A survey of 17α-ethinylestradiol and mestranol residues in Hawkesbury River, Australia, using a highly specific enzyme-linked immunosorbent assay (ELISA) demonstrates the levels of potential biological significance

- Synthesis of a hapten with a linker attachment at the C3 position of EE2.
- A limit of detection (LOD) is 0.04±0.01 µg L−1 EE2 and mestranol.
- A limit of quantitation (LOQ) is 0.05±0.01 ng/L with a pre-enrichment step.
- A good correlation between the ELISA and GC/MS (R2=0.934).
- The levels in Hawkesbury river could pose potential endocrine disrupting effects.

This study reports on the potential status of 17α-ethinylestradiol (EE2) and mestranol (MeEE2) residues in aquatic environments in New South Wales (NSW), Australia, based on the analysis by a specific ELISA we developed. Polyclonal antibodies were raised against the EE2 hapten with a linker attached at the C3-position to direct the antibody binding towards the ring D of EE2/MeEE2. Using this approach, an ELISA highly specific to EE2 and MeEE2 was successfully developed, showing less than 3.1% cross-reactivity (% CR) with other major steroidal sex hormones and their derivatives. The assay performed with the limit of detection (LOD) of 0.04 ± 0.01 µg/L for both EE2 and MeEE2, and the limit of quantitation (LOQ) of 0.05 ± 0.01 ng/L when it was coupled with the SM2-Biobeads solid phase extraction. Prior to conducting the survey study, it was validated against the gas chromatography-mass spectrophotometry (GC-MS) method, which showed high correlation with R2 of 0.934. Fresh surface water samples collected at different sites along Hawkesbury River in New South Wales (NSW) were analyzed for the EE2/ MeEE2 residues using the developed ELISA. The EE2/MeEE2 levels were found to range between 4.1 and 8.3 ng/L in Emigrant Creek, NSW, where the primary activity was macadamia plantation, and higher levels between 15 and 29 ng/L in South Creek, NSW, Greater Western Sydney at sites upstream and downstream of the municipal sewage treatment plants.

142