Are current regulatory log Kow cut-off values fit-for-purpose as a screening tool for bioaccumulation potential in aquatic organisms?

Persistent, Bioaccumulative and Toxic (PBT) and very Persistent and very Bioaccumulative (vPvB) are regulatory hazard categories that have been set to manage the possible risks to humans and the environment from these chemicals. In industrial chemicals regulations, their aquatic Bioaccumulation potential is usually assessed first with a screening based on the octanol/water partition coefficient (Kow). However, current log Kow cut-off values triggering classification, categorisation and/or further fish bioconcentration testing are not harmonised worldwide, and they have never been assessed for their regulatory relevance. In this study, the experimentally determined log Kow and fish bioconcentration factors (BCF) of 532 chemicals were compared. While the analysis underlined the robustness of using log Kow as a screening tool (5/532 were false negatives; log Kow: non-bioaccumulative, but BCF: bioaccumulative), it also demonstrated the conservatism of the cut-offs used worldwide. Indeed, many chemicals were deemed potentially Bioaccumulative based on log Kow when a fish bioaccumulation test showed no concern (false positives), therefore, leading to unnecessary use of vertebrate animals. Our analysis shows that the log Kow cut-off could be increased to 4.5 in all regions for all purposes without leading to a reduced protection of humans and the environment.

Intestinal Fish Cell Barrier Model to Assess Transfer of Organic Chemicals in Vitro: An Experimental and Computational Study.

We studied the role of the fish intestine as a barrier for organic chemicals using the epithelial barrier model built on the rainbow trout (Oncorhynchus mykiss) intestinal cell line, RTgutGC and the newly developed exposure chamber, TransFEr, specifically designed to work with hydrophobic and volatile chemicals. Testing 11 chemicals with a range of physicochemical properties (logKOW: 2.2 to 6.3, logHLC: 6.1 to 2.3) and combining the data with a mechanistic kinetic model enabled the determination of dominant processes underlying the transfer experiments and the derivation of robust transfer rates. Against the current assumption in chemical uptake modeling, chemical transfer did not strictly depend on the logKOW but resulted from chemical-specific intracellular accumulation and biotransformation combined with paracellular and active transport. Modeling also identified that conducting elaborate measurements of the plastic parts, including the polystyrene insert and the PET filter, is unnecessary and that stirring in the TransFEr chamber reduced the stagnant water layers compared to theoretical predictions. Aside from providing insights into chemical uptake via the intestinal epithelium, this system can easily be transferred to other cell-based barrier systems, such as the fish gill or mammalian intestinal models and may improve in vitro-in vivo extrapolation and prediction of chemical bioaccumulation into organisms.

Country-Specific Environmental Risks of Fragrance Encapsulates Used in Laundry Care Products.

Fragrance encapsulates (FEs) are designed to deliver fragrance components, notably in laundry care products. They are made of thermoset polymeric shells surrounding the fragrance content. These materials enter the environment mainly during laundry washing, but little is known about their distribution in and impact on the environment. The aim of the present study was to estimate the environmental concentrations of FE shells in freshwater, sediment, and soil compartments for 34 selected countries and to compare them with ecotoxicological effects. Probabilistic material flow analysis was used to estimate worst-case predicted environmental concentrations (PECs). The lowest freshwater PEC was predicted for Finland (0.00011 µg/L) and the highest for Belgium (0.13 µg/L). Accumulation of FE shells between 2010 and 2019 was considered for sediments and sludge-treated soils. The PECs in sediments ranged from 3.0 µg/kg (Finland) to 3400 µg/kg (Belgium). For sludge-treated soil, the concentration was estimated to be between 0 (Malta and Switzerland) and 3600 µg/kg (Vietnam). Ecotoxicological tests showed no effects for FE shells at any tested concentration (up to 2700 µg/L freshwater, 5400 µg/kg sediment, and 9100 µg/kg soil), thus not allowing derivation of a predicted-no-effect concentration (PNEC). Therefore, to characterize the environmental risks, the PEC values were compared with highest-observed-no-effect concentrations (HONECs) derived from ecotoxicological tests. The PEC/HONEC ratios were 9.3 × 10-6 , 0.13, and 0.04 for surface waters, sediments, and sludge-treated soils, respectively, which are much below 1, suggesting no environmental risk. Because the PEC values constitute an upper boundary (no fate considered) and the HONEC values represent a lower boundary (actual PNEC values based on NOECs will be higher), the current risk estimation can be considered a precautionary worst-case assessment. Environ Toxicol Chem 2022;41:905-916. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Investigations On the Fish Acute Toxicity of Fragrance Ingredients Involving Chinese Fish Species and Zebrafish Embryos.

