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Integrative water sampling for the detection and identification of antiandrogenic contaminants in European rivers

Final Report Summary - EDCSANTIANDROGENS (Integrative water sampling for the detection and identification of antiandrogenic contaminants in European rivers)

Project n: 254111.

Project Acronym: EDCsANTIANDROGENS

Project Full Name: Integrative water sampling for the detection and identification of antiandrogenic contaminants in European rivers.

Marie Curie Actions IEF Final Report

Contamination of natural waters is a major concern in many parts of the world, and the occurrence of emerging contaminants at trace levels which can potentially exert harmful effects on aquatic wildlife is an extremely timely issue. However, aquatic monitoring is an on-going challenge due to the ultra-trace levels and the significant differences in physical-chemical properties of the many environmental contaminants. A key issue is to identify the most important active compounds and their mixtures which contribute to detrimental health effects in aquatic organisms. The main research objectives of Dr. Camilla Liscio's project were to investigate the use of innovative water sampling techniques (passive sampling) for the monitoring of newly emerging anti-androgenic contaminants (Endocrine disrupting compounds, EDCs) in surface waters impacted by wastewater effluents.

In additional work, complex mixtures of chemical contaminants were profiled along the river catchment to determine their persistence in the aquatic environment and to investigate how contaminant chemistry differs at sites upstream and downstream of an effluent discharge. Within the fellowship period, the performance of a variety of passive sampling techniques in sampling relevant contaminants including anti-androgenic contaminants was evaluated. In Phase 1 of the project (1-18 months) key anti-androgens (chemicals able to bind to the cellular androgen receptor and therefore interfere with androgen action and cause feminization of fish) were identified using bioassay-directed fractionation and mass spectrometry techniques. In Phase 2 of the project (18-24 months), a selected combination of passive samplers were deployed along a river catchment and extracts of these passive samplers were analysed by novel chemical profiling techniques to obtain an overall picture of contamination in the river. Both phases involved the extensive use of passive sampling and mass spectrometry techniques but the first phase required further training of the fellow in bioanalytical techniques (bioassay-directed fractionation approach) and the second phase in chemical profiling analyses.

The monitoring of trace concentrations of biologically active compounds as anti-androgens in surface waters can be challenging, and thus may require sensitive analytical techniques, intensive sampling programs and large sample volumes. Analytical methods for the determination of EDCs in the aquatic environment are mostly based on spot sampling followed by laboratory-based extraction and analysis. This approach, however, allows only an instantaneous measurement of pollutant levels and suffers from the uncertainty of short and long term variations in concentrations. A recent alternative approach is the use of passive sampling devices, which allow continuous monitoring of aqueous pollutants by means of a time-integrated sampling of large volumes of water. Currently available passive sampling devices are applicable to monitor chemicals with a broad range of physicochemical properties but each device is usually meant to sample efficiently only a certain range of polarities.

Since anti-androgens in effluents are a complex mixture of polar and apolar chemicals, a battery of different passive samplers covering the broadest range of polarities must be used to guarantee an efficient sampling of the whole array of anti-androgens possibly present in the environment. During Phase 1, the fellow compared 4 different passive samplers in their efficiency to sampling anti-androgenic activity present in contaminated surface waters. Four canisters, each of them containing all the four types of passive samplers, were deployed for two weeks in river water 200m downstream of a domestic sewage effluent. Passive sampler extracts were screened by in vitro bioassay and differences in the sampled amount of anti-androgenic activity were statistically evaluated. Samples were then fractionated and profiles derived from the 4 passive samplers and grab samples were compared and characterized. Contaminants with anti-androgenic activity were putatively identified by means of mass spectrometry techniques, their identity and potency confirmed with commercial standards (if available).

The tested passive sampling devices showed significant differences in efficiency and selectivity of sampling of anti-androgenic activity. In addition, the amount of anti-androgenic activity in the tested passive sampler extracts were usually several times higher than the grab samples, increasing the likelihood of successful identification of the chemical structures of interest. Extracts of the samplers were separated into 50 fractions, and these were further tested for anti-androgenic activity. Two main anti-androgenic compounds were identified in the most active fractions and these belonged to the class of conazole fungicides namely propiconazole used agriculturally and miconazole applied topically to the skin or to mucus membranes to cure fungal infections. Many other anti-androgenic structures were identified including the germicides triclosan used in household products, the flame retardant tris-(2-chloropropanol)-phosphate TCPP and various pharmaceuticals (e.g. the anti-psycotics clozapine and clothiapine, the anti-platelet agent clopidogrel, the anti-fungal terbinafine). Examination of many of the fractions also revealed many other (non-anti-androgenic) contaminants such as pharmaceuticals, personal care products (e.g. sunscreen sulisobenzone), flame retardants (e.g triphenylphosphate TPP and tris(2-butoxyethyl)phosphate TBEP), pesticides (e.g. terbutryn and piperonyl butoxide) and household products as the artificial sweetener sucralose. This study revealed that passive sampling can be a very promising tool for screening of contaminants at ultra-trace concentrations yet toxicologically relevant such as anti-androgens. However a combination of different passive samplers is required to efficiently sample the whole complex mixture of anti-androgenic compounds present in the environment.
In Phase 2, a second experiment was planned in order to fully characterize the impact due to the wastewater contamination along the river catchment. Replicates (n=6) of two selected passive samplers were deployed for a 1 month period at five different sites along the same river catchment (upstream, wastewater effluent, 2km, 6km and 10 km downstream, respectively). The extracts were analysed by two mass spectrometry profiling techniques and the results have been statistically analysed using multivariate projection methods including principal component analysis (PCA) and partial least squares (PLS)). These analyses allowed an investigation on how contaminant profiles changed between the monitored sites. The projection method models revealed a distinct separation between upstream and the wastewater effluent impacted sites, suggesting that significant changes occurred in the catchment chemical profile due to the effluent discharge.

The wastewater effluent presented an extremely complex mixture of xenobiotics: some of the chemicals which had been already identified during Phase I of this project were further confirmed in this second study (e.g. clozapine, clothiapine, terbinafine, crotamiton, diltiazem and carbamazepine). These compounds were absent in the upstream profile but their amounts in the water were dramatically increased in correspondence of the effluent discharge, and then slowly decreased along the catchment. However, the above mentioned compounds were still present in the water 10 km downstream the effluent input, indicating that many of the xenobiotics released in the environment by the wastewater effluents might be very persistent and hazardous for the aquatic wildlife.

Overall, the research achievements attained under the IEF Marie Curie framework allowed Dr. Camilla Liscio to strengthen her expertise in trans-disciplinary fields (chemistry/biology/environmental science) as evidenced by the number of publications and presentations of the scientific results at international conferences (see below). The results of her work will provide new information for sampling and the identification of complex arrays of biologically active compounds in European surface waters. In addition to the research work, the fellow was actively involved in mentoring activities, including the co-supervision of one PhD student as well as lecturing and giving seminars on relevant topics (passive sampling techniques in water monitoring). Furthermore the fellow completed within the IEF granted period several training courses to further develop her knowledge in cutting edge analytical techniques (e.g. Advanced Interpretation of CID Mass Spectra from LC-MS-MS, IAEAC, Montreux, Switzerland, November 2010 and How to develop HPLC methods for challenging separations, Phenomenex, London, UK, September 2011), which gave her additional awareness on how to implement her research work. Dr Liscio also attended a number of training courses at the host institution to develop her skills and these included: Presentation skills, Train the trainer, Excel spreadsheets (charts, functions, pivot tables) and How to develop personal effectiveness and Research grant writing skills.