Spore inhibition-based enzyme substrate assay for monitoring of aflatoxin M1 in milk

A spore germination-based concept and its transformation into a field level prototype for monitoring aflatoxin M₁ (AFM₁) in milk was developed. Initially, 15 strains of Bacillus spp. procured from different culture collection were screened for AFM₁ sensitivity using spot assay and marker strain showing inhibition at 0.5 ppb was selected based upon maximum zone of inhibition. The selected strain B. megaterium 2949 was further screened for different enzymes activities and subsequently its spores were produced to an extent of 73.13% ± 3.197% in newly developed sporulation medium containing beef extract (0.0075% ± 0.0004%), yeast extract (0.015% ± 0.001%), peptone (0.0375% ± 0.0016%), and sodium chloride (0.0375% ± 0.0018%). A spore germination-based concept/ assay was optimized by immobilizing spores in eppendorf with pretreated milk (80°C/15 min) containing germinant and chromogenic substrate followed by incubation at 37°C. The appearance of sky blue color within real time of 45 min indicated spores germination and release of specific marker enzyme such as acetyl esterase and its specific action on chromogenic substrate which demonstrates absence of AFM₁ in milk. However, if there was no color change, presence of AFM₁ at 0.5 ppb MRL was denoted by Codex. The developed concept on AFM₁ detection was validated and a correlation of 0.97 was established with AOAC approved Charm 6602 and ELISA at Codex MRL with minimal false positive and negative results. The cost effective test has potential application in dairy farms, manufacturing, and R&D units for routine monitoring of AFM₁ in milk.     

Genotoxic effects of two different classes of insecticide in developing chick embryos

Pesticides provide considerable protection against pest population; however, rampant accumulation of these chemicals into varied habitats across the globe necessitates the need for a careful screening of each chemical due to toxic manifestations. In the current study, the genotoxic potential of two different classes of commercial insecticides – chlorpyrifos and cypermethrin combination and Spinosad, a naturalyte were compared. Rhode Island Red chick embryos were exposed to different doses of either of these insecticides individually, by in ovo treatment. Genotoxicity was then evaluated through micronucleus (MN) test and Comet assay. The combination insecticide exposure at low doses of 0.05 and 0.1 μg/egg induced DNA damage as evidenced by an increased tail moment in the Comet assay. Further, the presence of micronucleated erythrocytes and also various abnormal cells including dacryocytes, microcytes, erythroplastids, squashed/notched nuclei, and spindle-shaped erythrocytes in the blood smear consolidates indicate the presence of insecticide-induced genotoxicity. Spinosad, however, was found only mildly genotoxic but at a high dose of 1.5 mg/egg. The results indicate that usage of naturalyte insecticide may be a better option to minimize the harmful effects of chemical insecticides.     

Immunotoxic manifestations in rats following inhalative exposure to furfural vapors

Furfural is an industrial compound used as a process intermediate and as a solvent; it poses a potential inhalation hazard in occupational settings. This study was carried out to find furfural-induced immunotoxicity in Wistar rat following inhalative exposure. The weights of thymus and lymph node were found decreased, while the weights of the liver and the adrenal gland were significantly increased following furfural exposure. Delayed-type hypersensitivity response was found decreased in furfural vapors-exposed animals when compared to that of control animals. The phagocytic index of peritoneal and alveolar exudates showed significant decrease and was most prominent (90%) in 30 days-exposed groups. The number of anti-rat anti-sheep red blood cell immunoglobulin M plaque forming cells of spleen got decreased in furfural-exposed groups in comparison to control. Taken together, this study indicates that inhalation of furfural induces immunotoxic manifestations that could lead to severe immunological disorders.     

Carbofuran-induced effects on acetylcholinesterase (AChE) in erythrocytes and liver of Cyprinus carpio

A study was conducted to determine the effects of long-term exposure to carbofuran on erythrocyte and liver acetylcholinesterase (AChE) activity in Cyprinus carpio, and assess which tissue was more sensitive to pesticide exposure examining this enzyme. Fish were allowed to accilimatize in toxicant-free water for 24 days after 60-day exposure. AChE activity was determined with a spectrophotometer using acetylthiocholine iodide as a substrate in erythrocytes and liver. Enzymic activity of erythrocytes and liver decreased over time. A higher degree of enzyme inhibition was observed in erythrocyte compared to liver. The degree of enzyme inhibition was positively correlated with exposure time. When exposed fish were transferred to clean water, recovery was greater in erythrocytes than liver. Erythrocyte AChE activity recovered after 18 days, while it required 21 days in liver. The findings of this study indicate that erythrocyte AChE is more sensitive to carbofuran than liver. The greater sensitivity of erythrocyte AChE suggests that it may be more useful as a biomarker for monitoring status of pollution in freshwater bodies.     

