Ethanol and caffeine age-dependently alter brain and retinal neurochemical levels without affecting morphology of juvenile and adult zebrafish (Danio rerio).

Adolescent alcohol exposure in humans is predictive of adult development of alcoholism. In rodents, caffeine pre-exposure enhances adult responsiveness to ethanol via a pathway targeted by both compounds. Embryonic exposure to either compound adversely affects development, and both compounds can alter zebrafish behaviors. Here, we evaluate whether co-exposure to caffeine and/or alcohol in adolescence exerts neurochemical changes in retina and brain. Zebrafish (Danio rerio) were given daily 20 min treatments to ethanol (1.5% v/v), caffeine (25-100 mg/L), or caffeine + ethanol for 1 week during mid-late adolescence (53-92 days post fertilization (dpf)) or early adulthood (93-142 dpf). Immediately after exposure, anatomical measurements were taken, including weight, heart rate, pigment density, length, girth, gill width, inner and outer eye distance. Brain and retinal tissue were subsequently collected either (1) immediately, (2) after a short interval (2-4d) following exposure, or (3) after a longer interval that included an acute 1.5% ethanol challenge. Chronic ethanol and/or caffeine exposure did not alter anatomical parameters. However, retinal and brain levels of tyrosine hydroxylase were elevated in fish sacrificed after the long interval following exposure. Protein levels of glutamic acid decarboxylase were also increased, with the highest levels observed in 70-79 dpf fish exposed to caffeine. The influence of ethanol and caffeine exposure on neurochemistry demonstrates specificity of their effects during postembryonic development. Using the zebrafish model to assess neurochemistry relevant to reward and anxiety may inform understanding of the mechanisms that reinforce co-addiction to alcohol and stimulants.

Prolonged Hyperglycemia Causes Visual and Cognitive Deficits in Danio rerio.

The present study induced prolonged hyperglycemia (a hallmark symptom of Type 2 diabetes [T2DM]) in Danio rerio (zebrafish) for eight or twelve weeks. The goal of this research was to study cognitive decline as well as vision loss in hyperglycemic zebrafish. Fish were submerged in glucose for eight or twelve weeks, after which they were assessed with both a cognitive assay (three-chamber choice) and a visual assay (optomotor response (OMR)). Zebrafish were also studied during recovery from hyperglycemia. Here, fish were removed from the hyperglycemic environment for 4 weeks after either 4 or 8 weeks in glucose, and cognition and vision was again assessed. The 8- and 12-week cognitive results revealed that water-treated fish showed evidence of learning while glucose- and mannitol-treated fish did not within the three-day testing period. OMR results identified an osmotic effect with glucose-treated fish having significantly fewer positive rotations than water-treated fish but comparable rotations to mannitol-treated fish. The 8- and 12-week recovery results showed that 4 weeks was not enough time to fully recovery from the hyperglycemic insult sustained.

Developmental effects of siloxane exposure in zebrafish: A comparison study using laboratory-mixed and environmental water samples.

Siloxanes are used in personal care, biomedical, and industrial products. Their worldwide use and persistence in the environment cause consistent exposure for both humans and aquatic animals. Two siloxane congeners, decamethylcyclopentasiloxane (D5; CAS 541-02-6) and octamethylcyclotetrasiloxane (D4; CAS 556-67-2), are among the most prevalent, with measurable levels in air, sediment, water, and biological samples. However, few studies have examined the impact of developmental (embryo/larva) exposure. To address this gap, we performed parallel experiments using wildtype zebrafish (Danio rerio). One set of experiments used laboratory-mixed individual solutions containing either D4, D5, or 2,4,6,8-tetramethylcyclotetrasiloxane (D4 H ; CAS 2370-88-9); the other used environmental water samples containing a mixture of siloxanes, including D4 and D5. These samples were collected from Bladensburg Waterfront Park (BWP) a site along the Anacostia River, Washington, DC. In both experiments, zebrafish (24-48 h postfertilization, hpf) were exposed until 7 or 14 days (d)pf. Chronic exposure to D4, D5, or BWP water until 7 dpf caused stress-like behaviors and reduced swim velocities; anatomical differences were noted only in BWP-exposed larvae. At 14 dpf, BWP-treated larvae still showed slower swimming velocities and increased immobility; anatomical differences were no longer evident and thigmotactic behavior was reduced. D4 and D5-exposed larvae did not survive after 10 dpf. Larvae exposed to D4 H showed no decreases in behavior or growth at either age. These results suggest early developmental sensitivity to siloxane exposure and point to the need to consider embryonic/larval endpoints when assessing aquatic contaminants.

