The effects of commercial 2,4-D herbicide on game fish species: Natural lake water vs. laboratory system water.

Aquatic herbicides with active ingredient 2,4-dichlorophenoxyacteic acid (2,4-D) are commonly used to control and combat aquatic non-native species that cause detrimental impacts including habitat destruction, strained resources among biota, and biodiversity loss. While many (eco)toxicology studies are performed in the laboratory under highly controlled circumstances, these studies may disregard the nuances and disorder that come with the complexity of natural aquatic ecosystems. Therefore, we conducted a series of laboratory experiments using laboratory system water, different lake waters, and different water parameters to determine the effects of ecologically relevant concentrations of 2,4-D (0.00-4.00 ppm a.e.) on the development and survival of two freshwater game species (Sander vitreus and Esox lucius). For 2,4-D exposures using different water sources, there were significant main effects of 2,4-D concentration and water source on walleye embryo and larval survival, however, there was no significant interaction between 2,4-D exposure and water source. For 2,4-D exposures and pH (5-9 pH), there were significant main effects of 2,4-D concentration and pH on walleye and northern pike embryo survival and a significant interaction between 2,4-D exposure and pH. Our results indicate that 2,4-D exposures in controlled laboratory system water can predict similar outcomes as 2,4-D exposures in natural lake water. Moreover, individual water parameters, such as pH, play a significant role in the toxicity of 2,4-D. Taken together, these results suggest that highly controlled laboratory studies are a useful tool for predicting impacts on survival of non-target fish in natural waters, but it is crucial for management agencies to consider individual water sources and specific lake water parameters in herbicide risk assessments to minimize the impacts to non-target organism.

Elucidating the effects of acute and chronic exposure to 2,4-Dichlorophenoxyacetic acid on fathead minnow (Pimephales promelas) innate immunity.

Aquatic herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D) formulations, are commonly used for invasive species management throughout the United States. Ecologically relevant concentrations of 2,4-D can impair essential behaviors, reduce survival, and act as an endocrine disruptor; however, there is limited knowledge of its effects on the health of non-target organisms. Here, we investigate the acute and chronic exposure impacts of 2,4-D on adult male and female fathead minnow (Pimephales promelas) innate immune function. We exposed both adult male and female fathead minnows to three different ecologically relevant concentrations of 2,4-D (0.00, 0.40, and 4.00 mg/L) and took blood samples at three acute time points (6, 24, and 96 h) and one chronic time point (30 days). We found that male fatheads had higher total white blood cell concentrations when exposed to 2,4-D at the acute time points. For the females, only proportions of specific cell types were altered when exposed to 2,4-D at the acute time points. However, we did not observe any significant impacts of chronic exposure to 2,4-D on any innate immune responses for either males or females. Overall, this study is the first step in answering an important question for game fisheries and management agencies while providing insight to future studies that investigate the impacts of herbicide exposure to freshwater fish health and immunity.

Effects of subchronic exposure to environmentally relevant concentrations of a commercial fluridone formulation on fathead minnows (Pimephales promelas).

Invasive aquatic plants are a widespread problem in United States' waterways, and aquatic herbicide treatments are a common tool used in their management. Fluridone is an active ingredient in aquatic herbicides used globally to control aquatic plants. In order to be effective, fluridone requires a long contact time with plants resulting in extended exposure to non-target organisms. While there has been limited studies exploring the effects of fluridone on non-target aquatic organisms, the effects of subchronic commercial fluridone exposure at concentrations representative of operational use rates for plant management on fish are poorly understood. Therefore, we conducted a series of three exposure experiments using environmentally relevant concentrations on different life stages of the fathead minnow (Pimephales promelas). We exposed fathead minnows to a commercial fluridone formulation, Spritflo®, at environmentally relevant concentrations of 0.00, 3.00, 12.00, 25.00, and 100.00 µg/L. Exposure times included subchronic periods up to 35 days and a trans-generation exposure of 65 days, which is a likely residence time of fluridone when applied for plant management. Following 30 days of fluridone exposure, adult male fish had an increased presence of nuptial tubercules, an indicator of endocrine disruption, and an enlarged liver compared to the control. Additionally, we conducted larval fish behavior experiments and found fluridone exposure negatively affected prey capture ability, locomotion, and position preference. Our findings suggest fluridone treatment concentrations used in aquatic plant management do not directly cause mortality in fathead minnows, though sub-lethal effects observed could cause a decline in biological fitness and pose potential ecological implications.

Subchronic impacts of 2,4-D herbicide Weedestroy®AM40 on associative learning in juvenile yellow perch (Perca flavescens).

Aquatic herbicides are commonly used to control a wide variety of invasive and nuisance plants. One common active ingredient used in commercial herbicide formulations in Midwestern states is 2,4-dichlorophenoxyacetic acid (2,4-D). Due to the stability of 2,4-D in aquatic environments, many non-target aquatic species experience prolonged exposure throughout critical developmental life stages that can affect essential behaviors. However, the impacts of 2,4-D exposure on learning behaviors in juvenile fish are poorly understood. Therefore, we conducted a series of experiments using a maze environment to determine the effects of a commercial 2,4-D amine salt herbicide formulation (Weedestroy®AM40; WAM40; at 0.00, 0.50, 2.00, and 50.00 mg/L 2,4-D acid equivalent (a.e.)) exposure on juvenile yellow perch's ability to perform a feed associated learning behavior. We observed a significant decrease in the ability of yellow perch to correctly complete the feed associated learning behavior within 200 s when exposed to WAM40 at 2.00 and 50.00 mg/L 2,4-D as compared to controls (p = 0.0002; p < 0.0001, respectively) and within 600 s when exposed to WAM40 at 2.00 and 50.0 mg/L 2,4-D as compared to the controls (p = 0.0107 and p < 0.0001). These data suggest that exposure to 2,4-D in WAM40 can both increase the amount of time it takes for yellow perch to complete a feed associated learning behavior and/or obstruct the behavior altogether. Further experiments showed no significant decreases in locomotion (p > 0.05), hunger motivation (p > 0.05), and a visually guided startle response (p > 0.05), in all treatment groups tested as compared to controls. This suggests that 2,4-D in WAM40 does not inhibit feed associated learning behaviors via interaction with these mechanisms. Altogether, the results indicate that the use of 2,4-D herbicides for weed control in aquatic ecosystems could present risks to cognitive functions that control essential behaviors of yellow perch.

