Evaluation of genotoxic and oxidative stress response to dimethoate in freshwater fish Channa punctatus (Bloch)

Abstract

Dimethoate is one of the organophosphate insecticides widely used in agriculture throughout the world and is an inhibitor of cholinesterase in animals. The objective of the present study was to detect oxidative stress and DNA damage induced by dimethoate in the freshwater fish Channa punctatus (C. punctatus). The LC₅₀-96 h value of technical grade dimethoate was estimated at 19.10 μg L^-1 in a semi-static system and, on the basis of the LC₅₀ value, three concentrations were determined. The fish were exposed to these concentrations of dimethoate for 96 h and samplings were done at 24 and 96 h for assessment of oxidative stress and DNA damage. After exposure to dimethoate, the level of superoxide dismutase declined while lipid peroxidation, glutathione, induction of micronucleus and DNA damage were increased in C. punctatus as the concentration and exposure time increased. Thus our results suggest that dimethoate induces genotoxic effects which invariably accompanied and correlated with oxidative stress.     

Modified comet assay as a biomarker of sodium dichromate-induced oxidative DNA damage: optimization and reproducibility.

Hexavalent chromium (Cr[VI]) is a genotoxic carcinogen that has been associated with an increased risk of nasal and respiratory tract cancers following occupational exposure. Although the precise mechanism(s) remain to be elucidated, there is evidence for a role of oxidative DNA damage in the genotoxicity of Cr(VI). In the current study, human white blood cells were treated in vitro with non-cytotoxic concentrations of sodium dichromate (1-100 microM) for 1 h. Analysis by immunocytochemistry indicated the presence of elevated levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine at concentrations of sodium dichromate greater than 10 microM. In contrast, the lowest concentration of dichromate that resulted in a statistically significant increase in levels of formamidopyrimidine DNA glycosylase (FPG)-dependent DNA strand breaks was 100 nM (p<0.05). In addition, levels of both control and dichromate-induced FPG-dependent strand breaks from blood samples taken from the same individuals over 10 months proved remarkably reproducible in the individuals studied. The coefficients of variation over three different times of the year in control and dichromate-induced oxidative DNA damage for the four individuals were 54, 1, 37 and 4, and 45, 6, 21 and 18%, respectively. In summary, these results indicate that physiologically relevant, nanomolar concentrations of sodium dichromate cause DNA base oxidation in human white blood cells in vitro as assessed by the FPG-modified comet assay. Furthermore, comet assay data from an individual are reproducible over an extended period. This consistency is sufficient to suggest that the modified comet assay might prove to be a useful and sensitive biomonitoring tool for individuals occupationally exposed to hexavalent chromium.     

Investigating DNA damage in tannery workers occupationally exposed to trivalent chromium using comet assay

DNA damage of peripheral lymphocytes in 60 workers occupationally exposed to trivalent chromium [Cr(III)] in a tannery was studied using comet assay. The urinary and blood chromium levels were detected as a biomarker of internal exposure. The 90 subjects were divided into three groups: (i) exposure group I included 30 tannery workers highly exposed to chromium from tanning department; (ii) exposure group II included 30 tannery workers with moderate chromium exposure from finishing department; (iii) control group included 30 individuals without exposure to physical or chemical genotoxic agents. No significant difference was found among the three groups for age and smoking. The results showed that the medians of blood and urinary Cr of two exposure groups were significantly higher than those of control group (P < 0.01). And the medians of blood and urinary Cr of exposure group I were significantly higher than those of exposure group II (P < 0.05 or P < 0.01). The medians of mean tail length (MTL) of the three groups were 5.33 (2.90–8.50), 3.43 (2.31–8.29) and 2.04 (0.09–3.83) μm, respectively; The medians of mean tail moment (MTM) of the three groups were 6.28 (2.14–11.81), 3.41 (1.25–11.07) and 0.53 (0.13–3.29), respectively. The MTL and MTM of two exposure groups were significantly higher than those of control group (P < 0.01). The MTL and MTM of exposure group I were significantly higher than those of exposure group II (P < 0.01). The results of the present investigation suggest that occupational exposure to trivalent chromium can lead to a detectable DNA damage of human peripheral lymphocytes. Moreover, DNA damage was associated with chromium levels in blood. DNA damage may serve as a valuable effective biomarker and total chromium in blood may serve as a useful internal exposure biomarker in the population occupationally exposed to trivalent chromium.     

