title: From single chemicals to mixtures--reproductive effects of levonorgestrel and ethinylestradiol on the fathead minnow.
abstract: The aquatic environment is polluted with thousands of chemicals. It is currently unclear which of these pose a significant threat to aquatic biota. The typical exposure scenario is now represented by a widespread blanket of contamination composed of myriads of individual pollutants-each typically present at a low concentration. The synthetic steroids, 17α-ethinylestradiol and levonorgestrel, have been widely reported to be present in the aquatic environment in the low ng to sub-ng/l range. They are widely used in contraceptive formulations, both individually and in combination. Our research employed the fathead minnow (Pimephales promelas) 21 day 'pair-breeding' assay to assess reproductive output when pairs of fish were exposed to the single chemicals at low environmentally relevant concentrations, and then to a binary mixture of them. A variety of endpoints were assessed, including egg production, which was inhibited in a concentration-dependent manner by both the individual chemicals and the mixture. Significant, sex specific effects were also seen with both chemicals, at differing levels of biological organisation. Plasma concentrations of EE2 and levonorgestrel were predicted and in the case of levonorgestrel measured, and compared with the human therapeutic plasma concentrations (Read-Across approach) to support the interpretation of the results. A novel quantitative method was developed for the data analysis, which ensured a suitable endpoint for the comparative mixture assessment. This approach compares the reproductive performance from individual pairs of fish during chemical exposure to its pre-treatment performance. The responses from the empirical mixture study were compared to predictions derived from the single substance data. We hypothesised combined responses which were best described by the concept of concentration addition, and found no clear indications against this additivity expectation. However, the effect profiles support the current knowledge that both compounds act in different ways to reduce egg production in fish, and suggest that probably response addition (also called Independent action) is the more appropriate mixture model in this case.
authors.affiliation: Institute of Environment, Health and Societies, Brunel University London, Uxbridge, Middlesex, UB83PH, UK. Electronic address: Tamsin.Runnalls@Brunel.ac.uk.
authors.forename: Tamsin J
authors.initials: TJ
authors.lastname: Runnalls
authors.affiliation: Institute of Environment, Health and Societies, Brunel University London, Uxbridge, Middlesex, UB83PH, UK.
authors.forename: Nicola
authors.initials: N
authors.lastname: Beresford
authors.affiliation: Institute of Environment, Health and Societies, Brunel University London, Uxbridge, Middlesex, UB83PH, UK.
authors.forename: Subramaniam
authors.initials: S
authors.lastname: Kugathas
authors.affiliation: Institute of Environment, Health and Societies, Brunel University London, Uxbridge, Middlesex, UB83PH, UK.
authors.forename: Luigi
authors.initials: L
authors.lastname: Margiotta-Casaluci
authors.affiliation: Institute of Environment, Health and Societies, Brunel University London, Uxbridge, Middlesex, UB83PH, UK.
authors.forename: Martin
authors.initials: M
authors.lastname: Scholze
authors.affiliation: CEFAS Weymouth Laboratory, Barrack Road, Weymouth, Dorset, DT4 8UB, UK.
authors.forename: Alexander P
authors.initials: AP
authors.lastname: Scott
authors.affiliation: Institute of Environment, Health and Societies, Brunel University London, Uxbridge, Middlesex, UB83PH, UK.
authors.forename: John P
authors.initials: JP
authors.lastname: Sumpter
mesh_terms: D000818:Animals; D001681:Biological Assay; D003530:Cyprinidae / Q000502:physiology*; D004997:Ethinyl Estradiol / Q000097:blood / Q000633:toxicity*; D005260:Female; D016912:Levonorgestrel / Q000097:blood / Q000633:toxicity*; D008297:Male; D012098:Reproduction / Q000187:drug effects*; D014874:Water Pollutants, Chemical / Q000633:toxicity
chemical_list: D014874:Water Pollutants, Chemical; D004997:Ethinyl Estradiol; D016912:Levonorgestrel
keywords: EE2; Fathead minnow; Levonorgestrel; Mixtures toxicity; Read-Across; Reproduction