{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,8]],"date-time":"2026-05-08T05:10:12Z","timestamp":1778217012192,"version":"3.51.4"},"reference-count":47,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,4,29]],"date-time":"2026-04-29T00:00:00Z","timestamp":1777420800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Anhui Province Environment Science and Technology Project","award":["No. 2024hb009"],"award-info":[{"award-number":["No. 2024hb009"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>Pesticide manufacturing industry wastewater is a complex mixture of potentially harmful components. If not properly treated, discharged effluents may pose serious risks to environment and organisms. In this study, influent and effluent wastewater samples from a pesticide factory were comprehensively non-screened by liquid chromatography high-resolution mass spectrometry, coupled with zebrafish embryo toxicity testing to assess whole effluent toxicity. A total of eight chemical groups were identified, including pesticides, antibiotics, nitrogen compounds, ketones, esters, amines and derivatives, other drugs, and other organic compounds. While wastewater treatment processes reduced most of the analyzed groups of compounds, compounds (e.g., 2-aminophenol, N-Nitrosodipropylamine, and carbamazepine) increased during the treatments. The influent samples were more toxic to zebrafish than the effluent samples in terms of lethality, teratogenic effects, developmental impacts, locomotor behavior, and neurotoxicity. The results showed that locomotor behavior was the most sensitive phenotypic toxicity endpoint, with significantly higher sensitivity than traditional acute lethal or teratogenic endpoints. Through a multi-dimensional assessment approach combining chemical screening, literature-based, risk ranking, and targeted quantification, we identified three predominant pesticide residues in the wastewater samples (both influents and effluents): hexaconazole, fenobucarb and isoprocarb. All three compounds exhibited additive or synergistic toxicity in zebrafish embryos. Exposure to \u22650.08% influent or \u22652% effluent increased inflammation (interleukin-1 beta, IL-1\u03b2), oxidative stress (copper\/zinc superoxide dismutase, Cu\/Zn-Sod), apoptosis (tumor protein p53, p53), and significantly impaired neurodevelopment in zebrafish larvae by altering the expression of sonic hedgehog a (shha), synapsin IIa (syn2a), and glial fibrillary acidic protein (gfap). This study suggests the necessity of incorporating non-apical endpoint (locomotor behavior) into whole effluent toxicity test, as this approach is essential for reducing the environmental risks posed by pesticide factory wastewater.<\/jats:p>","DOI":"10.3390\/biology15090700","type":"journal-article","created":{"date-parts":[[2026,4,29]],"date-time":"2026-04-29T13:32:22Z","timestamp":1777469542000},"page":"700","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Multi-Dimensional Assessment Approach to Assess Pesticide Manufacturing Industry Wastewater Toxicity"],"prefix":"10.3390","volume":"15","author":[{"given":"Deling","family":"Fan","sequence":"first","affiliation":[{"name":"School of Biological and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, China"},{"name":"Nanjing Institute of Environmental Science, Ministry of Ecology and Environment, Nanjing 210042, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jian","family":"Wang","sequence":"additional","affiliation":[{"name":"Nanjing Institute of Environmental Science, Ministry of Ecology and Environment, Nanjing 210042, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lili","family":"Shi","sequence":"additional","affiliation":[{"name":"Nanjing Institute of Environmental Science, Ministry of Ecology and Environment, Nanjing 210042, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lei","family":"Wang","sequence":"additional","affiliation":[{"name":"Nanjing Institute of Environmental Science, Ministry of Ecology and Environment, Nanjing 210042, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zheng","family":"Fang","sequence":"additional","affiliation":[{"name":"School of Biological and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Tudi, M., Daniel Ruan, H., Wang, L., Lyu, J., Sadler, R., Connell, D., Chu, C., and Phung, D.T. (2021). Agriculture Development, Pesticide Application and Its Impact on the Environment. Int. J. Environ. Res. Public Health, 18.","DOI":"10.3390\/ijerph18031112"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"4720","DOI":"10.1021\/acs.est.0c06283","article-title":"Phase Transfer and Biodegradation of Pesticides in Water-Sediment Systems Explored by Compound-Specific Isotope Analysis and Conceptual Modeling","volume":"55","author":"Droz","year":"2021","journal-title":"Environ. Sci. Technol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"592","DOI":"10.1016\/j.scitotenv.2018.08.254","article-title":"Sunlight and UVC-254 irradiation induced photodegradation of organophosphorus pesticide dichlorvos in aqueous matrices","volume":"649","author":"Bustos","year":"2019","journal-title":"Sci. Total Environ."