Gene-TEQ--a standardized comparative assessment of effects in the comet assay using genotoxicity equivalents

Existing methods for the comparison of genotoxic effects in the comet assay bear considerable disadvantages such as the problem to link information about concentration dependence and severity of effects. Moreover, given the lack of standardized protocols and the use of various standards, it may be extremely difficult to compare different studies. In order to provide a method for standardized comparative assessment of genotoxic effects, the concept of genotoxicity equivalents (Gene-TEQ) was developed. As potential reference compounds for genotoxic effects, three directly acting (N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl-methanesulfonate, and N-methyl-N-nitrosourea) and three indirectly acting (cyclophosphamide, dimethylnitrosamine, and 4-nitroquinoline-oxide) genotoxic substances were compared with respect to their cytotoxic (neutral red) and genotoxic (comet assay) concentration-response profiles in the permanent fish cell line RTL-W1. For further comparison, two sediment extracts from the upper Danube River were investigated as environmental samples. Based on the results of cytotoxicity and genotoxicity testing, MNNG was selected as the reference compound. At several exposure levels and durations, genotoxic effects of both the other pure substances and the environmental samples were calculated as percentages of the maximum MNNG effect and related to the absolute MNNG effect (EC values). Thus, genotoxicity equivalent factors (Gene-TEQs) relative to MNNG could be calculated. Gene-TEQs can easily be applied to pure substances, mixtures and field samples to provide information about their toxicity relative to the reference compound. Furthermore, the Gene-TEQ concept allows a direct comparison of environmental samples from different laboratories.     

Uptake of arsenic by alkaline soils near alkaline coal fly ash disposal facilities

The attenuation of arsenic in groundwater near alkaline coal fly ash disposal facilities was evaluated by determining the uptake of arsenic from ash leachates by surrounding alkaline soils. Ten different alkaline soils near a retired coal fly ash impoundment were used in this study with pH ranging from 7.6 to 9.0, while representative coal fly ash samples from two different locations in the coal fly ash impoundment were used to produce two alkaline ash leachates with pH 7.4 and 8.2. The arsenic found in the ash leachates was present as arsenate [As(V)]. Adsorption isotherm experiments were carried out to determine the adsorption parameters required for predicting the uptake of arsenic from the ash leachates. For all soils and leachates, the adsorption of arsenic followed the Langmuir and Freundlich equations, indicative of the favorable adsorption of arsenic from leachates onto all soils. The uptake of arsenic was evaluated as a function of ash leachate characteristics and the soil components. The uptake of arsenic from alkaline ash leachates, which occurred mainly as calcium hydrogen arsenate, increased with increasing clay fraction of soil and with increasing soil organic matter of the alkaline soils. Appreciable uptake of arsenic from alkaline ash leachates with different pH and arsenic concentration was observed for the alkaline soils, thus attenuating the contamination of groundwater downstream of the retired coal fly ash impoundment.     

Genotoxicity evaluation of water soil leachates by Ames test, comet assay, and preliminary Tradescantia micronucleus assay

Combining genotoxicity/mutagenicity tests and physico-chemical methodologies can be useful for determining the potential genotoxic contaminants in soil samples. The aim of our study was to evaluate the genotoxicity of soil by applying an integrated physico-chemical-biological approach. Soil samples were collected at six sampling points in a Slovenian industrial and agricultural region where contamination by heavy metals and sulphur dioxide (SO₂) are primarily caused by a nearby power plant. The in vitro alkaline version of the comet assay on water soil leachates was performed with Caco-2 and HepG2 cells. A parallel genotoxicity evaluation of the samples was performed by Ames test using Salmonella typhimurium and the Tradescantia micronucleus test. Pedological analyses, heavy metal content determination, and different physico-chemical analyses, were also performed utilizing standard methodology. Water leachates of soil samples were prepared according to standard methods. Since only a battery of biotests with prokaryotic and eukaryotic organisms or cells can accurately estimate the effects of (geno)toxicants in soil samples and water soil leachates, a combination of three bioassays, with cells or organisms belonging to different trophic levels, was used. Genotoxicity of all six water soil leachates was proven by the comet assay on both human cell lines, however no positive results were detected by bacterial assay, Ames test. The Tradescantia micronucleus assay showed increase in micronuclei formation for three samples. According to these results we can assume that the comet assay was the most sensitive assay, followed by the micronucleus test. The Ames test does not appear to be sensitive enough for water soil leachates genotoxicity evaluations where heavy metal contamination is anticipated.     

