Supplementary Material I. Challenges for ecotoxicology and new research strategies in the context of stress-related toxicity studies

Currently, the need to reduce the testing with vertebrate animals is under serious debate and the 3Rs strategy (reduction, replacement and refinement) is accepted in majority of developed countries. The 3Rs concept means that laboratory animal use should be progressively reduced, refined and finally replaced without threatening human health and lowering the quality of research. The 3Rs strategy is entering the regulatory scientific practice, e.g. in the United States US EPA (Sterling and Rispin 2002) and in the European Union EU Directive 2010/63/EU specifically require use of replacement methods wherever possible.

Despite of that, in United Kingdom at 2013, about 3 million animal procedures were made, mostly on mice (75%), fish (12%) and rats (6%) ( The increasing need for toxicity testing creates a big challenge for scientists to work out relevant alternative methods to replace laboratory animals or fish. As fish test is also obligatory in regulatory testing for REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals, a regulation of the European Union, addressing the need to improve the protection of human health and the environment from the risks posed by chemicals in environment), it has been debated over the need for fish assay in the test battery of algae, Daphnia and fish (a simplified aquatic food chain model). According to the survey carried out by the European Chemicals Bureau (Weyers et al. 2000) to study the acute toxicity data for newly notified chemicals, for algae, Daphnia and fish, the algal growth inhibition test was the most sensitive in approximately 44%, the daphnid immobilization test in 22% and fish acute toxicity test in 16%of the cases. For the remaining 18% of the chemicals, the organisms were all equally sensitive. Analogously, Hutchinson et al. (2003), have also shown that for approximately 80% of active pharmaceutical ingredients tested in their study, the algal median effect concentration (EC50) and daphnid EC50 values were lower than or equal to the fish median lethal concentration (LC50). Thus, in general, Daphnia and algae may be considered as more sensitive organisms to predict possible toxicity of most chemicals. This research led to modifications of the widespread guidelines of acute toxicity testing of chemicals in fish (Rufli 2012): the fish test would be performed only at one concentration, the lowest between the EC50 concentrations obtained with previous testing with algae and Daphnia. If this test were to show that fish is more sensitive than algae and Daphnia, testing with fish would be continued at lower concentrations (step-down).

One not yet widely explored area is the use of simple prokaryotic and eukaryotic models for toxicity screens. Bacteria, fungi, protozoa, crustaceans, insects, algae, plants - test organisms for ecotoxicological studies - are well suited for short-listing of toxic chemicals/new materials (Aruoja et al. 2015; Bondarenko et al. 2016), and thus, potentially very relevant for implementation of 3Rs in scientific and regulatory research as this could save a lot of money, manpower and lives of experimental animals (Kahru 2006).

In field conditions, where multiple toxicants often simultaneously affect aqueous organisms and concentrations of individual xenobiotics significantly vary over time and in different parts of the aqueous ecosystem, quantification of the xenobiotic stress level is often complicated. In addition, new categories of potentially toxic contaminants are appearing throughout the world. Increased concentrations of nanoparticles, surfactants, personal care products and pharmaceuticals are found in waters while there is poor understanding of their impact on humans and on the environment and there are no in vitro toxicity test results available. Thus, different biomarkers that show either bioaccumulation or the effect of xenobiotics on organisms are used instead. The most promising fish bioaccumulation markers are contents of persistent organic pollutants like many chlororganics - PCBs, DDTs, PCDDs, and PCDFs. However, more easily biodegradable compounds as most of the polycyclic aromatic hydrocarbons (PAHs) and chlorinated phenols do not tend to accumulate in fish tissues in quantities that reflect the exposure. The most studied, commonly used and most valuable fish biomarkers up to now are hepatic biotransformation enzymes, e. g., cytochrome 1A (CYP1A) and glutathione S-transferase (GST), also antioxidants like catalase enzymes, as well as PAH metabolites in fish bile, reproductive (e. g., plasma VTG), and genotoxic parameters (Hedman et al. 2011). However, confounding climatic factors should be carefully considered when interpreting biomarker data because these factors also can alter the organism stress level and thus, can have substantial effects on biomarkers.

Additional References

Aruoja V, Pokhrel S, Sihtmäe M, Mortimer M, Mädler L, Kahru A (2015) Toxicity of 12 metal based nanoparticles to algae, bacteria and protozoa. Environmental Science: Nano 2:630-644, DOI: 10.1039/C5EN00057B

Bondarenko OM, Heinlaan M, Sihtmäe M, Ivask A, Kurvet I, Joonas E, Jemec A, Mannerström M, Heinonen T, Rekulapelly R, Singh S, Zou J, Pyykkö I, Drobne D, A. K (2016) Multilaboratory evaluation of 15 bioassays for (eco)toxicity screening and hazard ranking of engineered nanomaterials: FP7 project NANOVALID. Nanotoxicology 10:1229-1242, DOI: 10.1080/17435390.2016.1196251

Hedman JE, Rüdel H, Gercken J, Bergek S, Strand J, Quack M, Appelberg M, Förlin L, Tuvikene A, Bignert A (2011) Eelpout (Zoarces viviparus) in marine environmental monitoring. Marine Pollution Bulletin 62:2015- 2029, DOI: 10.1016/j.marpolbul.2011.06.028

Hutchinson H, Barrett S, Buzby M, Constable D, Hartmann A, Hayes E, Huggett D, Länge R, Lillicrap AD, Straub JO, Thompson RS (2003) A strategy to reduce the numbers of fish used in acute ecotoxicity testing of pharmaceuticals. Environmental Toxicology and Chemistry 22:3031-3036, DOI: 10.1897/02-558

Kahru A (2006) Ecotoxicological tests in non-ecotoxicological research: contribution to 3Rs. Use of luminescent photobacteria for evaluating the toxicity of 47 MEIC reference chemicals. ALTEX 23:302-308

Rufli H (2012) Introduction of moribund category to OECD fish acute test and its effect on suffering and LC50-values. Environmental Toxicology & Chemistry 31:1107-1112, DOI: 10.1002/etc.1779

Sterling S, Rispin A (2002) Incorporating the 3Rs into regulatory scientific practices. ILAR Journal 43:Suppl: S18-S20, DOI: 10.1093/ilar.43.Suppl_1.S18

Weyers A, Sokull-Klüttigen B, Baraibar-Fentanes J, Vollmer G (2000) Acute toxicity data: a comprehensive comparison of results of fish, daphnia, and algal tests with new substances notified in the European Union. Environmental Toxicology and Chemistry 19:1931-1933, DOI: 10.1002/etc.5620190731