Genotoxicity assay of landfill leachates

Environmental burden with genotoxic chemicals is increasingly becoming a serious problem mainly due to hazardous waste dumping and industrial effluents. The Mutatox® mutagenicity assay was validated with known mutagen for its efficacy in the detection of direct mutagens and S-9 activated promutagens and applied to the complex environmental samples of landfill leachates from the Al-Sulaibiyah solid waste disposal site in Kuwait. The leachate samples from borehole 3 collected between December 1994 and May 1995 (cold and rainy months) showed genotoxic response in direct assay, indicating the presence of direct acting mutagens, whereas the samples collected between June and September 1995 (hot and dry months) were active in both direct and S-9 activated assay, also showing the presence of promutagens. Thus the leachate from the landfill, which is not equipped with a leachate collection and treatment facility, was found capable of exerting genotoxic effects that may influence the biota of the receiving system. © 1998 John Wiley & Sons, Inc. Environ Toxicol Water Qual 13: 127–131, 1998     

Genotoxic effects of old landfill leachate on HepG2 cells after nitration/ultrafiltration/reverse osmosis membrane treatment process

Toxicity assessment of nitration/ultrafiltration/reverse osmosis (nitration/UF/RO) project, which has recently been widely used as an efficient process with applications in practical leachate treatment, was very limited. In the present study, DNA damage of leachates was investigated before and after the nitration/UF/RO process by a battery of assays with human hepatoma cells. Methyletrazolium assay showed a high cytotoxicity of 97.1% after being exposed to the highest concentration of raw leachate for 24 h, and a cytotoxicity of 26% in effluent at a concentration of 30% (v /v). Both comet assay (24 h) and γH2AX flow cytometer assay (3 h) showed increased levels of DNA damage in cells exposed to raw leachate and after nitration/UF‐treated leachate followed by a significant increase of 7‐ethoxyresorufin‐O‐deethylase activity. However, the effluent after nitration/UF/RO treatment showed no significant difference compared to negative control for γH2AX flow cytometer assay but slight DNA damage at concentrations of 20% and 30% (v /v) as well as increase of 7‐ethoxyresorufin‐O‐deethylase. Analysis showed that nitration/UF/RO process exhibited high removal of physicochemical indexes and significant reduction of toxic and genotoxic effects of leachate, but still demands an improvement to reduce all possible negative risks to the environment and humans. Copyright © 2017 John Wiley & Sons, Ltd.     

In vivo micronucleus test in the assessment of cytogenotoxicity of landfill leachates in three animal models from various ecological habitats

The in vivo micronucleus (MN) test, a standard test for the genotoxicity screening of xenobiotics, was used to evaluate the cytotoxic and genotoxic activities of landfill leachates in Clarias gariepinus, Coturnix coturnix japonica and Rattus norvegicus. These organisms were exposed to various sub-lethal concentrations (1–50 %) of Olusosun and Aba Eku landfill leachates. At post exposure, peripheral erythrocytes from catfish and quail, and bone marrow cells of quail and rat were subjected to MN analysis following standard protocols. The leachates induced significant increase in MN formation and total nuclear abnormalities (NAs) in the peripheral erythrocytes of catfish and quail. NAs occurred in the order; BN > BL > LB > NT in the catfish and BN > BudN > TLN > TN in quail. There was significant increase in MN formation in the bone marrow cells of quail, and micronucleated polychromatic erythrocytes and micronucleated normochromatic erythrocytes formation in the bone marrow of rats. The concentration dependent significant (p < 0.05) decrease in the PCE/NCE ratio in the bone marrow of the leachate treated rats suggest alterations in the bone marrow cell proliferation, leading to the suppression of immature erythrocytes (PCE). MN induction showed positive corrections with leachate concentrations in the test organisms; and it increased with exposure duration in the catfish. Indiscriminate disposal of solid waste generates leachates containing multiple xenobiotics that are capable of increasing genomic instability among vertebrates inhabiting various ecological habitats.     

