Plastic ingestion by fish in the coastal waters of the Hengchun Peninsula, Taiwan: Associated with human activity but no evidence of biomagnification.

Plastic pollution has become a global threat to the marine environment. Many studies have indicated that marine creatures are at risk of plastic ingestion, but relevant studies are still lacking in Taiwan. In this study, we quantified plastic debris ingestion by marine fish in the coastal waters of the Hengchun Peninsula, including the Kenting National park, located in southern Taiwan. We also investigated possible biotic and abiotic factors associated with the quantity of ingested plastic by fish. In the 117 fish samples we examined, 94.87% of them had ingested plastic debris, and all of the observed debris was microplastics (<5 mm). The average number of ingested microplastics was 5.6 ± 5.1 pieces per fish (ranged 0-32 pieces per fish). The major type and color of microplastics were fiber (96%) and blue (43%), respectively. The quantity of ingested microplastics was not significantly different between the reef and pelagic fish. However, reef fish from the more populated west and south coast ingested more microplastics than that from the east coast, suggesting that microplastic ingestion by fish is related to human activity. Regarding biotic factors, the size, trophic level, and taxonomic family of the fish were not significantly associated with the number of ingested microplastics. Our results, the first investigation of microplastic ingestion in marine fish of Taiwan, show a high prevalence of microplastic ingestion but no biomagnification of microplastics in the fish. More research is much needed to better characterize the biological and ecological impacts of plastic debris on fish.

UV-filter benzophenone-3 inhibits agonistic behavior in male Siamese fighting fish (Betta splendens).

Benzophenone-3 (BP-3) is a widely used organic UV-filter compound. Despite the frequent occurrence of BP-3 in aquatic environments, little is known about its effect on fish behavior. The aim of this study was to investigate the endocrine disrupting effects of BP-3 in male fighting fish (Betta splendens) with a focus on agonistic behavior. Male fighting fish were exposed to 10, 100, and 1000 µg/L BP-3, as well as a solvent control (0.1% ethanol) and a positive control (100 ng/L 17α-ethynylestradiol, EE2), for 28 days. At the beginning and the end of exposure, standard length and body mass of the fish were measured for calculating the condition factor (CF). In addition, spontaneous swimming activity (total distance moved) and agonistic behavior (maximum velocity and duration of opercular display in front of a mirror) were also quantified. At the end of exposure, the fish gonads were sampled for gonadosomatic index (GSI) measurement and histology. After the exposure, CF was significantly decreased in the 1000 µg/L BP-3 groups. Spontaneous swimming activity was not affected. However, maximum velocity was significantly reduced in the EE2 and 1000 µg/L BP-3 treatments; duration of opercular display was significantly decreased in the EE2 and 10 and 1000 µg/L BP-3 treatments. GSI was not significantly different between groups. There was a slight but statistically significant decrease of relative proportion of mature spermatozoa in testicular tissue in the 100 µg/L BP-3 treatment. Collectively, our results demonstrate that BP-3 can disrupt agonistic behavior of male fighting fish, indicating the endocrine disrupting activity of this compound.

Assessment of ichthyotoxicity and anthropogenic contamination in the surface waters of Kenting National Park, Taiwan.

Millions of tourists visit Kenting National Park (KNP) in southern Taiwan every year, causing great amount of sewage discharges in this area. This study aimed to assess the impact of sewage on KNP surface waters using zebrafish embryo-larval bioassays combined with chemical analyses of pharmaceuticals and personal care products (PPCPs). Surface water samples were collected monthly from May to October in 2010. During the monthly bioassays, zebrafish embryos were exposed to the water samples for 144 h. Hatchability, embryonic heart rate, larval survival rate, and deformities were recorded. Larval swimming behavior was also digitally quantified at the end of exposure. Significant decreases in hatchability and larval survival rate were observed at all sites. Both hatchability and larval survival rate were negatively correlated with nitrite and ammonia concentrations in the water. The field water had little effect on embryonic heart rate and morphology. However, lower swimming speeds and activity levels were observed in the larvae, suggesting neurobehavioral toxicity of the surface waters. The general detection frequency of the 28 target PPCPs was 75%. High levels of some PPCPs, particularly caffeine, N,N-diethyl-3-methylbenzamide (DEET), and nonylphenol, were measured in the water samples. Our results show that surface waters in KNP have been strongly impacted by human activities, resulting in lethal and behavioral toxicities in developing fish.

Organic ultraviolet filter mixture promotes bleaching of reef corals upon the threat of elevated seawater temperature.

