P corneus is a freshwater

P. corneus is a freshwater hermaphroditic gastropod that is distributed all over the world (Jopp, 2006). In previous works of our laboratory, we have characterized the activities of B-esterases and we have also reported that the exposure for 48h to chlorpyrifos inhibits ChE and CES of whole SGC 0946 soft tissue (Cacciatore et al., 2011, Cacciatore et al., 2012). It has been reported that P. corneus population declines constantly (Jopp, 2006, Wiese, 2005) so it is very important to study different effects of the subchronic or chronic exposure to low concentrations of contaminants. In particular, the alterations on reproduction which have an important ecological significance because the survival of the species is determined by the success in giving birth to new individuals. Chronic and subchronic exposures are relative terms especially in relation to the species. They are often designed according to the expected lifespans of the species involved (Barile, 2008). Generally, a chronic exposure implies an exposure of at least 10% of the lifetime. An acute exposure corresponds to until 96h while the subchronic exposure involves a duration between the acute and the chronic exposure (Barile, 2008). In laboratory conditions, it has been registered that P. corneus can live until 2 years (Collins Baker, 1945), so a 14 days exposure should be considered a subchronic one.
Materials and methods
Results
Discussion Exposure of organisms to contaminants may cause multiple toxic effects. The study of different effects in an exposed organism is essential for the understanding of the different biological responses and the mechanisms of toxicity of xenobiotics. Also, it is necessary to find the most sensitive biomarker in a bioindicator organism exposed to a particular toxic (Van der Oost et al., 2003). As cholinesterases are the primary target of organosphosphates and carbamates insecticides, their inhibition has been used as a specific biomarker of exposure and effect for these insecticides (Edwards and Fisher, 1991, Timbrell, 2000, Walker et al., 2001). However, some authors have found other parameters that could be used to complete a toxicity study or that they may be more sensitive than ChEs in aquatic organisms (Ferrari et al., 2007, Kristoff et al., 2008, Kristoff et al., 2011). In this work, we have observed that a 14 days exposure to 0.4μgL−1 chlorpyrifos produced only a decrease in CES activity measured with p-NPB and an increase in the number of egg masses without eggs while no inhibition of ChE was observed. Similarly, Kristoff et al. (2011) have also described that ChE was not significantly inhibited during a 14 days exposure of the gastropod B. glabrata to an environmental concentration of the OP azinphos-methyl; instead, Restriction Enzyme led to a smaller number of egg masses. Some authors have postulated that carboxylesterases are more sensitive than cholinesterases to inhibition by exposure to an OP insecticide (Kristoff et al., 2012, Vioque-Fernández et al., 2007). However in P. corneus exposed to chlorpyrifos, such results were only obtained by using p-NPB as substrate after a 14 days exposure. In contrast, after a 48h exposure, ChE inhibition was either similar or greater than CES inhibition depending on the substrate (Cacciatore et al., 2011). ChE inhibition and alterations in the reproduction parameters were observed with 5μgL−1 of chlorpyrifos. The main effects were on the hatching and survival as it has been observed by other authors in invertebrate species. For example, in the gastropod B. glabrata exposed for 14 days to 2.5mgL−1 or 5mgL−1 of azinphos-methyl, the authors reported a decrease in the number of hatchings per mass and non-living offspring after 1 month, respectively. However, in this case, azinphos-methyl concentrations were very high (Kristoff et al., 2011). In the present work and in previous works (Kristoff, 2010, Kristoff et al., 2011), the aim of our group was to study the effect that the pesticide had on reproduction from adult snails until the hatching of the offspring, exposing both the adult snails and egg masses. While doing this, we respected the times for hatching for each group simulating an environmental subchronic exposure. However, it would be interesting for future investigations to study the pesticide effect both on adult snails and egg masses separately in order to elucidate whether the effect of the pesticide is on the adult snail or upon the egg masses. Also, the offspring exposure to the pesticide could be evaluated in future works.