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"152863","DOI":"10.1016\/j.scitotenv.2021.152863","article-title":"Overarching issues on relevant pesticide transformation products in the aquatic environment: A review","volume":"815","author":"Anagnostopoulou","year":"2022","journal-title":"Sci. Total Environ."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1016\/j.envpol.2016.03.064","article-title":"Body burden of pesticides and wastewater-derived pollutants on freshwater invertebrates: Method development and application in the Danube River","volume":"214","author":"Inostroza","year":"2016","journal-title":"Environ. Pollut."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"115605","DOI":"10.1016\/j.envres.2023.115605","article-title":"Global distribution of pesticides in freshwater resources and their remediation approaches","volume":"225","author":"Singh","year":"2023","journal-title":"Environ. Res."},{"key":"ref_7","unstructured":"(2024). Discharge Standard of Water Pollutants for Pesticide Industry (Standard No. GB 21523-2024). Ministry of Ecology and Environment of the People\u2019s Republic of China, State Administration for Market Regulation."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"158094","DOI":"10.1016\/j.scitotenv.2022.158094","article-title":"Review of ecologically relevant in vitro bioassays to supplement current in vivo tests for whole effluent toxicity testing\u2014Part 2: Non-apical endpoints","volume":"851","author":"Finlayson","year":"2022","journal-title":"Sci. Total Environ."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"180875","DOI":"10.1016\/j.scitotenv.2025.180875","article-title":"Species sensitivity distribution modeling for ecotoxicity prediction of industrial chemicals","volume":"1006","author":"Khan","year":"2025","journal-title":"Sci. Total Environ."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1016\/bs.ctdb.2016.10.007","article-title":"Zebrafish in Toxicology and Environmental Health","volume":"124","author":"Bambino","year":"2017","journal-title":"Curr. Top. Dev. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"109439","DOI":"10.1016\/j.cbpc.2022.109439","article-title":"Impact of pharmaceutical products on zebrafish: An effective tool to assess aquatic pollution","volume":"261","author":"Porretti","year":"2022","journal-title":"Comp. Biochem. Physiol. C Toxicol. Pharmacol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Gu, J., Guo, L., Zhu, Y., Qian, L., Shi, L., Zhang, H., and Ji, G. (2023). Neurodevelopmental Toxicity of Emamectin Benzoate to the Early Life Stage of Zebrafish Larvae (Danio rerio). Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24043757"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"113397","DOI":"10.1016\/j.ecoenv.2022.113397","article-title":"Exposure of preschool-aged children to highly-concerned bisphenol analogues in Nanjing, East China","volume":"234","author":"Fan","year":"2022","journal-title":"Ecotoxicol. Environ. Saf."},{"key":"ref_14","unstructured":"OECD (2013). Test No. 236: Fish Embryo Acute Toxicity (FET) Test. OECD Guidelines for the Testing of Chemicals, OECD Publishing. Section 2."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"106154","DOI":"10.1016\/j.aquatox.2022.106154","article-title":"Assessing the toxicity of bisphenol A and its six alternatives on zebrafish embryo\/larvae","volume":"246","author":"Gao","year":"2022","journal-title":"Aquat. Toxicol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"749","DOI":"10.1016\/j.enceco.2025.12.014","article-title":"An integrated approach unveils novel mechanisms of nicotine-induced neurotoxicity in zebrafish larvae","volume":"8","author":"Kou","year":"2026","journal-title":"Environ. Chem. Ecotoxicol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1220","DOI":"10.1016\/j.envpol.2016.11.011","article-title":"Impact of untreated wastewater on a major European river evaluated with a combination of in vitro bioassays and chemical analysis","volume":"220","author":"Konig","year":"2017","journal-title":"Environ. Pollut."