Assessment of the radiological impacts of utilizing coal combustion fly ash as main constituent in the production of cement

The purpose of this study is to assess potential radiological impacts of utilizing pulverized fly ash (PFA) as a constituent in ordinary Portland cement. For this purpose, the activity concentrations of ²²⁶Ra, ²³²Th, and ⁴⁰K in samples of PFA and Portland cement containing 15%, 20%, and 25% by mass PFA were measured using gamma-ray spectrometry with HPGe detector. The mean activity concentrations of ²²⁶Ra, ²³²Th, and ⁴⁰K were found as 366.6, 113.7, and 460.2 Bq kg^???1, 94.2, 25.9, and 215.3 Bq kg^???1, 113.7, 34.3, and 238.3 Bq kg^???1, and 124.2, 41.8, and 279.3 Bq kg^???1 for the examined samples of PFA, Portland cement with 15%, 20%, and 25% by mass PFA, respectively. Radiological parameters such as radium equivalent activity, external exposure index (activity concentration index), internal dose index (alpha index), indoor absorbed gamma dose rate, and the corresponding the annually effective dose were assessed for Portland cement samples containing three percentages (15%, 20%, and 25%) by mass PFA. The results of assessment show that all Portland cement samples are within the safe limits recommended for building materials for dwellings.     

Immobilization of B,F, Cr,and As in alkaline coal fly ash through an aging process with water

Fourteen different alkaline coal fly ashes (CFAs) were used for the experiment, in which each sample was mixed with water to be 28.6 % of water content (wt/wt) and aged for 1–4 weeks at 10–30 °C. This simple treatment is advantageous for decreases in water-soluble B, F, Cr, and As. Compared to non-aged CFAs, their water-soluble fractions remained 0.56–88 %, 21–85 %, 0.37–93 % and 2.6–88 %, respectively, after aging for a week at 20 °C, although the amounts of Cr and As released from some CFA samples increased. Considering the significant decrease in elution of sulfate, Ca and Al after aging, the immobilization, namely prevention of toxic element elution, could be related to formation of secondary minerals such as portlandite, gypsum and ettringite. Immobilization of B and Cr tends to proceed preferentially under colder conditions. Aging at higher temperatures enhances the leachability of Cr in some CFA samples. Contrary to the behavior of B and Cr, water-soluble F effectively decreases under warmer conditions.     

Monitoring the species of arsenic, chromium and nickel in milled coal, bottom ash and fly ash from a pulverized coal-fired power plant in western Canada

The concentration of As, Cr and Ni and their speciation (As3+;5+, Cr3+;6+ and Ni0;2+) in milled coal, bottom ash and ash collected by electrostatic precipitator (ESP) from a coal fired-power plant in western Canada were determined using HGAAS, ICP-AES and XANES. The chemical fractionation of these elements was also determined by a sequential leaching procedure, using deionized water, NH4OAC and HCI as extracting agents. The leachate was analyzed by ICP-AES. Arsenic in the milled coal is mostly associated with organic matter, and 67% of this arsenic is removed by ammonium acetate. This element is totally removed from milled coal after extraction with HCI. Arsenic occurs in both the As3+ and the As5+ oxidation states in the milled coal, while virtually all (>90%) of the arsenic in bottom ash and fly ash appears to be in the less toxic arsenate (As5+) form. Both Ni and Cr in the milled coal are extracted by HCI, indicating that water can mobilize Ni and Cr in an acidic environment. The chromium is leached by water from fly ash as a result of the high pH of the water, which is induced during the leaching. Ammonium acetate removes Ni from bottom ash through an ion exchange process. Chromium in milled coal is present entirely as Cr3+, which is an essential human trace nutrient. The Cr speciation in bottom ash is a more accentuated version of the milled coal and consists mostly of the Cr3+ species. Chromium in fly ash is mostly Cr3+, with significant contamination by stainless-steel from the installation itself.     

Investigation on chemical species of arsenic, selenium and antimony in fly ash from coal fuel thermal power stations

Investigations of the existing chemical forms and the concentrations of arsenic (As), selenium (Se) and antimony (Sb) in samples of fly ash obtained from six coal-fired power stations in various countries were carried out. The concentration of As was found to vary from 5.4 to 22.3 mg kg(-1), and the most common mode of occurrence of As in the fly ashes is in association with carbonates or Fe-Mn oxides. The concentrations of Se and Sb ranged from 2.3-5.2 and 1.0-3.9 mg kg(-1), respectively. The dominant chemical forms of Se and Sb in the fly ashes were as extractable species. Also, water-soluble As, Se and Sb in the fly ashes were extracted, and the chemical species of As, Se and Sb in the extract determined using HPLC-ICP-MS. This was done as the potential release of soluble As, Se and Sb through leaching of fly ash is of environmental concern. The most abundant form of As in the extract was the low toxicity As(V). The main species of Se was Se(IV), although it was found that the ratio of Se(VI) to Se(IV) in acidic fly ashes is higher than in alkaline fly ash samples. Antimony was mostly present as Sb(III).     