Chromosomal aberrations induced in mouse bone marrow cells by municipal landfill leachate

The cytogenetic damage induced by municipal landfill leachate was studied using chromosomal aberration (CA) in mouse bone marrow assay. Results show that leachate samples collected in different seasons decreased the mitotic index (MI), and caused significant increases of CA frequencies in treatment concentration (Chemical oxygen demand (COD) measured by the method of potassium dichromate oxidation, COD(Cr))-dependent manners. Compared with the negative control, reductions of the MI of 54 and 38% were detected for the highest leachate concentration (COD(Cr) 320mg/L) in mouse bone marrow treated with both samples. The frequencies of CA increased significantly with increasing concentrations of sample 1 from COD(Cr) 40 to 320mg/L, and from 80 to 320mg/L after exposure to sample 2. In addition, a seasonal difference of MI and CA frequencies induced by leachate was observed. The results confirm that leachate is a genotoxic agent in mammalian cells, and imply that exposure to leachate in aquatic environment may pose a potential genotoxic risk to mammals and humans. The results suggest that the CA in mouse bone marrow bioassay is efficient in genotoxicity studies of leachate on mammals, and that there appears to be a correlation between the genotoxicity in mammal system and the chemical measurement (COD(Cr)) of leachate. The results also indicate that different discharge guidelines and environmental quality standards should be established for leachates discharged from landfills to aquatic environment in different seasons.     

The analysis of the possibility of using biological tests for assessment of toxicity of leachate from an active municipal landfill

One of the consequence of municipal waste deposition is the production of landfill leachate. Its volume and composition is determined by numerous factors, including waste composition, landfill age and the volume of precipitation. Leachate may contain a number of mineral and organic compounds, the volume of which must be controlled regularly. One of the methods of determining the toxicity of substances contained in landfill leachate is the use of biological tests, based − among others − on aquatic organisms sensitive to environmental contamination. The purpose of this study was to analyse the possibility of using ecotoxicological tests (supplementing the physical and chemical tests) for the purpose of assessment of landfill leachate toxicity. The tests were conducted at an operating municipal landfill in Stary Sącz (southern Małopolska Region, Poland N: 49°55’33”76, E: 20°65’68’70) from December 2015 to October 2016. The scope of the tests included the analysis of physical and chemical indicators as part of the landfill monitoring process, and also the analysis of additional selected indicators: namely the boron, barium and vanadium content. The selected ecotoxicological tests included tests using Daphnia magna Straus (Cladocera, Crustacea).Leachate tests conducted with the use of physical and chemical indicators have, for nearly twenty years, mainly demonstrated changes related to the age of the used landfill; besides increased boron and barium values, no evident contamination has been found, excluding the case of boron. However, a statistically significant correlation between the B and Ba contents and the amount of precipitation was determined. In two cases, the used biological tests have confirmed the toxicity of the leachate: in January and June 2016. In the same months, the highest and abnormal boron contents were measured, which could cause a toxic effect of leachates.     

Antiestrogenicity and estrogenicity in leachates from solid waste deposits

A great deal of effort has been devoted to developing new in vitro and in vivo methods to identify and classify endocrine disrupting chemicals that have been identified in environmental samples. In this study an in vitro test based on recombinant yeast strains transfected with genes for the human estrogen receptor α was adapted to examine the presence of estrogenic and antiestrogenic substances in six Swedish landfill leachates. Antiestrogenic effects were measured as inhibition of the estradiol induced response with the human estrogen receptor α, and quantified by comparison with the corresponding inhibitory effects of a known antiestrogen, hydroxytamoxifen. The estrogenicity was within the range of that determined in domestic sewage effluents, from below the limit of detection to 29 ng estradiol units L^?1. Antiestrogenicity was detected in some of the investigated landfill leachates, ranging between <38 and 3800 μg hydroxytamoxifen equivalents L^?1. There was no apparent relation between the type of waste deposited on the landfills and the antiestrogenic effect. Fractionation of a landfill leachate showed that estrogenic compounds were located in two dominant fractions. Three estrogenic compounds were found that accounted for the estrogenic activity in extracts of leachates: bisphenol A, estradiol, and ethinylestradiol. The bisphenol may have been released from decomposing plastic waste and the estrogenic steroids from earlier deposits of municipal sewage sludge and pharmaceutical waste. Fractionation of leachates from three parts of a landfill showed that the antiestrogenic activity was distributed in at least four fractions and somewhat different in different flows of leachate. This indicated a heterogeneous mixture of antiestrogenic substances. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2011.     

