Lipid nanocapsules for behavioural testing in aquatic toxicology: Time–response of Eurytemora affinis to environmental concentrations of PAHs and PCB

• We exposed estuarine copepods to sub-lethal concentrations of hydrophobic pollutants.
• We used lipid nanocapsules to obtain contact toxicity and trophic-like exposure.
• The initial hyperactivity and higher swimming velocity indicate contact toxicity.
• More complex responses at longer exposure durations suggest systemic effects.
• Lipid nanocarriers need to be considered further in behavioural ecotoxicology.

The increasing interest for behavioural investigations in aquatic toxicology has heightened the need for developing tools that allow realistic exposure conditions and provide robust quantitative data. Calanoid copepods dominate the zooplankton community in marine and brackish environments. These small organisms have emerged as attractive models because of the sensitivity of their behaviour to important environmental parameters and the significance of self-induced motion in their ecology. Estuarine copepods are particularly relevant in this context because of their incessant exposure to high levels of pollution. We used lipid nanocapsules to deliver sub-lethal concentrations of PAHs (pyrene, phenanthrene and fluoranthene) and PCB 153 into the digestive track of males and females Eurytemora affinis. This novel approach enabled us to achieve both contact and trophic exposure without using phytoplankton, and to expose copepods to small hydrophobic molecules without using organic solvent. We reconstructed the motion of many copepods swimming simultaneously by means of three-dimensional particle tracking velocimetry. We quantified the combined effects of contact and trophic toxicity by comparing the kinematic and diffusive properties of their motion immediately and after 3 h and 24 h of exposure. Despite the lack of toxicity of their excipients, both empty and loaded capsules increased swimming activity and velocity immediately after exposure. Laser microscopy imaging shows adhesion of nanocapsules on the exoskeleton of the animals, suggesting contact toxicity. The behavioural response resembles an escape reaction allowing copepods to escape stressful conditions. The contact toxicity of empty capsules and pollutants appeared to be additive and nanocapsules loaded with PCB caused the greatest effects. We observed a progressive accumulation of capsules in the digestive track of the animals after 3 h and 24 h of exposure, which suggests an increasing contribution of systemic toxicity. Nanocapsules filled with PAHs caused a smaller response compared to empty capsules, which we attribute to the narcotic properties of these toxicants. The sharp decrease in velocity after 24 h of exposure to capsules loaded with PCB suggests physiological incapacitation following systemic toxicity. Clear differences are visible between genders in their response to empty and loaded capsules, for all exposure durations. Females appear to be less sensitive than males, suggesting different tolerance to stress conditions. Our results confirm the feasibility of using lipid nanocapsules to identify pollutant-induced behavioural alteration in the plankton. They also add new insights into the contact and systemic toxicity of common pollutants. We expect that our results will assist and evoke further research to develop suitable nanocarrier systems for behavioural testing.