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The insecticide carbaryl is commonly found in indirectly exposed freshwater ecosystems at low concentrations considered safe for fish communities. In this study, we showed that after only 24 h of exposure to environmental concentrations of carbaryl (0.066-660 ng/L), zebrafish larvae exhibit impairments in essential behaviours. Interestingly, the observed behavioural effects induced by carbaryl were acetylcholinesterase-independent. To elucidate the molecular initiating event that resulted in the observed behavioural effects, in silico predictions were followed by in vitro validation. We identified two target proteins that potentially interacted with carbaryl, the α2B adrenoceptor (ADRA2B) and the serotonin 2B receptor (HTR2B). Using a pharmacological approach, we then tested the hypothesis that carbaryl had antagonistic interactions with both receptors. Similar to yohimbine and SB204741, which are prototypic antagonists of ADRA2B and HTR2B, respectively, carbaryl increased the heart rate of zebrafish larvae. When we compared the behavioural effects of a 24-h exposure to these pharmacological antagonists with those of carbaryl, a high degree of similarity was found. These results strongly suggest that antagonism of both ADRA2B and HTR2B is the molecular initiating event that leads to adverse outcomes in zebrafish larvae that have undergone 24 h of exposure to environmentally relevant levels of carbaryl. ; This work was supported by "Agencia Estatal de Investigación" from the Spanish Ministry of Science and Innovation (project PID2020-113371RB-C21), IDAEA-CSIC, Severo Ochoa Centre of Excellence (CEX2018-000794-S), which financed M.F. with Severo Ochoa funds. Juliette Bedrossiantz was supported by a PhD grant (PRE2018-083513) co-financied by the Spanish Government and the European Social Fund (ESF). This work was supported by Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF-FEDER European Fund (projects CTQ2017-89222-R and PID2020-120499RB-I00) and by the Catalan Government (2017 SGR 1604 and 2017-SGR-1807) to AL. XR research was financed by the Spanish Ministry of Economy, Industry and Competitiveness (SAF2015-74132-JIN). We thank Dr. Kees Jalink (The Netherlands Cancer Institute, Amsterdam, the Netherlands) for providing the plasmids encoding for the Epac-SH188 biosensor and Dr. Karen Martinez (University of Copenhagen, Copenhagen, Denmark) for providing the HEK 293 SNAP-β1AR). The findings of this report are not to be construed as an official Department of the Army position unless so designated by other authorized documents. ; Peer reviewed

Fenitrothion is an organophosphorus insecticide usually found in aquatic ecosystems at concentrations in the range of low ng/L. In this manuscript we show that 24 h exposure to environmental concentrations of fenitrothion, from ng/L to low μg/L, altered basal locomotor activity, visual-motor response and acoustic/vibrational escape response of zebrafish larvae. Furthermore, fenitrothion and expression of gap43a, gfap, atp2b1a, and mbp exhibited a significant non-monotonic concentration-response relationship. Once determined that environmental concentrations of fenitrothion were neurotoxic for zebrafish larvae, a computational analysis identified potential protein targets of this compound. Some of the predictions, including interactions with acetylcholinesterase, monoamine-oxidases and androgen receptor (AR), were experimentally validated. Binding to AR was the most suitable candidate for molecular initiating event, as indicated by both the up-regulation of cyp19a1b and sult2st3 and the non-monotonic relationship found between fenitrothion and the observed responses. Finally, when the integrity of the monoaminergic system was evaluated, altered levels of L-DOPA, DOPAC, HVA and 5-HIAA were found, as well as a significant up-regulation of slc18a2 expression at the lowest concentrations of fenitrothion. These data strongly suggest that concentrations of fenitrothion commonly found in aquatic ecosystems present a significant environmental risk for fish communities. ; We thank Dr. Harold A. Burgess for providing Flote software package and advice with the kinematic analysis setup for the acoustic/vibrational evoked escape response. This work was supported by the Spanish Government with FEDER Funds (CTM2017-83242- R; D.R.) and the network of recognized research groups by the Catalan Government (2017 SGR_902). Melissa Faria acknowledges financial support from the Beatriu de Pinós programme (grant Nº: 2016 BP 00233) provided by the Secretariat of Universities and Research department of the Ministry for Business and Knowledge, Catalonia Government. Maria Pagano benefited from a PhD grant (Avviso 24/2018) given by Sicily region, supported by FSE 2014-2020. Mention of specific products or trade names does not indicate endorsement by the US federal government. ; Peer reviewed