Genotoxic and cytotoxic in vitro evaluation of ivermectin and its formulation ivomec® on Aedes albopictus larvae (CCL-126™) cells

Genotoxic effects of ivermectin (IVM) and its commercial formulation ivomec® (IVM 1.0%) were studied on Aedes albopictus larvae (CCL-126?) cells by sister chromatid exchange (SCE) and single cell gel electrophoresis (SCGE) while cytotoxicity was determined by cell-cycle progression (CCP), proliferative rate index (PRI), mitotic index (MI), 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and neutral red (NR) endpoints within a 1–250 µg mL^?1 concentration range. While IVM and ivomec® did not markedly affect SCE frequencies, these agents induced DNA-strand breaks enhancing both slightly damaged and damaged cells at 25–50 and 5–50 µg mL^?1 IVM and ivomec®, respectively. Both compounds exerted a delay in CCP and reduction of PRI at 10 µg mL^?1. Cytotoxicity was observed at concentrations higher than 25 µg mL^?1. A marked reduction of about 98% and 94% of MI compared to controls was noted with 25 µg mL^?1 of IVM and ivomec®, respectively. NR and MTT assays revealed that both compounds induced a cell growth inhibition within the 1–250 µg mL^?1 concentration range. Data indicated that IVM and ivomec® exert both genotoxicity and cytotoxicity in insect cells in vitro, at least in A. albopictus larvae CCL-126? cells.     

Acute toxicity of roundup to the nontarget organism Hyalella curvispina. Laboratory and field study

Glyphosate is the most used pesticide in Argentina. Hyalella curvispina is a widely distributed and commonly abundant component of the invertebrate assemblages in shallow waters of southern South America. The aim of this study was to assess the acute toxicity of the increasingly common Roundup Full II®, commercial formulation of the herbicide glyphosate (66.2% active ingredient), to H. curvispina in laboratory and field assessments. The mean estimated 48-h LC₅₀ of Roundup was 9.9 ± 1.7 mg L^?1. In a field experiment Roundup was applied to soybean plots. Simulated rain was generated the following day by means of irrigation sprinkler equipment. H. curvispina was exposed to runoff water and soy leaves. No mortality was observed. It is suggested that Roundup crop applications represent a low risk of acute toxicity to H. curvispina adults inhabiting water bodies adjacent to crop fields.     

Photodegradation of the pure and formulated alpha-cypermethrin insecticide gives different products

Alpha-cypermethrin is a broad-spectrum insecticide widely used in the treatment of rice crops, mainly commercialized as a CONTEST^® formulation. The photodegradation of alpha-cypermethrin and of the commercial formulation has not yet been systematically investigated in paddy water under natural conditions. Here, paddy water solutions of alpha-cypermethrin and CONTEST^® formulation at 5.0 mg L^?1 were irradiated under simulated sunlight for 10 days. Hydrolysis experiments were carried out on the same solutions preserved in the dark. Analysis by ultra-high-performance liquid chromatography–tandem mass spectrometry was developed for the identification of photodegradation products. Results show that degradation of pure alpha-cypermethrin and the formulation counterpart produces both common and different photodegradation products. Five out eleven photodegradation products were identified for the first time, in particular three in the alpha-cypermethrin paddy water solution and four in the formulation. Our findings underline the importance of carrying out photodegradation experiments directly on the commercial formulation, since degradation products could be different from the pure insecticide.     

Combined effects of copper and cadmium on Chlorella pyrenoidosa H.Chick: subcellular accumulation,distribution, and growth inhibition

Disposal of waste into aquatic ecosystems may cause microalgae to be exposed to various metals, e.g. copper and cadmium. The effects caused by combinations of metals may be more serious. Evaluations of subcellular fate, bioaccumulation, and biological effects of metals on aquatic organisms are generally derived from experiments with individual metals. The present study aims to evaluate the effects of exposure of Chlorella pyrenoidosa to copper and cadmium in combination on subcellular accumulation, distribution, and growth. The algae were exposed for 72 h to copper at concentrations of 13 – 25 µmol L^?1, cadmium at about 6 µmol L^?1, and combinations thereof. The levels of copper and cadmium in subcellular organelles, heat-denaturated protein, metal-rich granules, and heat-stable protein were determined by atomic absorption spectrometry. Exposure of C. pyrenoidosa to copper and cadmium in combination inhibited growth more strongly than copper and cadmium individually. Highest accumulation was observed in metal-rich granules and heat-stable proteins. Administration of both metals in combination affected their subcellular distribution: copper was mainly distributed into the metal-rich granules (70%–80%) and heat-stable proteins (9%–24%), cadmium in the metal-rich granules (88%–98%).     