Mutagenic,genotoxic and bioaccumulative potentials of tannery effluents in freshwater fishes of River Ganga

The tannery industries are the reason of major environmental concerns as they release toxic heavy metals, like chromium, in rivers posing risks of genotoxicity and mutagenicity in aquatic organism and indirectly in humans through food chain. In the present analysis, the freshwater inhabitant fishes of River Ganges, viz., Labeo calbasu, Puntius sophore, and Mystus vittatus, were examined for assessing the genotoxic, mutagenic, and bioaccumulative potentials of tannery effluents. For genotoxicity assessment, the blood and gill samples of fishes prevailed from polluted sites of River Ganges adjoining Kanpur city were utilized for comet assay and micronucleus test. The present investigation revealed the presence of significantly (p < 0.05) higher micronuclei induction and % tail DNA in erythrocytes and gill cells of the fishes collected from the polluted sites. The bioaccumulation studies revealed chromium concentration in muscle (0.89 µg/g) and gill tissues (0.24 µg/g) of L. calbasu; muscle (0.44 µg/g) and gills (1.23 µg/g) of P. sophore; and muscle (0.9617 µg/g) and gills (0.3628 µg/g) of M. vittatus, quite higher than the permissible limits of the World Health Organization. Consequently, the present study indicates strongly that River Ganges is contaminated with harmful tannery pollutants causing genotoxicity and mutagenicity in freshwater fishes.     

DNA damage and repair after exposure to sevoflurane in vivo, evaluated in Swiss albino mice by the alkaline comet assay and micronucleus test

The relationship between DNA damage and repair of peripheral blood leukocytes, liver, kidney and brain cells was investigated in Swiss albino mice (Mus musculus L.) after exposure to sevoflurane (2.4 vol% for 2 h daily, for 3 days). Genetic damage of mouse cells was investigated by the comet assay and micronucleus test. To perform the comet assay, mice were divided into a control group and 4 groups of exposed mice sacrificed on day 3 of the experiment, at 0, 2, 6 or 24 h after the last exposure to sevoflurane. Mean tail length (TL), tail moment (TM), and tail intensity (TI) values were significantly higher in exposed mice (all examined organs) than in the control group. Significant DNA damage immediately after exposure to sevoflurane was observed in leukocytes. Damage induction in the liver, kidney, and brain occurred 6 h later than in leukocytes, as expected according to the toxicokinetics of the drug, where blood is the first compartment to absorb sevoflurane. However, none of the tested tissues revealed signs of repair until 24 h after the exposure. To distinguish the unrepaired genome damage in vivo, the micronucleus test was applied. Number of micronuclei in reticulocytes showed a statistically significant increase, as compared with the control group at all observed times after the treatment.     

DNA damage and micronucleus induction in human leukocytes after acute in vitro exposure to a 1.9 GHz continuous-wave radiofrequency field

Human blood cultures were exposed to a 1.9 GHz continuous-wave (CW) radiofrequency (RF) field for 2 h using a series of six circularly polarized, cylindrical waveguides. Mean specific absorption rates (SARs) of 0.0, 0.1, 0.26, 0.92, 2.4 and 10 W/kg were achieved, and the temperature within the cultures during a 2-h exposure was maintained at 37.0 +/- 0.5 degrees C. Concurrent negative (incubator) and positive (1.5 Gy (137)Cs gamma radiation) control cultures were run for each experiment. DNA damage was quantified immediately after RF-field exposure using the alkaline comet assay, and four parameters (tail ratio, tail moment, comet length and tail length) were used to assess DNA damage for each comet. No evidence of increased primary DNA damage was detected by any parameter for RF-field-exposed cultures at any SAR tested. The formation of micronuclei in the RF-field-exposed blood cell cultures was assessed using the cytokinesis-block micronucleus assay. There was no significant difference in the binucleated cell frequency, incidence of micronucleated binucleated cells, or total incidence of micronuclei between any of the RF-field-exposed cultures and the sham-exposed controls at any SAR tested. These results do not support the hypothesis that acute, nonthermalizing 1.9 GHz CW RF-field exposure causes DNA damage in cultured human leukocytes.     