While zebrafish (Danio rerio) have been accepted worldwide for evaluating chemical hazards to aquatic vertebrates, and in some countries it is mandated to generate fish toxicity data using native species, such as Chinese rare minnow (Gobiocypris rarus) in China. This represents an additional regulatory constraint that may cause redundant tests, additional animal uses, and higher costs. Previous studies showed that juvenile G. rarus was more sensitive than zebrafish juveniles and embryos to metals. To better understand the sensitivity of G. rarus to organic chemicals, we selected 29 fragrance ingredients belonging to various chemical classes and with differing physicochemical properties, for which good quality zebrafish acute toxicity data were available and tested them with juvenile G. rarus and embryo D. rerio using the Organisation of Economic Co-operation and Development test guidelines. Chemical toxicity distribution (CTD) and chemical ratio distribution (CRD) models were established to systematically compare the sensitivity between juveniles of G. rarus and D. rerio, as well as between D. rerio embryos and juveniles. The results of the CTD models showed that for tested chemicals, the sensitivity of juvenile G. rarus was similar to that of D. rerio juveniles and embryos. The CRD comparisons revealed that juvenile G. rarus was slightly less sensitive by a factor of ~2 than juvenile D. rerio to ingredients belonging to Verhaar class 3 and Ecological Structure Activity Relationship ester class, while comparable to other chemicals. These comparative experiments demonstrated that fish toxicity data with G. rarus can be submitted for use in chemical registrations outside China, which would avoid repeating animal tests using D. rerio. Meanwhile, the similar sensitivity of zebrafish juveniles and embryos to fragrance ingredients confirmed the suitability of replacing juveniles by zebrafish embryos. Environ Toxicol Chem 2022;41:2305-2317. © 2022 SETAC.

Species and Life-Stage Sensitivity of Chinese Rare Minnow (Gobiocypris rarus) to Chemical Exposure: A Critical Review.

Chemical production and consumption in Asia are increasing at an unprecedented rate, calling for regulations on chemical management. Under the New Chemical Substance Notification in China, information on ecotoxicological effects of chemicals is mandatory for the simplified registration of chemicals with the requirement that one ecotoxicological test is conducted locally. It is now mandatory to use the native fish species Chinese rare minnow (Gobiocypris rarus). However, its chemical sensitivity compared to that of fathead minnow (Pimephales promelas) or zebrafish (Danio rerio) is still unclear. We performed a holistic literature review on toxicity data with G. rarus from 1997 to 2020. Species sensitivity among G. rarus, P. promelas, and D. rerio and life-stage sensitivity of G. rarus were systematically investigated for various chemicals using both chemical ratio distribution and probabilistic chemical toxicity distribution approaches. Comparatively, the Chinese native fish species G. rarus was more sensitive than D. rerio, particularly to metals. Juvenile and adult G. rarus were more sensitive than its larvae and embryos. The observed lower sensitivity of G. rarus embryo was likely due to the thick embryonic chorion, discrepant methods of collecting embryos, and the paucity of toxicity data, implying the necessity to standardize G. rarus embryo tests and validate the sensitivity with various types of chemicals. This unique review allows us to conclude that G. rarus studies could be used in worldwide registrations and that further investigations are needed to use G. rarus embryos as alternatives to the fish test. Environ Toxicol Chem 2021;40:2680-2692. © 2021 SETAC.

In Vitro Biotransformation Assays Using Liver S9 Fractions and Hepatocytes from Rainbow Trout (Oncorhynchus mykiss): Overcoming Challenges with Difficult to Test Fragrance Chemicals.

In vitro metabolic stability assays using rainbow trout (Oncorhynchus mykiss) isolated hepatocytes (RT-HEP) or hepatic S9 fractions (RT-S9) were introduced to provide biotransformation rate data for the assessment of chemical bioaccumulation in fish. The present study explored the suitability of the RT-HEP and RT-S9 assays for difficult test chemicals, and the in vitro-based predictions were compared to in silico-based predictions and in vivo-measured bioconcentration factors (BCFs). The results show that volatile or reactive chemicals can be tested with minor modifications of the in vitro protocols. For hydrophobic chemicals, a passive dosing technique was developed. Finally, a design-of-experiment approach was used to identify optimal in vitro assay conditions. The modified assay protocols were applied to 10 fragrances with diverse physicochemical properties. The in vitro intrinsic clearance rates were higher in the S9 than in the hepatocyte assay, but the in vitro-in vivo (IVIV) predictions were comparable between the 2 assays. The IVIV predictions classified the test chemicals as nonbioaccumulative (BCF < 2000), which was in agreement with the in vivo data but in contrast to the in silico-based predictions. The findings from the present study provide strong evidence that the RT-HEP and RT-S9 assays can provide reliable estimates of in vivo biotransformation rates for test chemicals with difficult physicochemical properties. Environ Toxicol Chem 2020;39:2396-2408. © 2020 SETAC.

