Dietary Exposure to Environmentally Relevant Levels of Chemical Contaminants Reduces Growth and Survival in Juvenile Chinook Salmon.

Chemical pollution can degrade aquatic ecosystems. Chinook salmon in contaminated habitats are vulnerable to health impacts from toxic exposures. Few studies have been conducted on adverse health outcomes associated with current levels and mixtures of contaminants. Fewer still address effects specific to the juvenile life-stage of salmonids. The present study evaluated contaminant-related effects from dietary exposure to environmentally relevant concentrations and mixture profiles in juvenile Chinook salmon from industrialized waterways in the U.S. Pacific Northwest using two end points: growth assessment and disease susceptibility. The dose and chemical proportions were reconstituted based on environmental sampling and analysis using the stomach contents of juvenile Chinook salmon recently collected from contaminated, industrialized waterways. Groups of fish were fed a mixture with fixed proportions of 10 polychlorinated biphenyls (PCBs), 3 dichlorodiphenyltrichloroethanes (DDTs), and 13 polycyclic aromatic hydrocarbons (PAHs) at five concentrations for 35 days. These contaminant compounds were selected because of elevated concentrations and the widespread presence in sediments throughout industrialized waterways. Fork length and otolith microstructural growth indicators were significantly reduced in fish fed environmentally relevant concentrations of these contaminants. In addition, contaminant-exposed Chinook salmon were more susceptible to disease during controlled challenges with the pathogen Aeromonas salmonicida. Our results indicate that dietary exposure to contaminants impairs growth and immune function in juvenile Chinook salmon, thereby highlighting that current environmental exposure to chemicals of potential management concern threatens the viability of exposed salmon.

Roads to ruin: conservation threats to a sentinel species across an urban gradient.

Urbanization poses a global challenge to species conservation. This is primarily understood in terms of physical habitat loss, as agricultural and forested lands are replaced with urban infrastructure. However, aquatic habitats are also chemically degraded by urban development, often in the form of toxic stormwater runoff. Here we assess threats of urbanization to coho salmon throughout developed areas of the Puget Sound Basin in Washington, USA. Puget Sound coho are a sentinel species for freshwater communities and also a species of concern under the U.S. Endangered Species Act. Previous studies have demonstrated that stormwater runoff is unusually lethal to adult coho that return to spawn each year in urban watersheds. To further explore the relationship between land use and recurrent coho die-offs, we measured mortality rates in field surveys of 51 spawning sites across an urban gradient. We then used spatial analyses to measure landscape attributes (land use and land cover, human population density, roadways, traffic intensity, etc.) and climatic variables (annual summer and fall precipitation) associated with each site. Structural equation modeling revealed a latent urbanization gradient that was associated with road density and traffic intensity, among other variables, and positively related to coho mortality. Across years within sites, mortality increased with summer and fall precipitation, but the effect of rainfall was strongest in the least developed areas and was essentially neutral in the most urbanized streams. We used the best-supported structural equation model to generate a predictive mortality risk map for the entire Puget Sound Basin. This map indicates an ongoing and widespread loss of spawners across much of the Puget Sound population segment, particularly within the major regional north-south corridor for transportation and development. Our findings identify current and future urbanization-related threats to wild coho, and show where green infrastructure and similar clean water strategies could prove most useful for promoting species conservation and recovery.

Unexpectedly high mortality in Pacific herring embryos exposed to the 2007 Cosco Busan oil spill in San Francisco Bay.

In November 2007, the container ship Cosco Busan released 54,000 gallons of bunker fuel oil into San Francisco Bay. The accident oiled shoreline near spawning habitats for the largest population of Pacific herring on the west coast of the continental United States. We assessed the health and viability of herring embryos from oiled and unoiled locations that were either deposited by natural spawning or incubated in subtidal cages. Three months after the spill, caged embryos at oiled sites showed sublethal cardiac toxicity, as expected from exposure to oil-derived polycyclic aromatic compounds (PACs). By contrast, embryos from the adjacent and shallower intertidal zone showed unexpectedly high rates of tissue necrosis and lethality unrelated to cardiotoxicity. No toxicity was observed in embryos from unoiled sites. Patterns of PACs at oiled sites were consistent with oil exposure against a background of urban sources, although tissue concentrations were lower than expected to cause lethality. Embryos sampled 2 y later from oiled sites showed modest sublethal cardiotoxicity but no elevated necrosis or mortality. Bunker oil contains the chemically uncharacterized remains of crude oil refinement, and one or more of these unidentified chemicals likely interacted with natural sunlight in the intertidal zone to kill herring embryos. This reveals an important discrepancy between the resolving power of current forensic analytical chemistry and biological responses of keystone ecological species in oiled habitats. Nevertheless, we successfully delineated the biological impacts of an oil spill in an urbanized coastal estuary with an overlapping backdrop of atmospheric, vessel, and land-based sources of PAC pollution.

