With regards to what might represent a

With regards to what might represent a typical “baseline” ChE (or AChE) activity in Saccostrea sp., it 99026 should be remembered that the observed enzyme responses were obtained in laboratory exposures, over a narrow experimental window (96h), with specimens collected over the course of a week, during meteorologically stable conditions, and pre-acclimated to laboratory conditions for several days. Bearing in mind that biomarker responses often tend to vary significantly due to seasonal and oceanographic (i.e. temperature and salinity), nutritional and reproductive variation (Escartin and Porte, 1997, Doran et al., 2001, Choi et al., 2011), the range of ChE activity could, presumably, be much greater in field studies and at different times of the year. On the other hand, the relatively low concentration-dependent sensitivity of T-ChE and Es-ChE observed for Saccostrea sp. in the present study might, in part, be the consequence of high baseline contaminant concentrations of oysters sourced from Santa Marta marina, as evidenced by very high Cu and Zn tissue concentrations, and perhaps other contaminants as well. This could potentially favor selection or acclimation responses in oysters, which could reduce sensitivity to environmental stress, buffering the variation of observed biochemical responses upon experimental exposure to concentrations that are not markedly different than the environment concentrations to which they are acclimated or adapted to. Finally, our results suggest that current procedures for measuring cholinesterase activity appear to lump together the activity of more than one enzyme. We show that useful ecotoxicological insight can be gleaned from measuring different functional components of total cholinesterase using ChE inhibitors, such as eserine, as these may respond differentially to different toxicants. For example, Es-ChE activity in gills of Saccostrea sp. decreased upon exposure to chlorpyrifos and imidacloprid, whereas Er-ChE activity remained similar over the exposure concentration range. On the other hand, Er-ChE activity responded sensitively to metal exposure, diminishing significantly and in a concentration-dependent manner for Cd, whereas decreasing significantly at high Cu concentrations, indicative of a distinct sensitivity profile of this sub-component of total cholinesterase activity.
Conclusions The results from this short-term laboratory study corroborate the use of T-ChE and Es-ChE activity in gills and digestive gland (albeit, with higher control variance) of Saccostrea sp. as sensitive biomarkers of xenobiotic exposure. We show that a common organophosphate pesticide, chlorpyrifos, caused a concentration-dependent inhibition of T-ChE and Es-ChE activity in gills and digestive gland, and that the magnitude of this change was relatively greater for Es-ChE than for T-ChE. Nevertheless, reductions in T-ChE and Es-ChE activity were not an exclusive response to chlorpyrifos exposure, since other non-OP xenobiotics, such as the neonicotinoid imidacloprid and the non-essential metal Cd, similarly, caused inhibition of T-ChE and Es-ChE activity, as well as Er-ChE (for Cd). Further complexity in the interpretation of ChE activity results springs from the observation that T-ChE and Es-ChE activity increased in gills and digestive gland, rather than decreasing, upon exposure to Cu (1000µg/L), as well as upon exposure to imidacloprid (100mg/L, in digestive gland), showing that Es-ChE and T-ChE activity do not always decrease with increasing xenobiotic concentrations. Therefore, encountering high ChE activities should not be interpreted uncritically as indicating “pristine” of “healthy” (i.e. contaminant-free) condition. For Saccostrea sp., gills and digestive gland showed greater sensitivity (but also greater within-treatment variability) to xenobiotic exposure than adductor muscle, rendering them the tissues of choice for further studies. Whereas Es-ChE and T-ChE activity do show sensitivity to xenobiotic exposure at higher concentrations, replicate measurements for the same treatment level were characterized by high variance (i.e. large standard errors), which diminishes statistical power to distinguish significant differences. Moreover, results from controlled laboratory studies could be less variable than results from field-collected specimens, due to potential covariance caused by other environmental covariates, including temperature, salinity and other contaminants, acting in synergy. For this reason, pooling of tissues of several individuals (at least 5) and adequate replication (n ≥ 3) is highly recommended for further studies. Finally, the confirmation in Saccostrea sp. of at least two different cholinesterase fractions, one that is sensitive and one that is resistant to inhibition by eserine, each of which respond differentially to different xenobiotics, raises the question what might be the structural equivalents of these different fractions, operationally defined. We suspect the existence of at least two types of cholinesterase proteins in Saccostrea sp., one corresponding to Es-ChE whose activity tends to be reduced significantly by exposure to organophosphates (such as chlorpyrifos), neonicotinoids (such as imidacloprid) and Cd, and another one (Er-ChE) that is less sensitive to pesticides but particularly sensitive to Cd. Moreover, the apparent insensitivity of ChE activity in adductor muscle to all four toxicants tested might not merely be the consequence of lower contaminant accumulation in this tissue, but could indicate the presence of a third, unknown, type of cholinesterase that is highly resistant to xenobiotic inhibition.