Toxicological effects assessment for wildlife in the 21st century: Review of current methods and recommendations for a path forward.

Model species (e.g., granivorous gamebirds, waterfowl, passerines, domesticated rodents) have been used for decades in guideline laboratory tests to generate survival, growth, and reproductive data for prospective ecological risk assessments (ERAs) for birds and mammals, while officially adopted risk assessment schemes for amphibians and reptiles do not exist. There are recognized shortcomings of current in vivo methods as well as uncertainty around the extent to which species with different life histories (e.g., terrestrial amphibians, reptiles, bats) than these commonly used models are protected by existing ERA frameworks. Approaches other than validating additional animal models for testing are being developed, but the incorporation of such new approach methodologies (NAMs) into risk assessment frameworks will require robust validations against in vivo responses. This takes time, and the ability to extrapolate findings from nonanimal studies to organism- and population-level effects in terrestrial wildlife remains weak. Failure to adequately anticipate and predict hazards could have economic and potentially even legal consequences for regulators and product registrants. In order to be able to use fewer animals or replace them altogether in the long term, vertebrate use and whole organism data will be needed to provide data for NAM validation in the short term. Therefore, it is worth investing resources for potential updates to existing standard test guidelines used in the laboratory as well as addressing the need for clear guidance on the conduct of field studies. Herein, we review the potential for improving standard in vivo test methods and for advancing the use of field studies in wildlife risk assessment, as these tools will be needed in the foreseeable future. Integr Environ Assess Manag 2024;20:699-724. © 2023 His Majesty the King in Right of Canada and The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). Reproduced with the permission of the Minister of Environment and Climate Change Canada. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Wildlife ecological risk assessment in the 21st century: Promising technologies to assess toxicological effects.

Despite advances in toxicity testing and the development of new approach methodologies (NAMs) for hazard assessment, the ecological risk assessment (ERA) framework for terrestrial wildlife (i.e., air-breathing amphibians, reptiles, birds, and mammals) has remained unchanged for decades. While survival, growth, and reproductive endpoints derived from whole-animal toxicity tests are central to hazard assessment, nonstandard measures of biological effects at multiple levels of biological organization (e.g., molecular, cellular, tissue, organ, organism, population, community, ecosystem) have the potential to enhance the relevance of prospective and retrospective wildlife ERAs. Other factors (e.g., indirect effects of contaminants on food supplies and infectious disease processes) are influenced by toxicants at individual, population, and community levels, and need to be factored into chemically based risk assessments to enhance the "eco" component of ERAs. Regulatory and logistical challenges often relegate such nonstandard endpoints and indirect effects to postregistration evaluations of pesticides and industrial chemicals and contaminated site evaluations. While NAMs are being developed, to date, their applications in ERAs focused on wildlife have been limited. No single magic tool or model will address all uncertainties in hazard assessment. Modernizing wildlife ERAs will likely entail combinations of laboratory- and field-derived data at multiple levels of biological organization, knowledge collection solutions (e.g., systematic review, adverse outcome pathway frameworks), and inferential methods that facilitate integrations and risk estimations focused on species, populations, interspecific extrapolations, and ecosystem services modeling, with less dependence on whole-animal data and simple hazard ratios. Integr Environ Assess Manag 2024;20:725-748. © 2023 His Majesty the King in Right of Canada and The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). Reproduced with the permission of the Minister of Environment and Climate Change Canada. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Transdisciplinary and social-ecological health frameworks-Novel approaches to emerging parasitic and vector-borne diseases.

Ecosystem Health, Conservation Medicine, EcoHealth, One Health, Planetary Health and GeoHealth are inter-related disciplines that underpin a shared understanding of the functional prerequisites of health, sustainable vitality and wellbeing. All of these are based on recognition that health interconnects species across the planet, and they offer ways to more effectively tackle complex real-world challenges. Herein we present a bibliometric analysis to document usage of a subset of such terms by journals over time. We also provide examples of parasitic and vector-borne diseases, including malaria, toxoplasmosis, baylisascariasis, and Lyme disease. These and many other diseases have persisted, emerged or re-emerged, and caused great harm to human and animal populations in developed and low income, biodiverse nations around the world, largely because of societal drivers that undermined natural processes of disease prevention and control, which had developed through co-evolution over millennia. Shortcomings in addressing drivers has arisen from a lack or coordinated efforts among researchers, health stewards, societies at large, and governments. Fortunately, specialists collaborating under transdisciplinary and socio-ecological health umbrellas are increasingly integrating established and new techniques for disease modeling, prediction, diagnosis, treatment, control, and prevention. Such approaches often emphasize conservation of biodiversity for health protection, and they provide novel opportunities to increase the efficiency and probability of success.

