A dosage-effect assessment of acute toxicology tests of microplastic exposure in filter-feeding fish.

Abundant microplastics was found in aquatic ecosystem and aquatic organisms, which raised many concerns in public. Silver carp (Hypophthalmichthys molitrix), a species filter-feeding planktivorous fish, feed on particle between 4 and 85 µm in size, and the respiratory process works together with feeding mechanism when filtering plankton from water. The aim of this study was to assess the physiological response of silver carp exposed to 5 µm polystyrene microspheres during 48 h of exposure followed by 48 h of depuration through the gill histology, and oxidative stress biomarkers in intestine. The results revealed that microplastics can pass through the whole digestive tract of silver carp and be excreted by feces. Low microplastic concentration (80 µg/L) induced oxidative stress and up-regulation of TUB84 and HSP70 gene in intestine, and silver carp have ability to recover after the exposure to microplastic was removed. High microplastic concentration (800 µg/L) definitely cause significant damage to gills and intestines, in this situation, far beyond the possibility of fish own repair, and even when the threaten removed, silver carp can't recovery soon. Our studies assessed the dosage-effect relationship with physiological stress on silver carp when exposure to microplastics.

Comparative Acute Toxicity Assessment of Organophosphate and Avermectin Insecticides on a Freshwater Fish Oreochromis niloticus.

Oreochromis niloticus was exposed to 10.0 ppb of organophosphate insecticide chlorpyrifos (CPF) and avermectin insecticides abamectin (ABM) and emamectin benzoate (EB) for 48 and 96 h. RBC and Hb decreased in CPF- and ABM-exposed fish after 96-h. Plasma ALT, AST, cortisol, and glucose increased in 96-h CPF-, ABM- and EB-exposed fish, while plasma ions declined in 96-h CPF-exposed ones. Insecticides caused alterations in liver oxidative stress parameters. In fish exposed to CPF, CAT increased after 48-h whereas it decreased after 96-h. Also, CAT declined in 48- and 96-h ABM-exposed fish, whereas it elevated in 48-h EB-exposed ones. Insecticides caused decreases in SOD at 48- and 96-h and in GR after 96-h. GSH elevated in CPF-exposed fish after 48-h, while it decreased in all the tested insecticide exposures after 96-h. Malondialdehyde of fish exposed to insecticides for 96-h increased. Consequently, toxic effects of insecticides on O. niloticus were generally as CPF > ABM > EB.

Acute toxicity of copper to the larval stage of three species of ambystomatid salamanders.

Copper (Cu) appears to be consistently more toxic to anuran species relative to other vertebrate taxa. There are limited Cu toxicity data for salamanders; of the few studies conducted on salamanders, most examined Cu effects on the embryonic, but not the larval, stage. We performed acute toxicity experiments, to quantify LC50s, on Harrison stage 46 larvae (free swimming hatchlings with egg yolk completely absorbed) of three ambystomatid salamander species. Each LC50 experiment used exposure concentrations of 10, 20, 30, 40, 50, and 60 µg/L with 10 replicates per concentration each containing one larva. We found very high toxicity for all species compared to previously published research on the embryo stage. Specifically, the 4-d LC50s for Ambystoma tigrinum and A. opacum were 35.3 and 18.73 µg/L, respectively. The same Cu concentrations caused similar toxicity to A. talpoideum (LC50 = 47.88 µg/L), but exposures required up to 48 d to elicit the same level of mortality. A time-to-event analysis indicated that time to mortality was significantly affected by Cu concentration. Additionally, for A. talpoideum, we observed that elevated levels of Cu decreased growth rate. Comparisons with previously reported Cu toxicity for embryos suggest that, as with fish, Cu may be more toxic to larval salamander stages than for embryos. Further, our data suggest that Cu is an important environmental contaminant that deserves increased scrutiny on the potential for population-level effects where contamination has occurred in wetlands and streams inhabited by salamanders.

Acute Toxicity of Divalent Mercury Ion to Anguilla japonica from Seawater and Freshwater Aquaculture and Its Effects on Tissue Structure.