Biochemical and histological alterations induced by a formulation of dicofol in the embryonic liver of Gallus domesticus

In the present investigation, hepatotoxic effect of a commercially available insecticide formulation of dicofol (Colonel–S® 18.5% emulsified concentrate) was studied in the developing chick embryo. Fertilized eggs of Gallus domesticus were immersed in three different dose concentrations, i.e. 250, 500, and 1000 mg L^?1 of Colonel–S on “0” and fourth days of incubation for 60 min at 37 °C and incubated till embryonic day 16. Severe histopathological cellular lesions, such as extensive cell degeneration and necrosis with enlarged blood sinusoids, cytoplasmic vacuolization, and leucocyte infiltrations with congestion or dilation of central vein, appeared in dose-depended manner. Dicofol treatment also caused significant decrease in the levels of total protein, glycogen, and glutathione content and an increase in alkaline phosphatase activity of embryonic liver, whereas glutamic pyruvic transaminase activity showed mixed response.     

Analysis of the toxicity of glyphosate and Faena® using the freshwater invertebrates Daphnia magna and Lecane quadridentata

With the present contribution an evaluation of the toxicity of the pure herbicide glyphosate and its commercial formulation Faena® is reported using the cladoceran Daphnia magna Strauss and the rotifer Lecane quadridentata Ehrenberg. LC₅₀, EC₅₀, NOEC, and LOEC values for each toxicant and for both test organisms are obtained. Regarding acute toxicity, Faena was 11-fold more toxic to L. quadridentata than pure glyphosate and slightly more toxic to D. magna (1.7-fold). Inhibition of esterase activity in L. quadridentata by glyphosate was the most sensitive end-point; the EC₅₀ was 1500-fold smaller than the LC₅₀. The implications of these results and their comparison with established international and national limit values for glyphosate are discussed.     

Antioxidant rich diet supplements (Spirulina and tamarind fruit pulp) mitigate hematological disorders in fluoride exposed mice

The effect of antioxidant rich diet supplements of Spirulina and tamarind fruit pulp at 230 mg kg^?1 body weight, separately as well as in combination, on various hematological parameters of Swiss albino mice exposed to fluoride for seven days at daily doses of 190 mg kg^?1 body weight and for 90 days at daily doses of 94 mg kg^?1 body weight is reported. Compared with controls, fluoride exposure decreased erythrocyte (12%–18%), leukocyte (16%) and platelet counts (7%, only for short-term exposure), hemoglobin (2%–17%) and packed cell volume (2%–14%) in Swiss albino mice reared on standard feed but these were altered little in the diet supplement groups. Fluoride exposure decreased lymphocytes in both standard feed (16%–21%) and diet supplement groups (5%–19%, but only after long-term exposure). Fluoride withdrawal led to recovery of the post-treated mice from long-term exposure. Spirulina alone and also in combination with tamarind fruit pulp was found more effective than tamarind alone in reducing hematological disorders in fluoride treated mice.     

Green chemistry approach to analysis of formic acid and acetic acid in aquatic environment by headspace water-based liquid-phase microextraction and high-performance liquid chromatography

A simple and totally organic-free (green) method, viz. headspace water-based liquid-phase microextraction combined with high-performance liquid chromatography-ultraviolet detection has been successfully developed for analysis of formic acid and acetic acid in environmental water samples. A microdrop of an aqueous solution of sodium hydroxide was suspended from the tip of a microsyringe needle over the headspace of the stirred sample solution containing the analytes at pH 1.0 for a given time. The microdrop was then retracted into the microsyringe, diluted with HPLC mobile phase, and injected to HPLC. Optimum efficiency has been achieved for: 3.0 µL NaOH microdrop (0.1 mol L^?1) exposed for 15 min over the headspace of an aqueous sample of 6.5 mL at 55 °C, containing 15% w/v of Na₂SO₄, adjusted to pH = 1.0 and stirred at 750 rpm. Under these conditions, enrichment factors of 162 and 187, limits of detection of 0.3 and 0.1 µg L^?1 (S/N = 3) with dynamic linear ranges of 1–500 and 0.5–500 µg L^?1 were obtained for formic acid and acetic acid, respectively. A reasonable repeatability (5.8% ≤ RSD ≤ 8.8%, n = 6) and satisfactory linearity (r² ≥ 0.997) illustrated the performance of the method.     

