I don't have experience with this specific product, but there are ELISA test kits for detecting estrogenic hormones in water - http://www.biosense.com/render.asp?segment=3&ID=87

Other ELISA tests I have been involved with were relatively inexpensive, and appropriate for a grad student with lab experience.

I've seen one stat that claims 80% of US waters have detectable levels of estrogenics present, and that they're even in bottled waters. So don't be surprised by positive results. So, one problem for you to address is correlation versus causation.

Cheers,

Mike

Sean

We have used MCF-7 cell proliferation assays to screen for estrogenicity in effluents.  The techniques require basic laboratory skills so it should not be a problem for a graduate student.  See reference below.

Liu J, Carr S, Rinaldi K, Chandler W., Screening estrogenic oxidized by-products by combining ER binding and ultrafiltration Environmental Toxicology and Pharmacology 20(2):269-78 · September 2005

Good luck

Steve

There are several tests for estrogenicity, each with its own strengths and weaknesses.

Estrogenicity in the environment has many causes, e.g. the natural hormones produced by animals, synthetic "hormones" such as ethynylestradiol used in the contraceptive pill, industrial products such as bisphenol A or the alkylphenols (breakdown products of the alkylphenol ethoxylates used in some detergents), and natural estrogenic compounds like genistein produced by plants. There are also antiestrogens which will counteract the effects of estrogenic substances, and within the body you can get interactions with substances like testosterone or testosterone mimics, and various feedback mechanisms, which will influence the observed responses in an organism. Also, different species may have different relative sensitivities to the various estrogenic compounds.

So you need to think about exactly what you are trying to show, and what each type of test will demonstrate.

ELISAs will detect the specific compounds that the antibodies are targeted at, but won't pick up other substances or the interactions. There are many brands of ELISAs, with different sensitivities to the various hormones (E1, E2, EE2, E3 etc), so always check the specifications for the kits you choose.

Cell-based assays have the advantage that they are testing for effects in a somewhat more realistic model than simply assaying for different compounds, and they will respond to all estrogenic compounds in a sample (and in some cases, antiestrogens as well). Examples would be the Yeast Estrogen Screen (YES) developed by Sumpter's group at Brunel University (which uses GM yeast, so is not exactly equivalent to animal cell metabolism); the yeast two-hybrid assay developed by Fujio Shiraishi's group at NIES in Japan (which uses yeast with either human or medaka fish estrogen receptors so to some extent is more appropriate for fish studies than cells with human receptors); the MCF-7 method mentioned by Steve Carr; E-Screen, and the ER CALUX assays.

Microarray and other genomic/proteomic methods are also now becoming commercially available.

Testing on actual animals is more realistic than the various in-vitro assays above, but raises many ethical concerns. However, just because you can detect estrogenics in a sample, doesn't mean that they will actually affect organisms, because of all the interactions and feedback controls in living things. Whole-animal testing therefore has a place in demonstrating that estrogenic compounds that may be detected by extremely sensitive tests are actually a concern. Again, there are many models developed, from behavioural studies (e.g. spiggin test in sticklebacks), vitellogenin assays, and so on.

All these tests can be carried out by graduate students with appropriate training, although setting them up in a lab may involve some specialist help, and some require donated cell lines or licence fees. You could also investigate the possibility of sending your student to work in labs where the tests are already up and running; many labs are happy to accommodate this.

There are some helpful documents at: 

http://www.oecd.org/chemicalsafety/testing/seriesontestingandassessmenttestingforendocrinedisrupters.htm

Leusch et al Comparison of Five in Vitro Bioassays to Measure Estrogenic Activity in Environmental Waters. Environ. Sci. Technol., 2010, 44 (10), pp 3853–3860

Good luck with your project!

Another approach that has not been mentioned is using solid phase extraction (SPE) and GC-MS/MS or LC-MS/MS to actually measure the compounds in your waters. Plus sides: you know what you are measuring (in ELISA you only know activity); you can run ANOVA or other statistics to correlate changes in Hellbender decline with specific compounds or sums of compounds. Down sides: SPE is time-consuming and somewhat costly. A quick search for SPE+hormone+water will give many papers that use this technique.

Are you looking to measure estrogenicity (as in activity in a sample) or endocrine disruptors (the actual compounds and their concentration in water)? The only way you can measure actual compounds reliably is to use an LC-MS/MS or GC-MS/MS. This can be done with offline SPE or online automated SPE depending on the sensitivity and robustness of the analytical instrument and can require anywhere between 5-1000 mL of sample. You can check with contract labs like Eurofins etc. who normally charge a few hundred dollars per sample for this analysis (or find someone at a university that may do it for cheaper). To measure estrogenic bioactivity, you can use bioassays.  

Hi Tarun,

Would the analysis be for hormones only in a sample, or would the "few hundred dollars" include the phytoestrogens and industrial estrogenics as well? I haven't priced chemical analysis for some time, and don't know whether it is going up or down.

Also, we found significant antiestrogenic activity in our river waters around Sydney, Australia (by bioassay), but never had the money to try to characterise them. Do you know about any ongoing research into identifying antiestrogens? I'm a bit out of touch on that.

Cheers,

Anne

The Yeast Estrogen Screen (YES) assay would be the simplest and can be done by graduate students with some training. It has much simpler requirements than mammalian cell lines. It has been used by ecotoxicology teams from institutes around the word to look at oestrogenic endocrine activity in wastewaters and other environmental samples. In fact it was used in the effect directed analysis procedure that first identified the steroid oestrogens as the significant contributors to oestrogenic activity. Although the assay is very sensitive you may still need to use solid phase extraction to concentrate up your samples. 

Article Estrogenic Activity of Surfactants and Some of Their Degrada...

Article Identification of Estrogenic Chemicals in STW Effluent. 1. C...

Article Issues Arising When Interpreting Results from an in Vitro As...

Thank you everyone!

Yes, the YES assay is relatively simple and requires less equipment than the mammalian cell line assays, but it is not terribly sensitive (see the paper by Leusch et al 2010 I've listed above). You will almost certainly need to concentrate the samples; I've wasted a lot of time testing effluents and waters, and getting negative results. I later discovered that the effluent Routledge's group were testing in their effects-directed analysis study was SO estrogenic that they could get away with testing water samples directly, but it wouldn't work for most samples. To concentrate samples, we've used Waters Oasis HLB cartridges, and 3M Empore C18 disks, with fairly comparable results. If you try to concentrate too much to overcome the lack of sensitivity, however, you may run into problems with toxicity to the yeast.