The acute toxicity of divalent mercury ion to Anguilla japonica from seawater and freshwater aquaculture was assessed. In particular, the effects of toxicity on the microstructures of the gill and liver tissues were examined using the hydrostatic method, without feeding, at a water temperature of 20 °C. The median lethal concentrations (LC50) of divalent mercury ion to fishes in seawater and freshwater over various durations were: 24 h = 1.637 and 1.428 mg/L; 48 h = 1.562 and 1.377 mg/L; 72 h = 1.530 and 1.284 mg/L; and 96 h = 1.442 and 1.228 mg/L. The safety mass concentrations were 0.1442 and 0.01228 mg/L, respectively. After exposure to divalent mercury ion, adhesion between the gill lamellae and massive cellular disintegration and necrotic shedding were observed in the gill tissue sections. The liver tissues underwent hyperemia and swelling, with the appearance of blood spots, swelling of the hepatocyte mitochondria, dilation of the rough endoplasmic reticulum, and intercellular inflation.

Acute and sublethal effects in an Indian major carp Cirrhinus mrigala exposed to silver nitrate: Gill Na+/K+-ATPase, plasma electrolytes and biochemical alterations.

Due to prolonged use of silver in many applications, it enters into the freshwater and affects the aquatic organisms. Fingerlings of Cirrhinus mrigala were exposed to acute and sublethal concentrations of silver nitrate and the alterations of gill Na(+)/K(+)-ATPase, plasma electrolytes and biochemical parameters were assessed. The median lethal concentration of silver nitrate to the fish C. mrigala for 96 h was found to be 0.107 mg/l (with 95% confidence limits). 1/10th of LC 50 96 h value (0.0107 mg/l) was selected for sublethal study. During acute treatment branchial Na(+)/K(+)-ATPase activity was inhibited approximately 44.34% after 96 h of exposure. In sublethal treatment, silver nitrate could not produce a significant change in the activity of the enzyme at the end of 7th day. However, after 14th day, significant (p < 0.05) decrease was noted showing 22.52%-49.11% in rest of the study period. Silver intoxication resulted hyponatremia, hypokalemia, hypochloremia, and hypoproteinemia in both the treatments. Despite the decrease in these parameters, plasma glucose level was found to be increased in both the treatments to endure the silver toxicity. We suggest that the alterations in branchial Na(+)/K(+)-ATPase activity, plasma electrolytes, and biochemical parameters of fish may be useful in environmental biomonitoring and to assess the health of fish in freshwater habitat contaminated with silver.

A simple orchidometric method for the preliminary assessment of maturity status in male cynomolgus monkeys (Macaca fascicularis) used for nonclinical safety studies.

INTRODUCTION: The identification and use of mature male non-human primates in nonclinical toxicology studies could be important for evaluating candidate drugs for which the profile of toxicity may differ depending on sexual maturity. This investigation sought to establish operational criteria to complement the current standard of histological evaluation for defining sexual maturity in male cynomolgus monkeys (Macaca fascicularis) used for toxicology studies, and to identify a practical non-invasive measure to select mature males for study. METHOD: Retrospectively, the relationships between body weight, testicular weight and testis histology were established in control males (n=126) used in previous toxicology studies. Prospectively, testicular volumes were measured in-life by orchidometry using comparative scrotal palpation (n=23 males used for study), then compared to testicular weights measured at necropsy. RESULTS: Consistent with previous literature, a weak relationship was observed between body weight and testicular weight. There was, however, a very good relationship between testicular weight and histological maturation level, which was based upon microscopic examination of testes, epididymides and prostates. Orchidometric measurement of testicular volume was found to be a reasonable predictor of testicular weight and served to rapidly select sexually mature males for study, and a total testicular volume (left and right combined) of >20 ml correlated with the histological appearance of maturity. CONCLUSION: Based upon this preliminary exploratory study, the initial simple measurement of testicular volume by orchidometry may provide a non-invasive alternative approach for assessing the sexual maturity of male cynomolgus monkeys in research colonies or during toxicology studies that will require more thorough validation.

