David H. Baldwin

The Synergistic Toxicity of Pesticide Mixtures: Implications for Risk Assessment and the Conservation of Endangered Pacific Salmon

Background Mixtures of organophosphate and carbamate pesticides are commonly detected in freshwater habitats that support threatened and endangered species of Pacific salmon (Oncorhynchus sp.). These pesticides inhibit the activity of acetylcholinesterase (AChE) and thus have potential to interfere with behaviors that may be essential for salmon survival. Although the effects of individual anticholin-esterase insecticides on aquatic species have been studied for decades, the neurotoxicity of mixtures is still poorly understood. Objectives We assessed whether chemicals in a mixture act in isolation (resulting in additive AChE inhibition) or whether components interact to produce either antagonistic or synergistic toxicity. Methods We measured brain AChE inhibition in juvenile coho salmon (Oncorhynchus kisutch) exposed to sublethal concentrations of the organophosphates diazinon, malathion, and chlorpyrifos, as well as the carbamates carbaryl and carbofuran. Concentrations of individual chemicals were normalized to their respective median effective concentrations (EC50) and collectively fit to a nonlinear regression. We used this curve to determine whether toxicologic responses to binary mixtures were additive, antagonistic, or synergistic. Results We observed addition and synergism, with a greater degree of synergism at higher exposure concentrations. Several combinations of organophosphates were lethal at concentrations that were sublethal in single-chemical trials. Conclusion Single-chemical risk assessments are likely to underestimate the impacts of these insecticides on salmon in river systems where mixtures occur. Moreover, mixtures of pesticides that have been commonly reported in salmon habitats may pose a more important challenge for species recovery than previously anticipated.

Olfactory toxicity in fishes

Olfaction conveys critical environmental information to fishes, enabling activities such as mating, locating food, discriminating kin, avoiding predators and homing. All of these behaviors can be impaired or lost as a result of exposure to toxic contaminants in surface waters. Historically, teleost olfaction studies have focused on behavioral responses to anthropogenic contaminants (e.g., avoidance). More recently, there has been a shift towards understanding the underlying mechanisms and functional significance of contaminant-mediated changes in fish olfaction. This includes a consideration of how contaminants affect the olfactory nervous system and, by extension, the downstream physiological and behavioral processes that together comprise a normal response to naturally occurring stimuli (e.g., reproductive priming or releasing pheromones). Numerous studies spanning several species have shown that ecologically relevant exposures to common pollutants such as metals and pesticides can interfere with fish olfaction and disrupt life history processes that determine individual survival and reproductive success. This represents one of the pathways by which toxic chemicals in aquatic habitats may increasingly contribute to the decline and at-risk status of many commercially and ecologically important fish stocks. Despite our emerging understanding of the threats that pollution poses for chemical communication in aquatic communities, many research challenges remain. These include: (1) the determination of specific mechanisms of toxicity in the fish olfactory sensory epithelium; (2) an understanding of the impacts of complex chemical mixtures; (3) the capacity to assess olfactory toxicity in fish in situ; (4) the impacts of toxins on olfactory-mediated behaviors that are still poorly understood for many fish species; and (5) the connections between sublethal effects on individual fish and the long-term viability of wild populations. This review summarizes and integrates studies on fish olfaction-contaminant interactions, including metrics ranging from the molecular to the behavioral, and highlights directions for future research.

Sublethal exposure to crude oil during embryonic development alters cardiac morphology and reduces aerobic capacity in adult fish

Exposure to high concentrations of crude oil produces a lethal syndrome of heart failure in fish embryos. Mortality is caused by cardiotoxic polycyclic aromatic hydrocarbons (PAHs), ubiquitous components of petroleum. Here, we show that transient embryonic exposure to very low concentrations of oil causes toxicity that is sublethal, delayed, and not counteracted by the protective effects of cytochrome P450 induction. Nearly a year after embryonic oil exposure, adult zebrafish showed subtle changes in heart shape and a significant reduction in swimming performance, indicative of reduced cardiac output. These delayed physiological impacts on cardiovascular performance at later life stages provide a potential mechanism linking reduced individual survival to population-level ecosystem responses of fish species to chronic, low-level oil pollution.

Comparative thresholds for acetylcholinesterase inhibition and behavioral impairment in coho salmon exposed to chlorpyrifos

Chlorpyrifos is a common organophosphate insecticide that has been widely detected in surface waters that provide habitat for Pacific salmon in the western United States. Although chlorpyrifos is known to inhibit acetylcholinesterase (AChE) in the brain and muscle of salmonids, the relationship between sublethal AChE inhibition and more integrative indicators of neuro-behavioral impairment are poorly understood. This is particularly true for exposures that reflect the typical range of pesticide concentrations in the aquatic environment. To directly compare the effects of chlorpyrifos on AChE activity and salmon behavior, we exposed juvenile coho salmon (Oncorhynchus kisutch) to chlorpyrifos (0-2.5 microg/L) for 96 h. A computer-assisted, three-dimensional video imaging system was used to measure spontaneous swimming and feeding behaviors in control and chlorpyrifos-exposed fish. After the behavioral trials, brain and muscle tissues were collected and analyzed for AChE activity. Chlorpyrifos inhibited tissue AChE activity and all behaviors in a dose-dependent manner. Moreover, brain AChE inhibition and reductions in spontaneous swimming and feeding activity were significantly correlated. Benchmark concentrations for sublethal neurotoxicity (statistical departure values) were <0.5 microg/L and were similar for both neurochemical and behavioral endpoints. Collectively, these results indicate a close relationship between brain AChE inhibition and behavioral impairment in juvenile coho exposed to chlorpyrifos at environmentally realistic concentrations.

