Assessing nanomaterial exposures in aquatic ecotoxicological testing: Framework and case studies based on dispersion and dissolution.

The unique behavior of engineered nanomaterials (ENM) in aqueous media and dynamic changes in particle settling, agglomeration and dissolution rates is a challenge to the consistency, reliability and interpretation of standard aquatic hazard bioassay results. While the toxicological endpoints (e.g., survival, growth, reproduction, etc.) in ecotoxicity bioassays are largely applicable to ENMs, the standard methods as written for dissolved substances are confounded by the dynamic settling, agglomeration and dissolution of particulate ENMs during the bioassay. A testing framework was designed to serve as a starting point to identify approaches for the consistent conduct of aquatic hazard tests that account for the behavior of ENMs in test media and suitable data collection to support representative exposure metrology. The framework was demonstrated by conducting three case studies testing ENMs with functionally distinct characteristics and behaviors. Pretests with a temporal sampling of particle concentration, agglomeration and dissolution were conducted on each ENM in test media. Results indicated that a silver nanoparticle (AgNP) powder was not dispersible, a nano-TiO2 powder was dispersible but unstable, and a polyvinylpyrrolidinone-coated AgNP was relatively stable in test media. Based on these functional results, Ceriodaphnia dubia bioassays were conducted to compare different exposure summary methods (nominal, arithmetic average, geometric average, time-weighted average) for calculating and expressing toxicity endpoints. Results indicated that while arithmetic means were effective for expressing the toxicity of more stable materials, time-weighted averaged concentrations were appropriate for the unstable nano-TiO2.

Impact of natural organic matter on particle behavior and phototoxicity of titanium dioxide nanoparticles.

Due to their inherent phototoxicity and inevitable environmental release, titanium dioxide nanoparticles (nano-TiO2) are increasingly studied in the field of aquatic toxicology. One of the particular interests is the interactions between nano-TiO2 and natural organic matter (NOM). In this study, a series of experiments was conducted to study the impacts of Suwannee River natural organic matter (SRNOM) on phototoxicity and particle behaviors of nano-TiO2. For Daphnia magna, after the addition of 5mg/L SRNOM, LC50 value decreased significantly from 1.03 (0.89-1.20) mg/L to 0.26 (0.22-0.31) mg/L. For zebrafish larvae, phototoxic LC50 values were 39.9 (95% CI, 25.9-61.2) mg/L and 26.3 (95% CI, 18.3-37.8) mg/L, with or without the presence of 5mg/L SRNOM, respectively. There was no statistically significant change of these LC50 values. The impact of SRNOM on phototoxicity of nano-TiO2 was highly dependent on test species, with D. magna being the more sensitive species. The impact on particle behavior was both qualitatively and quantitatively examined. A global predictive model for particle behavior was developed with a three-way interaction of SRNOM, TiO2 concentration, and time and an additive effect of ionic strength. Based on power analyses, 96-h exposure in bioassays was recommended for nanoparticle-NOM interaction studies. The importance of reactive oxygen species (ROS) quenching of SRNOM was also systematically studied using a novel exposure system that isolates the effects of environmental factors. These experiments were conducted with minimal impacts of other important interaction mechanisms (NOM particle stabilization, NOM UV attenuation, and NOM photosensitization). This study highlighted both the particle stabilization and ROS quenching effects of NOM on nano-TiO2 in an aquatic system. There is an urgent need for representative test materials, together with key environmental factors, for future risk assessment and regulations of nanomaterials.

Adapting OECD Aquatic Toxicity Tests for Use with Manufactured Nanomaterials: Key Issues and Consensus Recommendations.

The unique or enhanced properties of manufactured nanomaterials (MNs) suggest that their use in nanoenabled products will continue to increase. This will result in increased potential for human and environmental exposure to MNs during manufacturing, use, and disposal of nanoenabled products. Scientifically based risk assessment for MNs necessitates the development of reproducible, standardized hazard testing methods such as those provided by the Organisation of Economic Cooperation and Development (OECD). Currently, there is no comprehensive guidance on how best to address testing issues specific to MN particulate, fibrous, or colloidal properties. This paper summarizes the findings from an expert workshop convened to develop a guidance document that addresses the difficulties encountered when testing MNs using OECD aquatic and sediment test guidelines. Critical components were identified by workshop participants that require specific guidance for MN testing: preparation of dispersions, dose metrics, the importance and challenges associated with maintaining and monitoring exposure levels, and the need for reliable methods to quantify MNs in complex media. To facilitate a scientific advance in the consistency of nanoecotoxicology test results, we identify and discuss critical considerations where expert consensus recommendations were and were not achieved and provide specific research recommendations to resolve issues for which consensus was not reached. This process will enable the development of prescriptive testing guidance for MNs. Critically, we highlight the need to quantify and properly interpret and express exposure during the bioassays used to determine hazard values.