Endosulfan acute toxicity and histomorphological alterations in Jenynsia multidentata (Anablepidae, Cyprinodontiformes).

Toxicity tests using adult specimens of Jenynsia multidentata were carried out during 96 hours in order to determine the lethal concentration (LC50) of endosulfan. Histological alterations were determined in gills and liver. Gill damage was quantified as secondary lamellae thickness. The 96 hr LC50 values were significantly different between males (0.719 microg x L(-1)) and females (1.317 microg x L(-1)). The sex difference was attributed to the dimorphism in the lipid content in females (2.16%) and males (1.79%). Histological alterations in gills included hypertrophy and lifting of the epithelium of the secondary lamellae and aneurisms. These alterations caused a significant increase of the secondary lamellae thickness in treatment versus control fish. Finally, reversible histological alterations (such as hydropic degeneration and dilation of sinusoids) were observed in the liver of exposed fish as well as an irreversible change such as necrosis at the highest concentrations.

Impaired terrestrial and arboreal locomotor performance in the western fence lizard (Sceloporus occidentalis) after exposure to an AChE-inhibiting pesticide.

We examined the effects of a commonly used AChE-inhibiting pesticide on terrestrial and arboreal sprint performance, important traits for predator avoidance and prey capture, in the western fence lizard (Sceloporus occidentalis). Lizards were exposed to carbaryl (2.5, 25, and 250 microg/g) and were raced before and 4, 24, and 96 h after dosing. In the terrestrial setting, exposure to low concentrations of carbaryl had stimulatory effects on performance, but exposure to the highest concentration was inhibitory. No stimulatory effects of carbaryl were noted in the arboreal environment and performance in lizards was reduced after exposure to both the medium and highest dose of carbaryl. Our findings suggest that acute exposure to high concentrations of carbaryl can have important sublethal consequences on fitness-related traits in reptiles and that arboreal locomotor performance is a more sensitive indicator of AChE-inhibiting pesticide poisoning than terrestrial locomotor performance.

Acute toxicity profile of cadmium revealed by proteomics in brain tissue of Paralichthys olivaceus: potential role of transferrin in cadmium toxicity.

An analytical approach using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separated proteins from the brain tissue of the fish Paralichthys olivaceus. Approximately 600 protein spots were detected from the brain sample when applying 600 microg protein to a 2D-PAGE gel in the pH range 3.5-10.0. Compared to a control sample, significant changes of 24 protein spots were observed in the fish tissue exposed to acute toxicity of seawater cadmium (SCAT) at 10 ppm for 24h. Among these spots, nine were down-regulated, nine were up-regulated, two showed high expression, and four showed low expression. The collected spots were identified by peptide mass fingerprinting (PMF) and database search, and they were further classified by LOC tree, a hierarchical system of support vector machines which predict their sub-cellular localization. The amount of transferrin expression in brain cells decreased linearly with the increase of SCAT concentration in seawater. Among the 24 proteins identified on a 2D-PAGE gel, 9 demonstrated a synchronous response to acute cadmium, suggesting that they might represent a biomarker profile. Based on their variable levels and trends on the 2D-PAGE gel this protein (likely to be transferrin) suggesting they might be utilized as biomarkers to investigate cadmium pollution levels in seawater and halobios survival, as well as to evaluate the degree of risk of human fatalities. The results indicate that the application of multiple biomarkers has an advantage over a single biomarker for monitoring levels of environmental contamination.

Larvicidal activity of extracts of Ginkgo biloba exocarp for three different strains of Culex pipiens pallens.