Differential effects of fluoxetine on the phototactic behavior of 3 amphipod species (Crustacea; Amphipoda).

We document phototactic responses in different amphipod populations of Gammarus minus, Stygobromus tenuis, and Crangonyx shoemakeri, each collected at 2-3 sites within the Washington DC area. We then assessed how baseline phototaxis was altered following either short-term (3-week) or long-term (6-week) exposure to 0.05 µg/L or 0.5 µg/L fluoxetine. Our results classify all species as significantly photonegative, a response that depended solely on the presence, not quality, of light. Short-term fluoxetine exposure caused some animals to become photoneutral, regardless of concentration, while others remained photonegative. Long-term exposure to 0.5 µg/L fluoxetine caused photoneutral behaviors in all surviving populations; exposure to 0.05 µg/L had variable effects. These differential effects were due to a significant effect of population/sampling location on photobehavior. Overall, these results identify species-specific effects of chronic fluoxetine exposure and underscore how the response to light in 7 geographically distinct populations is uniquely tuned to requirements for survival.

The Role of Estrogen and Thyroid Hormones in Zebrafish Visual System Function.

Visual system development is a highly complex process involving coordination of environmental cues, cell pathways, and integration of functional circuits. Consequently, a change to any step, due to a mutation or chemical exposure, can lead to deleterious consequences. One class of chemicals known to have both overt and subtle effects on the visual system is endocrine disrupting compounds (EDCs). EDCs are environmental contaminants which alter hormonal signaling by either preventing compound synthesis or binding to postsynaptic receptors. Interestingly, recent work has identified neuronal and sensory systems, particularly vision, as targets for EDCs. In particular, estrogenic and thyroidogenic signaling have been identified as critical modulators of proper visual system development and function. Here, we summarize and review this work, from our lab and others, focusing on behavioral, physiological, and molecular data collected in zebrafish. We also discuss different exposure regimes used, including long-lasting effects of developmental exposure. Overall, zebrafish are a model of choice to examine the impact of EDCs and other compounds targeting estrogen and thyroid signaling and the consequences of exposure in visual system development and function.

Neurochemical and Behavioral Consequences of Ethanol and/or Caffeine Exposure: Effects in Zebrafish and Rodents.

Zebrafish are increasingly being utilized to model the behavioral and neurochemical effects of pharmaceuticals and, more recently, pharmaceutical interactions. Zebrafish models of stress establish that both caffeine and ethanol influence anxiety, though few studies have implemented coadministration to assess the interaction of anxiety and reward-seeking. Caffeine exposure in zebrafish is teratogenic, causing developmental abnormalities in the cardiovascular, neuromuscular, and nervous systems of embryos and larvae. Ethanol is also a teratogen and, as an anxiolytic substance, may be able to offset the anxiogenic effects of caffeine. Co-exposure to caffeine and alcohol impacts neuroanatomy and behavior in adolescent animal models, suggesting stimulant substances may moderate the impact of alcohol on neural circuit development. Here, we review the literature describing neuropharmacological and behavioral consequences of caffeine and/or alcohol exposure in the zebrafish model, focusing on neurochemistry, locomotor effects, and behavioral assessments of stress/anxiety as reported in adolescent/juvenile and adult animals. The purpose of this review is twofold: (1) describe the work in zebrafish documenting the effects of ethanol and/or caffeine exposure and (2) compare these zebrafish studies with comparable experiments in rodents. We focus on specific neurochemical pathways (dopamine, serotonin, adenosine, GABA), anxiety-type behaviors (assessed with a novel tank, thigmotaxis, shoaling), and locomotor changes resulting from both individual and co-exposure. We compare findings in zebrafish with those in rodent models, revealing similarities across species and identifying conservation of mechanisms that potentially reinforce coaddiction.

Transient developmental exposure to tributyltin reduces optomotor responses in larval zebrafish (Danio rerio).

