A laboratory approach for future exploitation of woody plants in the phytoremediation of antibacterials. Oral presentation

Abstract: Actual veterinary practices are one of the major means through which antibiotics enter the environment. In fact, these medicines, both used to treat animal diseases and to promote a faster biomass growth, are slowly absorbed by cattle and the non-absorbed portion is eliminated unchanged in the excreta. As a consequence of manure application to arable lands followed by leaching processes, surface and ground waters result frequently polluted by a large variety of antibiotics. Several authors have raised concerns about the potential risks due to environmental presence of these drugs, such as the spread of medicine resistance among pathogenic bacteria. In opposite to numerous organic compounds (i.e. petroleum derivates, chlorinated substances, explosives etc.), little is known about the ability of woody plants to remove these contaminants from the environment. With the aim to investigate the application of phytoremediation technology to sulfonamides (SAs), one class of the most exploited veterinary medicines, we performed studies with Salix fragilis L. plants. Willow aptitude to remediate sulfadimethoxine (SDM) was evaluated by studying its ability to both absorb and tolerate doses of SDM found in soils and surface/ground waters. Genetically identical Salix fragilis L. cuttings (20-cm long and 1-cm diameter) grown in hydroponic conditions, were exposed to 0, 0.01, 0.1, 1 and 10 mg L-1 of SDM for one month. The nutrient solution, a 25 times diluted Hoagland’s nutrient solution, pH 7.0, was entirely replaced twice a week to prevent active molecule and nutrient depletion. During the experimental period, growth parameters were measured to address the effects of exposure on plant physiology. At the end of the treatment, plant tissues were analyzed for their SDM and glutathione content through HPLC, to check both antibiotic accumulation and SDM-induced oxidative stress. Finally, observed alterations in root morphology of treated plants were studied through light microscopy and further investigated by comparing roots alterations following both SDM and 3-indoleacetic acid exposure. Results showed that biometric data didn’t revealed alterations on leaf and stem length, while a hormetic effect on root length and weight was noticed. In fact, the antibiotic was found to stimulate the root development with 0.01 mg L-1 exposure and to inhibit it at 1 and 10 mg L-1 concentrations. Furthermore, at the dose 10 mg L-1 , important root morphology alterations were noticed: in particular, this treatment was found to promote an abnormal emission of numerous lateral roots. We hypothesized a growth hormones disturbance as explication of that hypogeal stress. For this reason, new Salix cuttings were exposed to SDM 10 mg L-1 and 3-indoleacetic acid (from 5 to 10 mg L-1 ) separately for one month. This assay revealed similar morphologic disturbance of hypogeal apparatus between the antibiotic and the auxin, suggesting strong hormonal disequilibrium due to SDM treatment. In spite of these evidences, root accumulation potential was quite high at every SDM dose, while the antibiotic was not detectable in leaves. Furthermore, results obtained after growing Salix with 10 mg L-1 SDM for two weeks at different plant development stages, showed differences in plants tolerance according to the developmental step. These results suggest that Salix fragilis L. could be a well exploitable species for phytoremediation purposes for SAs concentrations often found in agricultural ecosystems. However, more studies are necessary to define the response of willow in a soil growth medium, in view to applicative purposes. Furthermore, it would be interesting to scrutinize the answers of different Salix spp. to SDM and other sulfonamides, under laboratory and field conditions.