Hematoxicity and hepatoxicity attributed to zinc and cadmium in the Norwegian rat

This study was carried out to determine changes in the blood and liver of rats given oral doses of cadmium (Cd) and zinc (Zn). Thirty 3 month old rats were kept six to a compartment in a well-demarcated five compartment cage labeled A, B, C, D, and E. After 2 weeks of acclimatization, and with ad libitum administration of water and feed, group A and B rats were, respectively, exposed to 100 mg CdSO₄ and 2200 mg ZnSO₄, respectively, mixed with their feed. Group C rats were fed with a combination of 100 mg CdSO₄ and 2200 mg ZnSO₄ mixed with their feed, while group D rats were exposed to 100 mg CdSO₄ mixed with their feed for 48 h, and after they were given 2200 mg ZnSO₄ for 96 h. Group E rats served as control and were fed a normal diet. Analysis of blood samples after 5 days showed that in rats of groups A, B, C, and D, WBC count increased from 6800 to 12,400 mm^?3 and platelets from 26,000 to 88,000 mm^?3. Reduction in blood counts were found for RBC from 13.8 to 4.7 × 10⁶ mm^?3, hemoglobin from 15.6 to 11.4 dL^?1, and PCV from 47 to 34%. ESR was the same 1.0 mm h^?1 in all groups. Severe damage to the liver was also shown by severe degeneration of hepatocytes and increase in Kuppfer cells. The above observations were mildest in the liver of group C rats. A reduction of body weight was observed in all treatment groups. Cd and Zn were found to be hematoxic and hepatotoxic in the Norwegian rat with Cd exhibiting greater toxicity than Zn. Toxicity of Cd was suppressed by Zn only when both metals were admistered at the same time, but a delay in intake of Zn (after 48 h) had little effect on Cd toxicity.     

Optimisation of bisphenol A removal from water using chemically modified pine bark and almond shell

The ability of pine bark and almond shells to remove bisphenol A (BPA) from aqueous solutions was evaluated. Samples of these traditional agro-forestry by-products were milled, sieved into different particle size fractions (0.10–0.15 and 1.5–2.0 mm) and submitted to two different types of treatment. Sorption experiments were conducted in a batch system at room temperature and natural pH. Sorption equilibrium was attained after 48 h for all systems under study. Bisphenol A was adsorbed more effectively on the smaller particles of the sorbents. Pine bark and almond shell pretreated with formaldehyde presented higher sorption efficiency (95%), followed by almond shell (87%) and pine bark (82%) washed with hot water. All the sorption isotherms were found to fit a Freundlich equation, with correlation coefficients (R ²) between 0.823 and 0.989. The sorption coefficient (K _F) ranged from 0.06 to 0.74 (mg^1?1/n ·L ^1/n ·g ^?1). These results indicate that utilisation of both materials as an alternative sorbent for the removal of bisphenol A from contaminated waters is promising because they are available in large amounts and have an acceptable cost–efficiency ratio when compared with traditional adsorbents.     

Acute toxicity evaluation of triazophos,deltamethrin and their combination on earthworm,Eudrilus eugeniae and its impact on AChE activity

The acute toxicity of three formula grade pesticides namely, triazophos (an organophosphate, OP), deltamethrin (a pyrethroid) and combined pesticide (triazophos?+?deltamethrin) was determined in earthworm, Eudrilus eugeniae. They were exposed to different concentrations of these pesticides for 48 h by paper contact toxicity method. The LC₅₀ values for triazophos, deltamethrin and of combination were determined as 0.076, 0.031 and 0.065?μg/cm², respectively. To study the sublethal effect of these pesticides, E. eugeniae were exposed to 5% and 10% of LC₅₀ of pesticides for 48 h. Morphological alterations such as coiling, clitellar swelling, mucus release and bleeding followed by body segmentation were observed in exposed earthworm. Acetylcholinesterase (AChE) activity assayed in different regions of body segment exhibited a significant (p?<?0.05) decrease in its activity particularly in the pre-clitellar region as compared to other regions. The altered behavioural responses in pesticides exposed earthworms would have been due to decline in AChE activity of the nervous system.     