This study determined the effects of transient developmental exposure to tributyltin (TBT), a well-known anti-estrogenic environmental endocrine disrupting compound, on visual system development of larval zebrafish (Danio rerio). Zebrafish were exposed to either 0.2 µg/L or 20 µg/L TBT for 24 h when they were aged 24 h postfertilization (hpf), 72 hpf, or 7 days (d)pf. Immediately after exposure, larvae were transferred to system water for seven days of recovery followed by behavioral testing (startle and optomotor responses) and morphological assessment. TBT-treated larvae displayed age-dependent changes in morphology characterized by delayed/reduced growth and susceptibility to exposure. TBT exposure reduced the number of larvae displaying optomotor responses regardless of age of exposure; eye diameter was also decreased when exposure occurred at 24 hpf or 7 dpf. Startle responses were reduced only in TBT-treated larvae exposed when they were 24 hpf, suggesting transient TBT exposure during the early larval period may cause vision-specific effects.

Alternate Immersion in Glucose to Produce Prolonged Hyperglycemia in Zebrafish.

Zebrafish (Danio rerio) are an excellent model to investigate the effects of chronic hyperglycemia, a hallmark of Type II Diabetes Mellitus (T2DM). This alternate immersion protocol is a noninvasive, step-wise method of inducing hyperglycemia for up to eight weeks. Adult zebrafish are alternately exposed to sugar (glucose) and water for 24 hours each. The zebrafish begin treatment in a 1% glucose solution for 2 weeks, then a 2% solution for 2 weeks, and finally a 3% solution for the remaining 4 weeks. Compared to water-treated (stress) and mannitol-treated (osmotic) controls, glucose-treated zebrafish have significantly higher blood sugar levels. The glucose-treated zebrafish show blood sugar levels of 3-times that of controls, suggesting that after both four and eight weeks hyperglycemia can be achieved. Sustained hyperglycemia was associated with increased Glial Fibrillary Acidic Protein (GFAP) and increased nuclear factor Kappa B (NF-kB) levels in retina and decreased physiological responses, as well as cognitive deficits suggesting this protocol can be used to model disease complications.

The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System.

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or cognitive impairment. We have modified and developed a three-chamber choice cognitive task similar to that used with rodents for use with hyperglycemic zebrafish. The testing chamber consists of a centrally located starting chamber and two choice compartments on either side, with a shoal of conspecifics used as the reward. We provide data showing that once acquired, zebrafish remember the task at least 8 weeks later. Our data indicate that zebrafish respond robustly to this reward, and we have identified cognitive deficits in hyperglycemic fish after 4 weeks of treatment. This behavioral assay may also be applicable to other studies related to cognition and memory.

Zebrafish Optomotor Response and Morphology Are Altered by Transient, Developmental Exposure to Bisphenol-A.

Estrogen-specific endocrine disrupting compounds (EDCs) are potent modulators of neural and visual development and common environmental contaminants. Using zebrafish, we examined the long-term impact of abnormal estrogenic signaling by testing the effects of acute, early exposure to bisphenol-A (BPA), a weak estrogen agonist, on later visually guided behaviors. Zebrafish aged 24 h postfertilization (hpf), 72 hpf, and 7 days postfertilization (dpf) were exposed to 0.001 µM or 0.1 µM BPA for 24 h, and then allowed to recover for 1 or 2 weeks. Morphology and optomotor responses (OMRs) were assessed after 1 and 2 weeks of recovery for 24 hpf and 72 hpf exposure groups; 7 dpf exposure groups were additionally assessed immediately after exposure. Increased notochord length was seen in 0.001 µM exposed larvae and decreased in 0.1 µM exposed larvae across all age groups. Positive OMR was significantly increased at 1 and 2 weeks post-exposure in larvae exposed to 0.1 µM BPA when they were 72 hpf or 7 dpf, while positive OMR was increased after 2 weeks of recovery in larvae exposed to 0.001 µM BPA at 72 hpf. A time-delayed increase in eye diameter occurred in both BPA treatment groups at 72 hpf exposure; while a transient increase occurred in 7 dpf larvae exposed to 0.1 µM BPA. Overall, short-term developmental exposure to environmentally relevant BPA levels caused concentration- and age-dependent effects on zebrafish visual anatomy and function.

Observational learning and irreversible starvation in first-feeding zebrafish larvae: is it okay to copy from your friends?