Sublethal effects of copper on coho salmon: Impacts on nonoverlapping receptor pathways in the peripheral olfactory nervous system

The sublethal effects of copper on the sensory physiology of juvenile coho salmon (Oncorhynchus kisutch) were evaluated. In vivo field potential recordings from the olfactory epithelium (electro-olfactograms) were used to measure the impacts of copper on the responses of olfactory receptor neurons to natural odorants (L-serine and taurocholic acid) and an odorant mixture (L-arginine, L-aspartic acid, L-leucine, and L-serine) over a range of stimulus concentrations. Increases in copper impaired the neurophysiological response to all odorants within 10 min of exposure. The inhibitory effects of copper (1.0-20.0 micrograms/L) were dose-dependent and they were not influenced by water hardness. Toxicity thresholds for the different receptor pathways were determined by using the benchmark dose method and found to be similar (a 2.3-3.0 micrograms/L increase in total dissolved copper over background). Collectively, examination of these data indicates that copper is broadly toxic to the salmon olfactory nervous system. Consequently, short-term influxes of copper to surface waters may interfere with olfactory-mediated behaviors that are critical for the survival and migratory success of wild salmonids.

Effects of the synthetic estrogen, 17α-ethinylestradiol, on aggression and courtship behavior in male zebrafish (Danio rerio)

The synthetic estrogen, 17alpha-ethinylestradiol (EE(2)), is the active component in oral contraceptive pills. It is excreted from the human body in high amounts and released via sewage treatment plant effluent into aquatic environments. In fish, estrogen receptors have strong binding affinities for EE(2), and exposure raises the possibility of adverse neuroendocrine responses in aquatic animals. In the present study we explored the effects of dissolved-phase EE(2) on the dynamics of male-male aggression and courtship behaviors in adult zebrafish. Further, we assessed whether the behavioral effects of EE(2) result in changes in male offspring paternity. We scored the aggressive behaviors of individual unexposed males and categorized these fish as either dominant or subordinate. We then exposed dominant males to EE(2) at doses of 0, 0.5, 5.0, and 50.0ng/L for 48h. Subsequent trials examined the agonistic behaviors of males in two testing scenarios: (1) a dyadic encounter with another male alone, and (2) a competitive spawning interaction with another male and three adult females. Competitive spawning tests were also used to assess the impacts of EE(2) exposure on courtship behavior and paternity using males that were homozygous for green fluorescent protein (GFP) expression under the control of the islet-1 promoter. We found that EE(2) at all exposure concentrations reduced male aggression during male-male dyadic encounters and caused a social dominance reversal in 50% of the fish at the highest exposure dose (50ng/L EE(2)). The frequency of courtship-specific behavior decreased in dominant males exposed to the steroid, though this effect was only significant for the lowest dose group (0.5ng/L EE(2)). In the highest exposure group (50ng/L EE(2)), 50% of dominant males relinquished paternal dominance. Our results show that short-term exposure to EE(2) at environmentally relevant levels can alter aggression, and shift individual social status and reproductive success in male zebrafish.

A fish of many scales: extrapolating sublethal pesticide exposures to the productivity of wild salmon populations.

For more than a decade, numerous pesticides have been detected in river systems of the western United States that support anadromous species of Pacific salmon and steelhead. Over the same interval, several declining wild salmon populations have been listed as either threatened or endangered under the U.S. Endangered Species Act (ESA). Because pesticides occur in surface waters that provide critical habitat for ESA-listed stocks, they are an ongoing concern for salmon conservation and recovery throughout California and the Pacific Northwest. Because pesticide exposures are typically sublethal, a key question is whether toxicological effects at (or below) the scale of the individual animal ultimately reduce the productivity and recovery potential of wild populations. In this study we evaluate how the sublethal impacts of pesticides on physiology and behavior can reduce the somatic growth of juvenile chinook salmon (Oncorhynchus tshawytscha) and, by extension, subsequent size-dependent survival when animals migrate to the ocean and overwinter in their first year. Our analyses focused on the organophosphate and carbamate classes of insecticides. These neurotoxic chemicals have been widely detected in aquatic environments. They inhibit acetylcholinesterase, an enzyme in the salmon nervous system that regulates neurotransmitter-mediated signaling at synapses. Based on empirical data, we developed a model that explicitly links sublethal reductions in acetylcholinesterase activity to reductions in feeding behavior, food ration, growth, and size at migration. Individual size was then used to estimate size-dependent survival during migration and transition to the sea. Individual survival estimates were then integrated into a life-history population projection matrix and used to calculate population productivity and growth rate. Our results indicate that short-term (i.e., four-day) exposures that are representative of seasonal pesticide use may be sufficient to reduce the growth and size at ocean entry of juvenile chinook. The consequent reduction in individual survival over successive years reduces the intrinsic productivity (lambda) of a modeled ocean-type chinook population. Overall, we show that exposures to common pesticides may place important constraints on the recovery of ESA-listed salmon species, and that simple models can be used to extrapolate toxicological impacts across several scales of biological complexity.

