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Pseudoreplication is a widely discussed topic in the scientific community. Its principal critique concerns the lack of independence in flawed experimental designs and the use of inferential statistics to test the hypothesis of such experiments. Thirty years after its appearance, it remains misunderstood by many researchers, including ecotoxicologists. In the present study, we try to clarify some of its concepts by filling in what seems to be a gap in the terminology of manipulative experiments. We propose the term "experimental medium" to refer strictly to the relevant spatial scale of the experiment to preserve the specificity of the experimental and observational units and to display pseudoreplication as a kind of misinterpretation and/or misanalysis of inferential statistics. A classification of the types of experimental designs in ecotoxicology is offered, and the problems in using inferential statistics in suboptimal designs are discussed. We hope to shed some light on such a classic topic for ecotoxicologists. Environ Toxicol Chem 2017;36:2868-2874. © 2017 SETAC.

The sublethal effects of water-soluble fraction of gasoline (WSFG, 1.5 % v/v) were evaluated in the freshwater fish, Astynax altiparanae, after acute exposure (96 h) under a semi-static system. In addition, the recovery process was assessed in the fish following contaminant depuration. Recovery treatments were carried out with gradual depuration (GD), consisting of 7 days in the WSFG, followed by 8 days in clean water; and treatments with total depuration in clean water for 15 (DEP 15) and 30 days (DEP 30). The effects were evaluated through the piscine micronucleus test and by differential counting of organic defense cells. Acute exposure increased the frequency of neutrophils. In the GD treatment, the thrombocyte count and erythrocytic nuclear abnormalities (ENA) increased. In the DEP 15 treatment, there was a reduction of ENA; and following 30 days of depuration (i.e., DEP 30), the number of lymphocytes increased and the thrombocyte count remained high. These results indicate a long-term response to a condition of stress from WSFG.

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