Bioassays for the Evaluation of Landfill Leachate Toxicity

This article reviews the application of bioassays for assessing the toxicity hazard posed by landfill leachate discharged to an aquatic environment. Landfill leachate is a complex mixture of chemicals; thus it is difficult to assess the risk posed to aquatic wildlife using standard chemical identification techniques, such as gas chromatography–mass spectroscopy (GC-MS). From this review it is clear that toxicity testing, using species that represent the different trophic levels, is a superior way to predict the risk posed by discharge than chemical analysis. Previous studies assessed leachate toxicity using bacteria, algae, plants, invertebrates, fish, and genotoxicity. Studies showed that leachate exhibits a wide range of toxicities to the species tested. Ammonia, alkalinity, heavy metals, and recalcitrant organics were identified to be the cause of adverse responses from the test organisms. Concentrations of these chemicals were found to depend upon the types of waste landfilled. As part of this review, Slooff analysis was applied to published results to calculate the sensitivity of test species. It was concluded that Lemna minor and Thamnocephalus platyurus were the most sensitive tests and, Vibrio fischeri (Microtox) was the least sensitive test available. Little is known about the sensitivity of each species to the different types of waste that might have been landfilled. A battery of tests needed for a more accurate assessment of landfill leachate is proposed. Some of the more common tests have been replaced by more sensitive tests that produce more relevant results for the industry and regulators.     

Determining toxicity of leachates from Florida municipal solid waste landfills using a battery-of-tests approach

The toxicity and physicochemical parameters of municipal solid-waste (MSW) landfill leachates from six sites in north and north-central Florida, United States, were determined. Landfill leachates are complex mixtures of organic and inorganic constituents. Leachate toxicity was assayed using the acute C. dubia (48-h) and Microtoxtrade mark (15-min) assays and the chronic S. capricornutum (96-h) assay. The landfill leachates were shown to be highly toxic to both C. dubia and S. capricornutum with an EC(50) < 10% and < 15%, respectively. However, Microtoxtrade mark was not as sensitive to the leachates. Based on these results, the assays were ranked for their sensitivities to the landfill leachates; C. dubia > S. capricornutum > Microtoxtrade mark. Chemical analyses showed high concentrations of un-ionized ammonia and salts in the landfill leachates but low concentrations of heavy metals. The toxicity of the landfill leachates varied between the sites sampled and within each of the landfill sites. A significant relationship was found between the total ammonia content of the leachates and toxicity as determined by the C. dubia and S. capricornutum assays. Although the chemical constituents in Florida MSW landfill leachates may be more dilute, the toxicity of the leachates is equivalent to that from industrial waste landfills.     

Toxicity analysis of leachates from hazardous wastes via microtox and daphnia magna

One of the most common ways of surface and ground water contamination from hazardous wastes is through its leachates. Toxicity is a meaningful parameter allowing an integrated evaluation of the potential danger of leachates. Two bioassays were considered for measuring toxicity: Microtox and Daphnia magna. The toxicity was measured on leachates obtained, using different leaching procedures, from wastes of a pesticide manufacturing industry and sludge from electroplating wastewater. Twenty-two tests were carried out, measuring IC50 for D. magna and EC50 for Microtox. Both bioassays were compared using three toxicity criteria. The first criterium is classification on toxic/non-toxic at 25 and 50% leachate concentration. The second criterium is classification on percent ranks, and the third on log ranks. Considering these criteria, the agreement between both bioassays is within a 75–85%. It is shown that both, Microtox and D. magna assays could be used as toxicity indicators for the wastes considered. The sensitivity of the bioassays is different depending on leachate composition. In all samples, the leachate concentration of chemicals was measured.     

Increased reproductive toxicity of landfill leachate after degradation was caused by nitrite

Leachate from the landfill Lindbodarna was suspected to cause reproductive effects on fish in a Swedish lake, called Molnbyggen. The acute toxicity of this landfill leachate is caused by ammonia. In the present study the acute and chronic toxicity of the leachate from the landfill was tested with Ceriodaphnia dubia before and after treatment, either with (inoculated) or without addition of microorganisms from activated sludge, in both 2000 and 2001. On both occasions, the acute toxicity decreased after treatment, more rapidly with inoculum than without, and the cause of the decrease was mainly explained by decreasing concentrations of ammonia. However, the chronic toxicity decreased after treatment with inoculum but increased after treatment without inoculum. Therefore, we performed a series of acute and reproductive tests with ammonia, nitrite and nitrate on C. dubia, and the 24-h EC50s were 1.0, 2.7 and 59 mM, respectively, which are consistent with literature data. However, the chronic toxicity of these compounds gave quite a different picture with 8-day EC50s for reproduction of 3.0 mM for ammonia, 0.016 mM for nitrite and 1.5 mM for nitrate. Thus, the acute-chronic ratios for these compounds were 0.33 for ammonia, 170 for nitrite and 39 for nitrate. These findings show that reproduction is more sensitive than survival for both nitrite and nitrate, and that nitrite is the more hazardous of the two. This implies that the chronic and reproductive toxicity of nitrite and nitrate on zooplankton may in fact increase effects of eutrophication. In this study the toxicity of the fresh leachate was dominated by ammonia, but after treatment the contribution of nitrite increased, and especially the chronic toxicity of the treated landfill leachate was dominated by nitrite toxicity.     

