Respirometer experiments recording oxygen consumption and a behavioural choice bioassay of the hermit crab Pagurus bernhardus to the plastic additive oleamide

The physiological and behavioural responses of the hermit crab Pagurus bernhardus to the plastic additive oleamide was assessed. This was evaluated via respirometer experiments that recorded oxygen consumption, and a behavioural choice bioassay. In February 2019, 40 hermit crabs were collected from the rocky intertidal shore near Scarborough (54°25'19.6”N 0°31'43.6”W), North Yorkshire, UK, and then transported to the aquarium facilities at the University of Hull. Hermit crabs were housed in individual containers in three 200 L tanks as part of a recirculation system (1600 L) filled with artificial seawater and acclimatised to a twice weekly feeding rhythm with cooked blue mussels. A 12 h/ 12 h light/dark cycle period was in place to simulate day/night rhythms. Hermit crabs were kept at pH 8.1±0.1, an average temperature of 15.8±0.2°C and 35.9± 0.2 PSU. All collections and experiments were conducted following national and/or institutional guidelines. Approval was granted by the Ethics Committee of the University of Hull (No. UO20, FEC_2019_155). Experimental trials were conducted in March 2019 – April 2019. Dissolved oxygen saturation was recorded inside the respirometer chamber at the start and end of two-minute intervals (before and after the injection of the cue). The difference between the oxygen concentrations at the start and the end of the interval showed the oxygen consumption for the time period. For the statistical analysis, the computing software R (version 3.5.2; R Core Team. (2018). R: a language and environment of statistical computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org/) was used. The distribution of data proved to be not normal (Shapiro-Wilk test, p=0.02), so non-parametric paired Wilcoxon tests were used to determine whether the differences in oxygen consumption were significantly different before and after injection and between test and control conditions. P-values were adjusted using the Bonferroni correction method. To analyse the difference between time spent in each of the test conditions, the statistical software R (R Core Team. (2018). R: a language and environment of statistical computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org/) was used. The distribution of the data was firstly checked via the Shapiro-Wilk test and inspected using histograms and quantile-quantile (QQ) plots. All Shapiro-Wilk tests cross referenced with histograms and QQ plots confirmed that the data was not normally distributed. Hence, the data was analyzed using the non-parametric Friedman rank sum test. Where the Friedman test indicated a significant difference, the Nemenyi post-hoc test was then used for multiple comparisons. Hermit crabs that stayed in their shell for the entire duration of the seawater control or solvent control assay were deemed inactive and excluded from the dataset. Treatment and testing order were randomized for both experiments, as was the side of test and control zones for bioassay experiments. Each hermit crab was only tested on one cue per day of experimentation, with an interval of 24 hours between trials to minimize stress and likelihood of a decreased response to cues over time.