Investigating DNA damage in tannery workers occupationally exposed to trivalent chromium using comet assay

DNA damage of peripheral lymphocytes in 60 workers occupationally exposed to trivalent chromium [Cr(III)] in a tannery was studied using comet assay. The urinary and blood chromium levels were detected as a biomarker of internal exposure. The 90 subjects were divided into three groups: (i) exposure group I included 30 tannery workers highly exposed to chromium from tanning department; (ii) exposure group II included 30 tannery workers with moderate chromium exposure from finishing department; (iii) control group included 30 individuals without exposure to physical or chemical genotoxic agents. No significant difference was found among the three groups for age and smoking. The results showed that the medians of blood and urinary Cr of two exposure groups were significantly higher than those of control group (P<0.01). And the medians of blood and urinary Cr of exposure group I were significantly higher than those of exposure group II (P<0.05 or P<0.01). The medians of mean tail length (MTL) of the three groups were 5.33 (2.90-8.50), 3.43 (2.31-8.29) and 2.04 (0.09-3.83) microm, respectively; The medians of mean tail moment (MTM) of the three groups were 6.28 (2.14-11.81), 3.41 (1.25-11.07) and 0.53 (0.13-3.29), respectively. The MTL and MTM of two exposure groups were significantly higher than those of control group (P<0.01). The MTL and MTM of exposure group I were significantly higher than those of exposure group II (P<0.01). The results of the present investigation suggest that occupational exposure to trivalent chromium can lead to a detectable DNA damage of human peripheral lymphocytes. Moreover, DNA damage was associated with chromium levels in blood. DNA damage may serve as a valuable effective biomarker and total chromium in blood may serve as a useful internal exposure biomarker in the population occupationally exposed to trivalent chromium.     

Toxicity of intermittent chlorination to freshwater fish: Influence of temperature and chlorine form

Fingerling size Salmo gairdneri, Oncorhynchus kisutch, Notemigonus crysoleucas, Cyprinus carpio, and Ictalurus punctatus were exposed in the laboratory three times daily for up to seven days to pulses of either free chlorine or monochloramine. This regime simulated conditions often encountered in the outfall of steam electric generating plants which chlorinate intermittently. LC₅₀'s, LT₅₀'s and response isopleths giving various percentage mortalities, were computed from the bioassays. S. gairdneri, O. kisutch, and I. punctatus were the most sensitive to both types of chlorine. C. carpio were most resistant and the N. crysoleucas were intermediate in sensitivity. Temperature had relatively little effect on the toxicity of intermittent chlorine to the species tested. In this type of test regime, free chlorine was three to fourteen-fold more toxic (depending on the species) than monochloramine. Water quality criteria for the protection of fish should, in the future, take this differential toxicity into consideration.     

Association of HSP70 and genotoxic damage in lymphocytes of workers exposed to coke-oven emission

Heat shock proteins (Hsps) have been reported to protect cells, tissues, and organisms against damage from a wide variety of stressful stimuli. Whether they protect against deoxyribonucleic acid (DNA) damage in individuals exposed to environmental stresses and chemical carcinogens is unknown. In the study, we investigated the association between Hsp70 levels (the most abundant mammalian Hsp) and genotoxic damage in lymphocytes of workers exposed to coke-oven emission using Western dot blot and 2 DNA damage assays, the comet assay and the micronucleus test. The data show that there is a significant increase in Hsp70 levels, DNA damage score, and micronucleus rates in lymphocytes of workers exposed to coke-oven emission as compared with the control subjects. Furthermore, there was a significant negative correlation of Hsp70 levels with DNA damage scores in the comet assay (r = -0.663, P < 0.01) and with micronucleus rates (r = -0.461, P < 0.01) in the exposed group. In the control group, there was also a light negative correlation between Hsp70 with DNA damage and micronuclei rate (r = -0.236 and r = 0.242, respectively), but it did not reach a statistically significant level (P > 0.05). Our results show that individuals who had high Hsp70 levels generally showed lower genotoxic damage than others. These results suggest a role of Hsp70 in the protection of DNA from genotoxic damage induced by coke-oven emission.     