Extending the concept of predicting fish acute toxicity in vitro to the intestinal cell line RTgutGC.

Testing chemicals for fish acute toxicity is a legal requirement in many countries as part of environmental risk assessment. To reduce the numbers of fish used, substantial efforts have been focussed on alternative approaches. Prominently, the cell viability assay with the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, has been recognized, owing to its high predictive power and robustness. Like gills, the intestine is considered a major site of chemical uptake and biotransformation but, in contrast to gills, is expected to be exposed to rather hydrophobic chemicals, which enter the fish via food. In the present study, we therefore aimed to extend the cell bioassay to the rainbow trout epithelial cell line from intestine, RTgutGC. Using 16 hydrophobic and volatile chemicals from the fragrance palette, we showed that also the RTgutGC cell line can be used to predict fish acute toxicity of chemicals and yields intra-laboratory variability in line with other bioassays. By comparing the RTgutGC toxicity to a study employing the RTgill-W1 assay on the same group of chemicals, a fragrance specific relationship was established which reflects an almost perfect 1:1 relationship between in vitro and in vivo toxicity results. Thus, both cell lines can be used to predict fish acute toxicity, either by using the obtained in vivo-in vitro relationship or by taking the in vitro results at face value. We moreover demonstrate the derivation of non-toxic concentrations for downstream applications which rely on a healthy cell state, such as the assessment of biotransformation or chemical transfer.

A streamlined workflow to study direct photodegradation kinetic and transformation products for persistence assessment of a fragrance ingredient in natural waters.

Photodegradation can be an important abiotic degradation process to consider for the fate and persistence assessment of chemical substances in the environment. In this work, using a fragrance ingredient (FI, (E)-4-(2,2,3,6-tetramethylcyclohexyl)but-3-en-2-one) as an example, we developed a streamlined workflow to investigate direct photodegradation of chemicals in the aquatic environment, including laboratory investigation of kinetics and transformation products and estimation of its aquatic environmental half-lives. Direct photodegradation was determined to be the dominant photodegradation process for FI with a quantum yield of 0.25, which was supported by photodegradation experiments conducted in natural sunlight. Accounting for light attenuation by dissolved organic matter in natural waters of different depths resulted in aquatic half-lives of <31 days even at polar latitudes. Photoisomerization was shown to be a major photodegradation pathway along with the formation and subsequent degradation of constitutional isomers and photooxidation products. These results contributed to FI being assessed as non-persistent in the environment.

Vitellogenin: a review of analytical methods to detect (anti) estrogenic activity in fish.

Vitellogenin, the fish egg yolk precursor protein, is the most common biochemical endpoint in general use for the detection of (anti) estrogen active substances in fish and other oviparous species. This review aims to cover the major methods (both protein and nucleic acid) for vitellogenin determination. Comparisons are drawn between vitellogenin and other endpoint effect levels. This review highlights strengths and weaknesses of the various techniques, concentrating on practical considerations and problems in evaluating the data they generate.

Animal use replacement, reduction, and refinement: development of an integrated testing strategy for bioconcentration of chemicals in fish.

When addressing the use of fish for the environmental safety of chemicals and effluents, there are many opportunities for applying the principles of the 3Rs: Reduce, Refine, and Replace. The current environmental regulatory testing strategy for bioconcentration and secondary poisoning has been reviewed, and alternative approaches that provide useful information are described. Several approaches can be used to reduce the number of fish used in the Organization for Economic Cooperation and Development (OECD) Test Guideline 305, including alternative in vivo test methods such as the dietary accumulation test and the static exposure approach. The best replacement approach would seem to use read-across, chemical grouping, and quantitative structure-activity relationships with an assessment of the key processes in bioconcentration: Adsorption, distribution, metabolism, and excretion. Biomimetic extraction has particular usefulness in addressing bioavailable chemicals and is in some circumstances capable of predicting uptake. Use of alternative organisms such as invertebrates should also be considered. A single cut-off value for molecular weight and size beyond which no absorption will take place cannot be identified. Recommendations for their use in bioaccumulative (B) categorization schemes are provided. Assessment of biotransformation with in vitro assays and in silico approaches holds significant promise. Further research is needed to identify their variability and confidence limits and the ways to use this as a basis to estimate bioconcentration factors. A tiered bioconcentration testing strategy has been developed taking account of the alternatives discussed.