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"131369","DOI":"10.1016\/j.chemosphere.2021.131396","article-title":"Occurrence, spatial distribution and risk assessment of high concern endocrine-disrupting chemicals in Jiangsu Province, China","volume":"285","author":"Fan","year":"2021","journal-title":"Chemosphere"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Dai, P., Ding, M., Yu, J., Gao, Y., Wang, M., Ling, J., Dong, S., Zhang, X., Zeng, X., and Sun, X. (2024). The male reproductive toxicity caused by 2-naphthylamine was related to testicular immunity disorders. Toxics, 12.","DOI":"10.3390\/toxics12050342"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"109993","DOI":"10.1016\/j.cbpc.2024.109993","article-title":"Flusilazole induced developmental toxicity, neurotoxicity, and cardiovascular toxicity via apoptosis and oxidative stress in zebrafish","volume":"284","author":"Lee","year":"2024","journal-title":"Comp. Biochem. Physiol. C Toxicol. Pharmacol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1016\/j.neuro.2020.03.013","article-title":"Fenobucarb-induced developmental neurotoxicity and mechanisms in zebrafish","volume":"79","author":"Zhu","year":"2020","journal-title":"Neurotoxicology"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"113870","DOI":"10.1016\/j.ecoenv.2022.113870","article-title":"Isoprocarb causes neurotoxicity of zebrafish embryos through oxidative stress-induced apoptosis","volume":"242","author":"Wang","year":"2022","journal-title":"Ecotoxicol. Environ. Saf."},{"key":"ref_23","first-page":"129","article-title":"Can the learning health care system be educated with observational data?","volume":"312","author":"Gu","year":"2025","journal-title":"Jama"},{"key":"ref_24","first-page":"137830","article-title":"A novel framework for industrial pesticide effluent assessment: Integrating chemical screening, multi-endpoint responses and literature-based validation","volume":"490","author":"Ptak","year":"2018","journal-title":"J. Hazard. Mater."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"6419","DOI":"10.1021\/acs.est.5b01173","article-title":"Indoor and Outdoor Levels and Sources of Submicron Particles (PM1) at Homes in Edmonton, Canada","volume":"49","author":"Bari","year":"2015","journal-title":"Environ. Sci. Technol."},{"key":"ref_26","first-page":"27","article-title":"The triazole fungicides prochloraz and tebuconazole combination inhibits reproductive development in the rat","volume":"297","author":"Jacobsen","year":"2012","journal-title":"Toxicology"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"S70","DOI":"10.1016\/j.jped.2024.11.008","article-title":"Pesticides and human health","volume":"101","author":"Rodrigues","year":"2025","journal-title":"J. Pediatr."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"113612","DOI":"10.1016\/j.fct.2023.113612","article-title":"Enantioselective toxicity effect and mechanism of hexaconazole enantiomers to human breast cancer cells","volume":"173","author":"Yang","year":"2023","journal-title":"Food Chem. Toxicol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"141990","DOI":"10.1016\/j.scitotenv.2020.141990","article-title":"Treatment technologies for emerging contaminants in wastewater treatment plants: A review","volume":"753","author":"Rout","year":"2021","journal-title":"Sci. Total Environ."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1186\/s40246-025-00863-1","article-title":"Unraveling diethyl phthalate-induced prostate carcinogenesis: Core targets revealed by integrated network toxicology, machine learning, and structural validation","volume":"19","author":"Liu","year":"2025","journal-title":"Hum. Genom."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"6913","DOI":"10.1021\/jf3021128","article-title":"Occurrence and profiles of phthalates in foodstuffs from China and their implications for human exposure","volume":"60","author":"Guo","year":"2012","journal-title":"J. Agric. Food Chem."},{"key":"ref_32","first-page":"112313","article-title":"Degradation of nitrogen-containing hazardous compounds using advanced oxidation processes: A review on aliphatic and aromatic amines, dyes, and pesticides","volume":"287","author":"Gani","year":"2021","journal-title":"J. Environ. Manag."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"744","DOI":"10.1016\/j.scitotenv.2017.07.032","article-title":"Investigation of the presence and endocrine activities of pesticides found in wastewater effluent using yeast-based bioassays","volume":"607\u2013608","author":"Westlund","year":"2017","journal-title":"Sci. Total Environ."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1179\/174329211X13190184351680","article-title":"Autoimmune reactivity of IgM acquired after oxidation","volume":"16","author":"Omersel","year":"2011","journal-title":"Redox Rep."