Effects of hypoxia and petroleum on the genotoxic and morphological parameters of Hippocampus reidi

Hypoxia events are common in many aquatic systems, which may be a natural event or provoked by anthropogenic actions, as well as accidents involving oil occurring throughout the world are frequent. Thus, through the possibility of occurrence of these two situations in same place the purpose of this study was to evaluate if damage caused by crude oil on genotoxic and morphological parameters in the marine fish species Hippocampus reidi will be aggravated by events of severe hypoxia. Sea horses were exposed during 8h to the following conditions: crude oil (OIL), severe hypoxia (HYP), association of severe hypoxia and crude oil (HYP+OIL) and normoxia without contaminant (CONT). An increase in micronuclei observed in OIL and HYP+OIL groups indicates that the crude oil exposure was a determining factor in the micronuclei induction and hypoxia did not intensify this result. In comet assays, both petroleum and hypoxia provoke DNA damage. The most frequent histopathology in the control groups and in those exposed to OIL and HYP+OIL groups were: hypertrophy and capillary dilation; hypertrophy and hyperplasia; hypertrophy, epithelial "lifting" and epithelial hyperplasia. An elongation of the lamellae was observed in fish from the two groups exposed to hypoxia, probably due to the fact that these groups required a greater flow of blood in the gills to increase the efficiency of gas exchange, since they were in a hypoxic environment. In summary, the micronuclei test and comet assay can be used as a good biomarker of contamination by petroleum. The association of hypoxia with crude oil in some aspects may exacerbate the responses of fish, in the light of the increase in DNA damage and the alterations in thickness of the gill epithelium.     

Genotoxic ability of cadmium, chromium and nickel salts studied by kinetochore staining in the cytokinesis-blocked micronucleus assay

The aneugenic and clastogenic ability of cadmium chloride(II), cadmium sulfate(II), nickel chloride(II), nickel sulfate(II), chromium chloride(III) and potassium dichromate(IV) have been evaluated through kinetochore-stained micronucleus test. Traditional genotoxicity assays evaluate DNA damage, gene mutations and chromosome breakage. However, these tests are not adequate to detect aneugenic agents that do not act directly on DNA. Staining kinetochores in the cytokinesis-blocked micronucleus assay is a useful way to discriminate between clastogens and aneuploidogens and may allow a rapid identification of aneuploidy-inducing environmental compounds.Human diploid fibroblasts (MRC-5) were employed. All compounds increased micronuclei frequency in a statistically significant way. However, increases in kinetochore-positive micronuclei frequencies were higher than in kinetochore-negative ones. The present work demonstrates the genotoxic ability of the cadmium and chromium salts studied. Aneugenic as well as clastogenic ability could be observed with this assay. Nickel salts, as it was expected because of their known weak mutagenicity, showed lower genotoxic effects than the other metal salts studied. As the test employed only allows the detection of malsegregation, it is proposed that this mechanism is at least one of those by which the tested metal salts induced aneuploidy. On the other hand, visualization of kinetochores in all experiments suggests that the compounds studied did not act by damaging these structures.     