Impacts of subchronic exposure to a commercial 2,4-D herbicide on developmental stages of multiple freshwater fish species.

Invasive, nuisance aquatic species such as Eurasian watermilfoil (Myriophyllum spicatum) are rapidly spreading across the United States. One common active ingredient used to control this invasive species is 2,4-Dichlorophenoxyacetic acid (2,4-D). Application of 2,4-D to aquatic environments typically occurs while many freshwater fish are spawning and due to 2,4-D stability in aquatic environments, many non-target species experience prolonged exposure throughout embryogenesis and larval development. The impacts of 2,4-D exposure on phylogenetically distant fish species is poorly understood. Herein, we investigated the impacts of the 2,4-D commercial herbicide DMA4®IVM on nine freshwater fish species from six different families (four orders) at different points during ontogeny. Each species was exposed to ecologically relevant concentrations of a commercial 2,4-D herbicide (0.05, 0.50, and 2.00 ppm or mg/L 2,4-D a. e.), and effects on morphology, survival, and growth were evaluated. Our results demonstrate that exposure of embryonic and larval fish to ecologically relevant concentrations of a commercial 2,4-D herbicide reduced survival in early developmental stages of six freshwater species that spanned five phylogenetic families and three phylogenetic orders; however, sensitivity to 2,4-D exposure did not show correlation with phylogenetic proximity. Altogether, our results indicate that the use of 2,4-D herbicides in aquatic ecosystems at current recommended concentrations (≤2 ppm whole-lake treatment) could present risk to multiple freshwater fish species survival during early development.

2,4-Dichlorophenoxyacetic acid containing herbicide impairs essential visually guided behaviors of larval fish.

Aquatic herbicides are used worldwide to eradicate nuisance and invasive plants despite limited knowledge of their toxicity to non-target organisms. 2,4-Dichlorophenoxyacetic acid (2,4-D) is a common active ingredient in commercial herbicide formulations, which triggers plant cell death by mimicking the plant-specific hormone auxin. Application practices of 2,4-D commercial herbicides typically coincide with yearly freshwater fish spawning periods. This practice exposes fish to xenobiotics at their vulnerable larval stages. The full impacts of 2,4-D on larval fish remains poorly understood, and hence, whether it may alter larval survival, larval behavior, fish populations, and ecosystem dynamics. In the present study, we exposed embryonic and larval zebrafish (Danio rerio) to the active ingredient 2,4-D (pure 2,4-D) or a 2,4-D containing commercial herbicide DMA4®IVM (DMA4) and evaluated morphology, survival, behavior, and nervous system function. At 2,4-D concentrations producing no overt morphological defects during embryonic or early larval stages, we observed reduced survival throughout a 21-day larval assay (4-8 ppm DMA4 and 0.75-4 ppm pure 2,4-D). Notably, prey capture, a behavior essential to survival, was reduced in 2,4-D-exposed larval zebrafish (4-8 ppm DMA4 and 0.75-4 ppm pure 2,4-D) and yellow perch (Perca flavescens) (4-20 ppm DMA4). In zebrafish, 8 ppm DMA4 exposure reduced prey capture when exposure was restricted to the period of visual system development. Consistent with these results, larval zebrafish exposed to 8 ppm DMA4 showed reduced neural activity within the optic tectum following prey exposure. Together, our results suggest that 2,4-D alters the development and function of neural circuits underlying vision of larval fish, and thereby reduces visually guided behaviors required for survival.

Effects of low, subchronic exposure of 2,4-Dichlorophenoxyacetic acid (2,4-D) and commercial 2,4-D formulations on early life stages of fathead minnows (Pimephales promelas).

Aquatic herbicides are commonly used to control a wide variety of algae and plants, but they also have the potential to contaminate and affect nontarget organisms. However, the impacts of low-level 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide exposure on larval fish are not well understood. We conducted a series of experiments to determine the effects of low concentrations (0.05, 0.50, and 2.00 ppm) of 2 commercial 2,4-D amine salt herbicide formulations (Weedestroy® AM40 [WAM40] and DMA® 4 IVM [DMA4]) and pure 2,4-D on the development and survival of fathead minnows (Pimephales promelas) at various life cycle stages. Larval survival (30 d post hatch [dph]) was decreased following exposure of eggs and larvae to pure 2,4-D (0.50 ppm; p ≤ 0.001), as well as to WAM40 (0.50 and 2.00 ppm; p ≤ 0.001, p ≤ 0.001) and DMA4 (0.50 and 2.00 ppm; p ≤ 0.001, p ≤ 0.001). The results also narrowed the critical window of exposure for effects on survival to the period between fertilization and 14 dph. Development was not negatively altered by any of the compounds tested, although the commercial formulations increased larval total length and mass at 2.00 ppm. Altogether, the results indicate that the use of 2,4-D herbicides for weed control in aquatic ecosystems at current recommended concentrations (<2 ppm whole lake; <4 ppm spot treatment) could present risks to fathead minnow larval survival. Environ Toxicol Chem 2018;37:2550-2559. © 2018 SETAC.