Ethanolic extracts from the Ginkgo biloba L. exocarp from the Chinese ginkgo were assayed against larvae of three strains of Culex pipiens pallens Coquillett. The chemical compositions were detected using a Hewlett-Packard 6890/5973 mass spectrometric detector. The larvicidal bioassay was carried out according to the recommendations of the World Health Organization. The analysis of the essential oil of ginkgo exocarp showed that its major components are ginkgo acid (85.3%) and ginkgo phenolic (5.69%). The larvicidal bioassay showed that extracts of ginkgo exocarp have LC50 of 18.6, 12.7, and 25.0 mg/liter for deltamethrin-susceptible, deltamethrin-resistant, and field strains, respectively. The acute toxicity concentrations of the ginkgo extracts that killed 50% (LD50) of Wistar rats within 2 wk and young carp within 96 h were 4947.2 mg/kg and 557.9 mg/liter, respectively. These results are promising in creating new, effective, and affordable approaches to mosquito control.

Toxicity of silver to two freshwater algae, Chlamydomonas reinhardtii and Pseudokirchneriella sub-capitata, grown under continuous culture conditions: influence of thiosulphate.

In a test of the biotic ligand model (BLM), the uptake and toxicity of silver, in the absence or presence of the inorganic ligand, thiosulphate, were assessed for two freshwater green algae, Chlamydomonas reinhardtii and Pseudokirchneriella sub-capitata, using turbidostat continuous cultures. In the initial experiments, run in the absence of thiosulphate, the influent Ag concentration was varied from 0 to 75 nM in steps; for each influent concentration, silver uptake was calculated and the algal growth rate was determined. Silver uptake rates at low Ag concentrations were similar for both algae (e.g., 14-19 nmolm(-2)h(-1), for influent Ag(+) concentrations of approximately 9 nM) but at higher exposures uptake by P. sub-capitata exceeded that of C. reinhardtii. Despite this higher uptake rate, in the absence of thiosulphate P. sub-capitata was not more sensitive to free silver; 50% growth inhibition was reached at influent free Ag(+) concentrations of 15+/-7 and 22+/-13 nM for C. reinhardtii and P. sub-capitata, respectively. In the second series of experiments, the free Ag(+) concentration was held constant ( approximately 9 nM in the influent; 2-3 nM in the effluent) while the concentration of the silver thiosulphate complex, AgS(2)O(3)(-), was increased from 9 to 90 nM in steps. Under such conditions, the BLM would predict that silver uptake and toxicity should remain constant. On the contrary, both silver uptake and silver toxicity increased, indicating that the anionic silver thiosulphate complex enters the algal cells via a membrane-bound sulphate transporter and contributes to uptake and toxicity. However, for both algae there were indications that silver assimilated in this manner was somewhat less toxic to the algal cell than silver that entered via cation transport only. Physiological indicators of stress revealed possible different intracellular targets for these two freshwater algae, proteins and enzymes for C. reinhardtii and the photosynthetic apparatus for P. sub-capitata.

Bioaccumulation and toxicity of 4-nonylphenol (4-NP) and 4-(2-dodecyl)-benzene sulfonate (LAS) in Lumbriculus variegatus (Oligochaeta) and Chironomus riparius (Insecta).

The discharge of surfactants, such as 4-nonylphenol (4-NP) and linear alkylbenzene sulfonates (LAS), into water bodies leads to accumulation of the chemicals in the sediments and may thus pose a problem to benthic organisms. To study the bioaccumulation of surfactants, Oligochaeta worm Lumbriculus variegatus was exposed to sediment-spiked, [14C]-labeled 4-NP and 4-(2-dodecyl)-benzene sulfonate (C12-LAS) in three different sediments (S1-S3). The sediments were characterized for organic carbon (OC) content and particle size distribution. The acute toxicity was examined by exposing L. variegatus and three to four instar Chironomus riparius (Insecta) larvae in water-only exposure to 4-NP and LAS at different concentrations. After 48-h exposure, lethal water concentrations (LC50) and lethal body residues (LBR50) were estimated using liquid scintillation counting. Chronic toxicity was evaluated in two different sediments by exposing first instar C. riparius larvae to sediment-spiked chemicals at different concentrations. After 10 days, the sublethal effects of surfactants were observed by measuring wet weight and head capsule length. Finally, another 10-day test was set up in order to measure the LAS body residues associated with sublethal effects in C. riparius in S2 sediment. The bioaccumulation test revealed that the bioaccumulation of both 4-NP and LAS increased as the sediment organic matter content decreased. It is assumed that the chemical binding to organic material decreased chemical bioavailability. The acute toxicity tests showed that L. variegatus was more tolerant of 4-NP, and C. riparius was more tolerant of LAS when based on water exposure concentration. The LBR-estimates revealed, however, that L. variegatus tolerated clearly higher tissue residues of both chemicals compared with C. riparius. Both chemicals had sublethal effects on C. riparius growth in sediment exposure, reducing larvae wet weight and head capsule size. 4-NP, however, showed an irregular dose-response pattern. The characteristics of the exposure media affected the bioaccumulation potential of both chemicals. Thus, exposure concentrations offered no prediction of body residue, and therefore it is proposed that organism body residue offered a more accurate dose-metric for chemical exposure than the chemical concentration of the environment.