Global reef degradation is a critical environmental health issue that has triggered intensive research on ocean warming, but the implications of emerging contaminants in coral habitats are largely overlooked. Laboratory experiments assessing organic ultraviolet (UV) filter exposure have shown that these chemicals negatively affect coral health; their ubiquitous occurrence in association with ocean warming may pose great challenges to coral health. We investigated both short- (10-day) and long-term (60-day) single and co-exposures of coral nubbins to environmentally relevant organic UV filter mixtures (200 ng/L of 12 compounds) and elevated water temperatures (30 °C) to investigate their effects and potential mechanisms of action. The initial 10-day exposure of Seriatopora caliendrum resulted in bleaching only under co-exposure conditions (compounds + temperature). The 60-day mesocosm study entailed the same exposure settings with nubbins of three species (S. caliendrum, Pocillopora acuta and Montipora aequituberculata). Bleaching (37.5 %) and mortality (12.5 %) of S. caliendrum were observed under UV filter mixture exposure. In the co-exposure treatment, 100 % S. caliendrum and P. acuta bleached associating with 100 % and 50 % mortality, respectively, and significant increase of catalase activities in P. acuta and M. aequituberculata nubbins were found. Biochemical and molecular analyses indicated significant alteration of oxidative stress and metabolic enzymes. The results suggest that upon the adverse effects of thermal stress, organic UV filter mixture at environmental concentrations can cause bleaching in corals by inducing a significant oxidative stress and detoxification burden, suggesting that emerging contaminants may play a unique role in global reef degradation.

Assessing the effects of polychlorinated biphenyls (Aroclor 1254) on a scleractinian coral (Stylophora pistillata) at organism, physiological, and molecular levels.

Polychlorinated biphenyls (PCBs) are a group of widespread contaminants, and accumulation of PCBs has been observed in corals in the field. However, the toxic effects of PCBs on corals have not been investigated. In this study, we tested short and long term toxicity of Aroclor 1254, a commercial PCB mixture, on the scleractinian coral Stylophora pistillata. Coral nubbins were incubated in either control seawater or seawater dosed with PCBs (approximately 300ng/L) for 96h. The effect of PCB exposure on coral gene expression at 4h post exposure was tested with the suppression subtractive hybridization (SSH) and quantitative PCR methods. Photosystem II activity of the zooxanthellae was measured at 96h. After the exposure, nubbins were moved into clean seawater and their survival and growth were observed for another 50 days. All nubbins survived during the exposure and the following 50-d recovery period. Photosystem II activity and coral growth were not affected by PCB exposure in this study. Fifty-four clones were sequenced for gene expression analysis, and 15% of these sequences were identified, including genes involved in general stress response, peptide metabolism, cellular receptor, cytoskeleton organization, membrane trafficking, and oxidative stress response. However, the quantitative PCR did not show significant difference in the five selected genes. In conclusion, acute exposure of S. pistillata to Aroclor 1254 at 300ng/L did not affect coral survival, photosynthesis or growth but may alter the expression of certain genes involved in various important cellular functions. The nubbin technique proved to be an efficient approach to simultaneously characterize the impact of PCBs on the corals at multiple biological levels.

Concentrations of polycyclic aromatic hydrocarbon in the surface sediments from inter-tidal areas of Kenting coast, Taiwan.

The main objective of this study was to investigate the concentrations of polycyclic aromatic hydrocarbons (PAHs) in inter-tidal sediments of the Kenting coast, Taiwan, to assess the levels and origin of PAHs, and to provide useful information on the potential ecological risk of PAHs to benthic organisms. The total concentrations of 38 PAHs ranged from 0.2 to 493 ng/g dry weight. The high variation in total PAH concentrations was caused by the sand content of the sediment in the area. Compared with other coasts and bays in the world, the concentrations of PAHs in the inter-tidal surface sediment of the Kenting coast is low to moderate. Based on the sediment quality guidelines, the total PAH concentrations were below the effects range low value, indicating that the PAH levels in the Kenting area were within minimal effects ranges for benthic organisms. Principal component analysis and isomer ratios were analyzed to identify the contamination source in the inter-tidal surface sediment of the Kenting coast. The results of compounds' pattern and origin analysis suggest that the source of PAHs in the inter-tidal surface sediment of the Kenting coast is the combustion of petroleum and biomass.

Maternal effect and dietary supplementation of estradiol-17ß on female zebrafish (Danio rerio) affects the swimming behavior and stress-coping styles of its offspring.