One health-Transdisciplinary opportunities for SETAC leadership in integrating and improving the health of people, animals, and the environment.

One Health is a collaborative, transdisciplinary effort working locally, nationally, and globally to improve health for people, animals, plants, and the environment. The term is relatively new (from ∼2003), and it is increasingly common to see One Health included by name in interinstitutional research partnerships, conferences, communications, and organizational frameworks, particularly those championed by the human health and veterinary medical communities. Environmental quality is arguably the least developed component within the One Health framework, but can be guided by expertise within the Society of Environmental Toxicology and Chemistry (SETAC). Despite SETAC's long history of tripartite (academic, government, business) interdisciplinary environmental science activities, the term "One Health" is seldom used in SETAC communications (i.e., many of SETAC's activities are guided by One Health, but it is called by other names in SETAC's journals, newsletters, and presentations). Accordingly, the objective of this Focus article is to introduce the One Health concept to the SETAC membership. The article discusses the origins, evolution, and utility of the One Health approach as an organizational framework and provides key examples of ways in which SETAC expertise can benefit the One Health community. The authors assert that One Health needs SETAC and, to be most effective, SETAC needs One Health. Given that One Health to date has focused too little on the environment, on ecosystems, and on contaminants, SETAC's constructive involvement in One Health presents an opportunity to accelerate actions that will ultimately better protect human and ecosystem health. Environ Toxicol Chem 2016;35:2383-2391. © 2016 SETAC.

One health and cyanobacteria in freshwater systems: animal illnesses and deaths are sentinel events for human health risks.

Harmful cyanobacterial blooms have adversely impacted human and animal health for thousands of years. Recently, the health impacts of harmful cyanobacteria blooms are becoming more frequently detected and reported. However, reports of human and animal illnesses or deaths associated with harmful cyanobacteria blooms tend to be investigated and reported separately. Consequently, professionals working in human or in animal health do not always communicate findings related to these events with one another. Using the One Health concept of integration and collaboration among health disciplines, we systematically review the existing literature to discover where harmful cyanobacteria-associated animal illnesses and deaths have served as sentinel events to warn of potential human health risks. We find that illnesses or deaths among livestock, dogs and fish are all potentially useful as sentinel events for the presence of harmful cyanobacteria that may impact human health. We also describe ways to enhance the value of reports of cyanobacteria-associated illnesses and deaths in animals to protect human health. Efficient monitoring of environmental and animal health in a One Health collaborative framework can provide vital warnings of cyanobacteria-associated human health risks.

Predator diversity, intraguild predation, and indirect effects drive parasite transmission.

Humans are altering biodiversity globally and infectious diseases are on the rise; thus, there is interest in understanding how changes to biodiversity affect disease. Here, we explore how predator diversity shapes parasite transmission. In a mesocosm experiment that manipulated predator (larval dragonflies and damselflies) density and diversity, non-intraguild (non-IG) predators that only consume free-living cercariae (parasitic trematodes) reduced metacercarial infections in tadpoles, whereas intraguild (IG) predators that consume both parasites and tadpole hosts did not. This likely occurred because IG predators reduced tadpole densities and anticercarial behaviors, increasing per capita exposure rates of the surviving tadpoles (i.e., via density- and trait-mediated effects) despite the consumption of parasites. A mathematical model demonstrated that non-IG predators reduce macroparasite infections, but IG predation weakens this "dilution effect" and can even amplify parasite burdens. Consistent with the experiment and model, a wetland survey revealed that the diversity of IG predators was unrelated to metacercarial burdens in amphibians, but the diversity of non-IG predators was negatively correlated with infections. These results are strikingly similar to generalities that have emerged from the predator diversity-pest biocontrol literature, suggesting that there may be general mechanisms for pest control and that biocontrol research might inform disease management and vice versa. In summary, we identified a general trait of predators--where they fall on an IG predation continuum--that predicts their ability to reduce infections and possibly pests in general. Consequently, managing assemblages of predators represents an underused tool for the management of human and wildlife diseases and pest populations.

