Combined toxicity of graphene oxide and wastewater to the green alga Chlamydomonas reinhardtii

The toxic action of graphene oxide (GO) and the micropollutants contained in a biologically-treated wastewater were studied alone and in combination. For the toxicity assays, the unicellular green alga Chlamydomonas reinhardtii was used and the toxic mechanism was assessed by studying changes in esterase activity, cytoplasmic membrane potential, membrane integrity, the generation of intracellular reactive oxygen species (ROS), changes in intracellular calcium, mitochondrial membrane potential, and mitochondrial ROS formation. The joint toxic effect was quantified using the combination index (CI)-isobologram method using 72 h growth rate data. The pollutants monitored were polar pharmaceuticals, metabolites and artificial sweeteners with concentrations from tens of ng L-1 to several g L-1. The amount of compounds adsorbed on GO reached 89.5 g g(-1) with preferential adsorption for the more hydrophilic. The most adsorbed compounds were azithromycin, clarithromycin, erythromycin, metoclopramide, ofloxacin, and ranitidine. GO induced cell damage due to oxidative stress. Short-term defence response was observed consisting of an increase in intracellular calcium levels and higher metabolic activity. Long-term damage was evidenced by mitochondrial ROS increase and strong depolarisation of the mitochondrial membrane. Cells exposed to GO-wastewater mixtures were considerably less affected with lower or non-significant damage in comparison with GO or wastewater alone. GO-wastewater mixtures displayed considerable antagonism for the lower values of assayed concentrations. The antagonism was attributed to the adsorption of toxic chemicals on the surface of GO nanoparticles and to the higher aggregation of GO in wastewater. The results show that non-additive interactions at low effect levels cannot be generally neglected.