We stored water samples at -20°C until they were thawed for extraction in Spain. We extracted hormones from water using C 18 solid phase extraction (SPE) columns (Waters Inc.). After extracting into the column, we stored the SPE columns at -20°C until we were ready for methanol elution at Texas State University. All methods follow from [5 (link)]. We measured CORT release rates using enzyme-immunoassay (EIA) kit (Cayman Chemicals Inc.) in duplicate with a 96 well plate reader set at 415 nm (BioTek Powerwave XS). We did not subtract the CORT level measured in the water because we used the same water for all populations in Experiment 1.
We previously validated the use of water-borne hormones on EIA plates and the positive correlation between plasma CORT and water-borne CORT release rates for A. obstetricans [33 ]. Gabor et al. [5 (link)] additionally validated the use of the CORT EIA kits with water-borne hormones for A. muletensis.
We previously validated the use of water-borne hormones on EIA plates and the positive correlation between plasma CORT and water-borne CORT release rates for A. obstetricans [33 ]. Gabor et al. [5 (link)] additionally validated the use of the CORT EIA kits with water-borne hormones for A. muletensis.
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