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"174388","DOI":"10.1016\/j.scitotenv.2024.174388","article-title":"Pesticides in wastewater treatment plant effluents in the Yeongsan River Basin, Korea: Occurrence and environmental risk assessment","volume":"946","author":"Kim","year":"2024","journal-title":"Sci. Total Environ."},{"key":"ref_36","first-page":"466","article-title":"Occurrence and behavior of pesticides in wastewater treatment plants and their environmental impact","volume":"458\u2013460","author":"Villagrasa","year":"2013","journal-title":"Sci. Total Environ."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"619","DOI":"10.1016\/j.scitotenv.2013.12.065","article-title":"A review on the occurrence of micropollutants in the aquatic environment and their fate and removal during wastewater treatment","volume":"473","author":"Luo","year":"2014","journal-title":"Sci. Total Environ."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"5762","DOI":"10.1021\/ie030229y","article-title":"Oxidation of acetamide herbicides in natural waters by ozone and by the combination of ozone\/hydrogen peroxide: kinetic study and process modeling","volume":"42","author":"Acero","year":"2003","journal-title":"Ind. Eng. Chem. Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2577","DOI":"10.1080\/10643389.2013.829765","article-title":"Advanced oxidation processes in water\/wastewater treatment: Principles and applications. A review","volume":"44","author":"Oturan","year":"2014","journal-title":"Crit. Rev. Environ. Sci. Technol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1016\/j.ecoenv.2016.09.035","article-title":"Occurrence, distribution and risk assessment of suspected endocrine- disrupting chemicals in surface water and suspended particulate matter of Yangtze River (Nanjing section)","volume":"135","author":"Liu","year":"2017","journal-title":"Ecotoxicol. Environ. Saf."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"517","DOI":"10.1016\/j.chemosphere.2018.12.061","article-title":"Developmental toxicity of kresoxim-methyl during zebrafish (Danio rerio) larval development","volume":"219","author":"Jiang","year":"2019","journal-title":"Chemosphere"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"158780","DOI":"10.1016\/j.scitotenv.2022.158780","article-title":"Ethalfluralin induces developmental toxicity in zebrafish via oxidative stress and inflammation","volume":"854","author":"Hong","year":"2023","journal-title":"Sci. Total Environ."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"104896","DOI":"10.1016\/j.pestbp.2021.104896","article-title":"Neurotoxicity of fipronil affects sensory and motor systems in zebrafish","volume":"177","author":"Wu","year":"2021","journal-title":"Pestic. Biochem. Physiol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"629","DOI":"10.1016\/j.chemosphere.2018.10.218","article-title":"Neurobehavioral effects of bisphenol S exposure in early life stages of zebrafish larvae (Danio rerio)","volume":"217","author":"Gu","year":"2019","journal-title":"Chemosphere"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1022","DOI":"10.1016\/j.scitotenv.2017.01.156","article-title":"Quantitative investigation of the mechanisms of microplastics and nanoplastics toward zebrafish larvae locomotor activity","volume":"584\u2013585","author":"Chen","year":"2017","journal-title":"Sci. Total Environ."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"22873","DOI":"10.1002\/jbt.22873","article-title":"Bisphenol A exposure induces neurobehavioral deficits and neurodegeneration through induction of oxidative stress and activated caspase-3 expression in zebrafish brain","volume":"35","author":"Sahoo","year":"2021","journal-title":"J. Biochem. Mol. Toxicol."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Toni, M., Arena, C., Cioni, C., and Tedeschi, G. (2023). Temperature- and chemical-induced neurotoxicity in zebrafish. Front. Physiol., 14.","DOI":"10.3389\/fphys.2023.1276941"}],"container-title":["Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-7737\/15\/9\/700\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,8]],"date-time":"2026-05-08T04:22:08Z","timestamp":1778214128000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-7737\/15\/9\/700"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,29]]},"references-count":47,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["biology15090700"],"URL":"https:\/\/doi.org\/10.3390\/biology15090700","relation":{},"ISSN":["2079-7737"],"issn-type":[{"value":"2079-7737","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,29]]}}}