Kamal K. Panda

In vitro biosynthesis and genotoxicity bioassay of silver nanoparticles using plants

Silver nanoparticles (AgNP-P) from AgNO(3) were synthesized by using the broth prepared from the aromatic spath of male inflorescence of screw pine, Pandanus odorifer (Forssk.) Kuntze AgNP-P was then characterized by UV-visible spectroscopy, transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDS). Functional groups in the broth were analyzed by Fourier Transform infrared spectroscopy (FTIR). Genotoxicity of AgNP-P was assessed by utilizing our well-established Allium cepa assay system with biomarkers including the generation reactive oxygen species (ROS: O(2)(·-) and H(2)O(2)), cell death, mitotic index, micronucleus, mitotic aberrations; and DNA damage by Comet assay. Other chemical forms of silver such as Ag(+) ion, colloidal AgCl, and AgNP-S at doses 0-80 mg L(-1) were included for comparison with AgNP-P. The results revealed that AgNP-P and AgNP-S exhibited similar biological effects in causing lesser extent of cytotoxicity and greater extent of genotoxicity than that was exhibited by Ag(+) ion alone. Among different tested chemical forms of silver, colloidal AgCl was identified to be the least cytotoxic and genotoxic. Cell death and DNA-damage induced by AgNP-P were prevented by Tiron and dimethyl thiourea that scavenge O(2)(·-) and H(2)O(2), respectively. The present findings demonstrated the role of ROS in the AgNP-induced cell death and DNA damage.

Biomonitoring of low levels of mercurial derivatives in water and soil by Allium micronucleus assay

The Allium micronucleus (MNC) assay was developed to monitor low levels of mercury in aquatic and terrestrial environments. Four mercurial derivatives namely mercuric chloride (MC), methyl mercuric chloride (MMC), phenyl mercuric acetate (PMA) and a methoxy ethyl mercuric chloride based fungicide, Emisan-6, were tested to assess the sensitivity and versatility of the Allium MNC assay. Allium bulbs were set directly on water and soil contaminated with known levels of mercurial derivatives (0.0001-10.00 ppm). On the 5th day the endpoints measured were root length, mitoses with spindle abnormality and cells with MNC in root meristems. The effective concentrations of the test chemicals that cause 50% of root length as compared to control (EC50) were determined from dose-response curves so obtained. The lowest effective concentration tested (LECT) and highest ineffective concentration tested (HICT) for each of the mercurial derivatives for the induction of spindle malfunction and MNC were determined. It was found that EC50, LECT and HICT values for mercurial derivatives in soil were higher than those in water. The frequencies of cells with MNC and mitoses with spindle abnormality were highly correlated indicating that MNC is a good parameter of spindle malfunction. The present approach increased the sensitivity of the Allium assay by 10-fold, the detection limit being 0.001-0.1 ppm and 0.1-1.0 ppm in aquatic and terrestrial environments respectively, depending on the species of mercury.

Monitoring and assessment of mercury pollution in the vicinity of a chloralkali plant. IV. Bioconcentration of mercury in in situ aquatic and terrestrial plants at Ganjam, India

In situ aquatic and terrestrial plants including a few vegetable and crop plants growing in and around a chloralkali plant at Ganjam, India were analyzed for concentrations of root and shoot mercury. The aquatic plants found to bioconcentrate mercury to different degrees included Marsilea spp., Spirodela polyrhiza, Jussiea repens, Paspalum scrobiculatam, Pistia stratiotes, Eichhornia crassipes, Hygrophila schulli, Monochoria hastata and Bacopa monniera. Among wild terrestrial plants Chloris barbata, Cynodon dactylon, Cyperus rotundus and Croton bonplandianum were found growing on heavily contaminated soil containing mercury as high as 557 mg/kg. Analysis of mercury in root and shoot of these plants in relation to the mercury levels in soil indicated a significant correlation between soil and plant mercury with the exception of C. bonplandianum. Furthermore, the tolerance to mercury toxicity was highest with C. barbata followed by C. dactylon and C. rotundus, in that order. The rice plants analyzed from the surrounding agricultural fields did not show any significant levels of bioconcentrated mercury. Of the different vegetables grown in a contaminated kitchen garden with mercury level at 8.91 mg/kg, the two leafy vegetables, namely cabbage (Brassica oleracea) and amaranthus (Amaranthus oleraceous), were found to bioconcentrate mercury at statistically significant levels. The overall study indicates that the mercury pollution is very much localized to the specific sites in the vicinity of the chloralkali plant.