The feline thyroid gland: a model for endocrine disruption by polybrominated diphenyl ethers (PBDEs)?

The role of polybrominated diphenyl ethers (PBDE) was investigated in the occurrence of feline hyperthyroidism (FH) by evaluating 15 PBDE congeners in serum from 62 client-owned (21 euthyroid, 41 hyperthyroid) and 10 feral cats. Total serum PBDE concentrations in euthyroid cats were not significantly different from those of hyperthyroid cats. Total serum PBDE in feral cats were significantly lower than in either of the groups of client-owned cats. Total serum PBDE did not correlate with serum total T4 concentration. Ten samples of commercial canned cat food and 19 dust samples from homes of client-owned cats were analyzed. Total PBDE in canned cat food ranged from 0.42 to 3.1 ng/g, and total PBDE in dust from 510 to 95,000 ng/g. Total PBDE in dust from homes of euthyroid cats ranged from 510 to 4900 ng/g. In dust from homes of hyperthyroid cats, total PBDE concentrations were significantly higher, ranging from 1100 to 95,000 ng/g. Dust PBDE and serum total T4 concentration were also significantly correlated. Estimates of PBDE exposures calculated from canned cat food and dust data suggest that domestic cats are primarily exposed through ingestion of household dust. These findings indicate further study of the role of PBDE is needed in the development of FH, which might identify the cat as a model and sentinel for humans with toxic nodular goiter (TNG).

Effects of wetland vs. landscape variables on parasite communities of Rana pipiens: links to anthropogenic factors.

The emergence of several diseases affecting amphibian populations worldwide has prompted investigations into determinants of the occurrence and abundance of parasites in frogs. To understand the spatial scales and identify specific environmental factors that determine risks of parasitism in frogs, helminth communities in metamorphic frogs of the northern leopard frog (Rana pipiens) were examined in relation to wetland and landscape factors at local (1 km) and regional (10 km) spatial extents in an agricultural region of Minnesota (USA) using regression analyses, ordination, and variance partitioning techniques. Greater amounts of forested and woody wetland habitats, shorter distances between woody wetlands, and smaller-sized open water patches in surrounding landscapes were the most consistently positive correlates with the abundances, richness, and diversity of helminths found in the frogs. Wetland and local landscape variables were suggested as most important for larval trematode abundances, whereas local and regional landscape variables appeared most important for adult helminths. As previously reported, the sum concentration of atrazine and its metabolite desethylatrazine, was the strongest predictor of larval trematode communities. In this report, we highlight the additional influences of landscape factors. In particular, our data suggest that anthropogenic activities that have resulted in the loss of the availability and connectivity of suitable habitats in the surrounding landscapes of wetlands are associated with declines in helminth richness and abundance, but that alteration of wetland water quality through eutrophication or pesticide contamination may facilitate the transmission of certain parasite taxa when they are present at wetlands. Although additional research is needed to quantify the negative effects of parasitism on frog populations, efforts to reduce inputs of agrochemicals into wetlands to limit larval trematode infections may be warranted, given the current high rates of amphibian declines and extinction events.

Demasculinization and feminization of male gonads by atrazine: consistent effects across vertebrate classes.

Atrazine is the most commonly detected pesticide contaminant of ground water, surface water, and precipitation. Atrazine is also an endocrine disruptor that, among other effects, alters male reproductive tissues when animals are exposed during development. Here, we apply the nine so-called "Hill criteria" (Strength, Consistency, Specificity, Temporality, Biological Gradient, Plausibility, Coherence, Experiment, and Analogy) for establishing cause-effect relationships to examine the evidence for atrazine as an endocrine disruptor that demasculinizes and feminizes the gonads of male vertebrates. We present experimental evidence that the effects of atrazine on male development are consistent across all vertebrate classes examined and we present a state of the art summary of the mechanisms by which atrazine acts as an endocrine disruptor to produce these effects. Atrazine demasculinizes male gonads producing testicular lesions associated with reduced germ cell numbers in teleost fish, amphibians, reptiles, and mammals, and induces partial and/or complete feminization in fish, amphibians, and reptiles. These effects are strong (statistically significant), consistent across vertebrate classes, and specific. Reductions in androgen levels and the induction of estrogen synthesis - demonstrated in fish, amphibians, reptiles, and mammals - represent plausible and coherent mechanisms that explain these effects. Biological gradients are observed in several of the cited studies, although threshold doses and patterns vary among species. Given that the effects on the male gonads described in all of these experimental studies occurred only after atrazine exposure, temporality is also met here. Thus the case for atrazine as an endocrine disruptor that demasculinizes and feminizes male vertebrates meets all nine of the "Hill criteria".