Modeling TiO2 nanoparticle phototoxicity: The importance of chemical concentration, ultraviolet radiation intensity, and time.

As a semiconductor with wide band gap energy, TiO2 nanoparticles (nano-TiO2) are highly photoactive, and recent efforts have demonstrated phototoxicity of nano-TiO2 to aquatic organisms. However, a dosimetry model for the phototoxicity of nanomaterials that incorporates both direct UV and photo-activated chemical toxicity has not yet been developed. In this study, a set of Hyalella azteca acute toxicity bioassays at multiple light intensities and nano-TiO2 concentrations, and with multiple diel light cycles, was conducted to assess how existing phototoxicity models should be adapted to nano-TiO2. These efforts demonstrated (a) adherence to the Bunsen-Roscoe law for the reciprocity of light intensity and time, (b) no evidence of damage repair during dark periods, (c) a lack of proportionality of effects to environmental nano-TiO2 concentrations, and (d) a need to consider the joint effects of nano-TiO2 phototoxicity and direct UV toxicity.

Chronic TiO2 nanoparticle exposure to a benthic organism, Hyalella azteca: impact of solar UV radiation and material surface coatings on toxicity.

There is limited information on the chronic effects of nanomaterials to benthic organisms, as well as environmental mitigating factors that might influence this toxicity. The present study aimed to fill these data gaps by examining various growth endpoints (weight gain, instantaneous growth rate, and total protein content) for up to a 21 d sediment exposure of TiO2 nanoparticles (nano-TiO2) to a representative benthic species, Hyalella azteca. An uncoated standard, P25, and an Al(OH)3 coated nano-TiO2 used in commercial products were added to sediment at 20 mg/L or 100 mg/L Under test conditions, UV exposure alone was shown to be a greater cause of toxicity than even these high levels of nano-TiO2 exposure, indicating that different hazards need to be addressed in toxicity testing scenarios. In addition, this study showed the effectiveness of a surface coating on the decreased photoactivity of the material, as the addition of an Al(OH)3 coating showed a dramatic decrease in reactive oxygen species (ROS) production. However, this reduced photoactivity was found to be partially restored when the coating had been degraded, leading to the need for future toxicity tests which examine the implications of weathering events on particle surface coatings.

Comparison of TiO2 nanoparticle and graphene-TiO2 nanoparticle composite phototoxicity to Daphnia magna and Oryzias latipes.

With a dramatic rise in complexity, needs of nanotoxicology research go beyond simple forms of nanomaterials. This study compared the phototoxicity of nano-TiO2 and graphene-TiO2 nanocomposite (GNP). GNP was synthesized based on a hydrothermal method, which simultaneously performed the reduction of graphene oxide and nano-TiO2 loading. A series of acute toxicity tests of nano-TiO2, graphene and GNP was performed on two aquatic organisms, Daphnia magna and Oryzias latipes. Fast and substantial agglomeration and sedimentation of nanoparticles in test media and surface attachment of nano-TiO2 and GNP on D. magna surface was observed. Similar phototoxicity of nano-TiO2 and GNP for both species existed, though compared with nano-TiO2, GNP had a 2.3-fold increase in visible light photocatalytic ROS generation. In summary, this study demonstrated the significance of illumination spectrum, particle behavior, and species sensitivity on nanophototoxicity, and the needs for research on increasingly sophisticated functional materials.

Species sensitivity and dependence on exposure conditions impacting the phototoxicity of TiO2 nanoparticles to benthic organisms.