Transport of metanil yellow in the rat plasma and interaction of its metabolite,p‐aminodiphenylamine with serum proteins

Binding affinity of metanil yellow and its breakdown product p‐aminodiphenylamine to serum proteins has been studied employing chromatographic separation on Sephadex G‐200 and by paper and polyacrylamide gel electrophoresis. Metanil yellow has more affinity towards albumin than to globulins. The complexing is presumably through electrostatic forces. p‐Aminodiphenylamine on the other hand, preferably binds to globulin fractions of serum protein. However, a stable binding with BSA alone was also observed. The binding was quite stable and was accompanied by a shift in absorbance from 430 nm to 500 nm. Aspartic acid moiety of protein was found to be one of the units involved in the binding of p‐ADPA to proteins.     

Detection and quantitation of trace levels of low molecular weight halocarbons (LMHs) associated with commercial sodium humate

Examination of vapor above commercial sodium humate and above 10wt% aqueous humate solution by gas chromatography (GC) revealed the presence of chloroform and trichloroethylene. Further examination of the aqueous humate by the purge and trap procedure confirmed the association of chloroform and trichloroethylene with the humate. GC/mass spectrometry analyses of methylene chloride extract of the humate established the association of LMHs with the humate. Improved headspace capillary GC technique showed that the humate contained not only chloroform, trichloroethylene but several other LMHs in the range 190 to 4000ng·kg^‐1.     

Lead uptake by Eichhornia Crassipes

Factors influencing uptake of lead(II) by the water hyacinth (Eichhornia Crassipes), were examined. Two phases of uptake were observed for the concentration range investigated (0.01–1000 ppm). The initial, rapid uptake phase of about 4 hours is followed by a slower, near linear phase extending past 24 hours. Stirring the solution enhanced uptake, suggesting that lead removal is in part diffusion limited. In the range of 4–8, pH has little effect on uptake where as outside this range, uptake is reduced. Increased solution volume or rootmass results in more metal being removed by the plant. The presence of strong complexers blocks the initial rapid uptake phase as does the presence of Zn(II), Cd(II), Hg(II), and Fe(III). Strong complexers can also strip a portion of any lead already removed from solution by the plant.     

Preliminary environmental study of New Karvali Bay (Kavala Gulf,North Greece)

In this presentation the results of an environmental study of New Karvali bay, in Kavala Gulf, North Greece, are announced. In the studied bay the main industrial activity is a Factory of Chemical Fertilizers (FCF) and its wastes have been discharged, practically without any treatment, to the sea. The study has shown that the concentrations of phosphates and nitrates in the area around the place of the waste ejection from the factory are relatively high. Other remarkable points of the study are the presence of fluoride anions in higher level than normal and the low concentration of heavy metals (Cu, Pb and Zn).     

Databanks of data sources for environmental chemicals

There exist a vast variety of data sources, either manual sources or computerized databases in the field of environmental chemicals. To facilitate the access and to retrieve the information wanted efficiently, we built up two “databanks of data sources”, one called “Databank of Manual Sources (DMAS)” and the other named “Databank of Databases (DADB)”. The set‐up of these databanks is explained and the load status statistics are shown. DMAS has 164 and DADB 106 hits.     

Effects of the level of dietary protein on the toxicity of hexachlorocyclohexane in rats

Male albino rats were fed for 28 days from weaning on diets containing 5% (group 1), 10% (group 2) and 21% (group 3, normal protein) protein as casein. At the end of dietary period, HCH was administered daily for 30 days to investigate the interaction between protein deficiency and pesticide toxicity. The results indicated that rats fed a lower protein diet and HCH had a higher mortality, lower rate of growth, increased liver weight and deposition of the pesticide in blood and tissues in larger amounts. Blood pressure (systolic and diastolic) was significantly increased and the heart rate showed tachycardia in low protein exposed animals. A significant increase of total lipids, cholesterol, triglycirides, free fatty acids in serum and tissues of animals exposed to low protein was observed. A close correlation existed between lipid accumulation and storage of HCH in tissues and dietary protein seemed to play an important role in detoxification.