It is a challenge to determine how best to protect aquatic biota from pollution within the context of the increasing pressures on our planetary freshwater resources. Aquatic environmental regulations based on single toxicant concentrations have helped protect biota. There are concerns, however, that these regulations do not adequately protect against chronic exposure to toxicant mixtures. Advances have been made to assist regulators in identifying chemicals of concern, as well as the construction of conceptual frameworks for mixture and ecosystem risk assessment. The basis of these new initiatives is a greater understanding of the biological effects of toxicants on individuals, populations and ecosystems. This chapter focuses on bioavailability, uptake, metabolism, excretion and mode of action of toxicants (metal, pharmaceuticals, organic pollutants, pesticides and nanomaterial) and how knowledge of these processes assists environmental risk assessment. There is an urgent need for guidance on allowable toxicant loads to protect freshwater biota before irreparable damage is done.