Toxicity of titanium dioxide nanoparticles (nano-TiO2 ) to aquatic organisms can be greatly increased after exposure to ultraviolet (UV) radiation. This phenomenon has received some attention for water column species; however, investigations of nano-TiO2 phototoxicity for benthic organisms are still limited. In the present study, bioassays of 3 representative benthic organisms (Hyalella azteca, Lumbriculus variegatus, and Chironomus dilutus) were conducted to evaluate nano-TiO2 phototoxicity. When exposed to 20 mg/L of nano-TiO2 and various light intensities (0-30 W/m(2)), H. azteca was the most sensitive, with a median lethal dose of 40.7 (95% confidence interval, 36.3-44.7) Wh/m(2), and hence is a potential model organism in future toxicological guidelines for photoactive nanomaterials to freshwater benthos. Without the presence of nano-TiO2 , no mortality was observed in L. variegatus and C. dilutus exposed to UV intensity ranging from 0 W/m(2) to 41 W/m(2). However, a sharp drop of H. azteca survival was observed when UV intensity was higher than 9.4 W/m(2), demonstrating the importance of UV-only effects on the ultimate phototoxicity of nanomaterials. Furthermore, both bioavailability and surface attachment of nano-TiO2 onto organisms were affected by the exposure scenario, supported by the exposure scenario-dependent phototoxicity seen in H. azteca and C. dilutus. Overall, the present study demonstrates the importance of species sensitivity and exposure scenarios in future test guidelines of nano-phototoxicity.

Phototoxicity of TiO2 nanoparticles to a freshwater benthic amphipod: are benthic systems at risk?

This study investigated phototoxicity of TiO2 nanoparticles (nano-TiO2) to a freshwater benthic amphipod (Hyalella azteca) using 48-h and 96-h bioassays. Thorough monitoring of particle interactions with exposure media (Lake Superior water, LSW) and the surface of organisms was performed using dynamic light scattering, UV/Vis spectroscopy, and Scanning Electron Microscopy. Large agglomeration and sedimentation (>77%) in LSW was observed after 0.5h. A simulated solar radiation (SSR)-favored surface attachment of nanoparticles was observed, indicating enhanced phototoxicity with the increased attachment. A 96-h median lethal concentration (LC50) of 29.9 mg/L in H. azteca was calculated, with a daily 4-h UV exposure of 2.2 W/m(2). Phototoxicity of nano-TiO2 under SSR had a 21-fold increase as compared to that under ambient laboratory light. This phototoxicity was also dependent on UV dose, with calculated LC50s around 22.9 (95% CI, 20.5-23.3)Wh/m(2) when exposed to 20 mg/L nano-TiO2. Also, H. azteca exhibited negative phototaxis in the presence of shelters, indicating that other factors might play a role in environmental systems. Finally, the environmental implications of nano-TiO2 to benthic organisms were illustrated, emphasizing the importance of various environmental factors in the ultimate phototoxicity. This increased phototoxicity and its complex interactions with various environmental factors suggest further investigations are needed for future risk assessment of photoactive nanomaterials to benthic organisms.

Phototoxicity of TiO2 nanoparticles to zebrafish (Danio rerio) is dependent on life stage.

Zebrafish embryos have been used increasingly to evaluate nanomaterial toxicity. The present study compared phototoxicity of TiO2 nanoparticles with zebrafish at 4 life stages (embryos, yolk-sac larvae, free-swimming larvae, and juvenile) under simulated sunlight using the 96-h standard toxicity assay. Yolk-sac larvae were found to be the most sensitive to TiO2 phototoxicity, suggesting that the widely used zebrafish embryo test may not fully or accurately predict hazard and risk of these nanoparticles to small fish.

Ecotoxicity of manufactured ZnO nanoparticles--a review.

This report presents an exhaustive literature review on the toxicity of manufactured ZnO nanoparticles (NPs) to ecological receptors across different taxa: bacteria, algae and plants, aquatic and terrestrial invertebrates and vertebrates. Ecotoxicity studies on ZnO NPs are most abundant in bacteria, and are relatively lacking in other species. These studies suggest relative high acute toxicity of ZnO NPs (in the low mg/l levels) to environmental species, although this toxicity is highly dependent on test species, physico-chemical properties of the material, and test methods. Particle dissolution to ionic zinc and particle-induced generation of reactive oxygen species (ROS) represent the primary modes of action for ZnO NP toxicity across all species tested, and photo-induced toxicity associated with its photocatalytic property may be another important mechanism of toxicity under environmentally relevant UV radiation. Finally, current knowledge gaps within this area are briefly discussed and recommendations for future research are made.

Photocatalytic reactive oxygen species production and phototoxicity of titanium dioxide nanoparticles are dependent on the solar ultraviolet radiation spectrum.