Extrinsic estradiol-17ß (E2) is an environmental hormone. Female fish exposure to waterborne E2 might affect the development of craniofacial cartilage of its offspring. The present study investigates the effects of maternal E2 on larval craniofacial cartilage development by administering oral feed containing E2 (F-E2) to female zebrafish, and examines whether the swimming behavior and their stress coping style are influenced by maternal E2. The results showed that E2 contents responded to dosage and time in male fish after being fed with a diet containing E2. In addition, the E2 contents in female ovaries showed a significant increase after 250 mg of E2/kg treatment for 14 d. On the other hand, the fecundity rate of F-E2 group was lower around 2 folds than FC (female fed 0 mg of E2/kg) group. Craniofacial chondrogenesis on 72 hpf (hours of post fertilization) of F-E2 larvae were abnormalities, and a recovery to a normal developmental pattern was observed at the 96 hpf stage. The swimming speed was slower for F-E2 larvae compared to the FC larvae; and the F-E2 juvenile seems to be less responsive to cortisol (LRC) after cold stress. According to the results, we suggested that F-E2 larvae might have worse environmental adaptability than FC larvae.

Effects of bisphenol A or diethyl phthalate on cartilage development and the swimming behavior of zebrafish (Danio rerio) through maternal exposure.

Waterborne bisphenol A (BPA) and diethyl phthalate (DEP) are endocrine disruptive chemicals that impact the reproductive system of fish. The present study checks the effectiveness of the reproductive capacity on zebrafish after BPA and DEP exposure, and consequently investigates its effect on their development and the swimming behavior of its offspring. The exposure of BPA and DEP to zebrafish reveals that the levels of ovarian 17ß-estradiol (E2) and relative mRNA expression (RRE) ratios (Treatment/Control) of hepatic vitellogenin (vtg1) could be induced and decreased. Liver RRE levels in estrogen receptors (ERs) are also affected. Among the ERs, esr2a significantly increased upon BPA exposure, and esr1 and esr2b decreased upon DEP exposure. In addition, the ceratohyal cartilage (CH) angle of larvae whose mothers were exposed to BPA (F-BPA) was significantly bigger, but the CH angle of larvae whose mothers were exposed to DEP (F-DEP) was significantly smaller than the control. The swimming performance of larvae from F-DEP was more compromised than the control, but the situation did not appear in the larvae from the F-BPA group. The success rate of larvae hatching from F-BPA and F-DEP was lower than control group. Moreover, the successful rate of female spawns was higher in the control group compared to the treatment groups exposed to BPA and DEP. We suggested that both maternal BPA and DEP disrupt E2 levels, and influence the CH development of larvae, resulting in a decrease in successful hatching. Only the swimming behavior of larvae from maternal DEP was disrupted.

Growth and transcriptional effect of dietary 2,2',4,4'-tetrabromodiphenyl ether (PBDE-47) exposure in developing zebrafish (Danio rerio).

In this study, zebrafish (Danio rerio) were fed food dosed with pure PBDE-47 (2,2',4,4'-tetrabromodiphenyl ether) congener or a blank from 20 to 60 day post-hatch (dph). At 38 dph, half of the fish were sampled for body size measurements and gene expression analyzes (CYP1A1, VTG, TTR, D1, and TSH-beta). At 60 dph, body size was measured again for all fish remaining. Whole-fish histology was performed and the PBDE levels in fish were determined. PBDE treated fish was significantly smaller at 38 dph but not at 60 dph. No apparent histopathological effect was observed. In the PBDE treated fish, there was a weak induction of CYP1A1 mRNA transcription, but not of the other genes. The tissue levels of PBDE-47 were comparable to that found in other wild fish reported in the literature, indicating that our exposure level was ecologically relevant.

A preliminary assessment of polycyclic aromatic hydrocarbon distribution in the kenting coral reef waters of southern Taiwan.

This distribution and variation of 45 polycyclic aromatic hydrocarbons (PAHs) in the waters of Kenting coral reefs were investigated by using sensitive research-grade sampling and analytical methods. The concentration levels of total PAHs (including particulate and dissolved PAHs) in the Kenting waters were relatively low, ranging from 2.2 to 34.4 ng/L. There was no significant spatial difference in PAH concentrations among the sampling stations, even between the surface and bottom waters. The fraction of dissolved PAHs on average was greater than that of particulate PAHs due to the small amount of total suspended particles. The partition coefficients between the particulate and dissolved phase (log K(oc)) were positively correlated with their log K(ow) values (r(2) = 0.67, n = 80), except for the more hydrophobic PAHs with log K(ow) values greater than 5.91. Results of principal component analysis indicate that the patterns of particulate PAH compositions were similar through the Kenting waters in the fall and winter, whereas there was a significant spatial difference between the spring and summer samples, which might be affected by a combination of land runoff and coastal currents. PAHs along the Kenting coasts were dominated by low-molecular PAHs, which have higher solubility than high-molecular PAHs. Based on isomer ratios, analysis of the source of PAHs in the Kenting coastal waters demonstrated that the PAHs come mostly from petroleum and, to a lesser degree, from combustion.