Oxidative stress and genotoxicity of 1-methyl-3-octylimidazolium chloride on loach (Misgurnus anguillicaudatus)

This study was a preliminary step to evaluate the acute toxicity of 1-methyl-3-octylimidazolium chloride ([C₈mim]Cl) on loach (Misgurnus anguillicaudatus) by determining the effects on hepatic antioxidant enzyme activities and by the comet assay. The results showed that [C₈mim]Cl had acute toxicity at concentrations above 20 mg L^?1, inducing oxidative stress and genotoxicity on fish liver cells. In respect to enzyme activities, [C₈mim]Cl induced changes in the activities of superoxide dismutase, catalase, and glutathione content the livers of fish exposed at 20–80 mg L^?1. [C₈mim]Cl at the same exposure level caused a remarkable increase in malondialdehyde level. The comet assay indicated that [C₈mim]Cl at 20–80 mg L^?1 induced genotoxicity in liver cells. With increased exposure concentration and time, the two comet parameters trailing rate and tail moment were significantly increased, with significant differences (P < 0.05) observed between control group and each treatment group. The present study shows that ionic liquids can be a threat to the health of aquatic organism when accidentally released to aquatic ecosystems.     

Bioaccumulation and toxic effects of copper in common onion Allium cepa L.

The aim of this study was to investigate the potential utility of Allium cepa L. as a bioindicator organism for measuring copper bioaccumulation and toxicity in laboratory conditions. Onions were exposed to increasing concentrations of the metal (0, 0.1, 0.5, 1, 5 and 10 μg mL^?1) for 7 days. Root and leaf development were chosen as biological endpoints, while bioaccumulation was evaluated in roots, bulbs and leaves. Copper caused inhibition of root elongation with increasing effects at the higher doses, growth being reduced by almost 60% at 0.1 μg mL^?1 and up to 95% at 10 μg mL^?1. The elongation of leaves was significantly lower only in specimens exposed at 0.5 μg mL^?1, but a total absence of newly formed tissues was observed at 10 μg mL^?1. A marked bioaccumulation of copper was measured in roots, with values increasing up to almost four orders of magnitude compared to controls; only slight or even no significant differences were observed for copper levels in leaves and bulbs of treated A. cepa. Multiple linear correlations revealed a significant inverse relationship between copper concentrations and tissue length in both the roots and leaves, evidencing a sensitive responsiveness of this biological model. The overall results suggest the suitability of A. cepa as a robust species for easy and simple ecotoxicological bioassays to test the toxic effects and bioavailability of environmental pollutants, especially trace metals.     

Response of mussel Brachidontes variabilis to chlorination

Brachidontes variabilis is a common fouling mussel species in cooling water systems of tropical coastal power stations. However, there are hardly any data available on the response of B. variabilis to chlorine, a commonly used antifouling biocide. Therefore, lethal and sublethal responses of this mussel to chlorine are of considerable interest to the industry. The response of mussels in terms of mortality pattern (LT₅₀ and LT₁₀₀) and physiological activities (oxygen consumption, filtration rate, foot activity and byssus thread production) in different size groups (with shell lengths of 7–24 mm) of B. variabilis was studied in the laboratory under different chlorine concentrations (0.25, 0.50, 0.75 and 1.00 mg l^?1 for sublethal responses and 1, 2, 3 and 5 mg l^?1 for mortality). The results showed that the exposure time for 100% mortality of mussels decreased significantly with increasing chlorine concentration. However, mussel size was not a determinant of its chlorine tolerance: all size groups tested (with shell lengths of 7–24 mm) took comparable exposure times to reach 100% mortality at a given chlorine concentration (1–5 mg l^?1). All size groups of B. variabilis showed a progressive reduction in physiological activities such as oxygen consumption, filtration rate, foot activity and byssus thread production, when chlorine residuals were increased from 0 to 1 mg l^?1. The data generated in the present work are compared with similar data available for other tropical fouling mussel species to see how far relative chlorine toxicity could have influenced the relative distribution of the mussels inside the seawater intake tunnel of a power station at Kalpakkam in India. It is shown that in this insufficiently chlorinated system, the relative distribution of Brachidontes striatulus, B. variabilis and Modiolus philippinarum reflects the relative tolerance of the species to chlorine.     