Agrochemicals increase trematode infections in a declining amphibian species.

Global amphibian declines have often been attributed to disease, but ignorance of the relative importance and mode of action of potential drivers of infection has made it difficult to develop effective remediation. In a field study, here we show that the widely used herbicide, atrazine, was the best predictor (out of more than 240 plausible candidates) of the abundance of larval trematodes (parasitic flatworms) in the declining northern leopard frog Rana pipiens. The effects of atrazine were consistent across trematode taxa. The combination of atrazine and phosphate--principal agrochemicals in global corn and sorghum production--accounted for 74% of the variation in the abundance of these often debilitating larval trematodes (atrazine alone accounted for 51%). Analysis of field data supported a causal mechanism whereby both agrochemicals increase exposure and susceptibility to larval trematodes by augmenting snail intermediate hosts and suppressing amphibian immunity. A mesocosm experiment demonstrated that, relative to control tanks, atrazine tanks had immunosuppressed tadpoles, had significantly more attached algae and snails, and had tadpoles with elevated trematode loads, further supporting a causal relationship between atrazine and elevated trematode infections in amphibians. These results raise concerns about the role of atrazine and phosphate in amphibian declines, and illustrate the value of quantifying the relative importance of several possible drivers of disease risk while determining the mechanisms by which they facilitate disease emergence.

Measuring agreement and discord among hemagglutination inhibition assays against different ophidian paramyxovirus strains in the Eastern massasauga (Sistrurus catenatus catenatus).

At present, the hemagglutination inhibition (HI) assay is the sole commercially available serologic method available to detect exposure to ophidian paramyxovirus (OPMV) in snakes. During 2006, 26 eastern massasaugas (Sistrurus catenatus catenatus) were collected, and blood was sampled to determine their OPMV status. Samples from each snake were divided into 3 aliquots and tested by using commercially available HI assays against the 4 OPMV isolates used in the 3 laboratories that offer the service. All snakes were tested for antibodies by using HI assays against the green tree python (GTP), San Lucan rattlesnake (SLR), and Aruba Island rattlesnake (AIR) isolates. Twenty-five snakes were tested for antibodies against the western diamondback rattlesnake (WDR) isolate. All samples tested against the GTP and SLR were positive (26/26), whereas 56% (14/25) of the WDR assays were positive, and none (0/26) of the AIR assays yielded a positive result. There was 100% agreement between the GTP and SLR assays, and complete disagreement between the SLR and AIR, as well as the GTP and AIR assays. Kappa statistics for the GTP-WDR, SLR-WDR, GTP-AIR, SLR-AIR, and WDR-AIR indicated that the assays had less than chance agreement. The results demonstrate that current HI assays are not reliable as a sole diagnostic assay in the eastern massasauga. Furthermore, HI assays need to be evaluated by using other parameters to determine OPMV exposure in eastern massasaugas.

Ribeiroia ondatrae cercariae are consumed by aquatic invertebrate predators.

Trematodes amplify asexually in their snail intermediate hosts, resulting in the potential release of hundreds to thousands of free-living cercariae per day for the life of the snail. The high number of cercariae released into the environment undoubtedly increases the probability of transmission. Although many individual cercariae successfully infect another host in their life cycle, most fail. Factors that prevent successful transmission of cercariae are poorly understood. Microcrustaceans and fish have been observed to eat cercariae of some species, although the possibility that predation represents a significant source of mortality for cercariae has been largely unexplored. We tested the cercariophagic activity of several freshwater invertebrates on Ribeiroia ondatrae, a trematode that causes limb deformities in amphibians. Individuals of potential predators were placed into wells of multiwell plates with 10-15 cercariae, and numbers of cercariae remaining over time were recorded and compared with numbers in control wells that contained no predators. Of the species tested, Hydra sp., damselfly (Odonata, Coenagrionidae) larvae, dragonfly (Odonata, Libellulidae), larvae, and copepods (Cyclopoida) consumed cercariae. In some cases, 80-90% of the cercariae offered to damselfly and dragonfly larvae were consumed within 10 min. In most cases, predators continued to consume cercariae at the same average rates when offered cercariae together with individuals of an alternate prey item. Hydra sp. ate fewer cercariae in these trials. Our findings suggest the need for field and laboratory studies to further explore the effects of predators on transmission of R. ondatrae to amphibian larvae. In addition, the results suggest that conservation of the biodiversity and numbers of aquatic predators may limit adverse impacts of trematode infections in vertebrate hosts.