Spatial and seasonal distribution of microplastics on sandy beaches along the coast of the Hengchun Peninsula, Taiwan.

Here we present the first report on microplastic pollution on the beaches along the coast of the Hengchun Peninsula, which is one of the major tourist attractions in Taiwan. By using a standard operating procedure, sand samples were collected from eight beaches in June and November in 2017, and the microplastics in the sand samples were quantified and characterized in the laboratory. The average density of microplastics ranged from 80 to 480 particles/kg dry weight sand. There was no apparent seasonal difference but there were significant spatial differences among sampling sites. No significant difference in microplastic levels was observed among the west, south, and east coasts, but microplastic density was higher on beaches with higher tourism activity levels. The most abundant type of microplastics was fiber (>97%) and the most common color was white/transparent (57%). In addition, using a Fourier Transform Infrared (FTIR) spectrophotometer we identified microplastics as polyethylene (PE) and polypropylene (PP). Our results show that microplastics are ubiquitous along the coast of the Hengchun Peninsula, and the major factor associated with the abundance of microplastics is tourism activity.

Chronic exposure to pentavalent arsenic of larval leopard frogs (Rana pipiens): bioaccumulation and reduced swimming performance.

Arsenic (As) is mainly released to the environment from anthropogenic sources, with inorganic pentavalent As (As [V]) predominant in surface water. In this study, Rana pipiens were exposed to As (V) in water at environmentally relevant concentrations (control, 10, 20, 150, 500, and 1,000 microg l(-1)) in a static-renewal system from post-hatch stage through metamorphosis for 113 days. There was no significant effect of As exposure on tadpole survival, growth, and percent metamorphosis. Maximum swimming speed was significantly slower in the As-treated groups compared with the control. During the period of tail resorption (Gosner stage 42-46), no significant differences in age at metamorphosis, survival, length of tail resorption period, snout-vent length of metamorphs, and sex ratio were found among treatments. Whole body As concentrations ranged from <0.6 to 5.31 mg kg(-1) dry mass, and were significantly higher in the 150, 500, and 1,000 microg l(-1) treatments than the control. Based on our data, larval tissue concentrations of As close to that reported in previous field studies were not associated with any significant effects except decreased tadpole swimming speed.

Comparative toxicities of four benzophenone ultraviolet filters to two life stages of two coral species.

The benzophenone (BP) organic ultraviolet (UV) filters have been measured in seawater at ng/L to µg/L levels, but more data on their effects in non-target marine organisms are needed. Corals can be exposed to BPs due to wastewater discharges and coastal recreational activities. In this study, toxicities and bioaccumulation of BP-1 (2,4-dihydroxybenzophenone), BP-3 (oxybenzone), BP-4 (sulisobenzone) and BP-8 (dioxybenzone) to larvae and adults of two coral species, Pocillopora damicornis and Seriatopora caliendrum, were assessed at concentrations ranging from 0.1-1000 µg/L. BP-1 and BP-8 exposure caused significant settlement failure, bleaching and mortality of S. caliendrum larvae [lowest observed effect concentration (LOEC): ≥10 µg/L] compared to the other BPs, while none of the tested compounds and concentrations affected P. damicornis larvae. Nubbins were more sensitive to BP-3, BP-1 and BP-8 than larvae. Overall, BP-1 and BP-8 were more toxic to the two tested species than BP-3 and BP-4, which matches the relative bioaccumulation potential of the four BPs (BP-8 > BP-1 ≈ BP-3 > BP-4). A conservative risk assessment using the effect concentrations derived from this study showed that BP-3, BP-1 and BP-8 pose high or medium risk to the health of corals in popular recreational areas of Taiwan and Hong Kong. Our study suggests that future ecotoxicological studies of corals should take their sensitivities, life stages and metabolic capacities into consideration.

Toxicological effects of two organic ultraviolet filters and a related commercial sunscreen product in adult corals.