Generation of reactive oxygen species (ROS) by titanium dioxide nanoparticles (nano-TiO(2)) and its consequent phototoxicity to Daphnia magna were measured under different solar ultraviolet (UV) spectra by applying a series of optical filters in a solar simulator. Removing UV-B (280-320 nm) from solar radiation had no significant impact on photocatalytic ROS production of nano-TiO(2), whereas removal of UV-A (320-400 nm) decreased ROS production remarkably. Removal of wavelengths below 400 nm resulted in negligible ROS production. A linear correlation between ROS production and D. magna immobilization suggests that photocatalytic ROS production may be a predictor of phototoxicity for nano-TiO(2). Intracellular ROS production within D. magna was consistent with the immobilization of the organism under different solar UV spectra, indicating that oxidative stress was involved in phototoxicity. The dependence of nano-TiO(2) phototoxicity on environmentally realistic variations in solar radiation suggests that risk assessment of these nanomaterials requires careful evaluation of exposure conditions in the environment.

Phototoxicity of TiO2 nanoparticles under solar radiation to two aquatic species: Daphnia magna and Japanese medaka.

One target of development and application of TiO(2) nanoparticles (nano-TiO(2) ) is photochemical degradation of contaminants and photo-killing of microbes and fouling organisms. However, few ecotoxicological studies have focused on this aspect of nano-TiO(2) , specifically whether this photoreactivity might significantly increase hazard and risk of the materials in the natural environment. In the present study, we evaluated acute phototoxicity of nano-TiO(2) under simulated solar radiation (SSR) to two aquatic species-Daphnia magna and Japanese medaka, using 48-h and 96-h assays, respectively. A thorough characterization of the exposure system was performed by measuring particle agglomeration and TiO(2) concentration in suspension in a time-course manner. Sedimentation and loss of bulk concentration of nano-TiO(2) particles occurred at all concentrations above 2 mg/L and was more significant as concentration increased. Phototoxicity of nano-TiO(2) under SSR was enhanced by two to four orders of magnitude as compared to toxicity under ambient laboratory light, with a 48-h median lethal concentration (LC50) of 29.8 µg/L in D. magna and a 96-h LC50 of 2.2 mg/L in medaka. Our results also indicate that these effects are dependent on simultaneous exposure of the organisms to nanoparticles and SSR. This dramatic increase in toxicity of nano-TiO(2) at environmentally realistic levels of SSR indicates the need to incorporate this mode of action into risk assessment for nano-TiO(2) and other photoreactive nanomaterials.

Relating daily solar ultraviolet radiation dose in salt marsh-associated estuarine systems to laboratory assessments of photoactivated polycyclic aromatic hydrocarbon toxicity.

Estuaries of the southeastern United States not only serve an important nursery function but also are common repositories of polycyclic aromatic hydrocarbons (PAHs) derived from upland activities. Thus, these habitats may be at risk for PAH phototoxicity. To better characterize this risk, a daily survey of ultraviolet-A (UV-A; 320-400 nm) irradiance was performed at Leadenwah Creek (Wadmalaw Island, SC, USA) on June 27 and August 1, 2003. In addition, laboratory assays were completed using two light exposure regimes: One that was typical of historical phototoxicity assessments (continuous light [C-UV]), and a more environmentally realistic regime (ER-UV). On both survey days, irradiance at a depth of 10 cm exhibited a pattern generally similar to that observed at the surface, whereas irradiance at the bottom of the creek was a function of both tidal height and time of day. Total UV-A dose at a 10-cm depth on June 27 and August 1, 2003 was 4.37 and 4.78 J/cm2, respectively. Attenuation coefficients on both days varied as a function of tidal height. In the laboratory, larval grass shrimp (Palaemonetes pugio) exposed to an ER-UV regime for these habitats (photoperiod, 12:12-h light:dark; total daily UV-A dose, 4.40 J/cm2) exhibited a 2.5-fold decrease in toxicity compared with those exposed to the C-UV regime (photoperiod, 24:0-h light:dark; total daily UV dose, 1.50 J/cm2), despite a threefold higher UV dose in the ER-UV regime. The lower potency under the ER-UV regime likely is attributable to the presence of a 12-h dark period allowing for recovery. The consequences of these results are discussed in the context of habitat-specific UV-A dose and its relevance to future laboratory assessments of PAH phototoxicity.

Photoactivated polycyclic aromatic hydrocarbon toxicity in medaka (Oryzias latipes) embryos: relevance to environmental risk in contaminated sites.

