‘Kissing the frog into a handsome fish’: complete recovery from the feminising effects of xenoestrogen exposure after upgrades of WWTP operations in the Southern Biscay Bay

The Plentzia Marine Station has been long assessing the effects of xenoestrogens bioavailable in the Basque estuaries draining into the Southern Biscay Bay. Significant effects have been observed in fish, the most notable being the development of oocytes in the testes of males (intersex-testis). The UNESCO Biosphere Reserve of Urdaibai embraces the Oka estuary crossing Gernika and in 2007 male Chelon labrosus mullets exhibiting intersex testes were identified downstream its Waste Water Treatment Plant. Then, the PiE-EHU´s ‘Biscay-Bay-Environmental-Biospecimen-Bank’ (BBEBB) launched a monitoring program to annually sample mullets in the outlet of this malfunctioning WWTP. Overexpression of estrogenicity marker-genes was evident and up to 50% of males showed intersex-testis with accumulation of xenoestrogens (mainly alkylphenols) in bile and WWTP effluent. Under the EU Water Framework Directive the WWTP was closed in 2021 and activities deviated to a modern coastal facility, representing an inflexion point. Here we present an evaluation of xenoestrogenicity in the Biosphere Reserve yearly from 2007 to 2024. Intersex-condition was identified every year until 2021, when 53% of males showed oocytes in testis. The intersex-severity-index (ISI) began to increase in 2014 and 40% of 2021 intersex-males displayed ISI values above 3. Situation reversed with WWTP closure, no intersex-males appearing after 2021. Recovery was corroborated through molecular 5S/18S-rRNA, 5S/5.8S-rRNA and tRNA/5.8S-rRNA indexes, showing high values in ovaries and intersex-testes and low in testes of 2022-2024. Estrogenicity (cyp19a1a) and oocyte (gtf3ab, 42sp43) marker-genes displayed significantly higher transcription levels in ovaries and intersex-testes than in testes, while testis specific amh showed the inverse profile. All testes after 2021 showed a typical male transcription pattern for these genes. Hepatic levels of alkylphenols (HPLC-MS/MS) also decreased significantly from 2020 to 2021 and onwards, although bioaccumulated bisphenol-A persists. Recovery is total, because sometimes good things happen to handsome fish!Funded: Basque-Government (IT1743-22), Spanish MCIU&EU-FEDER/ERDF (PID2023-146085NB-I00).