Starvation is one cause of high mortality during the early life stages of many fish species. If larvae do not learn to feed, or if no food is available during early stages, irreversible starvation occurs and larvae reach the Point of No Return (PNR), the developmental period/age when they will not feed even if food is available. Fish larvae may learn to how to feed by observing conspecifics or through personal/individual experience with prey items that are encountered. We examined food acquisition in first-feeding zebrafish larvae to determine the impact of delayed feeding and identify the time of irreversible starvation and the PNR. Next, we examined how feeding ability, and the PNR, is altered by either observational learning or previous experience, to determine which paradigm facilitates successful feeding.Our data indicate that zebrafish larvae learn to feed, with the PNR at 7-8 days postfertilization (dpf). Exposure to prey items immediately after hatching (3-5 dpf) results in the highest survival rates. Zebrafish larvae learning to feed by observing conspecifics also had high survival, though the PNR was not changed. In contrast, previous experience with prey items caused an earlier PNR and reduced survival. Overall, these results that indicate feeding is a learned behavior in zebrafish larvae and interacting with/observing conspecifics during the early larval period is a better predictor of feeding ability than previous experience with food.

Using a variant of the optomotor response as a visual defect detection assay in zebrafish.

We describe a visual stimulus that can be used with both larval and adult zebrafish (Danio rerio). This protocol is a modification of a standard visual behavior analysis, the optomotor response (OMR). The OMR is often used to determine the spatial response or to detect directional visuomotor deficiencies. An OMR can be generated using a high contrast grated pattern, typically vertical bars. The spatial sensitivity is measured by detection and response to a change in grating bar width and is reported in cycles per degree (CPD). This test has been used extensively with zebrafish larvae and adults to identify visual- and/or motor-based mutations. Historically, when tested in adults, the grated pattern was presented from a vertical perspective, using a rotating cylinder around a holding tank, allowing the grating to be seen solely from the sides and front of the organism. In contrast, OMRs in zebrafish larvae are elicited using a stimulus projected below the fish. This difference in methodology means that two different experimental set-ups are required: one for adults and one for larvae. Our visual stimulus modifies the stimulation format so that a single OMR stimulus, suitable for use with both adults and larvae, is being presented underneath the fish. Analysis of visuomotor responses using this method does not require costly behavioral tracking software and, using a single behavioral paradigm, allows the observer to rapidly determine visual spatial response in both zebrafish larvae and adults.

Anatomical and Behavioral Assessment of Larval Zebrafish (Danio rerio) Reared in Anacostia River Water Samples.

Rapid urbanization, industrial activity, and runoff have all played a role in transforming the Anacostia River from a biologically rich ecosystem to an ecologically threatened environment facing extensive pollution. In recent decades, numerous groups have worked to document and begin to address pollution in the waterway, but few have examined the biological impact of these contaminants. To assess water quality, the current study examined the effects of Anacostia water on early fish development and behavior using zebrafish (Danio rerio). Zebrafish embryos and larvae were reared in water samples collected from the Washington Navy Yard from 0-30dpf (days post fertilization). At 7, 15, 20, and 30dpf, larvae were subsampled for morphological (length, girth, eye diameter, inter-eye distance) and behavioral (angular velocity, total distance traveled, swimming velocity, total activity duration, time immobile, frequency and duration of burst swimming, time at the edge of the dish) assessment. Water samples were processed using gas chromatography-mass spectroscopy (GC-MS) to identify major organic contaminants. Results indicated the presence of 13 bioactive organic contaminants, including siloxane species and hormone derivatives, and accelerated growth and altered swim behaviors in Anacostia-exposed fish after 30 days of exposure. These findings emphasize sublethal but significant impacts of exposure to organic contaminants experienced by fish residing in urban waterways.

Acute exposure to 4-OH-A, not PCB1254, alters brain aromatase activity but does not adversely affect growth in zebrafish.