Evaluation of surrogate tests in toxicant impact assessments

A surrogate test battery was evaluated at several stream and landfill sites where toxicant contamination had occurred. The battery consisted of microbial activity assays and macrofaunal organisms: alkaline phosphatase, amylase, arylsulfatase, electron transport system activity, β-galactosidase, β-glucosidase, protease, metal resistance, heterotrophic uptake (¹⁴C), zooplankton (Daphnia magna, Ceriodaphnia), amphipods (Hyalella azteca), and fathead minnows (Pimephales promelas). Waters, leachates, and sediments were tested for toxicity from sites in 5 states. Surrogate responses were correlated with in situ biological and/or chemical parameters. At each test site, statistically significant relationships were observed between microbial test responses and in situ biological or chemical responses, revealing both toxicant impacts and natural variations. Field validation of this test approach revealed its sensitivity and versatility, supporting its use in routine toxicant impact assessments.     

Effect-directed investigation and interactive effect of organic toxicants in landfill leachates combining Microtox test with RP-HPLC fractionation and GC/MS analysis

Landfill leachates contain a large amount of unknown harmful compounds derived from domestic and industrial sources. A toxicity effect-directed approach was used to identify biologically active compounds in three landfill leachate samples (S1–S3) by combining the Microtox test with reverse-phase high performance liquid chromatography (RP-HPLC) fractionation and gas chromatography/mass spectrometry (GC/MS) analysis. Organic toxicants were recovered from coarse fractions only in S1 and in S2. Fine fractionation exhibited a somewhat different toxicity pattern in S1 and S2. GC/MS analysis positively identified Bisphenol A (BPA) and 4-t-butylphenol (4-t-BP) in both samples, N-ethyltoluenesulfoneamide (NETSA) was detected only in S1. However, their concentrations were not high enough to be responsible for the observed toxicity in original samples. A synergistic effect among detected organic compounds (BPA, NETSA, and 4-t-BP) was demonstrated. Each compound present at 1/7 of its individual EC₅₀, might lead to undesirable mixture toxicity, which indicated that interactive effects may, to a certain extent, play a role in landfill leachates with complex matrices. The results from further hydrophobicity analysis and estrogen receptor (ER) competitive binding assays of fraction 13 of both samples gave evidence that some possible toxicants that failed to be identified by GC/MS might be endocrine disrupting chemical(s) (EDC) with a log K _ow range of 3.5–3.7 in both samples.     

Chemical composition and toxicity of waste dump leachates using Selenastrum capricornutum Printz (Chlorococcales,Chlorophyta)

Chemical composition and toxicity of leachates from a municipal waste dump in Buenos Aires Province were analyzed. Three sets of samples, obtained in 1996 and 1997, were compared. Levels of nitrogen, phosphorus, and organics (expressed as COD) were distinctly lower in 1997. Such differences were mainly attributed to higher rainfall in that year. The Selenastrum capricornutum assay showed a high sensitivity to heavy metals (Cd=0.049 mg L⁻¹; Cr hexavalent=0.247 mg L⁻¹ and Zn=0.037 mg L⁻¹). Cu, Zn, and Pb concentrations were toxic to algae in leachate samples of the first year, while the EC₅₀ leachates were slightly toxic (86.6 and 99.31%). Most likely, high levels of organics (COD=4640 and 3470 mg L⁻¹) form complex mixtures with metals reducing their toxicity. The EC₅₀ leachate=48.5% in the last year may be explained by high concentrations of Pb and Cu. © 2000 John Wiley & Sons, Inc. Environ Toxicol 15: 76–80, 2000     

Sperm functional parameters and erythrocytes oxidant–antioxidant imbalance during municipal landfill leachate treatment withdrawal in rats