Hematology, plasma biochemistry, and antibodies to select viruses in wild-caught Eastern massasauga rattlesnakes (Sistrurus catenatus catenatus) from Illinois.

During the 2004 field season, blood was collected from Eastern massasauga rattlesnakes (Sistrurus catenatus catenatus) in the Carlyle Lake (Carlyle, Illinois, USA) and Allerton Park (Monticello, Illinois, USA) populations to derive baseline complete blood count and plasma biochemistry data and to assess the prevalence of antibodies to West Nile virus (WNV) and ophidian paramyxovirus (OPMV). Massasaugas were located for sampling through visual encounter surveys. Body weight, snout-vent length, total protein, globulins, sodium, and potassium were normally distributed among the survey population. Aspartate aminotransferase, creatine kinase, albumin, calcium, uric acid, white blood cell count, heterophils, lymphocytes, monocytes, eosinophils, and basophils were non-normally distributed within these animals. Female snakes had significantly shorter tail lengths; lower blood glucose, packed cell volumes, and absolute azurophil counts; and higher plasma calcium and phosphorus concentrations than did males. None of the snakes tested (n=21) were seropositive for WNV, whereas all (n=20) were seropositive for OPMV.

Fate of tritiated didemnin B in mice: excretion and tissue concentrations after an intraperitoneal dose.

Didemnin B has undergone trials in cancer patients, and has antiviral and immunosuppressive properties. [(3)H]didemnin B was administered intraperitoneally (i.p.) to mice at 320 or 1280 microg/kg. Urine and feces were collected until 168 h, at which time the mice were killed and tissues collected. Additionally, [(3)H]didemnin B was given i.p. at 320 microg/kg, and mice were killed at 1-120 h post-dosing. Radiolabel increased rapidly in blood then rapidly declined. Most radiolabel in urine, feces and tissues represented parent compound. Concentrations of [(3)H]didemnin B were greatest in the liver > gallbladder > lower digestive tract congruent with pancreas > spleen > kidney congruent with adipose tissue congruent with urinary bladder with urine. The pancreas had the longest terminal half-life of the tissues and the highest radioactivity at 7 days. Intermediate concentrations were in the duodenum congruent with jejunum > lung > iliopsoas > stomach congruent with testes congruent with skin > heart. Low concentrations were in the humerus congruent with femur congruent with quadriceps congruent with triceps >> brain. Fecal excretion accounted for 45.9%-58.3% of the dose and declined after 24 h, followed by an increase, suggesting possible enterohepatic recycling or an impact of circadian rhythms. Urinary excretion accounted for 18.4%-25.2% of the dose, but was minimal after 24 h. The concentrations were highest in organs previously found to be sensitive in animals and humans. Didemnin B should be evaluated in animal models for treatment of pancreatic cancer.

Intersexuality and the cricket frog decline: historic and geographic trends.

Exposure to anthropogenic endocrine disruptors has been listed as one of several potential causes of amphibian declines in recent years. We examined gonads of 814 cricket frogs (Acris crepitans) collected in Illinois and deposited in museum collections to elucidate relationships between the decline of this species in Illinois and the spatial and temporal distribution of individuals with intersex gonads. Compared with the preorganochlorine era studied (1852-1929), the percentage of intersex cricket frogs increased during the period of industrial growth and initial uses of polychlorinated biphenyls (PCBs) (1930-1945), was highest during the greatest manufacture and use of p,p-dichlorodiphenyltrichloroethane (DDT) and PCBs (1946-1959), began declining with the increase in public concern and environmental regulations that reduced and then prevented sales of DDT in the United States (1960-1979), and continued to decline through the period of gradual reductions in environmental residues of organochlorine pesticides and PCBs in the midwestern United States (1980-2001). The proportion of intersex individuals among those frogs was highest in the heavily industrialized and urbanized northeastern portion of Illinois, intermediate in the intensively farmed central and northwestern areas, and lowest in the less intensively managed and ecologically more diverse southern part of the state. Records of deposits of cricket frog specimens into museum collections indicate a marked reduction in numbers from northeastern Illinois in recent decades. These findings are consistent with the hypothesis that endocrine disruption contributed to the decline of cricket frogs in Illinois.

