Contents. 1. Introduction: endocrine disruption and antiandrogenic. 3. Case study 1: profiling anti-androgens in surface waters

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2 Contents 1. Introduction: endocrine disruption and antiandrogenic contaminants 2. Bioassay- directed analysis 3. Case study 1: profiling anti-androgens in surface waters 4. Case study 2: Identification and activity of bioavailable anti-androgens in fish. 5. Case study 3: Anti-androgenic contaminants in the coastal environment 6. Conclusions/research needs.

3 Exposure of fish to wastewater effluents causes intersex and feminization of the testes Caused by exposure to Estrogens or Estrogens + Anti-androgens? Picture courtesy C.R.Tyler, and A. Lange University of Exeter.

4 Why investigate the nature of anti-androgenic compounds in the environment? Anti-androgens block the male androgen receptor and can inhibit masculinisation and promote feminisation of fish. Receptor assays reveal that most UK wastewater finals effluents contain high concentrations of anti-androgen activity ( mg flutamide equivalents/l). Modelling studies suggest anti-androgens in combinations with estrogens may be related to fish intersexuality (Jobling et al; EHP ) The structures of the anti-androgens are unknown.

5 Expected classes of anti-androgenic contaminants in UK wastewater effluents o Selected PCBs, PBDEs Certain insecticides; e.g. pirimiphos-methyl Fungicides; o-phenylphenol Herbicides; linuron Bisphenol A, parabens Sunscreen agents: benzophenones Naphthenic acids and some PAHs. Perfluorooctane sulfonate (PFOS) Musk chemicals BUT concentrations are too low to account for the antiandrogen receptor activity in effluent samples.

6 How do we identify unknown anti-androgens? Bioassay directed analysis of environmental samples Sometimes termed effects-directed analysis, or toxicity identification and evaluation (TIE)

7 Bioassay directed analysis- a tool to identify unknown contaminants with a selected biological activity Extract sample for ALL chemical types Fractionate extract Bioassay should be relevant to the target species, Test each and fraction high for biological activity throughput. Identification of unknown structures often difficult and time consuming Activity Identify the chemicals present in the biologically active fractions Desbrow et al Environ Sci Technol 1998; Gibson et al EST 2005 Absorbance (ABS) Time (minutes) Fraction

8 Case study 1: What is the profile of the antiandrogen mixture in surface water downstream of a wastewater effluent? Passive sampling: Sampling over a time period avoids variability due to grab sampling Sample is preconcentrated prior to analysis BUT Each sampler is designed for sampling a specific range of log Kow A combination of passive samplers maybe needed

9 Silicone strips or Low Density Polyethylene (LDPE) lay flat tubing for compounds POCIS A: Pharmaceuticals, Sorbent Phase: OASIS HLB. POCIS B: pesticides, sorbent ENV+/Ambersorb/BioBeads Both for compounds

10 Sampling site 4 x canisters 200 m downstream effluent sewage for 2 weeks. Influent Population Equivalent : 107,250, 95% domestic Input Primary treatment + biological (percolating) filters and humus tanks Grab samples of water taken during deployment period Grab and passive sampler extracts fractionated on RP-HPLC, and fractions tested for anti-androgen activity. Active fractions analysed by GC-MS and LC-TOFMS

11 Anti-androgenic activity was detected in extracts from both silicone and POCIS passive samplers. μg flutamide equivalents/cm 2 mean ± standard deviation. Single-phase devices Biphasic devices Silicone (n=8) LDPE (n=8) POCIS A (n=4) Pharmaceutical POCIS B (n=2) Pesticides 3.60 ± 0.33 <LOD ± ± 0.70 Field blanks were below LOD values. LOD single phase devices: 0.28 μg FEq/cm 2 LOD biphasic devices: 2.03 μg FEq/cm 2.