Low‐level copper exposures increase visibility and vulnerability of juvenile coho salmon to cutthroat trout predators

Copper contamination in surface waters is common in watersheds with mining activities or agricultural, industrial, commercial, and residential human land uses. This widespread pollutant is neurotoxic to the chemosensory systems of fish and other aquatic species. Among Pacific salmonids (Oncorhynchus spp.), copper-induced olfactory impairment has previously been shown to disrupt behaviors reliant on a functioning sense of smell. For juvenile coho salmon (O. kisutch), this includes predator avoidance behaviors triggered by a chemical alarm cue (conspecific skin extract). However, the survival consequences of this sublethal neurobehavioral toxicity have not been explored. In the present study juvenile coho were exposed to low levels of dissolved copper (5-20 microg/L for 3 h) and then presented with cues signaling the proximity of a predator. Unexposed coho showed a sharp reduction in swimming activity in response to both conspecific skin extract and the upstream presence of a cutthroat trout predator (O. clarki clarki) previously fed juvenile coho. This alarm response was absent in prey fish that were exposed to copper. Moreover, cutthroat trout were more effective predators on copper-exposed coho during predation trials, as measured by attack latency, survival time, and capture success rate. The shift in predator-prey dynamics was similar when predators and prey were co-exposed to copper. Overall, we show that copper-exposed coho are unresponsive to their chemosensory environment, unprepared to evade nearby predators, and significantly less likely to survive an attack sequence. Our findings contribute to a growing understanding of how common environmental contaminants alter the chemical ecology of aquatic communities.

Very low embryonic crude oil exposures cause lasting cardiac defects in salmon and herring.

The 1989 Exxon Valdez disaster exposed embryos of pink salmon and Pacific herring to crude oil in shoreline spawning habitats throughout Prince William Sound, Alaska. The herring fishery collapsed four years later. The role of the spill, if any, in this decline remains one of the most controversial unanswered questions in modern natural resource injury assessment. Crude oil disrupts excitation-contraction coupling in fish heart muscle cells, and we show here that salmon and herring exposed as embryos to trace levels of crude oil grow into juveniles with abnormal hearts and reduced cardiorespiratory function, the latter a key determinant of individual survival and population recruitment. Oil exposure during cardiogenesis led to specific defects in the outflow tract and compact myocardium, and a hypertrophic response in spongy myocardium, evident in juveniles 7 to 9 months after exposure. The thresholds for developmental cardiotoxicity were remarkably low, suggesting the scale of the Exxon Valdez impact in shoreline spawning habitats was much greater than previously appreciated. Moreover, an irreversible loss of cardiac fitness and consequent increases in delayed mortality in oil-exposed cohorts may have been important contributors to the delayed decline of pink salmon and herring stocks in Prince William Sound.

Dose‐additive inhibition of chinook salmon acetylcholinesterase activity by mixtures of organophosphate and carbamate insecticides

Organophosphate and carbamate insecticides are widely detected in surface waters of the western United States. These chemicals interfere with acetylcholine-mediated synaptic transmission in the nervous systems of fish and other aquatic animals via the inhibition of AChE (acetylcholinesterase) enzyme activity. Anticholinesterase insecticides commonly co-occur in the environment. This raises the possibility of antagonistic, additive, or synergistic neurotoxicity in exposed fish, including threatened and endangered species of Pacific salmon. We extracted AChE from the olfactory nervous system of chinook salmon (Oncorhynchus tshawytscha) and investigated the inhibitory effects of organophosphates (the oxon derivatives of diazinon, chlorpyrifos, and malathion) and carbamates (carbaryl and carbofuran), alone and in two-way combinations. We found that the joint toxicity of anticholinesterase mixtures can be accurately predicted from the inhibitory potencies of individual chemicals within a mixture. This indicates that organophosphate and carbamate insecticides are noninteractive in terms of AChE inhibition and that it might be possible to estimate the cumulative neurotoxicity of mixtures by simple dose addition. Because organophosphates and carbamates are likely to have additive effects on the neurobehavior of salmon under natural exposure conditions, ecological risk assessments that focus on individual anticholinesterases might underestimate the actual risk to salmon in watersheds in which mixtures of these chemicals occur.