Monitoring genotoxicity among gasoline station attendants and traffic enforcers in the City of Manila using the micronucleus assay with exfoliated epithelial cells

Some types of occupations involve high levels of exposure to potentially genotoxic gaseous and particulate substances from internal combustion engines used in motor vehicles. These occupational exposures may contribute to the development of many illnesses, usually through chromosomal change mechanisms that include strand breakage, deletions, sister chromatid exchange and non-disjunction. To determine the effect of occupational exposure in gasoline station attendants and traffic enforcers, the micronucleus test was used. Exfoliated oral mucosa cells from 18 gasoline station attendants, 18 traffic enforcers and 18 control subjects in the City of Manila were examined for micronucleated cell (MNC) frequency. Analysis of buccal cells showed that MNC frequencies in exposed individuals were significantly greater than in control subjects (p < or = 0.05). However, between gasoline station attendants and traffic enforcers, MNC frequencies of the two exposed groups exhibited no significant difference. No relation was also found between MNC frequency and any of the factors such as age, smoking habits, alcohol habits and working period. This was further confirmed in the multiple regression analysis which showed that only occupational exposure was a good predictor of MNC frequency. The results of this study suggest that gasoline station attendants and traffic enforcers, compared to the control individuals, are at a greater risk of chromosomal damage. For the assessment of chromosomal damage, the study, development, and standardization of tests are recommended for public institutions concerned with matters regarding environmental quality and community health.     

Assessment of oxidative stress markers and concentrations of selected elements in the leaves of Cassia occidentalis growing wild on a coal fly ash basin

Assessment of oxidative stress levels and tissue concentrations of elements in plants growing wild on fly ash basins is critical for realistic hazard identification of fly ash disposal areas. Hitherto, levels of oxidative stress markers in plants growing wild on fly ash basins have not been adequately investigated. We report here concentrations of selected metal and metalloid elements and levels of oxidative stress markers in leaves of Cassia occidentalis growing wild on a fly ash basin (Badarpur Thermal Power Station site) and a reference site (Garhi Mandu Van site). Plants growing on the fly ash basin had significantly high foliar concentration of As, Ni, Pb and Se and low foliar concentration of Mn and Fe compared to the plants growing on the reference site. The plants inhabiting the fly ash basin showed signs of oxidative stress and had elevated levels of lipid peroxidation, electrolyte leakage from cells and low levels of chlorophyll a and total carotenoids compared to plants growing at the reference site. The levels of both protein thiols and nonprotein thiols were elevated in plants growing on the fly ash basin compared to plants growing on the reference site. However, no differences were observed in the levels of cysteine, reduced glutathione and oxidized glutathione in plants growing at both the sites. Our study suggests that: (1) fly ash triggers oxidative stress responses in plants growing wild on fly ash basin, and (2) elevated levels of protein thiols and nonprotein thiols may have a role in protecting the plants from environmental stress.     

Investigation into the relationship between major and minor element contents and particle size and leachability of boron in fly ash from coal fuel thermal power plants

A basic investigation of boron in discharged fly ash by coal fuel thermal power plants in several worldwide locations was carried out. Eight kinds of fly ash sample were prepared from eight coal fuel thermal power plants. Two of the fly ash samples were used to examine the relationship between the concentration of boron in fly ash and the particle size. When the particle size of fly ash is smaller, there is a possibility that it will be released into the air and spread over a wide area in the environment. However, it has become apparent that fly ash of smaller particle size has a higher concentration of boron and a higher enrichment factor. In other fly ash samples, the boron contents were examined and leaching tests were carried out. There is acidic fly ash as well as alkaline fly ash that contains larger amounts of acidic or basic salts. On alkaline fly ash, when the concentration of boron bound to Fe-Mn oxide is low; it has become apparent that leaching boron is increased in a solution with lower pH of approximately 4 which is nearly the pH of acid rain.     