Modifications of photochemical efficiency,cellular viability and total phenolic content of Prosopis glandulosa leaves exposed to copper

The effects of copper (Cu²⁺) on photochemical efficiency, cellular viability and total phenolic content in Prosopis glandulosa leaves were evaluated after 4, 8 or 12 h exposure to 0.1, 10 or 100 mM Cu²⁺ using the tissue tolerance test. The results showed that significant reductions (p<0.003) of photochemical efficiency in P. glandulosa leaves were found after 8 h exposure to 100 mM Cu²⁺ compared with controls leaves. The results also showed a significant increase (p<0.05) on electrolyte leakage values in P. glandulosa leaves after 4 h exposure to 100 mM Cu²⁺ compared with control leaves. Total phenolic content in P. glandulosa leaves only differed significantly when exposed 12 h to the highest concentration of Cu²⁺. These results suggest that Cu²⁺ can produce reactive oxygen species that may form hydroxyl radicals that damage proteins and lipids, which in turn causes loss of integrity and reduce photochemical efficiency of leaves. In conclusion, the photochemical efficiency and cellular viability analyses can be used as useful physiological tools to assess early changes in photosynthetic performance of P. glandulosa in response to copper pollution.     

胚胎期草甘膦铵盐暴露对中华鳖幼体的表型与生理表现的影响

草甘膦是全球范围内生产与使用量最大的除草剂。虽然草甘膦制剂被分于低毒等级,但是许多研究显示其对动物的生理、行为表达具有广泛的干扰作用。本研究将中华鳖胚胎暴露于不同浓度的草甘膦铵盐溶液以评估草甘膦除草剂对其胚胎发育及孵出幼体表型及生理表现的影响。结果显示:一定浓度范围(5～5 000 mg·L~(-1))内,草甘膦铵盐对中华鳖胚胎发育速率、孵出幼体大小、游泳能力、肝脏甘油三酯含量、超氧化物歧化酶活性、丙二醛含量以及热激蛋白70 mRNA相对表达量无显著影响。结果表明:一定浓度范围草甘膦除草剂暴露对中华鳖胚胎发育的影响较小,不改变孵出幼体的表型及其生理表现。     

Biomass and carbon budgeting of sustainable agroforestry systems as ecosystem service in Indian Himalayas

Adoption of agroforestry is paramount as a climate change mitigation and adaptation strategy. The assessment of plant biomass is crucial for understanding the vulnerability of biological systems to climate change. In the present study, agroforestry systems viz., agrisilviculture (AS), agrihorticulture (AH), agrihortisilviculture (AHS) and agrisilvihorticulture (ASH) were investigated for biomass production and carbon stock in vegetation as well as in soil in the Indian central Himalaya along the elevation i.e. E₁ (<1100 m), E₂ (1100–1400 m), E₃ (1400–1700 m), E₄ (1700–2000 m) and E₅ (>2000 m). Mean aboveground and belowground biomass were 73.9% and 26.1%, respectively, of total biomass (64.4 t ha^?1) in agroforestry systems. Fodder and/or timber trees accounted for 31% (in AHS) to 74% (in AS) of total biomass, while fruit trees accounted for 18% (in ASH) to 73% (in AH) of total biomass. The contribution of agriculture crops to total biomass fluctuated between 19% (in ASH) and 26% (in AH). Total vegetation biomass, soil carbon and total carbon density in agroforestry systems increased significantly along the elevation, with maximum biomass at elevation E₅ (32.0 t ha^?1, 64.7 t C ha^?1 and 96.7 t C ha^?1). Total biomass of vegetation among agroforestry systems differed significantly. Soil carbon stock was highest in AHS (59.5 t C ha^?1) and total carbon density (vegetation + soil) was highest in ASH (93.0 t C ha^?1). Thus, in Indian Himalayas, vegetation biomass, carbon stock, soil and total carbon (vegetation + soil) stock increased along the elevation.