Acute developmental exposure to pharmaceuticals or environmental contaminants can have deleterious, long lasting effects. Many of these compounds are endocrine disruptors (EDCs) that target estrogen signaling, with effects on reproductive and non-reproductive tissues. We recently reported that zebrafish larvae transiently exposed to the pharmaceutical EDC 4-OH-A display visual deficits as adults. Here, we examine whether these long-term effects are due to compound-induced morphological and/or cellular changes. Zebrafish aged 24 h, 48 h, 72 h, or 7 days post-fertilization (larvae) or 3-4mos (adults) were exposed to either 4-OH-A or PCB1254 for 24 h. After that time, notochord length, eye diameter, inter-eye distance, and heart rate were measured from larvae; and aromatase (estrogen synthase) activity was measured in homogenates of adult brain tissue. In general, indices of larval growth and development were not altered by 24 h exposure to either compound. 4-OH-A potently inhibited aromatase activity, while PCB1254 did not, with inhibition continuing even after removal from treatment. These results support differential function of EDCs and indicate that developmental exposure to 4-OH-A causes sustained inhibition of aromatase, which could be associated with altered adult behaviors.

Color Processing in Zebrafish Retina.

Zebrafish (Danio rerio) is a model organism for vertebrate developmental processes and, through a variety of mutant and transgenic lines, various diseases and their complications. Some of these diseases relate to proper function of the visual system. In the US, the National Eye Institute indicates >140 million people over the age of 40 have some form of visual impairment. The causes of the impairments range from refractive error to cataract, diabetic retinopathy and glaucoma, plus heritable diseases such as retinitis pigmentosa and color vision deficits. Most impairments directly affect the retina, the nervous tissue at the back of the eye. Zebrafish with long or short-wavelength color blindness, altered retinal anatomy due to hyperglycemia, high intraocular pressure, and reduced pigment epithelium are all used, and directly applicable, to study how these symptoms affect visual function. However, many published reports describe only molecular/anatomical/structural changes or behavioral deficits. Recent work in zebrafish has documented physiological responses of the different cell types to colored (spectral) light stimuli, indicating a complex level of information processing and color vision in this species. The purpose of this review article is to consolidate published morphological and physiological data from different cells to describe how zebrafish retina is capable of complex visual processing. This information is compared to findings in other vertebrates and relevance to disorders affecting color processing is discussed.

One month of hyperglycemia alters spectral responses of the zebrafish photopic electroretinogram.

Prolonged hyperglycemia can alter retinal function, ultimately resulting in blindness. Adult zebrafish adults exposed to alternating conditions of 2% glucose/0% glucose display a 3× increase in blood sugar levels. After 4 weeks of treatment, electroretinograms (ERGs) were recorded from isolated, perfused, in vitro eyecups. Control animals were exposed to alternating 2% mannitol/0% mannitol (osmotic control) or to alternating water (0% glucose/0% glucose; handling control). Two types of ERGs were recorded: (1) native ERGs measured using white-light stimuli and medium without synaptic blockers; and (2) spectral ERGs measured with an AMPA/kainate receptor antagonist, isolating photoreceptor-to-ON-bipolar-cell synapses, and a spectral protocol that separated red (R), green (G), blue (B) and UV cone signals. Retinas were evaluated for changes in layer thickness and for the inflammatory markers GFAP and Nf-κB (RelA or p65). In native ERGs, hyperglycemic b- and d-waves were lower in amplitude than the b- and d-waves of mannitol controls. Alteration of waveshape became severe, with b-waves becoming more transient and ERG responses showing more PIII-like (a-wave) characteristics. For spectral ERGs, waveshape appeared similar in all treatment groups. However, a1- and b2-wave implicit times were significantly longer, and amplitudes were significantly reduced, in response to hyperglycemic treatment, owing to the functional reduction in signals from R, G and B cones. Nf-κB increased significantly in hyperglycemic retinas, but the increase in GFAP was not significant and retinal layer thickness was unaffected. Thus, prolonged hyperglycemia triggers an inflammatory response and functional deficits localized to specific cone types, indicating the rapid onset of neural complications in the zebrafish model of diabetic retinopathy.

Acute developmental exposure to 4-hydroxyandrostenedione has a long-term effect on visually-guided behaviors.

