PHARMACEUTICALS AND ILLICIT DRUGS AS NEW ENVIRONMENTAL CONTAMINANTS: ECOTOXICOLOGICAL EFFECTS AND NEW DEPURATION METHODOLOGIES

Pharmaceuticals and personal care products (PPCPs) and illicit drugs are now widely considered emerging aquatic contaminants because of their high usage worldwide and their consequent presence in surface water with concentrations ranging from ng/L to μg/L. In fact, once hired, these substances are eliminated by urine and faeces in their unaltered form or as metabolites and poured in sewage system. However, the traditional wastewater treatment plants (WWTPs) are not able to eliminate these compounds from wastewaters, consequently spilled placed in the aquatic environment. Thus, this research project suggests a natural process based on the natural bivalve filtration as a complementary method to traditional wastewater treatment, seeking thereby to remove the abovementioned contaminants from the aqueous matrix. In detail, we chose the invasive freshwater mussel Dreissena polymorpha to study this possible alternative depuration method. For this purpose, we built a pilot-plant within the Milano-Nosedo WWTP in which twenty removal Plexiglas® panels were placed. Moreover, we placed about 40,000 D. polymorpha specimens on these substrates appointed to the wastewater filtration. During the experiment we tested the abatement of 13 pharmaceutical compounds (atenolol, carbamazepine, ciprofloxacin, clarithromycin, dehydro-erythromycin, diclofenac, furosemide, ibuprofen, hydrochlorothiazide, ketoprofen, naproxen, paracetamol and ofloxacin), 4 illicit drugs (cocaine, benzoylecgonine, methamphetamine and methadone) and 7 heavy metals (Al, Cr, Cu, Fe, Mn, Ni and Pb) after D. polymorpha exposition. Because of the well-known presence of PPCPs and illicit drugs in the environment, the second part of this project was devoted to the assessment of their ecotoxicological effects. We decided to evaluate the potential chronic toxicity of these chemicals on the same model organism used in the bio-filtration process since D. polymorpha is particularly representative of the aquatic ecosystem and, at the same time, sensitive to environmental changes. The illicit drugs and PPCPs assayed at environmental concentrations in this study were the following: morphine, 3,4-methylenedioxymethamphetamine (MDMA), tonalide and galaxolide. Furthermore, because in the environment such substances form complex mixtures, we also tested the toxicity of an illicit drugs’ mixture composed by cocaine, benzoylecgonine, morphine, MDMA and amphetamine administered at environmental concentrations. The chronic toxicity of these substances was investigated by a biomarker battery: cytotoxicity was evaluated by the Neutral Red Retention Assay (NRRA) while, we assessed the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) for monitoring the raise of oxidative stress, the phase II detoxifying enzyme glutathione-Stransferase (GST), as well as the levels of lipid peroxidation (LPO) and protein carbonylation content (PCC) in order to highlight eventual oxidative damage. The potential injury at the genetic level induced by PPCPs and illicit drugs was assessed on D. polymorpha haemocytes by Comet Test, Micronucleus Test (MN test), percentage of apoptotic frequencies (DNA Diffusion Assay) and DNA fragmentation (DNA Precipitation Assay). Finally, the filtration rate of D. polymorpha has been tested as a physiological biomarker. Data obtained on the D. polymorpha removal ability due to the bio-filtration process are very encouraged. The bivalve activity has shown a great capacity in the concentration reduction of various PPCPs, illicit drugs and mainly heavy metals. The ecological importance of these results is high, taking into account the ecotoxicological effects of these substances on the aquatic communities. The main results for the evaluation of ecotoxicological effects of some emerging environmental contaminants highlighted that morphine and MDMA seem to induce a significant increase of oxidative stress in the bivalve, but not followed to genetic fixed damage; these results were partially confirmed by the exposure to the illicit drugs’ mixture that showed a significant increase in antioxidant enzyme activity and in protein carbonylation content. Finally, the exposure to the musks galaxolide and tonalide caused a significant increase in the DNA fragmentation percentage on bivalve haemocytes. Therefore, the results obtained during this research project contributed to point out the importance of new wastewater depuration methods allowing the removal of emerging contaminants whose ecotoxicological effects must not be underestimated, as they may ultimately affect the higher biological levels. Moreover, the use of an invasive species such as D. polymorpha in the depuration context may also have interesting implications for the economic sector and initiate an exploitation processes of alien species, that, being difficult to remove, constitute a serious problem for ecosystems. Despite the good results obtained, further studies are necessary to clarify both the bio-filtration treatment and toxic effects of illicit drugs and PPCPs on aquatic communities.