Genotoxicity and Mutagenicity of Metals in Plants

Metals are ubiquitous in nature. Metals are emitted from a wide spectrum of natural and man-made sources such as volcanic eruption, mining, fossil burning, industrial emissions and automobile exhausts and sewage disposals. Distribution of metals in the environment, however, is uneven (Moore and Ramamoorthy 1984, Nriagu and Pacyna 1988, Nriagu 1990). Upon entering into the environment in a variety of organic and inorganic forms and being neither degradable nor recoverable, metals get incorporated into biogeochemical cycles where they can exert long-term effect (Nriagu 1990). The problem of metal pollution is a global phenomenon. This, however, is accentuated in third world countries that have been offering the dumping grounds for toxic wastes and have become the hot spots of metal pollution because of population explosion coupled with poor economic conditions, use of outdated technologies in industries and lack of stringent anti-pollution laws (Anonymous 1991).

Studies on the Ability of Water Hyacinth (Eichhornia crassipes) to Bioconcentrate and Biomonitor Aquatic Mercury

Water hyacinth (Eichhornia crassipes, Mart solms) plants were employed to assess bioconcentration and genotoxicity of aquatic mercury. Plants were exposed to water contaminated with mercuric chloride (MC) or phenyl mercuric acetate (PMA) at 0.001 to 1.0 mg litre(-1), or mercury contaminated effluent from a chloralkali plant for various periods of 4 t0 96 h. Root samples taken after 4, 8, 12, 24, 48, 72 and 96 h of exposure were analysed for bioconcentration of mercury spectrophotometrically, and the root meristems were fixed in aceto-ethanol for cytological analysis to determine the frequencies of cells with micronuclei (MNC). Ethyl methane sulfonate and tap water served as positive and negative controls, respectively. The results indicated that bioconcentration of mercury in root tissue was both time- and concentration-dependent, providing evidence that water hyacinth is a good absorbant of aquatic mercury. The frequency of root meristematic cells with MNC followed a concentration-response. The findings indicate the potential of water hyacinth plants for in situ monitoring and for mitigation of aquatic mercury pollution.

Monitoring and assessment of mercury pollution in the vicinity of a chloralkali plant. I, Distribution, availability and genotoxicity of sediment mercury in the Rushikulya estuary, India

Industrial effluent discharged into the Rushikulya estuary from a chloralkali plant has been identified as the source of mercury which has polluted the estuary for the past two decades. Sediment samples taken from eight sites representing the effluent channel, effluent discharge point and the course of the estuary, covering a distance of 2 km, were analyzed for total mercury. Phthalate buffer (pH 2–6) was used to study leachability of mercury from sediments as a function of pH. Chemical extraction with 10% HNO3, ammonium acetate-EDTA and 0.1 and 0.05 M CaCl2 was used to assess bioavailability of mercury from the sediments. Further, sediment samples were bioassayed by the Allium micronucleus (MNC) assay. The endpoints measured were root length, root mercury (nioconcentrated mercury) and frequency of root meristematic cells with MNC. The mercury concentrations in sediment samples ranged from 1.6 to 192 mg kg−1. The results indicated that pH was an important factor regulating the availability of mercury; the lower the pH the higher the availability of mercury. Of the different chemical extractants used, 0.05 M CaCl2 extractable mercury showed the highest correlation with bioconcentrated mercury (r2 = 0.91, P ⩽ 0.01), emphasizing the fact that 0.05 M CaCl2 was the best predictor of bioavailable mercury from the sediment. Similarly, the frequency of root meristematic cells with MNC was highly correlated not only with bioconcentrated mercury (r2 = 0.92, P ⩽ 0.01), but also with bioavailable mercury, as evidenced by 0.05 M CaCl2 extractable mercury (r2 = 0.85, P ⩽ 0.01). The Allium MNC assay can assess mercury pollution and takes into account the bioavailability of the metal. The overall assessment indicated that, although localized, the sediment of the Rushikulya estuary is contaminated with high concentrations of mercury with respect to both bioavailability and toxicity.

Monitoring and assessment of mercury pollution in the vicinity of a chloralkali plant. II Plant-availability, tissue-concentration and genotoxicity of mercury from agricultural soil contaminated with solid waste assessed in barley (Hordeum vulgare L.).