Corals are exposed to organic ultraviolet (UV) filters and other personal care product (PCP) ingredients in the environment, but the toxicities of organic UV filters and their related PCP to corals are not well understood. In this study, 7-day exposures were conducted to evaluate the toxicities and bioaccumulation of two organic UV filters, ethylhexylmethoxy-cinnamate (EHMC; octinoxate) and octocrylene (OC) (single- and combined-chemical tests), and diluted sunscreen wash-off water containing both active ingredients to the adult life stage of two hard coral species, Seriatopora caliendrum and Pocillopora damicornis. In the single-chemical tests, death (33.3%) and bleaching (83.3%) were only observed in the 1000 µg/L EHMC treatment of S. caliendrum. In the sunscreen product exposures, 5% sunscreen water (containing 422.34 ±â€¯37.34 µg/L of EHMC and 33.50 ±â€¯7.60 µg/L of OC at Day 0) caused high mortality in S. caliendrum (66.7-83.3%) and P. damicornis (33.3-50%), and tissue concentrations were up to 10 times greater than in the single-chemical exposures; co-exposure to EHMC and OC at similar levels to those in the sunscreen product resulted in bioaccumulation similar to the single-chemical tests. These results confirm the bioaccumulation potential of EHMC and OC and show that other ingredients in sunscreen products may increase the bioavailability of active ingredients to corals and exacerbate the toxicity of sunscreen products. Future studies on the toxicities of PCPs to aquatic organisms should not only focus on the toxicities of active ingredients.

Effect of the UV-filter benzophenone-3 on intra-colonial social behaviors of the false clown anemonefish (Amphiprion ocellaris).

The UV-filter benzophenone-3 (BP-3) is widely used and is environmentally stable, lipophilic, and bioaccumulative. Previous in vitro and in vivo studies have shown that BP-3 can cause endocrine disrupting effects. However, little information is available on its ecotoxicity on coral reef fish. Agonistic behavior, which is regulated by the endocrine system, is crucial to the social structure of some coral reef fish species. Endocrine disruptors may disturb fish agonistic behavior and social interactions. In this study, we tested whether chronic BP-3 exposure can affect social behaviors in coral reef fish. Juvenile false clown anemonefish (Amphiprion ocellaris) were exposed to BP-3 via diet (0 and 1000 ng/g food) for 90 d. Through the experiment, each tank was videotaped and behavioral indicators of social status, including threatening, attacking, and submissive behaviors were quantitatively analyzed from the videos. Survival and growth were not affected by the BP-3 exposure except that the body weight of the dominant fish was higher in the BP-3 group. Social rankings were not changed by BP-3. Intra-colonial social behaviors were significantly affected only by rank but not by the BP-3 exposure. Our results suggest that BP-3 at environmental levels may not cause significant harm to social behavior of coral reef fish. However, more research is needed to better understand the behavioral effects of BP-3 in fish.

Persistent organic pollutants in Antarctic notothenioid fish and invertebrates associated with trophic levels.

Notothenioid fish and invertebrate samples from Antarctica were collected in the austral summer of 2009, and analyzed for persistent organic pollutants (POPs), including polycyclic aromatic hydrocarbons (PAHs), organochlorine pesticides (OCPs), and polybrominated diphenylethers (PBDEs), as well as δ13C and δ15N stable isotopes for trophic level determination. In this study, the POP levels in the Antarctic biota samples were found to be ranked in the following order: OCPs > PAHs >> PBDEs. The POP levels in notothenioid fish and krill correlate to trophic levels; however, the POP concentrations in intertidal benthic invertebrates are higher than in notothenioid fish implying that specific biogeochemical factors may affect bioaccumulation in the Antarctica ecosystem. Biomagnification of POPs may have a smaller role than bioconcentration in Antarctica environment. In addition to the source, transport, exposure, and absorption for each group of POPs in the short food chain in Antarctica, the biological variation among species, interaction habitats, diet and metabolism are also factors for future studies on contaminant bioaccumulation.

Adverse effects of chronic copper exposure in larval northern leopard frogs (Rana pipiens).