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.

Interactive neurobehavioral toxicity of diazinon, malathion, and ethoprop to juvenile coho salmon.

In western North America, mixtures of current use pesticides have been widely detected in streams and other aquatic habitats for threatened and endangered Pacific salmon and steelhead (Oncorhynchus sp.). These include organophosphate insecticides that inhibit acetylcholinesterase (AChE) enzyme activity in the salmon nervous system, thereby disrupting swimming and feeding behaviors. Several organophosphates have been shown to interact as mixtures to produce synergistic AChE inhibition at concentrations near or above the upper range of surface water detections in freshwater systems. To evaluate potential synergism at lower concentrations (near or below 1 part per billion), juvenile coho (Oncorhynchus kisutch) were exposed to a range of mixtures of diazinon-malathion and ethoprop-malathion below a cumulative 0.05 of the predicted EC50 for AChE inhibition, as determined from single chemical concentration-response curves. Brain enzyme inhibition was concentration-dependent, with a 90% reduction and a significant decrease in spontaneous swimming speed at the highest binary mixture concentrations evaluated (diazinon-malathion at 2.6 and 1.1 µg/L, respectively; ethoprop-malathion at 2.8 and 1.2 µg/L, respectively). Brain enzyme activity gradually recovered over six weeks. Our findings extend earlier observations of organophosphate synergism in salmon and reveal an unusually steep concentration-response relationship across a mere 2-fold increase in mixture concentration.

Crude oil exposure of early life stages of Atlantic haddock suggests threshold levels for developmental toxicity as low as 0.1 µg total polyaromatic hydrocarbon (TPAH)/L.

Atlantic haddock (Melanogrammus aeglefinus) embryos bind dispersed crude oil droplets to the eggshell and are consequently highly susceptible to toxicity from spilled oil. We established thresholds for developmental toxicity and identified any potential long-term or latent adverse effects that could impair the growth and survival of individuals. Embryos were exposed to oil for eight days (10, 80 and 300 µg oil/L, equivalent to 0.1, 0.8 and 3.0 µg TPAH/L). Acute and delayed mortality were observed at embryonic, larval, and juvenile stages with IC50 = 2.2, 0.39, and 0.27 µg TPAH/L, respectively. Exposure to 0.1 µg TPAH/L had no negative effect on growth or survival. However, yolk sac larvae showed significant reduction in the outgrowth (ballooning) of the cardiac ventricle in the absence of other extracardiac morphological defects. Due to this propensity for latent sublethal developmental toxicity, we recommend an effect threshold of 0.1 µg TPAH/L for risk assessment models.

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.

Sublethal neurotoxicity of organophosphate insecticides to juvenile coho salmon.

For decades, organophosphate (OP) insecticides have been used as chemical control agents in watersheds that support at-risk populations of Pacific salmon throughout western North America. Spray drift, runoff, and other processes transport OPs to critical surface water habitats for migratory salmonids. While most OPs share a common mechanism of action (i.e., inhibition of neuronal acetylcholinesterase, or AChE), they typically vary in toxic potency. Moreover, dose-response relationships for exposure and sublethal neurotoxicity (e.g., brain AChE inhibition) in salmonids have not been defined for many OPs. Here we exposed juvenile coho salmon (Oncorhynchus kisutch) to five common anticholinesterase insecticides (dimethoate, ethoprop, naled, phorate and phosmet) that are widely used on agricultural, commercial, residential, and public lands. Each of the five pesticides produced a concentration-dependent inhibition of AChE enzyme activity. The effective concentration for 50 % AChE inhibition (96-hr EC50) indicated the highest toxicity for phorate (EC50 = 0.57 µg/L) followed by phosmet (3.3 µg/L), naled (7.8 µg/L), ethoprop (90.6 µg/L) and dimethoate (273 µg/L). These findings can inform 1) relative hazard analyses for OP use near sensitive aquatic habitats, 2) predictions of sublethal OP mixture toxicity, and 3) ecological risk assessments for threatened or endangered species of Pacific salmon.

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.

Effects of water hardness, alkalinity, and dissolved organic carbon on the toxicity of copper to the lateral line of developing fish.

