wileyonlinelibrary.com/ETC © 2020 SETAC Environmental Toxicology and Chemistry—Volume 00, Number 00—pp. 1–7, 2020 Received: 4 November 2019 | Revised: 26 November 2019 | Accepted: 10 April 2020 1 Environmental Toxicology The Effect of Herbicide Formulations and Herbicide–Adjuvant Mixtures on Aquatic Food Web Species of the Sacramento–San Joaquin Delta, California, USA Marie E. Stillway* and Swee J. Teh School of Veterinary Medicine, Department of Anatomy, Physiology, and Cell Biology, Aquatic Health Program Laboratory, University of California, Davis, California, USA Abstract: Herbicides have recently been designated as one of the most frequently detected chemical types in aquatic systems. We evaluated the effect of the herbicide formulations Clearcast ® (active ingredient imazamox) and Galleon ® SC (active ingredient penoxsulam), in conjunction with the adjuvant Agri‐dex ® , on the diatom Thalassiosira pseudonana and the copepod Eurytemora affinis and whether their application in invasive aquatic weed control has acutely negative effects on important delta food web species. Herbicide–adjuvant mixtures were more toxic than herbicide formulations alone. For the diatom, 50% inhibition concentration (IC50) were 84.4 mg/L for Clearcast, >100 mg/L for Galleon SC, and 38.5 mg/L for Agri‐dex. In the herbicide mixture diatom tests, IC50s decreased to 2.8 mg/L for Clearcast + 64 mg/L Agri‐dex and to 6.6 mg/L for Galleon SC + 41 mg/L Agri‐dex. In the copepod tests, 50% effect concentrations (EC50s) were 45.4 mg/L for Agri‐dex, 31.2 mg/L for Galleon SC, and >100 mg/L for Clearcast. When tested together, EC50s were reduced to 24.1 for Galleon SC and 67.6 mg/L for Clearcast in the presence of 50mg/L Agri‐dex. Environmentally relevant herbicide–adjuvant mixture ratios were at the no‐observed‐effect level. Mixture interactions between herbicides and adjuvants indicate the potential for increased toxicity in herbicide formulations and tank mixes, especially in consideration of the unlisted, proprietary ingredients which are included in herbicide formulations, making predicting nontarget effects challenging. Environ Toxicol Chem 2020;00:1–7. © 2020 SETAC Keywords: Herbicides; Zooplankton; Microalgae; Mixture toxicology INTRODUCTION Herbicides are generally considered to be of low toxicity to animals because they target biosynthetic pathways of plants not typically present in mammals and other higher organisms (Netherland et al. 2009; Hamel 2012). Therefore, it is consid- ered rare to observe acute effects in fish and other vertebrates after exposure to environmentally relevant concentrations, which often designates these herbicides as low risk (Hamel 2012; National Marine Fisheries Service 2013). Direct applica- tion to plants and water bodies for aquatic weed control is one way these chemicals enter waterways; terrestrial applications and associated runoff from storm events are other major con- tributors of these types of pollutants in the aquatic environ- ment. Recently, herbicides have been the most frequently detected contaminant type in California water bodies (Orlando et al. 2014); for example, hexazinone was detected in 100% of samples evaluated between 2012 and 2013, with other herbi- cides such as diuron, simazine, and metolachlor following suit (Orlando et al. 2014). The Sacramento–San Joaquin delta, California, USA, is one of the most invaded estuaries in the world (Cohen and Carlton 1998), with invasive aquatic vegetation such as water hyacinth (Eichhornia crassipes) and Brazilian waterweed (Egeria densa) playing a major role in the decline in water quality and delta aquatic habitat. Increasing temperatures due to climate change and a lack of natural predators allow these species to thrive, which in turn causes damage to marinas, affects tur- bidity, and reduces habitat for endangered fish species such as the delta smelt (Hypomesus transpacificus). Invasive aquatic weed control is governed by California state agencies through the use of timed herbicide applications throughout the year to manage these nuisance species. Imazamox and penoxsulam are 2 herbicides planned for use in the management of invasive aquatic weed control in the Sacramento–San Joaquin delta. Penoxsulam, the active ingredient in Galleon ® SC received approval from the US Environmental Protection Agency This article contains online‐only supplemental data. * Address correspondence to mstillway@ucdavis.edu Published online 2020 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.4725