Genotoxic ability of cadmium, chromium and nickel salts studied by kinetochore staining in the cytokinesis-blocked micronucleus assay

The aneugenic and clastogenic ability of cadmium chloride(II), cadmium sulfate(II), nickel chloride(II), nickel sulfate(II), chromium chloride(III) and potassium dichromate(IV) have been evaluated through kinetochore-stained micronucleus test. Traditional genotoxicity assays evaluate DNA damage, gene mutations and chromosome breakage. However, these tests are not adequate to detect aneugenic agents that do not act directly on DNA. Staining kinetochores in the cytokinesis-blocked micronucleus assay is a useful way to discriminate between clastogens and aneuploidogens and may allow a rapid identification of aneuploidy-inducing environmental compounds.Human diploid fibroblasts (MRC-5) were employed. All compounds increased micronuclei frequency in a statistically significant way. However, increases in kinetochore-positive micronuclei frequencies were higher than in kinetochore-negative ones. The present work demonstrates the genotoxic ability of the cadmium and chromium salts studied. Aneugenic as well as clastogenic ability could be observed with this assay. Nickel salts, as it was expected because of their known weak mutagenicity, showed lower genotoxic effects than the other metal salts studied. As the test employed only allows the detection of malsegregation, it is proposed that this mechanism is at least one of those by which the tested metal salts induced aneuploidy. On the other hand, visualization of kinetochores in all experiments suggests that the compounds studied did not act by damaging these structures.     

In vivo genotoxicity of the pyrethroid pesticide beta-cyfluthrin using the comet assay in the fish Bryconamericus iheringii

Environmental pollution by pesticide residues is a major environmental concern due to the extensive use of these substances in agriculture. The insecticide beta-cyfluthrin is a synthetic pyrethroid widely used in agricultural and other domestic activities. The aim of the present study was to assess the genotoxic effects of a sublethal exposure of the fish Bryconamericus iheringii (Characidae) to a commercial formulation of beta-cyfluthrin using the comet assay. Fish were exposed to sublethal concentrations (4.2 and 5.6 microg/L) of beta-cyfluthrin under static conditions during 24- and 48-h exposure periods. Fish in tap water were used as negative controls. Results obtained by the comet assay revealed genotoxic effects of the pyrethroid in the higher concentration and at the longer exposure period. The mean DNA damage index of fish exposed to 5.6 microg/L beta-cyfluthrin for 48 h was significantly higher (145.9 +/- 51.8) than in the control group (69.3 +/- 39.5). These findings indicate that native fish species might be at risk for genotoxic damage in waters contaminated with beta-cyfluthrin.     

Oral chromium(VI) does not affect the frequency of micronuclei in hematopoietic cells of adult mice and of transplacentally exposed fetuses

Chromium(VI) compounds are genotoxic in a variety of cellular systems. Their potential carcinogenicity is affected by toxicokinetic patterns restricting bioavailability to certain targets, and by metabolic pathways affecting interaction of chromate-derived reactive species with DNA. Epidemiological data indicate that chromium(VI) can be carcinogenic to the human respiratory tract following inhalation at doses that are only achieved in certain occupational settings. However, concern has been raised that adverse effects may also result from oral intake. In order to further explore this issue, we performed studies in BDF1 and Swiss mice of both genders and various age. Sodium dichromate dihydrate and potassium dichromate were administered either with the drinking water, up to a concentration of 500 mg chromium(VI)/l for up to 210 consecutive days, or in a single intragastric dose of 17.7 mg/kg body weight. Under these conditions, no increase of the micronucleus frequency was observed in either bone marrow or peripheral blood erythrocytes. Conversely, the same compounds induced a clastogenic damage following intraperitoneal injection, which by-passes detoxification mechanisms. In addition, due to the hypothesis that susceptibility may be increased during the period of embryogenesis, we treated pregnant mice, up to a concentration of 10 mg chromium(VI)/l drinking water. There was no effect on the numbers of fetuses/dam and on body weight of fetuses. Again, no toxic or genotoxic effect was observed either in bone marrow of pregnant mice or in liver and peripheral blood of their fetuses. Thus, even at doses that largely exceed drinking water standards (up to 10,000 times) or by massive intragastric administration, chromium(VI) is not genotoxic to hematopoietic cells of either adult mice or transplacentally exposed fetuses. These conclusions are consistent with the poor toxicity and lack of carcinogenicity of oral chromium(VI), and are mechanistically explained by the high efficiency of chromium(VI) detoxification processes in the gastrointestinal tract.     