Conventional water chemistry parameters such as hardness, alkalinity, and organic carbon are known to affect the acutely lethal toxicity of copper to fish and other aquatic organisms. In the present study, we investigate the influence of these water chemistry parameters on short-term (3 h), sublethal (0-40 microg/L) copper toxicity to the peripheral mechanosensory system of larval zebrafish (Danio rerio) using an in vivo fluorescent marker of lateral line sensory neuron (hair cell) integrity. We studied the influence of hardness (via CaCl2, MgSO4, or both at a 2:1 molar ratio), sodium (via NaHCO3 or NaCl), and organic carbon on copper-induced neurotoxicity to zebrafish lateral line neurons over a range of environmentally relevant water chemistries. For all water parameters but organic carbon, the reductions in copper toxicity, although statistically significant, were small. Increasing organic carbon across a range of environmentally relevant concentrations (0.1-4.3 mg/L) increased the EC50 for copper toxicity (the effective concentration resulting in a 50% loss of hair cells) from approximately 12 microg/L to approximately 50 microg/L. Finally, we used an ionoregulatory-based biotic ligand model to compare copper toxicity mediated by targets in the fish gill and lateral line. Relative to copper toxicity via the gill, we find that individual water chemistry parameters are less influential in terms of reducing cytotoxic impacts to the mechanosensory system.

Crude oil cardiotoxicity to red drum embryos is independent of oil dispersion energy.

The potential bioavailability of toxic chemicals from oil spills to water column organisms such as fish embryos may be influenced by physical dispersion along an energy gradient. For example, a surface slick with minimal wave action (low energy) could potentially produce different toxic effects from high energy situations such as pressurized discharge from a blown wellhead. Here we directly compared the toxicity of water accommodated fractions (WAFs) of oil prepared with low and high mixing energy (LEWAFs and HEWAFs, respectively) using surface oil samples collected during the 2010 Deepwater Horizon spill, and embryos of a representative nearshore species, red drum (Sciaenops ocellatus). Biological effects of each WAF type was quantified with several functional and morphological indices of developmental cardiotoxicity, providing additional insight into species-specific responses to oil exposure. Although the two WAF preparations yielded different profiles of polycyclic aromatic hydrocarbons (PAHs), cardiotoxic phenotypes were essentially identical. Based on benchmark thresholds for both morphological and functional cardiotoxicity, in general LEWAFs had lower thresholds for these phenotypes than HEWAFs based on total PAH measures. However, HEWAF and LEWAF toxicity thresholds were more similar when calculated based on estimates of dissolved PAHs only. Differences in thresholds were attributable to the weathering state of the oil samples.

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.

Coho salmon spawner mortality in western US urban watersheds: bioinfiltration prevents lethal storm water impacts.

Adult coho salmon Oncorhynchus kisutch return each autumn to freshwater spawning habitats throughout western North America. The migration coincides with increasing seasonal rainfall, which in turn increases storm water run-off, particularly in urban watersheds with extensive impervious land cover. Previous field assessments in urban stream networks have shown that adult coho are dying prematurely at high rates (>50%). Despite significant management concerns for the long-term conservation of threatened wild coho populations, a causal role for toxic run-off in the mortality syndrome has not been demonstrated.We exposed otherwise healthy coho spawners to: (i) artificial storm water containing mixtures of metals and petroleum hydrocarbons, at or above concentrations previously measured in urban run-off; (ii) undiluted storm water collected from a high traffic volume urban arterial road (i.e. highway run-off); and (iii) highway run-off that was first pre-treated via bioinfiltration through experimental soil columns to remove pollutants.We find that mixtures of metals and petroleum hydrocarbons - conventional toxic constituents in urban storm water - are not sufficient to cause the spawner mortality syndrome. By contrast, untreated highway run-off collected during nine distinct storm events was universally lethal to adult coho relative to unexposed controls. Lastly, the mortality syndrome was prevented when highway run-off was pretreated by soil infiltration, a conventional green storm water infrastructure technology.Our results are the first direct evidence that: (i) toxic run-off is killing adult coho in urban watersheds, and (ii) inexpensive mitigation measures can improve water quality and promote salmon survival. Synthesis and applications. Coho salmon, an iconic species with exceptional economic and cultural significance, are an ecological sentinel for the harmful effects of untreated urban run-off. Wild coho populations cannot withstand the high rates of mortality that are now regularly occurring in urban spawning habitats. Green storm water infrastructure or similar pollution prevention methods should be incorporated to the maximal extent practicable, at the watershed scale, for all future development and redevelopment projects, particularly those involving transportation infrastructure.

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.

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.

Corresponding morphological and molecular indicators of crude oil toxicity to the developing hearts of mahi mahi.

Crude oils from distinct geological sources worldwide are toxic to developing fish hearts. When oil spills occur in fish spawning habitats, natural resource injury assessments often rely on conventional morphometric analyses of heart form and function. The extent to which visible indicators correspond to molecular markers for cardiovascular stress is unknown for pelagic predators from the Gulf of Mexico. Here we exposed mahi (Coryphaena hippurus) embryos to field-collected crude oil samples from the 2010 Deepwater Horizon disaster. We compared visible heart defects (edema, abnormal looping, reduced contractility) to changes in expression of cardiac-specific genes that are diagnostic of heart failure in humans or associated with loss-of-function zebrafish cardiac mutants. Mahi exposed to crude oil during embryogenesis displayed typical symptoms of cardiogenic syndrome as larvae. Contractility, looping, and circulatory defects were evident, but larval mahi did not exhibit downstream craniofacial and body axis abnormalities. A gradation of oil exposures yielded concentration-responsive changes in morphometric and molecular responses, with relative sensitivity being influenced by age. Our findings suggest that 1) morphometric analyses of cardiac function are more sensitive to proximal effects of crude oil-derived chemicals on the developing heart, and 2) molecular indicators reveal a longer-term adverse shift in cardiogenesis trajectory.