Abbrviations: AG: aboveground; BG: belowground; WD: wood density; VOB: volume over bark; BEF: biomass expansion factor; AS: agrisilviculture; AH: agrihorticulture; ASH: agrisilvihorticulture; AHS: agrihortisilviculture; E: elevation; C: carbon; CO₂: carbon-di-oxide; IPCC: Intergovernmental Panel on Climate Change; DBH: diameter at breast height; AGBD: aboveground biomass density; BGBD: belowground biomass density; GSVD: growing stock volume density     

Short-term effects of sucralose on Calanus finmarchicus and Calanus glacialis in Disko Bay,Greenland

The potential effects of sucralose on the Arctic copepods Calanus finmarchicus and Calanus glacialis were studied in Disko Bay, Greenland. Sucralose is a non-calorie sweetener and chlorine derivate of sucrose containing three chlorine atoms. Scandinavian screening studies of sucralose in 2007, revealed sucralose in all effluent samples. To investigate whether sucralose is harmful to the Arctic aquatic ecosystems, possible short-term effects were investigated on egg production, hatching rate, food intake and mortality of two species of Arctic copepods. The copepods were exposed to six different concentrations (0–50,000 ng · L^?1) of sucralose, which spans the range of concentrations found in the screening studies. Exposure led to no mortality among the copepods. Food intake by C. glacialis increased with increasing concentrations of sucralose. In C. finmarchicus, food intake did not differ with increasing concentrations. No effect of sucralose was observed on egg production of C. finmarchicus. Despite increased food intake with increasing concentrations of sucralose, C. glacialis did not increase its egg production. The results show that both species responded weakly to sucralose, but with C. glacialis being possibly slightly more sensitive to sucralose than C. finmarchicus.     

Biological effects of water soluble fraction of crude oil on the Arctic sea ice amphipod Gammarus wilkitzkii

With increasing petroleum related activity in the Barents Sea and the subsequent risk of accidents, there is a demand for knowledge about the effect of oil pollution in Arctic ecosystems. In the present study, the sea ice amphipod Gammarus wilkitzkii was exposed to a water soluble fraction (WSF) of oil in two experiments, using a rock column system. First, three groups of adult females were exposed for 36 days for control (n=9), low dose (n=10) (initial and final oil concentration: 14, 115 and 120 μ g/l) and high dose (n=10) (initial and final oil concentration: 78, 764 and 395 μ g/l). Total oxyradical scavenging capacity (TOSC), malondialdehyde (MDA), catalase activity (CAT), respiration rate and mortality were measured. In the second experiment, moulting rate was measured in immature individuals of two groups, control (n=10) and exposed (n = 20), over 113 days. No mortality was observed in either of the experiments. There was a dose-related significant increase in respiration rate (40.69 (±22.82), 55.63 (±20.98), 94.57 (±22.80)) mg O₂ h^?1g ww^?1 in control, low dose and high dose, respectively. A higher MDA level was detected in the low dose group (25.04 (±6.00) nmol g^?1 of tissue) compared to both control (20.44 (±2.62) nmol g^?1 of tissue) and high dose groups (20.93 (±4.79) nmol g^?1 of tissue). Likewise, the low dose group had the highest value of TOSC towards hydroxyl (727.74 (±475.58), 1157.58 (±278.62), 1067.30 (±369.22) TOSC unit value mg^?1 of protein for control, low and high dose, respectively). Although no difference in the catalase activity between control and exposed groups was detected, higher activity was measured at 0 °C (average: 248 μ mol^?1 min^?1 mg^?1 of protein) than at the standard temperature for catalase measurement of 25 °C (average: 140 μ mol^?1 min^?1 mg^?1 of protein), indicating a need to optimise the standard operational procedure when working with Arctic organisms. No effect of WSF exposure on moulting rate was detected. In conclusion, little mortality was observed during the experiments, indicating some degree of tolerance levels. However, the biomarker results indicated sub-lethal effects in G. wilkitzkii after exposure to WSF.     

Acute toxicity of ammonia to a freshwater teleost,Labeo bata larvae

Ammonia toxicity tests were performed with Labeo bata (bata) larvae of three different size groups. One hundred percent survival of larvae (500.0?±?4.0?mg) was recorded when exposed to ammonia concentrations of 1.0–13.56?mg?L^?1 at 96?h of exposure. Bata larvae exposed to ammonia concentrations of 15.8–25?mg?L^?1 showed 10–74% mortalities. The 96?h LC₅₀ value for 200 (±5), 250 (±2) and 500 (±4) mg bata larvae were 11.5, 16.8 and 22.5?mg?L^?1 un-ionised ammonia concentrations, respectively. When fish were exposed to different doses of ammonia, behavioural changes immediately occurred even at the lowest dose. At first, the fish became hyperexcitable, the skin darkened and they showed an increased ventilation frequency, fish behaviour became normal, 24?h after exposure. A 96?h LC₅₀ value of un-ionised ammonia showed direct relationship with the increasing size of bata larvae.