Adequate information on how leachates affect hematological and reproductive functions is necessary to help in linking causality with predictable response. The present study investigated the effects of Olushosun municipal landfill leachate (OMLL) exposure and withdrawal on sperm characteristics and erythrocytes oxidant–antioxidant balance in rats. Adult male Wistar rats were exposed to 0%, 12.5% and 25% OMLL in drinking water for 28 days. One-half of the rats in each group were sacrificed on day 29 while the remaining one-half stayed an additional 28 days without treatment. OMLL exposure significantly decreased sperm functional parameters, disrupted antioxidant systems with concomitant elevation in hydrogen peroxide and malondialdehyde levels in erythrocytes and sperm. Following withdrawal of treatment, OMLL-mediated decrease in sperm count and daily sperm production were reversed to near control. However, erythrocytes and sperm oxidative damage, increased sperm abnormalities, decreased epididymis weight, sperm progressive motility and testicular sperm number persisted and were consistent with results obtained from rats sacrificed immediately after OMLL treatment. Collectively, OMLL-induced irreversible oxidative damage to erythrocytes and sperm in rats within the time course of investigation. These findings highlight potential adverse effects of OMLL on individuals unduly exposed to leachates contaminated substances.     

In vitro evaluation of the cytotoxic and genotoxic effects of artemether,an antimalarial drug,in a gastric cancer cell line (PG100)

Artemisinin is a sesquiterpene lactone endoperoxide, obtained from Artemisia annua, and extensively used as an antimalarial drug. Many studies have reported the genotoxic and cytotoxic effects of artemisinins; however, there are no studies that compare such effects between cancer cell lines and normal human cells after treatment with artemether, an artemisinin derivative. Gastric cancer is the fourth most frequent type of cancer and the second highest cause of cancer mortality worldwide. Thus, the aim of this study was to evaluate the in vitro genotoxic and cytotoxic effects induced by artemether in gastric cancer cell line (PG100) and compare them with the results obtained in human lymphocytes exposed to the same conditions. We used MTT (3‐(4,5‐methylthiazol‐2‐yl)‐2, 5‐diphenyl‐tetrazolium bromide) assay, comet assay and ethidium bromide/acridine orange viability staining to evaluate the cytotoxic and genotoxic effects of artemether in PG100. MTT assay showed a decrease in the survival percentages for both cell types treated with different concentrations of artemether (P < 0.05). PG100 also showed a significant dose‐dependent increase in DNA damage index at concentrations of 119.4 and 238.8 µg ml^?1 (P < 0.05). Our results showed that artemether induced necrosis in PG100 at concentrations of 238.8 and 477.6 µg ml^?1, for all the tested harvest times (P < 0.05). In lymphocytes, artemether induced both apoptosis and necrosis at concentrations of 238.8 and 477.6 µg ml^?1, for all the tested harvest times (P < 0.05). In conclusion, human lymphocytes were more sensitive to the cytotoxic effects of the antimalarial drug than the gastric cancer cell line PG100. Copyright © 2011 John Wiley & Sons, Ltd.     

Acute aquatic toxicity of tire and road wear particles to alga,daphnid, and fish

Previous studies have indicated that tire tread particles are toxic to aquatic species, but few studies have evaluated the toxicity of such particles using sediment, the likely reservoir of tire wear particles in the environment. In this study, the acute toxicity of tire and road wear particles (TRWP) was assessed in Pseudokirchneriella subcapita, Daphnia magna, and Pimephales promelas using a sediment elutriate (100, 500, 1000 or 10000 mg/l TRWP). Under standard test temperature conditions, no concentration response was observed and EC/LC₅₀ values were greater than 10,000 mg/l. Additional tests using D. magna were performed both with and without sediment in elutriates collected under heated conditions designed to promote the release of chemicals from the rubber matrix to understand what environmental factors may influence the toxicity of TRWP. Toxicity was only observed for elutriates generated from TRWP leached under high-temperature conditions and the lowest EC/LC₅₀ value was 5,000 mg/l. In an effort to identify potential toxic chemical constituent(s) in the heated leachates, toxicity identification evaluation (TIE) studies and chemical analysis of the leachate were conducted. The TIE coupled with chemical analysis (liquid chromatography/mass spectrometry/mass spectrometry [LC/MS/MS] and inductively coupled plasma/mass spectrometry [ICP/MS]) of the leachate identified zinc and aniline as candidate toxicants. However, based on the high EC/LC₅₀ values and the limited conditions under which toxicity was observed, TRWP should be considered a low risk to aquatic ecosystems under acute exposure scenarios.     

