Reliability evaluation of biomarker reference ranges for mesocosm and field conditions: Cellular innate immunomarkers in Gasterosteus aculeatus

Due to their sensitivity to environmental contamination and their link with fish health status, innate immunomarkers are of great interest for environmental risk assessment studies. Nevertheless, the lack of knowledge about the effect of confounding factors can lead to data misinterpretation and false diagnostics. So, the determination of reference values was of huge interest for the integration of biomarkers in biomonitoring programs. Laboratory immunomarker reference ranges (including cellular mortality, leucocyte distribution, phagocytosis activity, respiratory burst and lysosomal presence) that consider three confounding factors (season, sex and body size) were previously developed in three-spined stickleback, Gasterosteus aculeatus, from our husbandry. Usefulness of these reference ranges in biomonitoring programs depends on how they can be transposed to various experimental levels, such as mesocosm (outdoor artificial pond) and field conditions. Immunomarkers were therefore measured every 2 months over 1 year in one mesocosm and in one site assumed to uncontaminated (Houdancourt, field). Differences between immunomarker seasonal variations in mesocosm and field fish on one side and laboratory fish on the other side were quantified: in some cases, seasonal trends were not significant or did not differ between mesocosm and laboratory conditions, but overall, models developed based on data obtained in laboratory conditions were poorly predictive of data obtained in mesocosm or field conditions. To propose valuable field reference ranges, mesocosm and field data were integrated in innate immunomarker modelling in order to strengthen the knowledge on the effect of confounding factors. As in laboratory conditions, sex was overall a confounding factor only for necrotic cell percentage and granulocyte-macrophage distribution and size was a confounding factor only for cellular mortality, leucocyte distribution and phagocytosis activity. Confounding factors explained a large proportion of immunomarker variability in particular for phagocytosis activity and lysosomal presence. Further research is needed to test the field models in a biomonitoring program to compare the sensitivity of immunomarkers to the confounding factors identified in this study and the sensitivity to various levels of pollution