Estrogenic and anti-estrogenic endocrine disrupting compounds (EDCs) are recognized as critical modulators of neural development, including sensory system development. Using the zebrafish model, we tested the effect of transient developmental exposure to a known anti-estrogenic EDC on adult visually-guided behavior. In particular, we exposed zebrafish aged 24-hour post-fertilization (hpf), 72 hpf, or 7-days post-fertilization (dpf) to the aromatase inhibitor 4-hydroxyandrostenedione (4-OH-A) for 24h. After this time, the fish were removed from treatment, placed into control conditions, and reared until adulthood (3-4months) when visually-guided optomotor responses (OMR) were assessed. Our results show significant decreases in positive OMR in adults exposed to 4-OH-A at 72 hpf and 7 dpf. These deficits were not accompanied by changes in overall swimming behaviors and startle responses, suggesting 4-OH-A specifically effected the visual system. Overall, this study identified long-term, quantifiable effects in visually-guided adult behaviors resulting from transient developmental exposure to the anti-estrogenic EDC, 4-OH-A. Further, these effects were noted when 4-OH-A exposure occurred after hatching, suggesting estrogen signaling is important for visual system maturation.

Differential behavioral effects of ethanol pre-exposure in male and female zebrafish (Danio rerio).

Alcohol exposure in adolescence is a contributing factor toward reward-seeking behavior in adulthood. This reward-seeking behavior is assessed in animal models using the conditioned place preference (CPP) paradigm. In this study, ethanol-induced change in time spent by zebrafish on the initially non-preferred tank side was studied by conditioning adult zebrafish to ethanol dissolved in water (0.00% 1.00%; 1.25%; 1.50%; 1.60%; 1.75% vol/vol) paired with an initially non-preferred environment. Following a single conditioning cycle, fish swam unrestricted in the CPP chamber to assess changes in preference. Daily 20-min pre-exposure to ethanol for 1 week during the juvenile stage starting at either 20days post fertilization (dpf) or 40 dpf altered percent time spent on the ethanol-paired side in adulthood in a dose-dependent and sex-dependent manner. The results suggest that male and female zebrafish are an effective model in which to investigate behavioral correlates of ethanol-induced changes in neural circuits implicated in reward and anxiety.

Mercury-induced epigenetic transgenerational inheritance of abnormal neurobehavior is correlated with sperm epimutations in zebrafish.

Methylmercury (MeHg) is a ubiquitous environmental neurotoxicant, with human exposures predominantly resulting from fish consumption. Developmental exposure of zebrafish to MeHg is known to alter their neurobehavior. The current study investigated the direct exposure and transgenerational effects of MeHg, at tissue doses similar to those detected in exposed human populations, on sperm epimutations (i.e., differential DNA methylation regions [DMRs]) and neurobehavior (i.e., visual startle and spontaneous locomotion) in zebrafish, an established human health model. F0 generation embryos were exposed to MeHg (0, 1, 3, 10, 30, and 100 nM) for 24 hours ex vivo. F0 generation control and MeHg-exposed lineages were reared to adults and bred to yield the F1 generation, which was subsequently bred to the F2 generation. Direct exposure (F0 generation) and transgenerational actions (F2 generation) were then evaluated. Hyperactivity and visual deficit were observed in the unexposed descendants (F2 generation) of the MeHg-exposed lineage compared to control. An increase in F2 generation sperm epimutations was observed relative to the F0 generation. Investigation of the DMRs in the F2 generation MeHg-exposed lineage sperm revealed associated genes in the neuroactive ligand-receptor interaction and actin-cytoskeleton pathways being effected, which correlate to the observed neurobehavioral phenotypes. Developmental MeHg-induced epigenetic transgenerational inheritance of abnormal neurobehavior is correlated with sperm epimutations in F2 generation adult zebrafish. Therefore, mercury can promote the epigenetic transgenerational inheritance of disease in zebrafish, which significantly impacts its environmental health considerations in all species including humans.

Developmental exposure to heavy metals alters visually-guided behaviors in zebrafish.

This laboratory-based study determined the consequences of heavy metal exposure using zebrafish Danio rerio. Embryos were transiently exposed to environmentally-relevant concentrations of cadmium or nickel until 72 h postfertilization (hpf), then they were returned to system water and allowed to grow until 7, 9, and 11 days postfertilization (dpf), when they were examined morphologically and behaviorally. Morphological measures of notochord length, eye diameter, and inter-eye distance were not different across treatments; however, significant differences in optomotor responses were observed in treated larvae at all ages tested. These results suggest that initial developmental exposure to cadmium and nickel significantly impacts visually-guided larval behavior. The absence of significant differences in gross morphology suggests that the effects of these metals are subtle and may occur at the cellular level. By using this ecologically relevant model and pollutant type, this study has broad application and implications with regard to safe levels of contaminant in drinking water and freshwater ecosystems.