The hazard for photoactivated toxicity of polycyclic aromatic hydrocarbons (PAHs) has been clearly demonstrated; however, to our knowledge, the risk in contaminated systems has not been characterized. To address this question, a median lethal dose (LD50) for fluoranthene photoactivated toxicity in medaka (Orvzias latipes) embryos was determined experimentally and then compared with ultraviolet-A (UV-A; 320-400 nm) radiation exposures in a PAH-contaminated field site. The dose metric, J/cm2/ microg fluoranthene/g egg wet weight, provided the means to estimate risk as the depth where the LD50 level would be exceeded at realistic field PAH concentrations, based on estimates of UV-A exposure. The estimates were made using 30 years of solar radiation data for Duluth (MN, USA) and measurements of water-column UV-A transmittance in a PAH-contaminated field site. Medaka embryo failure was strongly related to tissue PAH concentration and UV-A exposure. The LD50 was estimated to be 12.64 J/cm2/ microg fluoranthene/g egg wet weight; the 95% confidence interval was 8.46 to 19.7 J/cm2/microg fluoranthene/g egg wet weight. Embryo failures were characterized by undifferentiated cell proliferation that occurred very early in development. No partial effects or embryo/larval malformations were observed. Estimates of the depth at which the LD50 would be exceeded in the contaminated field site ranged from 10.7 cm (clear-sky conditions and lowest attenuation) to 0.0 cm (cloudy conditions and highest attenuation). Similar calculations were done using water-column attenuation estimates from 12 sites across the Great Lakes (USA). For these, the depths at which the LD50 would be exceeded ranged from 0.00 to 271.6 cm under the conditions described above. These results suggest that PAH phototoxicity may be a risk factor in specific contaminated sites, and they provide a framework for assessing that risk.

Photoactivated toxicity in amphipods collected from polycyclic aromatic hydrocarbon-contaminated sites.

The risk of photoactivated PAH toxicity in contaminated aquatic systems has not been well characterized. To document risk, amphipods (Gammarus spp.) were collected from two polycyclic aromatic hydrocarbon (PAH)-contaminated sites in the lower St. Louis River and Duluth Harbor, USA (Hog Island and USX) as well as a reference site (Chipmunk Cove) and were exposed in two separate, replicate tests to controlled intensities of solar radiation for 3 d. Contaminated site organisms died significantly faster compared to control site organisms. In all tests, mortality was strongly related to ultraviolet-A (UV-A; 320-400 nm) dose. Ultraviolet-B (280-320 nm) radiation did not increase mortality. To compare susceptibility among populations, regressions of arcsine-transformed, proportionate mortality versus UV dose were completed for each, and the slopes were statistically compared. Response slopes for the two contaminated site populations were both significantly greater than the reference site population (p = 0.0001 for test 1; p = 0.0002 for test 2). These results indicate that organisms residing in PAH-contaminated environments can accumulate PAH concentrations sufficient to be at risk for photoactivated toxicity. Although amphipods are not typically at risk of PAH-photoactivated toxicity because they are largely protected from exposure to sunlight, they are representative surrogates for species that may be similarly protected at some life stages (and thus able to accumulate significant PAH tissue concentrations) but not at others.

Assessment of the risk of solar ultraviolet radiation to amphibians. I. Dose-dependent induction of hindlimb malformations in the northern leopard frog (Rana pipiens).

A number of environmental stressors have been hypothesized as responsible for recent increases in limb malformations in several species of North American amphibians. The purpose of this study was to generate dose-response data suitable for assessing the potential role of solar ultraviolet (UV) radiation in causing limb malformations in a species in which this phenomenon seemingly is particularly prevalent, the northern leopard frog (Rana pipiens). Frogs were exposed from early embryonic stages through complete metamorphosis to varying natural sunlight regimes, including unaltered (100%) sunlight, sunlight subjected to neutral density filtration to achieve relative intensities of 85%, 75%, 65%, 50%, and 25% of unaltered sunlight, and sunlight filtered with glass or acrylamide to attenuate, respectively, the UVB (290-320 nm) and UVB plus UVA (290-380 nm) portions of the spectrum. The experiments were conducted in a controlled setting, with continual monitoring of UVB, UVA, and visible light to support a robust exposure assessment. Full sunlight caused approximately 50% mortality of the frogs during early larval development; no significant treatment-related mortality occurred under any of the other exposure regimes, including 100% sunlight with glass or acrylamide filtration. There was a dose-dependent (p < 0.0001) induction of hindlimb malformations in the frogs, with the percentage of affected animals ranging from about 97% under unaltered sunlight to 0% in the 25% neutral density treatment. Malformations were comprised mostly of missing or truncated digits, and generally were bilateral as well as symmetrical. Filtration of sunlight with either glass or acrylamide both significantly reduced the incidence of malformed limbs. The estimated sunlight dose resulting in a 50% limb malformation rate (ED50) was 63.5%. The limb ED50 values based on measured sunlight intensities corresponded to average daily doses of 4.5 and 100 Wh x m(-2) for UVB and UVA, respectively. Exposure to sunlight also resulted in increased eye malformations in R. pipiens, however, the dose-response relationship for this endpoint was not monotonic. The results of this study, in conjunction with measured or predicted exposure data from natural settings, provide a basis for quantitative prediction of the risk of solar UV radiation to amphibians.