A 28‐day Sub‐acute Genotoxic and Behavioural Assessment of Garcinielliptone FC

Garcinielliptone FC (GFC) is a polyisoprenylated benzophenone isolated from Platonia insignis Mart (Clusiaceae) with promising anticonvulsant properties. However, its safe use and other effects on the central nervous system require assessment. This study assessed the toxicological effects of GFC using the comet assay and the micronucleus test in mice treated for 28 days. A behavioural model was employed to detect possible injuries on the central nervous system. Mice treated with GFC (2, 10 and 20 mg/kg; i.p.) daily for 28 days were submitted to rotarod test, open‐field test and tail suspension test (TST). After the behaviour tasks, biological samples were assessed to evaluate genotoxic and mutagenic effects using the comet assay and the micronucleus test. Garcinielliptone FC did not impair the performance of the animals in the rotarod and open‐field tests, with no antidepressant‐like effect in TST. No genotoxic effects in blood and cerebral cortex were observable in the comet assay; however, there was a significant increase in index and frequency of damage in liver after treatment with GFC 20 mg/kg. Garcinielliptone FC did not increase micronucleus frequency in bone marrow. At the tested doses, GFC was not toxic to the CNS and did not induce genotoxic damage to blood or bone narrow cells. DNA damage to liver tissue was caused only by the highest dose, although no mutagenic potential was observed.     

Evaluation of the genotoxicity and cytotoxicity of fexofenadine in cultured human peripheral blood lymphocytes

Fexofenadine (FXF) is a new non-sedating antihistamine used in the treatment of seasonal allergic rhinitis and chronic idiopathic urticaria. Studies on FXF genotoxicity and cytotoxicity in cultured human peripheral blood lymphocytes have not been reported so far. Therefore, the present study is the first report investigating the genotoxic and cytotoxic effects of FXF in cultured human peripheral blood lymphocytes in vitro. Cultures were treated with FXF at three concentrations (50, 100 and 150 μg/ml) for 24 and 48 h. Endpoints analyzed included: mitotic index (MI), nuclear division index (NDI), chromosomal aberrations (CA) and micronucleus (MN) assay. Mitomycin C (MMC) was used as a positive control. The results of CA and MN assays showed that FXF was not genotoxic at all the concentrations tested, meanwhile MI and NDI results showed dose-dependent decrease and significant differences were found for at least one concentration. In conclusion, the results of this study suggest that FXF has a cytotoxic effect but not genotoxic effect on human peripheral blood lymphocyte cultures. Further cytogenetic studies, especially about the cell cycle kinetics of FXF are required to elucidate the decreases in dividing cells, and biomonitoring studies should also be conducted with patients receiving therapy with this drug.     

Evaluation of genotoxic and antigenotoxic effects of boron by the somatic mutation and recombination test (SMART) on Drosophila

Objective: In this study, different concentrations of boron have been evaluated for genotoxic and antigenotoxic properties by using the somatic mutation and recombination test (SMART) on Drosophila melanogaster. Study Design: The treatment concentrations were chosen to a pretest. Third-instar larvae trans-heterozygous for two genetic markers, multiple wing hair (mwh) and flare (flr3), were treated at different concentrations (0.1, 5, 10, 20, and 40?mg/mL) of boron. In addition to investigating antigenotoxic effects, the same boron concentrations were co-administered with 0.1?mM Ethyl Methane Sulfonate (EMS). Distilled water was used as a negative control; 0.1?mM of EMS was used as a positive control. For the chronic feeding study, small plastic vials were prepared with 1.5?g of dry Drosophila Instant Medium and 5?mL of the respective test solution. Hundreds of trans-heterozygous larvae were embedded into this medium. Feeding ended with pupation of the surviving larvae. After metamorphosis, all surviving flies were collected and stored in a 70% ethanol solution. Preparation and microscopic analyses of wing were made after the treatment. Then the observed mutations were classified according to size and type of mutation per wing. Results: Results indicated that there is no significant genotoxic effect with all of the boron concentrations. In addition, the antigenotoxic activities of boron against EMS were tested. Results indicated that all boron concentrations (0.1, 5, 10, 20 and 40?mg/mL) were able to abolish the genotoxic effects induced by the EMS. Conclusion: It is suggested that the observed effects can be linked to the antioxidant properties of boron. Moreover, these in vivo results will contribute to the antigenotoxicity database of boron.