Evaluation of genotoxicity in Rhamdia quelen (Pisces, Siluriformes) after sub-chronic contamination with Fipronil

Diverse genetic biomarkers have been used to evaluate the effects of pollution by mutagenic agents such as metals and pesticides, as well as a large variety of chemical substances derived from human activities. This work researched the effects that an exposure of 60 days to the insecticide Fipronil (concentrations of 0.05, 0.10 and 0.23 μg/L) can cause in the fish Rhamdia quelen using Comet assay with gills, histopathological analysis of gills and the Piscine Micronucleus test and Nuclear Morphological Alterations. The results for the Comet assay and for gills histopathological injuries showed no difference between the control group and the contaminated groups. In the Piscine Micronucleus test, the smallest concentration of Fipronil (0.05 μg/L) was similar as the control group, while concentrations of 0.10 and 0.23 μg/L caused more damage to the DNA. These results suggested that only the highest concentrations of Fipronil tested cause damage in erythrocytes, but none of these concentrations was sufficient to alter the DNA in the gill cells. R. quelen may be a less sensitive bioindicator than other fish that have been tested. On the other hand, the concentrations used may not have been sufficient to detect alterations in the DNA of R. quelen with the chosen tests. Works like this take on great importance given the enormous quantity of substances that are thrown daily into the environment in an uncontrolled way, without evaluation of the consequences. The application of these tests with other concentrations, tissues and exposure times is suggested for future works.     

Evaluation of lot-to-lot repeatability and effect of assay media choice in the recombinant Factor C assay

Measurement of environmental endotoxin exposures is complicated by variability encountered using current biological assay methods arising in part from lot-to-lot variability of the Limulus-amebocyte lysate (LAL) reagents. Therefore, we investigated the lot-to-lot repeatability of commercially available recombinant Factor C (rFC) kits as an alternative to LAL. Specifically, we compared endotoxin estimates obtained from rFC assay of twenty indoor dust samples, using four different extraction and assay media, to endotoxin estimates previously obtained by Limulus amebocyte lysate (LAL) assay and amounts of 3-hydroxy fatty acids (3-OHFA) in lipopolysaccharide (LPS) using gas-chromatography mass spectroscopy (GC-MS). We found that lot-to-lot variability of the rFC assay kits does not significantly alter endotoxin estimates in house dust samples when performed using three of the four assay media tested and that choice of assay media significantly altered endotoxin estimates obtained by rFC assay of house dust samples. Our findings demonstrate lot-to-lot reproducibility of rFC assay of environmental samples and suggest that use of rFC assay performed with Tris buffer or water as the extraction and assay medium for measurement of endotoxin in dust samples may be a suitable choice for developing a standardized methodology.     

Assessing the potential impact of fly ash amendments on Indian paddy field with special emphasis on growth, yield, and grain quality of three rice cultivars

Proper disposal and/or recycling of different industrial waste materials have long been recognized as a prime environmental concern throughout the world, and fly ash is major amongst them. In the present study, we tried to assess the feasibilities of possible effective and safe utilization of fly ash as soil amendment in Indian paddy field and its impact on rice plants, especially at growth and yield level. Our results showed that certain doses of fly ash amendments have significantly improved the physico-chemical and mineralogical properties of paddy field soil, and at lower level of amendments, fly ash induced the growth performances of three rice cultivars too. Grain yield and grain quality also responded similarly as per the growth responses. However, differential cultivar response was observed accordingly, and cultivar Sugandha-3 showed higher yield as compared with cultivars Sambha and Saryu-52. Based on the observed results, it was concluded that up to a certain level, fly ash amendments could be beneficial for Indian paddy field and can be utilized as feasible management strategy for the disposal of this major industrial waste.     

Evaluation of the SKC DPM cassette for monitoring diesel particulate matter in coal mines

In a previous study, the efficacy of commercial and prototype impactors for sampling diesel particulate matter (DPM) in coal mines was investigated. Laboratory and field samples were collected on quartz-fiber filters and analyzed for organic and elemental carbon. Coal dust contributed a minimal amount of elemental carbon when commercial cascade impactors and prototype impactors, designed by the University of Minnesota (UMN) and the US Bureau of Mines (BOM), were used to collect submicrometer dust fractions. Other impactors were not as effective at excluding coal dust. The impactors evaluated in that study were either not commercially available or were multi-stage, expensive, and difficult to use for personal measurements. A commercial version of the BOM impactor, called the DPM Cassette, was recently introduced by SKC. Tests were conducted to evaluate the performance of the DPM Cassette for measuring diesel-source elemental carbon in the presence of coal dust. Bituminous coals from three mines in two different coal provinces were examined. The dust particle diameters were small and the coal dust contained a high percentage of carbon, thereby giving a worst-case condition for non-anthracite coal mines. Results for the DPM Cassette were essentially identical to those obtained by the BOM impactors in a previous study. At a respirable coal dust concentration of 5.46 mg m(-3), which is 3.8 times the regulatory limit, the DPM Cassette collected only 34 microg m(-3) of coal-source elemental carbon.     