Prediction of rodent carcinogenesis: an evaluation of prechronic liver lesions as forecasters of liver tumors in NTP carcinogenicity studies.

The National Toxicology Program (NTP) developed the chronic 2-year bioassay as a mechanism for predicting the carcinogenic potential of chemicals in humans. The cost and duration of these studies has limited their use to small numbers of selected chemicals. Many different short-term methods aimed at increasing predictive accuracy and the number of chemicals evaluated have been developed in attempts to successfully correlate their results with evidence of carcinogenicity (or lack of carcinogenicity) are assessed. Using NTP studies, the effectiveness of correlating prechronic liver lesions with liver cancer encompassing multiple studies using mice (83 compounds) and rats (87 compounds). These lesions include hepatocellular necrosis, hepatocellular hypertrophy, hepatocellular cytomegaly, bile duct hyperplasia, and hepatocellular degeneration, along with increased liver weight. Our results indicate that pooling 3 of these prechronic data points (hepatocellular necrosis, hepatocellular hypertrophy, and hepatocellular cytomegaly) can be very predictive of carcinogenicity in the 2-year study (p < 0.05). The inclusion of increased liver weight as an endpoint in the pool of data points increases the number of rodent liver carcinogens that are successfully predicted (p < 0.05), but also results in the prediction of increased numbers of noncarcinogenic chemicals as carcinogens. The use of multiple prechronic study endpoints provides supplementary information that enhances the predictivity of identifying chemicals with carcinogenic potential.

Effect of oral gavage dosing regimens in female Tg rasH2 transgenic mice.

Female Tg rasH2 (CB6F1/Jic-TgrasH2@Tac) mice were administered water once daily, water twice daily with 8 or 12 hours between doses, 1% sodium dodecyl sulfate in water (1% SDS) once daily, or 1% SDS twice daily with 12 hours between doses by oral gavage at volumes of 10 ml/kg/day for 28 or 29 consecutive days. A control group of mice received no treatment and no sham manipulation. There were no significant differences in body weight or food consumption between treated groups and untreated control mice. Mean weights of spleens, livers, and thymuses were lower than control values in most groups of mice subjected to gavage. Focal or multifocal loss of thymic cortical architecture was observed in 13 of 50 mice distributed among all groups (including naïve controls), however only in one instance was this finding suggestive of a precursor to neoplasia. This study demonstrated that Tg rasH2 mice can tolerate once or twice daily gavage dosing with water or vehicle containing 1% SDS. Loss of thymic cortical architecture was a common incidental finding in female Tg rasH2 mice.

Relationships between organ weight and body/brain weight in the rat: what is the best analytical endpoint?

Analysis of organ weight in toxicology studies is an important endpoint for identification of potentially harmful effects of chemicals. Differences in organ weight between treatment groups are often accompanied by differences in body weight between these groups, making interpretation of organ weight differences more difficult. Using data from control rats that were part of 26 toxicity studies conducted under similar conditions, we have evaluated the relationship between organ weight and body/brain weight to determine which endpoint (organ weight, organ-to-body weight ratio, or organ-to-brain weight ratio) is likely to accurately detect target organ toxicity. This evaluation has shown that analysis of organ-to-body weight ratios is predictive for evaluating liver and thyroid gland weights, and organ-to-brain weight ratios is predictive for evaluating ovary and adrenal gland weights. Brain, heart, kidney, pituitary gland, and testes weights are not modeled well by any of the choices, and alternative analysis methods such as analysis of covariance should be utilized.

