Biomonitoring of environmental stress in Pollicipes pollicipes from the northern coast of Portugal: a non-destructive approach using haemolymph

• The influence of seasonal variation on the response elicited by anthropogenic compounds was studied.
• The biomarkers analyzed in this study showed a seasonal pattern.
• The use of the haemolymph as a non-destructive biomarker was validated.

In the intertidal area, the interactions between anthropogenic contaminants and natural variations (biotic and abiotic factors) are poorly understood. Consequently, there is a great need for new assessment procedures to characterize the biological responses occurring in organisms from this extreme environment. Considering the intrinsic inter-individual variations among organisms from a single population, it is important to propose new methods that address this variability, by validating a sampling strategy in target groups of organisms, encompassing seasonal fluctuations. This strategy must however be less invasive than traditional methods, avoiding the mandatory sacrifice of the sampled organisms. By doing so, it is also possible to increase the ecological relevance of obtainable data, and contribute to minimize damage to endangered species. The main purpose of the present study was to assess the influence of seasonal variations in the responses elicited by anthropogenic compounds on a marine crustacean species, by using a biomarker-based approach. According to this purpose, the seasonal variations in key physiological responses (biomarkers) were investigated in the crustacean Pollicipes pollicipes from the Northern coast of Portugal. Biomarkers used for this purpose were the activity of the phase II biotransformation isoenzymes glutathione-S-transferases (GSTs), the activity of cholinesterases (ChEs), and the levels of lipid peroxidation (TBARS). All biomarkers were quantified in distinct tissues (such as cirri, and peduncle) and haemolymph (a non-destructive source of biological samples). The glycogen content in peduncle tissue, and the variation in haemocyte number in haemolymph were also analyzed. Samples were collected monthly, during a year, in Lavadores, located in the proximity of an estuarine area (Douro River). The results showed a seasonal pattern in all tested biomarkers. The results also showed a significant increase in GSTs activities, and in peroxidative damage, especially during warmer months. The lowest ChEs values were recorded during the rainy season. The results also showed a similar pattern among all tested tissues, validating the proposed use of the haemolymph as a source tissue for non-lethal sampling strategy for biomarker determinations. Glycogen content was apparently related to the reproductive cycle, with lower values being observed in spring and summer. Total haemocyte count (THC) increased during summer months. The results pointed to an influence of natural variations in the levels of biomarkers in P. pollicipes, highly dependent upon abiotic factors, a factor that must be considered when interpreting biological responses elicited by anthropogenic contaminants from the marine coastal environment. The validation of haemolymph as a non-lethal source tissue for the quantification of biomarkers was successfully attained, opening the possibility of less invasive and almost unlimited sampling in a small number of organisms.