Stereotyped neuropil branching of an identified stomatogastric motor neuron.

Anatomical studies of the crab stomatogastric ganglion (STG) have suggested only minimal organization within the neuropil of this structure. Here, we present evidence that, for at least one intrinsic neuron type, the ventricular dilator (VD) neuron, a highly organized and stereotyped branching structure exists within the stomatogastric neuropil. Specifically, we show the morphology of the VD neuron consists of a single primary neurite that projects from the soma into the neuropil and bifurcates into a pair of subprimary neurites, which in turn exit the neuropilar region, one entering the left and the other the right medial ventricular nerve. Nearly all secondary neurite branching of the VD neuron is from the subprimary neurites. There are approximately 22 secondary branches/neuron (range 14-28), with no significant difference between the number of secondary branches off the right vs. the left subprimary neurite, although the ratio of secondary branches between subprimaries varies (range 0.4-1.6). The fine neurites that branch from the secondary processes segregate hemispherically within the neuropil, based on the subprimary neurite of origin. Within this hemispherical organization, another level of fine neurite segregation is present, namely, the fine neurites derived from each secondary branch are restricted to discrete regions of the hemisphere with only minimal overlap with those derived from other secondary branches. Monte Carlo simulations show that this segregation differs significantly from a random distribution. The organization of branching seen in the VD neuron may play a critical role in the electrotonic and local computational organization of this neuron and sets the stage for physiological experimentation addressing these issues.

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.

Elevated temperatures increase the toxicity of pesticide mixtures to juvenile coho salmon.

Pesticide mixtures and elevated temperatures are parallel freshwater habitat stressors for Pacific salmon in the western United States. Certain combinations of organophosphate (OP) insecticides are known to synergistically increase neurotoxicity in juvenile salmon. The chemicals interact to potentiate the inhibition of brain acetylcholinesterase (AChE) and disrupt swimming behavior. The metabolic activation and detoxification of OPs involve temperature-sensitive enzymatic processes. Salmon are ectothermic, and thus the degree of synergism may vary with ambient temperature in streams, rivers, and lakes. Here we assess the influence of water temperature (12-21°C) on the toxicity of ethoprop and malathion, alone and in combination, to juvenile coho salmon (Oncorhynchus kisutch). A mixture of ethoprop (0.9 µg/L) and malathion (0.75 µg/L) produced synergistic AChE inhibition at 12°C, and the degree of neurotoxicity approximately doubled with a modest temperature increase to 18°C. Slightly lower concentrations of ethoprop (0.5 µg/L) combined with malathion (0.4 µg/L) did not inhibit brain AChE activity but did produce a temperature-dependent reduction in liver carboxylesterase (CaE). The activity of CaE was very sensitive to the inhibitory effects of ethoprop alone and both ethoprop-malathion combinations across all temperatures. Our findings are an example of how non-chemical habitat attributes can increase the relative toxicity of OP mixtures. Surface temperatures currently exceed water quality criteria in many western river segments, and summer thermal extremes are expected to become more frequent in a changing climate. These trends reinforce the importance of pollution reduction strategies to enhance ongoing salmon conservation and recovery efforts.

Toward enhanced MIQE compliance: reference residual normalization of qPCR gene expression data.

Normalization of fluorescence-based quantitative real-time PCR (qPCR) data varies across quantitative gene expression studies, despite its integral role in accurate data quantification and interpretation. Identification of suitable reference genes plays an essential role in accurate qPCR normalization, as it ensures that uncorrected gene expression data reflect normalized data. The reference residual normalization (RRN) method presented here is a modified approach to conventional 2(-ΔΔCt)qPCR normalization that increases mathematical transparency and incorporates statistical assessment of reference gene stability. RRN improves mathematical transparency through the use of sample-specific reference residuals (RR i ) that are generated from the mean Ct of one or more reference gene(s) that are unaffected by treatment. To determine stability of putative reference genes, RRN uses ANOVA to assess the effect of treatment on expression and subsequent equivalence-threshold testing to establish the minimum permitted resolution. Step-by-step instructions and comprehensive examples that demonstrate the influence of reference gene stability on target gene normalization and interpretation are provided. Through mathematical transparency and statistical rigor, RRN promotes compliance with Minimum Information for Quantitative Experiments and, in so doing, provides increased confidence in qPCR data analysis and interpretation.