Necrotizing enterotyphlocolitis in dogs treated with a potent antimuscarinic.

To assess the safety of a potent muscarinic receptor antagonist (antimuscarinic) for human clinical testing, repeat-dose oral toxicity studies were conducted in the Beagle dog. Treatment resulted in unexpected gastrointestinal system effects that were likely mediated by the exaggerated pharmacologic effects of the antimuscarinic on intestinal motility. Dogs developed profound anorexia followed by severe bloody diarrhea, and at necropsy, the intestinal tract was distended, filled with red-tinged fluid, and the mucosa was reddened. Histologically, intestinal lesions consisted of severe epithelial necrosis and mucosal atrophy localized predominantly to the ileum and large intestine. Feces from dogs with diarrhea were culture-positive for Clostridium perfringens (CP), suggesting that CP might be the etiologic agent; however, the intestinal lesions were not consistent with histologic findings reported in dogs with hemorrhagic canine enteritis.

Acute toxicity of lead, steel, and an iron-tungsten-nickel shot to mallard ducks (Anas platyrhynchos).

Twenty mallards (Anas platyrhynchos) of both sexes were dosed by oral gavage with Heavi-Shot (H-S; Environ-Metal, Inc., Sweet Home, Oregon, USA) pellets, 20 with steel shot, and 10 with lead (Pb) pellets, all of equal size. All pellets were fired from a shotgun into an absorbent material, retrieved, and weighed prior to introduction into the ducks. Birds were fed whole kernel corn and grit and observed for signs of toxicity for 30 days following dosing. Hevi-Shot pellets lost an average of 6.2% of their mass and steel shot pellets lost 57% of their mass in the birds' gizzards. Almost all (90%) of the Pb shot dosed birds died before the end of the study, while no mortality was observed in the steel or H-S dosed groups. Even though total food consumption differed between the H-S and steel shot groups, mean bird weight change was not different. There were no significant morphologic or histopathologic abnormalities of the liver and kidney in the H-S and steel shot groups. Results indicated that mallards dosed orally with eight No. 4 H-S pellets were not adversely affected over a 30-day period, and that H-S provides another environmentally safe nontoxic shot for use in waterfowl hunting.

Effects of phosphorothionate on the reproductive system of male rats.

Acute and Sub-acute toxic effects of a new pesticide phosphorothionate coded as RPR-V on testis of albino rat were studied. For the acute study, rats received a single dose of 30 mg/kg of RPR-V and sacrificed after 24 hours. For the Subacute study, 1.42 mg/kg/day was administered orally to rats for 10 days and 21 days. Acute exposure of rats to RPR-V brought no change either in the Gonadosomatic Index (GSI) or in the structure of testis or in the serum levels of Testosterone. Similarly, no significant change was observed in the Glutathione S-transferase (GST) activity. But, in testis there was significant increased in the reduced Glutathione (GSH) and Acid Phosphatase (AcP), whereas Alkaline Phosphatase (AkP) levels decreased significantly at 24hr post treatment. On 7th day (withdrawal period) after the cessation of the treatment the GSH, AcP, and AkP levels reached to near control. The sub-acute study revealed a significant post treatment. Due to RPR-V treatment the testis AcP levels increased significantly at 21st day of medication but AkP levels decreased both at 10th and 21st day of post treatment. Histopathological studies showed that after 10th day testis showed considerable loss of spermatozoids and at 21st day complete derangement of cellular organization was observed. Testosterone levels decreased significantly after 10th day and remained significantly low at 21st day. However, withdrawal studies showed a recovery in testis of rat treated with RPR-V. GST, GSH, GSI, AcP and AkP values were recovered, testosterone levels were also recovered but recovery in testis structure remained at a low profile.