Development of a biopolymer-based Tier-1 assay for effects of plant-incorporated protectants (PIPs) on leaf-consuming shredders in aquatic systems

Movement of transgenic crop debris into streams may expose non-target aquatic shredding organisms to plant-incorporated protectants (PIPs). Tier-1 testing, which uses purified PIPs applied to food to directly assess effects on non-target organisms, is complicated in aquatic systems because PIPs will dissolve off food once submerged in water. As a solution we developed an assay based on encapsulation of PIPs by chitosan and zein biopolymer nanoparticles. Rhodamine B (RB) dye was used as a PIP substitute inside the nanoparticles. RB fluorescence was measured in water samples from treatments receiving either nanoparticle-coated food or dyed food with no nanoparticles to determine retention efficacy of the nanoparticles. Water sample fluorescence levels of the treatment receiving food coated in RB-containing zein nanoparticles were significantly lower (p<0.05) than those of both the chitosan-coated food and the uncoated food. To determine potential toxic effects of the nanoparticles, food coated in nanoparticles with no RB was fed to the freshwater amphipod Hyalella azteca. Amphipod performance was measured as survivorship, change in mass, and feeding frequency to determine effects of the biopolymers with no PIPs present. No significant change in amphipod survival, change in mass, or feeding frequency was detected when fed RB-free nanoparticle-coated food compared to amphipods fed uncoated food (p>0.05). This biopolymer nanoparticle-based Tier-1 system does not affect commonly measured toxicity endpoints like growth and survival and effectively retains the chemical to be delivered, making it a useful system for oral toxicity studies of PIPs and other chemicals on aquatic arthropods.