Biological Removal and Fate Assessment of Diclofenac Using Bacillus subtilis and Brevibacillus laterosporus Strains and Ecotoxicological Effects of Diclofenac and 4′-Hydroxy-diclofenac

Since bacterial consortia involved in conventional wastewater treatment processes are not efficient in removing diclofenac (DCF), an emerging pollutant frequently detected in water bodies, the identification of microorganisms able to metabolise this pharmaceutical compound is relevant. Thus, DCF removal was investigated using bacteria isolated from aqueous stock solutions of this micropollutant and identified as Bacillus and Brevibacillus species using 16S rRNA gene sequencing. A 100% DCF removal was achieved after 17 hours of experiment at 20 degrees C in a nutrient medium; the biodegradation kinetic followed a pseudo-first order (k(biol) = 11 L center dot g(SS)(-1)center dot d(-1)). Quantitative assessment of DCF removal showed that its main route was biotic degradation. The main degradation product of DCF, 4 '-hydroxy-diclofenac (4 '-OH-DCF), was identified using liquid chromatography-electrospray ionisation high-resolution mass spectrometry. Since the ecotoxicological impact of 4 '-hydroxy-diclofenac was not reported in the literature, the ecotoxicity of DCF and its metabolite were tentatively evaluated using Vibrio fischeri bioassays. Results from these tests showed that this metabolite is not more toxic than its parent compound and may hopefully be an intermediate product in the DCF transformation. Indeed, no significant difference in ecotoxicity was observed after 30 min between DCF (50 should be writtten in subscript all along the manuscript in EC50 = 23 +/- 4 mg center dot L-1) and 4 '-hydroxy-diclofenac (EC50 = 19 +/- 2 mg center dot L-1). Besides, the study highlighted a limit of the Microtox (R) bioassay, which is largely used to assess ecotoxicity. The bioluminescence of Vibrio fischeri was impacted due to the production of microbial activity and the occurrence of some carbon source in the studied medium.