Genotoxicity of municipal landfill leachate on root tips of Vicia faba

The genotoxicity of municipal landfill leachate was studied using the Vicia faba root-tip cytogenetic bioassay. Results show that landfill leachates collected in different seasons decreased the mitotic index (MI) and caused significant increases of micronucleus (MN) frequencies and anaphase aberration (AA) frequencies in a concentration-dependent manner (concentration expressed as ‘chemical oxygen demand’ measured by the method of potassium dichromate oxidation (COD_Cr)). In addition, a seasonal difference in genotoxicity induced by leachate was observed. The results confirm that leachate is a genotoxic agent in plant cells and imply that exposure to leachate in the aquatic environment may pose a potential genotoxic risk to organisms. The results also show that the V. faba cytogenetic bioassay is efficient, simple and reproducible in genotoxicity studies of leachate, and that there is a correlation between the genotoxicity and the chemical measurement (COD_Cr) of leachate.     

Evaluation of genotoxicity in a group of workers from a petroleum refinery aromatics plant

Petroleum refinery workers are potentially exposed to a wide range of petroleum-derived hydrocarbons and chemical substances used in the manufacturing of petroleum derivatives. Benzene, toluene and xylene (BTX) are produced by distillation in the aromatics units and used as raw materials for petrol and petrochemical products. The aim of this study was to evaluate the genotoxic effects of occupational exposure to BTX in a petroleum refinery in the North of Portugal. The exposed group consisted of 48 workers from the aromatics plant and the control group consisted of 30 persons matched for various confounding factors. Chromosome aberrations (CA), micronuclei (MN), and DNA damage (evaluated by means of the comet assay) were measured in peripheral blood leukocytes. t,t-Muconic acid (t,t-MA), hippuric acid (HA) and methylhippuric acid (MHA) concentrations were measured in urine samples collected at the end of the workshift. The results suggest that occupational exposure to toluene and xylene is very low. A statistically significant increase in t,t-MA excretion was found in the exposed group although t,t-MA levels were found to be lower than the biological exposure index (BEI). Significant increases were found for CA, MN and comet tail length (TL) in the exposed group (p<0.05). No association was found between tobacco smoking and the effect biomarkers analysed. A positive association was found between CA and MN with age in the control group (p<0.05).     

Comparative studies of the genotoxic activity of a new palladium (II) acidocomplex and cisplatin in human blood lymphocytes in vitro

Comparative studies on the genotoxic activity of cisplatin and morfozol, the first representative of a new class of cation-anion complexes of palladium [AH]₂[PdCl₄] (where A—methylmorpholine), have been performed using human lymphocytes in vitro. The results of the DNA-DNA cross-linking activity investigations showed that both compounds studied exhibited biphasic dose-effect relationship: a linear decrease of the DNA percent in the comet tail and a plateau region. However, in the plateau region, morfozol caused a 6-fold reduction of the DNA percent in the comet tail while cisplatin caused a 2-fold decrease only. Morfozol, like cisplatin, inducing DNA-protein cross-linking and generating reactive oxygen species, was more effective than cisplatin.     

The use of cyprinodont fish, Aphanius fasciatus, as a sentinel organism to detect complex genotoxic mixtures in the coastal lagoon ecosystem

