In the Cu bioassay notwithstanding the
In the Cu bioassay, notwithstanding the absence of significant Cu bioaccumulation in bulk tissues over the 96-h exposure period (less than 2-fold difference in tissue Cu between controls and the second-highest treatment), T-ChE activity increased with Cu concentration in gill and, to a lesser extent, in digestive gland, even though the proportion of Es-ChE vs. T-ChE activity remained approximately the same (30–60%). Although this apparent concentration-dependent activity increase was statistically significant, there is a conceptual difficulty to reconcile it with the observation that oysters did not differentially accumulate Cu in their tissues over the 96h exposure period (Cu concentration varied by a mere 1243 µg/g for controls and 1998µg/g dw for the 100mg/L treatment), unlike the study of Al-Subiai et al. (2011) who reported increasing Cu accumulation in adductor muscle, digestive gland and gills of M. edulis with increasing Cu concentrations. Experimental oysters used in the present study were sourced from a local marina, where use of Cu-based antifouling paints is likely to be high. This might explain the high baseline (pre-exposure) tissue Cu concentrations and the finding of no significant additional Cu accumulation for the selected exposure concentration range (1–1000µg/L, 96h). Unlike Es-ChE and T-ChE activity, metallothionein concentrations in gills, digestive gland and adductor muscle of Saccostrea sp. were insensitive to Cu exposure up to 1000µg/L over 96h (Moncaleano-Niño et al., 2017), consistent with absence of significant Cu accumulation over the exposure period. Nevertheless, although total soft tissue Cu concentrations did not increase over the 96h exposure, Cu allocation might, possibly, vary between individual tissues. Furthermore, the observed differential response of T-ChE and Es-ChE activity in the order gills > digestive gland > adductor muscle for the highest Cu exposure concentration (1000µg/L) might reflect a differential degree of tissue exposure to dissolved Cu, being highest for gill tissue (i.e. constantly exposed) and lowest for adductor muscle (i.e. intermittent, indirect exposure via hemolymph). Nevertheless, while this conjecture would explain a differentially greater response in gills, it still begs the question why Es-ChE and T-ChE activities increased in this tissue, rather than being inhibited, when exposed to higher dissolved Cu concentrations. With regard to tissue sensitivity, previous studies on bivalves have reported AChE activity to be particularly concentrated in the gills, PTP Inhibitor IV and adductor muscle (Monserrat et al., 2002, Damiens et al., 2004; Bernal-Hernández et al., 2010). In the present study, highest ChE activities (for total and eserine sensitive cholinesterase) were, indeed, found in gills, but ChE activity was similarly elevated in digestive gland, corroborating results of Bocchetti et al. (2008). In contrast, T-ChE and Es-ChE activities were significantly lower in adductor muscle and, moreover, largely insensitive to changes in exposure concentrations for all four toxicants tested. This contrasts with other studies, such as Choi et al. (2011) and Doran et al. (2001), who reported a significant 40-60% reduction of AChE activity in the adductor muscle of Amblema plicata exposed to 2 mg/L chlorpyrifos for 96h. The apparent insensitivity of Es-ChE and T-ChE activity in adductor muscle of Saccostrea sp. might be reconciled with the conjecture that under acute experimental conditions (96h), adductor muscle tissue (and the associated neurons) might not receive exposure to the dissolved toxicants to the same degree as do gills (via water) or digestive gland (via food). To test this hypothesis, it would be interesting to quantify metal and pesticide concentrations in specific tissues (rather than just bulk tissue, as was done here), in parallel to ChE activity measurements. On the other hand, adductor muscle cholinesterases could also have different structural and functional characteristics compared to other soft tissue ChEs, explaining their relative insensitivity to the toxicants tested. Whatever the reason for this insensitivity, we may conclude from the present study that for Saccostrea sp., adductor muscle tissue is not a sensitive tissue for measuring of ChE activity. In the case of digestive gland, the strong variation in T-ChE activity among control groups is puzzling and disconcerting, considering the substantial effort invested into the study design to minimize internal variance: oysters were sourced from the same location, pre-acclimated to laboratory conditions, tissues were separated into three tissue fractions (instead of analyzing whole organisms) and 3 replicates, each consisting of tissues from 5 individuals, were analyzed to reduce intra-treatment variability. While this approach worked well for gills, it was, apparently, not sufficient for digestive glands, where strong variance between control means persisted. Digestive gland tissue composition might be inherently more variable, perhaps due to admixtures of other tissue types (e.g. gonad). Future experiments might be able to overcome this problem by using a greater number of replicates and/or discriminating between age groups or size classes. Based on our comparison of three tissue types, we conclude that gills are best suited for the measurement of cholinesterases (in short-term exposures), owing to their low variance among controls and the large contact area afforded by gills to their surrounding and their key role in feeding, retaining detritus, food and a wide variety of pollutants, in addition to being highly permeable, giving gills the highest potential susceptibility to dissolved or suspended contaminants, corroborating similar conclusions by Lowe and Day (2002), Gold- Bouchot et al. (2007), Bernal-Hernández et al. (2010) and Andrade- Brito et al. (2012).