Northern leopard frogs (Rana pipiens) were exposed to environmentally relevant concentrations of copper in water (control and 5, 25, and 100 microg/L, as CuSO4) in a static renewal system for 154 d from posthatch stage to metamorphosis. After 10 d of copper exposure (from Gosner stages 19-25), survival of tadpoles was not significantly different between treatments (averages range from 96.7 to 99.3%), but a significantly higher incidence of deformities (p < 0.05) was observed in the 100-microg/L treatment. Tadpole growth rate was decreased in the 25- and 100-microg/L treatments relative to controls. Tadpole survival, swimming performance, percent metamorphosis, time to metamorphosis, and survival during and time required for tail resorption were also negatively affected in the 100-microg/L treatment. Copper concentrations in tadpole tissues ranged from 46.5 to 769.5 mg/ kg dry mass and were positively related to copper concentrations in the water. In conclusion, copper was shown to affect development, growth, survival, and behavior in larval northern leopard frogs at ecologically relevant concentrations. The existing U.S. Environmental Protection Agency freshwater-quality chronic criterion for copper appears to be protective for larval Rana pipiens.

Lethal and sublethal effects of chronic cadmium exposure on northern leopard frog (Rana pipiens) tadpoles.

Data on the chronic effects of cadmium on amphibians are lacking in spite of widespread anthropogenic contamination of terrestrial and aquatic systems. We exposed embryos and tadpoles of northern leopard frogs (Rana pipiens) to Cd (control, 0.25, 5.0, and 20.0 microg/L as CdCl2, nominal concentrations) in a static renewal system from embryonic stages to complete tail resorption. Survival of embryos (Gosner stage [GS] 17-25) was greater than 97% in all treatments. Tadpole survival was negatively correlated with dose and was significantly reduced in the 5.0 and 20.0 microg/L treatments compared with controls. Tadpole survival was greater than 80% through GS 42, forelimb emergence, for all other treatments. Tadpoles exposed to 0.25 and 5.0 microg/L exhibited increased growth by week 11; tadpoles exposed to 5.0 microg/L were significantly younger at forelimb emergence. Whole-tadpole body burdens of Cd were positively correlated with increasing Cd treatments. Cadmium was shown to alter growth and development in a native amphibian species at ecologically relevant concentrations. The existing chronic water quality criterion for Cd appears to be protective of amphibians. However, additional studies with other chemicals are needed to further explore the potential for adverse effects of contaminants on the complex life cycle of amphibians.

Fighting Nemo: Effect of 17α-ethinylestradiol (EE2) on aggressive behavior and social hierarchy of the false clown anemonefish Amphiprion ocellaris.

Aggressive behavior is crucial for maintaining social hierarchy in anemonefish. Endocrine disrupting chemicals such as EE2 may affect fish social hierarchy via disrupting their aggression. In this study, we aimed to characterize the effects of 17α-ethinylestradiol (EE2) on aggressive behavior and social hierarchy in the false clown anemonefish (Amphiprion ocellaris). In the laboratory experiment, juvenile anemonefish were randomly distributed to separated tanks to form small colonies of three individuals and were fed with EE2-dosed diet (100ng/g food) or a control diet for 90d. Through the experiment, each tank was videotaped and behavioral indicators of social status, including aggressive behavior, submissive response, and shelter utilization, were quantitatively analyzed from the videos. The EE2 exposure caused a higher frequency of intra-colonial aggressive interactions and a less stable social hierarchy. Our findings demonstrate the importance of examining the effects of endocrine disrupting chemicals on the social behavior of coral reef fish.

Sublethal effects of lead on northern leopard frog (Rana pipiens) tadpoles.

Northern leopard frogs (Rana pipiens) were exposed to environmentally relevant concentrations of lead in water (3, 10, and 100 microg/L as lead nitrate) from the embryonic stage to metamorphosis. Survival, growth, deformity, swimming ability, metamorphosis, and lead tissue concentrations were evaluated. Tadpole growth was significantly slower in the early stages (Gosner stages 25-30) in 100 microg/L treatment. More than 90% of tadpoles in the 100 microg/L treatment developed lateral spinal curvature, whereas almost all the tadpoles in the other groups were morphologically normal. Spinal deformity was associated with abnormal swimming behavior. Maximum swimming speed of tadpoles in the 100 microg/L treatment was significantly lower than that in the other groups. No significant effect of lead exposure was found on percentage metamorphosis, snout-vent length, mortality, and sex ratio of metamorphs. Time to metamorphosis was delayed in 100 microg/L treatment. Lead tissue concentrations in the tadpoles ranged from 0.1 to 224.5 mg/kg dry mass, were positively related to lead concentrations in the water, and fell within the range of tissue concentrations of wild tadpoles found in previous studies. According to our results, the current U.S. Environmental Protection Agency freshwater-quality chronic criterion for lead (2.5 microg/L) is appropriate to protect northern leopard frog larvae.