Publisher Summary Estuarine environments are rich and diverse ecological systems potentially under threat by intense humanization. This is the case of the Ebro Delta, an alluvial plain devoted to intensive agricultural activities, that requires of effective monitoring tools to assess the exposure of non-target organisms to operationally used insecticides and other anthropogenic contaminants. This chapter discusses the use of cholinesterases and carboxylesterases. Mussels -Mytilus galloprovincialis- from the marine farms, and red swamp crayfish-Procambarus clarkii- from the Delta irrigation channels, were selected as sentinel species for the study. After characterization of esterase activity in different tissues, the sensitivity of these enzymes to inhibition by model organophosphorus and carbamate compounds was investigated in vitro (12,000-g supematant) and in vivo (exposure experiments). The metabolism of fenitrothion, a model organophosphorus pesticide, was assessed by using primary cell cultures isolated from digestive gland of mussels and microsomal fractions isolated from red swamp crayfish hepatopancreas. The results support the existence of a cytochrome P450 mediated metabolism of fenitrothion, which may have toxicological consequences for the organisms, probably in terms of cholinesterase inhibition. Field work showed low acetylcholinesterase and carboxylesterase activities in mussels over the summer period, coinciding with the maximum use of pesticides in the rice crop fields, and a clear influence of temperature—on both enzymes. Reactivation techniques were attempted to distinguish between natural and pesticide induced changes, these techniques were successfully applied in crayfish neuromuscular tissue and mussel gill cells as an evidence of cholinesterase inhibition.