Prevalence of skeletal and eye malformations in frogs from north-central United States: estimations based on collections from randomly selected sites.

Skeletal malformation rates for several frog species were determined in a set of randomly selected wetlands in the north-central USA over three consecutive years. In 1998, 62 sites yielded 389 metamorphic frogs, nine (2.3%) of which had skeletal or eye malformations. A subset of the original sites was surveyed in the following 2 yr. In 1999, 1,085 metamorphic frogs were collected from 36 sites and 17 (1.6%) had skeletal or eye malformations, while in 2000, examination of 1,131 metamorphs yielded 16 (1.4%) with skeletal or eye malformations. Hindlimb malformations predominated in all three years, but other abnormalities, involving forelimb, eye, and pelvis were also found. Northern leopard frogs (Rana pipiens) constituted the majority of collected metamorphs as well as most of the malformed specimens. However, malformations were also noted in mink frogs (R. septentrionalis), wood frogs (R. sylvatica), and gray tree frogs (Hyla spp.). The malformed specimens were found in clustered sites in all three years but the cluster locations were not the same in any year. The malformation rates reported here are higher than the 0.3% rate determined for metamorphic frogs collected from similar sites in Minnesota in the 1960s, and thus, appear to represent an elevation of an earlier baseline malformation rate.

Relationship of tadpole stage to location of echinostome cercariae encystment and the consequences for tadpole survival.

The effect of echinostome infections on the survival of Rana pipiens tadpoles was examined in relation to developmental stage of tadpoles. Individual tadpoles of Gosner stages 25, 27, 32-33, and 37-39 were exposed to 1 of 4 levels of cercariae (0, 20, 50, or 100). Only tadpoles at stage 25, the earliest stage infected, died within a 5-day experimental period. This stage-specific mortality rate could be explained, in part, by the stage-specific location of encystment of cercariae, which was documented in a separate experiment. In accordance with kidney development, cercariae predominately encysted in the pronephroi during early stages of tadpole development (stages 25 through 31-32) and only in the mesonephroi and associated ducts at later stages (stages 37 through 46). As the mesonephros develops, renal capacity presumably increases. Thus, tadpoles died only when metacercariae concentrated in the functional portion of the kidney with the most limited renal capacity. As tadpoles aged, they also became less susceptible to infections. On average, 69.5% of cercariae that were exposed to stage 25-26 tadpoles successfully encysted. compared with only 8.4% of cercariae exposed to stage 37-38 tadpoles. Exposures of metamorphic frogs (poststage 46) to cercariae revealed that these individuals can become infected with echinostomes. Collectively, our data highlight the host stage-dependent dynamics of tadpole-echinostome interactions.

Toxic and feeding deterrent effects of native aquatic macrophytes on exotic grass carp (Ctenopharyngodon idella).

Declines of amphibians have been attributed to many factors including habitat degradation. The introduction of grass carp (Ctenopharyngodon idella) as a biological agent for aquatic plant control in ponds and lakes managed narrowly for human recreation has likely contributed to amphibian declines through massive plant removal and associated habitat simplification and thus degradation. This research examined the interactions among grass carp and three Midwestern aquatic plants (Jussiaea repens, Ranunculus longirostris, and R. flabellaris) that may be of value in rehabilitation of habitats needed by amphibians. The feeding preference study found that C. idella avoided eating both J. repens and R. longirostris. Ranunculus species studied to date contain a vesicant toxin called ranunculin that is released upon mastication. The study that compared the effects of R. flabellaris, J. repens and a control food administered by tube feeding to C. idella found significant lesions only in the mucosal epithelium of the individuals exposed to R.flabellaris. The avoidance by C. idella of J. repens and R. longirostris in the feeding preference study, and the significant toxicity of R. flabellaris demonstrated by the dosing study, indicate these plants warrant further examination as to their potential effectiveness in aquatic amphibian habitat rehabilitation.