Bioaccumulation and translocation of metals in the natural vegetation growing on fly ash lagoons: a field study from Santaldih thermal power plant, West Bengal, India

A field study was conducted in the fly ash lagoons of Santandih Thermal Power Plant located in West Bengal (India) to find out total, EDTA and DTPA extractable metals in fly ash and their bioaccumulation in root and shoot portion of the naturally growing vegetation. Fly ash sample has alkaline pH and low conductivity. The concentration of total Cu, Zn, Pb and Ni were found higher than weathered fly ash and natural soil, where as Co, Cd and Cr were found traces. Five dominant vegetation namely, Typha latifolia, Fimbristylis dichotoma, Amaranthus defluxes, Saccharum spontaenum and Cynodon dactylon were collected in the winter months (November–December). Bioaccumulation of metals in root and shoot portions were found varied significantly among the species, but all concentration were found within toxic limits. Correlation between total, DTPA and EDTA extractable metals viz. root and shoot metals concentration were studied. Translocation factor (TF) for Cu, Zn and Ni were found less than unity, indicates that these metals are immobilized in the root part of the plants. Metals like Mn have TF greater than unity. The study infers that natural vegetation removed Mn by phytoextraction mechanisms (TF > 1), while other metals like Zn, Cu, Pb and Ni were removed by rhizofiltration mechanisms (TF < 1). The field study revealed that T. latifolia and S. spontaenum plants could be used for bioremediation of fly ash lagoon.     

Evaluation of the presence-absence (P-A) test: A simplified bacteriological test for detecting coliforms in rural drinking water of India

One thousand three-hundred and ninety-four drinking water sources comprising ground water, surface water and piped supplies were tested in order to compare the presence-absence (P-A) test with standard MPN method to detect coliforms as indicators of water quality. Out of 1394 samples, 1074 (77.04%) and 1030 (74.88%) were positive by the MPN and P-A test, respectively. The P-A test detected 96% of the positives detected by the MPN test. The P-A test may be effectively used as a rapid screening method to detect coliform contamination in less polluted sources such as ground water and piped supplies.     

微波消解-ICP法测定土壤中重金属元素的不确定度评定

对微波消解-电感耦合等离子发射光谱法测定土壤样品中Cu、Pb、Zn、Cr、Ni的不确定度进行评估,在测定过程中,对样品称量、消解、定容体积、标准溶液的配制、曲线拟合、仪器测量重复性等影响不确定度的分量进行分析,按《测量不确定度评定与表示》(JJF 1059—1999)的规定进行合成,最终给出扩展不确定度,为电感耦合等离子发射光谱法测量土壤中重金属元素的实验室质量控制和不确定度评定提供参考依据。     

PM10 in the ambient air of Chandrapur coal mine and its comparison with other environments

This study compares the ambient air particulate matter (PM10) data of 15 different coal mine environments. For most of these mine environments, the monitoring was carried out by different researchers using respirable dust sampler (RDS) that separates PM10 by centrifugal inertial separation. At two sites — Padmapur and Ghugus (Chandrapur, Maharashtra, India) — mass inertial impaction-based sampler was used for PM10 monitoring. It is observed that the spatiotemporal average value of ambient air PM10 monitored using mass inertial impactor reports relatively higher values (240–372 μg/m³) compared to those monitored using RDS (<227 μg/m³). In order to realize the severity of mine area pollution, it is compared with PM10 values found in an urban area (Delhi, India). It is found that PM10 values in Delhi (using mass inertial impactor) are much higher (300–400 μg/m³) than those reported for the mine environment. The data seems to indicate that the mine environment is relatively cleaner than urban air and therefore raises doubt about the appropriateness of using either mass impactor or RDS for PM10 sampling.     

火焰原子吸收法测定水样铅含量的不确定度评定

实验室认可和资质认定都要求检测实验室具备评定测量不确定度的能力,现依据JJF1059-1999《测量不确定度评定与表示》,对火焰原子吸收法测定水样中铅含量进行不确定度评估,分析测定过程中不确定度来源,量化不确定度分量,求出合成不确定度和扩展不确定度,给出测定结果的表示式。