12 RP-HPLC profiles of anti-androgenic activity in surface water are very complex! Recoveries of anti-androgenic activity after HPLC fractionation of the extract were between 70-80%

13 Identity of anti-androgens in passive sampler extracts. HPLC fraction Identity Use Silicone POCIS A POCIS B Log P Potency relative to flutamide 11 Clopidogrel anticlotting Clothiapine antipsychotic Clozapine antipsychotic Clothiapine antipsychotic Terbinafine antifungal MIconazole antifungal TCPP flame retardant Diclofenac amide antiinflammatory Propiconazole fungicide Triclosan antibacterial Over 80 compounds identified. Triclosan was the most potent; 5 fold more active than the standard anti-androgen flutamide.

14 Case study 2: Identification of bioavailable anti-androgens in fish exposed to a wastewater effluent Juvenile female trout held in tanks containing final effluent or clean river water for 10 days. Fish bile profiled for anti-androgenic contaminants using two different and androgen receptor screens: YAS and AR CALUX. Active fractions analysed by GC-MS

15 Bioassay directed analysis of anti-androgenic fractions in fish bile

16 Contribution of identified contaminants to the antiandrogenic activity measured in bile of fish exposed to effluent. Identified Compounds Contribution to total AA activity of bile (%) Triclosan Chlorophene Abietic acid nonylphenol 0.1 Bisphenol A Others <1.0 % Do the compounds contributing significantly to the total anti-androgenic activity in the bile induce expected effects in vivo?

17 Do anti-androgens feminize fish? Proportion of individuals with male and female reproductive ducts Percentage Juvenile roach exposed to estrogens or antiandrogens for 120 days, 50 fish per treatment 0 control AAmix EE2 AAmix+EE2 male-like duct female-like duct unidentified Feminisation of males is enhanced by a combination of antiandrogens with estrogen!

18 Case study 3: Are anti-androgenic contaminants present in coastal environments? Scrobicularia plana, sediment dwelling clam Intersex in Scrobicularia gonad Eggs present in male gonads photo by Dr. W Langston, MBA. Upto 40% intersex incidence in some UK estuaries. Intersex populations present in France and Portugal. In the lab, intersex can be induced by estrogens. Are clams also exposed to anti-androgens?

19 Anti-androgenic activity is widespread in coastal sediments. Extracts of sediment and clams sampled from Southampton estuary were profiled for anti-androgenic activity

20 Bioavailable anti-androgens present in clam tissues and sediment extracts. Anti-Androgenicity (ug\ml of fraction) 12, ,0000 8,0000 6,0000 4,0000 2,0000 0, Time (minutes) The same antiandrogenic fractions were detected in extracts of sediments and clam tissues. Identified compound Potency relative to flutamide in receptor assay (AR CALUX) carbazole 0.29 benzanthrone 0.41 phenanthrene 0.33 fluoranthene 0.95 pyrene 0.41 benz(a)anthracene 1.3 2,3,benzofluorene 1.9 benzo[k]fluoranthene 2.6 benzo(a)pyrene 1.0 PAHs were major anti-androgenic contaminants Planar molecules can be potent antiandrogens in vitro. Relevance to aquatic organisms?

21 Conclusions and future research needs. 1. Over 84,000 man made chemicals in common use. There is only have exposure information on approximately 2000 of them. 2. Bioassay- directed analyses can be a critical tool to identify causative agents of toxicity, especially for endocrine disrupters. Fish/reptile/amphibian steroid and thyroid receptor screens now becoming available. 3. Proposed Water Framework Directive regulations for estrogens (E2, EE2) maybe ineffective if other chemical mixtures potentiate feminisation of male fish. 4. Bioassay-directed analyses is widely used in ecotoxicology but less so in human studies. 5. We need better open-source contaminant identification databases. Compulsory mass spectrometry data with chemical registration (REACH)

22 Acknowledgments University of Sussex : Pawel Rostkowski, Camilla Liscio Julia Horwood Charles Tyler/ Anke Lange University of Exeter Bill Langston/Nick Pope, Marine Biological Association, Plymouth.

23 Thank you for your attention!