Barley (Hordeum vulgare L.) was used to assess plant-availability, tissue-concentration and genotoxicity of mercury from the solid waste deposits of a chloralkali plant. Seeds of H. vulgare, presoaked in distilled water, were allowed to germinate and grow on agricultural soil mixed with solid waste containing 2550+/-339 mg Hg kg(-1) at different proportions (0.75, 1.5, 2.5 and 5%). Plants raised from germinating seeds on uncontaminated agricultural soil served as controls. On day 7, germination counts and seedling heights were recorded. The concentration of mercury in soil, and plant tissue (dry weight) were determined at different stages of plant growth from day 7 till maturity and harvest. The availability of mercury from the soil was assessed by extracting mercury at a range of pH values (2-6) and by chemical methods. The embryonic shoots excised at 36 h of germination were subjected to cytological analysis to determine mitotic index and frequency of mitoses with chromosomal aberrations. The pollen mother cells from anthers of young M1-spikes were analysed to score meiotic aberrations. Subsequently, the pollen fertility and seed set were determined. Furthermore, M2-seedlings were analysed for chlorophyll-deficient mutations. The cytogenetic analysis revealed the effects of mercury on the mitotic and meiotic chromosomes which were significantly correlated with soil-mercury. The bioconcentration of mercury in aerial tissues decreased with the age of the plant. Roots, in particular, were found to concentrate most of the mercury taken up by the plant. At the time of harvest, the bioconcentration of mercury in straw was at a minimum. The accumulation of mercury in grain, which was significant, did not increase with the increase in concentration of mercury in soil but maintained a plateau, indicating a restriction of transport of mercury through the phloem. The unique advantage with the use of Hordeum assay is that, besides assessing the germline toxicity, the assay takes into account the possible contamination of the agricultural food-chain.

Prophylaxis of antioxidants against the genotoxicity of methyl mercuric chloride and maleic hydrazide in Allium micronucleus assay

Antioxidants, namely cysteine, 2.46 x 10(-5) M; glutathione 9.75 x 10(-6), 9.75 x 10(-4) M; vitamin C, 10(-2) M; mannitol, 5 x 10(-2) M; potassium iodide, 5 x 10(-2) M and sodium selenite, 1.73 x 10(-6) M; were tested for their prophylactic activity against the genotoxicity of methylmercuric chloride, 1.26 x 10(-6) M and maleic hydrazide, 3 x 10(-4) M in Allium micronucleus assay. Antioxidants doses were administered simultaneously or prior to the genotoxic exposures. The results from the present experiments indicated that antioxidants conferred protection against the genotoxicity of both methyl mercuric chloride and maleic hydrazide. Furthermore, the protection of GSH against methyl mercuric chloride depending on the concentration persisted for 12 h.

Allium micronucleus (MNC) assay to assess bioavailability, bioconcentration and genotoxicity of mercury from solid waste deposits of a chloralkali plant, and antagonism of l-cysteine

Samples of solid waste from a chloralkali plant were bioassayed employing the Allium micronucleus (MNC) assay. The endpoints measured were root length, root mercury and frequency of root meristematic cells with MNC. Chemical extraction methods such as 10% HNO3, ammonium acetate-EDTA and 0.05 or 0.1 M CaCl2 were used to assess the bioavailability of mercury from soil contaminated with solid waste. Analysis of mercury was by cold vapour atomic absorption spectrophotometry. The frequency of MNC induced by contaminated soil, which followed a concentration-response curve, was statistically correlated with soil mercury, extractable mercury and root mercury. The antagonism of L-cysteine against the suppression of root growth and induction of MNC by solid waste suggested the involvement of mercury as well as thiol groups in the process. The threshold assessment values, such as the median effective concentration (EC50) for root growth, gross toxicity concentration tested (GTCT) as indicated by complete inhibition of sprouting of roots, lowest effective concentration tested (LECT) and highest ineffective concentration tested (HICT) for induction of MNC, were determined. These assessment values may be useful for environmental management and regulatory purposes. Furthermore, the detection limit of the Allium MNC assay for solid waste mercury as indicated by LECT was 9.6 mg kg-1 which corresponded to 0.13 mg kg-1 of 0.05 M CaCl2 extractable or 8.3 mg kg-1 dry weight bioconcentrated mercury.

Residual mercury in seed of barley (Hordeum vulgare L.) confers genotoxic adaptation to ethyl methanesulfonate, maleic hydrazide, methyl mercuric chloride and mercury-contaminated soil

Seeds of barley, Hordeum vulgare L., with or without residual mercury were exposed to concentrations of ethyl methanesulfonate (EMS), maleic hydrazide (MH), methyl mercuric chloride (MMCl) and mercury-contaminated soil. Subsequently the endpoints measured were germination, seedling height, mitotic index, mitotic chromosome or spindle aberrations in embryonic shoot cells and meiotic chromosome aberration in pollen mother cells. The results unequivocally demonstrated that the seed-residual mercury conferred protection against the genotoxicity of EMS, MH, MMCl as well as mercury-contaminated soil in barley. The genotoxic adaptation to MH and MMCl was significantly prevented by pre-exposing the Hg-seeds to buthionine sulfoximine, an inhibitor of phytochelatin synthesis. Furthermore, compared to normal seedlings, the seedlings grown from Hg-seeds exhibited a higher amount of non-protein SH. The findings indicated a possible involvement of phytochelatins in the mercury-induced adaptive response.