos.

Measuring the internal concentration of volatile organic compounds in small organisms using micro-QuEChERS coupled to LVI-GC-MS/MS.

There is an increasing demand for analytical tools to measure the internal concentrations of xenobiotic pollutants in small organisms. Such tools are required to determine exposure in ecotoxicological studies yet avoid sophisticated clean-up and enrichment techniques or large-scale experimental design. Thus, this paper presents a modified QuEChERS method coupled to gas chromatography tandem mass spectrometry (GC-MS/MS) for small volume organic samples. Ten zebrafish (Danio rerio) embryos were exposed to a 46-compound mixture at 10 ng/mL. After 72 h of exposure, they were extracted in 200 µL glass inserts using 70 µL of both acetonitrile and water. Volumes of 50 µL of extract were injected into a GC-MS/MS with a multi-mode inlet. Internal concentrations of zebrafish embryos could be reproducibly quantified in the lower nanogram per millilitre range at detection limits of 1-25 ng/mL and with recoveries of 63-133%. Internal concentrations varied over the tested range of compounds between 5.88 ± 0.616 ng/mL for dicofol and 232 ± 18.6 ng/mL for diflufenican. Detectability and recovery were best for compounds with a log D greater than four. As internal concentrations did not seem to exclusively depend on log D, biochemical transport processes could play an important role in the uptake kinetics of early zebrafish life stages. Graphical Abstract This paper presents an extraction and quantification method for 46 volatile organic compounds in zebrafish embryos. After exposure, pools of ten embryos were extracted with 70 µL acetonitrile applying a micro-QuEChERS approach. Internal embryo concentrations were analytically determined and quantified by large volumen injection gas chromatography tandem mass spectrometry (GC-MS/MS).

Is chemosensitisation by environmental pollutants ecotoxicologically relevant?

The active cellular efflux of toxicants is an efficient biological defense mode present in all organisms. By blocking this so-called multixenobiotic resistance transport-a process also referred to as chemosensitisation-, cellular bioaccumulation and the sensitivity of organisms towards environmental pollutants can increase. So far, a wide range of compounds, including pesticides, pharmaceuticals, fragrances, and surfactants, have been identified as chemosensitisers. Although, significant on a cellular level, the environmental impact of chemosensitisation on the organism level is not yet understood. Critically evaluating existing data, this paper identifies research needs to support our tentative conclusion that chemosensitisation may well enhance the risks of chemical exposure to aquatic organisms. Our conclusion is based on studies investigating the impact of individual chemicals and complex environmental mixtures on aquatic wildlife and a chemosensitiser mixture toxicity model which, however, is subject to great uncertainty due to substantial knowledge gaps. Those uncertainties include the inconsistent reporting of effect data, the lack of representative environmental contaminants tested for chemosensitisation, and the publishing of highly unreliable nominal exposure concentrations. In order to confirm the tentative conclusion of this paper, we require the significant and systematic investigation of a broader set of chemicals and environmental samples with a harmonised set of bioassays and rigorously controlled freely dissolved effect concentrations.

Comparative toxicokinetics of organic micropollutants in freshwater crustaceans.

Exposure and depuration experiments for Gammarus pulex and Daphnia magna were conducted to quantitatively analyze biotransformation products (BTPs) of organic micropollutants (tramadol, irgarol, and terbutryn). Quantification for BTPs without available standards was performed using an estimation method based on physicochemical properties. Time-series of internal concentrations of micropollutants and BTPs were used to estimate the toxicokinetic rates describing uptake, elimination, and biotransformation processes. Bioaccumulation factors (BAF) for the parents and retention potential factors (RPF), representing the ratio of the internal amount of BTPs to the parent at steady state, were calculated. Nonlinear correlation of excretion rates with hydrophobicity indicates that BTPs with lower hydrophobicity are not always excreted faster than the parent compound. For irgarol, G.pulex showed comparable elimination, but greater uptake and BAF/RPF values than D.magna. Further, G. pulex had a whole set of secondary transformations that D. magna lacked. Tramadol was transformed more and faster than irgarol and there were large differences in toxicokinetic rates for the structurally similar compounds irgarol and terbutryn. Thus, predictability of toxicokinetics across species and compounds needs to consider biotransformation and may be more challenging than previously thought because we found large differences in closely related species and similar chemical structures.

Biotransformation pathways of biocides and pharmaceuticals in freshwater crustaceans based on structure elucidation of metabolites using high resolution mass spectrometry.

So far, there is limited information on biotransformation mechanisms and products of polar contaminants in freshwater crustaceans. In the present study, metabolites of biocides and pharmaceuticals formed in Gammarus pulex and Daphnia magna were identified using liquid chromatography-high resolution mass spectrometry. Different confidence levels were assigned to the identification of metabolites without reference standards using a framework based on the background evidence used for structure elucidation. Twenty-five metabolites were tentatively identified for irgarol, terbutryn, tramadol, and venlafaxine in G. pulex (21 via oxidation and 4 via conjugation reactions) and 11 metabolites in D. magna (7 via oxidation and 4 via conjugation reactions), while no evidence of metabolites for clarithromycin and valsartan was found. Of the 360 metabolites predicted for the four parent compounds using pathway prediction systems and expert knowledge, 23 products were true positives, while 2 identified metabolites were unexpected products. Observed oxidative reactions included N- and O-demethylation, hydroxylation, and N-oxidation. Glutathione conjugation of selected biocides followed by subsequent reactions forming cysteine conjugates was described for the first time in freshwater invertebrates.