In the present work we aimed to standardise the alkaline comet assay with erythrocytes of the cyprinodont, Mediterranean Killifish, Aphanius fasciatus. The aims of the study were to explore the suitability of this fish to assess biomarkers of genotoxic effects and as a sentinel organism to detect complex genotoxic mixtures in coastal lagoon ecosystems. Following proper optimisation, the application and effectiveness of the comet assay in erythrocytes of A. fasciatus were tested by measuring the tail DNA (%) induced by (a) in vivo exposure of individual fish to X-rays (dose, 3Gy) and (b) following in vitro challenge of erythrocytes with restriction endonucleases Fok-I and Eco-RI, which selectively induce double-strand breaks with cohesive and blunt termini, respectively. Furthermore, in order to evaluate whether circulating fish blood contained actively proliferating cells that could influence the extent of DNA damage in control (untreated) fish, we measured the number of "comets" positive for 5-bromo-2'-deoxyuridine (BrdU) by the use of anti-BrdU antibody and immuno-histochemical methods. Both treatments (i.e. with X-rays and restriction endonucleases) induced statistically significant increases in tail DNA (%) values compared with the relevant untreated controls, indicating the effectiveness of the comet assay in the erythrocytes of A. fasciatus to detect different types of DNA lesions. Results from anti-BrdU antibody labelling of erythrocytes indicated a very low percentage (5%) of "comets" positive for BrdU. Following optimisation and validation of the assay under laboratory conditions, fish were collected in the Orbetello lagoon (Tuscany, Italy), considered to be a significantly polluted site. The results showed statistically significant increases for tail DNA (%) compared with corresponding values observed in erythrocytes of fish caught in the unpolluted reference site "Saline di Tarquinia". The effects of physico-chemical parameters of the water (i.e., salinity, pH and oxygen content) did not significantly influence the induction of DNA damage. These results indicate that the comet assay provides a reliable parameter and that A. fasciatus is a promising "sentinel organism" to detect the genotoxic impact of complex mixtures in coastal lagoon ecosystems.     

Boric acid‐induced immunotoxicity and genotoxicity in model insect Galleria mellonella L. (Lepidoptera: Pyralidae)

Boric acid (BA) is widely used in various industrial process and can be accessed to nontarget organisms. This study aimed to investigate the insecticidal effects of BA and its toxic activities with respect to immunologic and genotoxic effects using Galleria mellonella larvae as a model. BA concentrations (78.125–10,000 ppm) were administrated to the larvae using the feeding method. Concentration‐dependent mortality was observed in all larval groups. Probit analysis revealed LC₃₀, LC₅₀, and LC₇₀ values to be 112.4, 320.1, and 911.4 ppm, respectively. These concentrations were used in all bioassays. Drastic reductions in total hemocyte counts along with changes in differential hemocyte counts were observed following BA treatment. Cell viability assays showed dose‐dependent reductions in viable cells and an increase in the necrotic and apoptotic ratios after BA treatment. However, mitotic indices of larval hemocytes did not change at all BA concentrations. The cytotoxic effect of BA led to a significant reduction in cellular immune responses such as encapsulation, melanization, and nodulation activities of treated larvae. While BA increased micronucleus ratios at the highest concentration, comet parameters indicating DNA damage increased in G. mellonella larval hemocytes at all concentrations. These report that BA suppresses the immune system of G. mellonella and also poses risks of genotoxicity at high concentrations.     

Water quality requirements for first-feeding in marine fish larvae. II. pH,oxygen, and carbon dioxide

Previously unfed larvae representing eight species of marine teleosts (a soleid, Heteromycteris capensis Kaup; a cynoglossid. Trulla capensis Kaup; a gadid, Gaidropsarus capensis (Kaup); a congiopodid, Congiopodus spinifer (Smith); three sparids, Diplodus sargus (L.), Pachymetopon blochi (Val.), and an unidentified species; and a centracanthid, Pterosmaris axillaris (Boulenger)) were exposed for 24 h to various pH's and concentrations of dissolved oxygen and carbon dioxide and then tested for their ability to first-feed. Mortalities recorded during the 24-h exposure went into the fitting of response curves and the calculation of the LC₅₀ and LC₁₀. The incidence of successful first-feeding during the subsequent 4-h exposure to food yielded values for the EC₅₀ and EC₁₀. The exposure to sub-optimal conditions continued during the 4-h feeding test. The sensitivity of first-feeding larvae to high pH and dissolved oxygen may pose hazards for the marine fish culturist; low pH appears to be harmful (at least in the short term) only at levels below about pH 6.0; carbon dioxide poisoning is of doubtful importance in practical fish culture. There were only minor interspecific differences in calculated 24-h first-feeding EC₅₀'s, which suggests general applicability of the results to a wide variety of first-feeding marine fish larvae.