Assessment of the risk of solar ultraviolet radiation to amphibians. II. In situ characterzation of exposure in amphibian habitats.

Ultraviolet B (UVB) radiation has been hypothesized as a potential cause of amphibian population declines and increased incidence of malformations. Realistic studies documenting UV irradiance or dose have rarely been conducted in wetlands used by amphibians. Our data indicates that 99% of UVB is attenuated in the top 5-20 cm of wetlands in our study region (northern Minnesota and Wisconsin). Furthermore, vegetation and other habitat features have substantial impacts on local UVB irradiance levels and dose. UVB attenuation in the water columns of our wetlands is controlled by the specific absorption of dissolved organic carbon (DOC), and consequently, UVB attenuation is best predicted by simple laboratory absorbance measurements such as bulk water color (absorbance at 440 nm) or wavelength-specific absorbance coefficients. Seasonal data indicate thatthe UVB absorption by early and mid-season DOC is higher than that of late summer and fall DOC, suggesting increased protection from UVB during the potentially sensitive stages of amphibian development. In addition to dissolved components, our model indicates that suspended solids play a small role in UVB attenuation in our wetlands but apparently only at high concentrations. Models predicting UV attenuation in wetlands should be used cautiously and should consider temporal variability, given the volatility and dynamic nature of water column characteristics in wetlands. Organism behavior is a critical but poorly understood phenomenon that must be addressed for development of an accurate UV exposure risk model for amphibians.

Assessment of the risk of solar ultraviolet radiation to amphibians. III. Prediction of impacts in selected northern midwestern wetlands.

Solar ultraviolet radiation, especially UVB (280-320 nm), has been hypothesized to be at least partially responsible for adverse effects (e.g., declines and malformations) in amphibian species throughout the world. Evaluation of this hypothesis has been limited by the paucity of high-quality UV dose-response data and reliable estimates of typical UV doses that occur in amphibian habitats. In this preliminary risk assessment for effects of UV radiation on amphibians, dose-response relationships quantified in outdoor experiments were compared with UV exposure estimates for 26 wetlands in northern Minnesota and Wisconsin. A comparison of wetland doses, derived from model prediction, historical data, and dissolved organic carbon (DOC) characterization, with experimental effects levels for green (R. clamitans), northern leopard (R. pipiens), and mink (R. septentrionalis) frogs indicated that the risk of mortality and malformations due to UV exposure is low for the majority of wetlands evaluated. Wetland UV dose, averaged over the entire breeding season, exceeded effects doses for mortality for all three species in two of the 26 wetlands examined and for one species in an additional wetland. On the basis of evidence that shorter term doses caused mortality in amphibian larvae, 3-day doses were also evaluated. In three of the wetlands examined, 3-day doses in excess of 85% of full sunlight (the level that appeared to trigger effects in controlled experimentation) occurred at frequencies ranging 22-100% for all three species and at frequencies ranging from 15% to 58% for R. pipiens and R. septentrionalis in three additional wetlands. Risk of malformation in R. pipiens was apparent in five of the 26 wetlands evaluated. Overall, estimated UVB doses in 21 of the wetlands never exceeded experimental effects doses for mortality or malformations. These results suggest that most amphibians are not currently at significant risk for UVB effects in northern Minnesota and Wisconsin wetlands. However, continued reduction of ozone and other global climate change effects may increase UV doses in wetlands, suggesting that the risk of UV to amphibians should continue to be monitored and studied.