Determination of aflatoxin-B1 in poultry feed and its components employing enzyme-linked immunosorbent assay (ELISA)

A highly sensitive enzyme immunoassay was standardized for aflatoxin B₁ determination in poultry feed and its components using Riedel-de-Haen, ELISA Systems_. A microtitration plate method was optimized using anti-aflatoxin B₁ antibodies and peroxidase – aflatoxin B₁ conjugate, based on competitive enzyme immunoassay principle. Standards of concentrations of 5, 10, 20, 50, 100, 500?ng/L aflatoxin B₁, prepared in phosphate-buffered saline, were used. Standard curves showed that as the concentration of antigen decreased, absorbance values increased. Fifty percent inhibition was observed at 37?ng/L. Regression analysis showed that log concentration was inversely related to %B/B₀, with a highly significant negative correlation (?0.980). The lowest detection limit for aflatoxin B₁ was 5?ng/L. Using this standardized ELISA, aflatoxin B₁ was detected in most of the commercially available poultry feed samples and their components.

The data suggest that this test is suitable for the accurate determination of aflatoxin B₁ concentrations in poultry feed and its components.     

α1-Microglobulin levels and correlation with cadmium and other metals in urine of non-smoking women among general populations in Japan

The present study was initiated to examine urinary α₁-microglobulin (α₁-MG-U) levels among non-smoking women in the general population in Japan. A previously established database on spot urine samples from adult woman volunteers in 10 non-polluted areas all over Japan was re-examined. The data examined were on α₁-MG-U, cadmium, calcium, magnesium and zinc levels in urine (Cd-U, Ca-U, Mg-U and Zn-U, respectively), urinary creatinine (CR or cr), urine specific gravity (SG or sg), smoking habits and age. Thus, 8975 never-smoking women were selected for statistical analyses. The grand geometric mean (GM) for α₁-MG-U among the population was 2.1?mg/L or 2.5?mg/g?cr, depending on the correction for urine density. It was 1.1?µg/L or 1.3?µg/g?cr for GM Cd-U. The inter-area difference in α₁-MG-U was <1.5?mg/g?cr or <0.7?mg/L; the area with the highest or lowest GM Cd-U was not always highest or lowest in GM α₁-MG-U. The correlation coefficient (0.53) between log?Cd-U and log?α₁-MG-U (both without urine density correction) became substantially smaller when the analyte levels were corrected for CR (0.25) or SG (0.26). In multiple regression analysis, the power of influence of the five independent variables (log?Cd-U, Ca-U, Mg-U, Zn-U and age) in combination was small (R ²?≦?0.13). In contrast, logistic regression analysis suggested that α₁-MG-U might be elevated as a function of an increase in Cd-U, depending on the cut-off values. Discussion was made on dose (Cd-U_cr) and response (α₁-MG-U_cr) relationship based on information available in literatures to show that the increment in α₁-MG-U_cr per Cd-U_cr was much greater when Cd-U_cr was large, e.g., in excess of 10?µg/g?cr.     

Decolorization and degradation of acid dyes mediated by salt fractionated turnip ( Brassica rapa ) peroxidases

Peroxidases from turnip roots (524?U?g^?1 of vegetable) were highly effective in decolorizing acid dyes having wide spectrum chemical groups. Dye solutions, containing 40–170?mg?dye?L^?1, were treated by turnip peroxidases (TP) (specific activity of 122.0?U?mg^?1 proteins). These enzymes were able to decolorize most of the acid dyes in the presence of 2.0?mM 1-hydroxybenzotriazole (HOBT). Increasing concentration of enzyme and time in the absence of HOBT did not influence dye decolorization. The rate of decolorization was significantly enhanced when HOBT was added to the decolorizing solutions. The decolorization of all the used dyes was maximum at pH 5.0 and 40°C. Complex mixtures of dyes were significantly decolorized when treated with enzyme in the presence of HOBT (2.0?mM). Phytotoxicity test based on Allium cepa root growth inhibition has shown that majority of the TP-treated dye product were not more toxic than their parent dye. Kinetic parameters of the TP with various dyes showed that this enzyme has highest affinity for Acid Yellow 42. This study demonstrates that the peroxidase/mediator system was an effective biocatalyst for the treatment of industrial effluents from textile, dye manufacturing, dyeing and printing industries or complex mixtures of dyes.     

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.     

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.     

Interactions between metals accumulated in the narrow-clawed crayfish Astacus leptodactylus (Eschscholtz, 1823) in Dikilitaş Lake,Turkey

The accumulations of Al, Cd, Cr, Cu, Fe, Ni, Pb and Zn in the exoskeleton, gills, hepatopancreas and abdominal muscles of crayfish Astacus leptodactylus (Eschscholtz, 1823) were determined. The strongest correlation observed was between Cr and Ni in the gills (r?=?0.904); moderate to strong correlations between Al, Cr, Fe, Ni and Cu were also observed in gill tissue. Disregarding the gills, the strongest correlation was found between Cu and Zn in the hepatopancreas (r?=?0.808); the correlation between these two metals might have been a result of metallothionein activity. The accumulation of Pb was found to correlate with that of Cd in the exoskeleton, Cd and Zn in the gills, Zn and Cu in the hepatopancreas and Cu in the abdominal muscle. None of these correlations were present in lakewater and sediment samples, suggesting that the crayfish metabolism may be responsible for the co-accumulation of metal–metal pairs. As all correlations in non-gill tissues are observed between divalent metals, a shared transporter such as divalent metal transporter 1 might be involved in the accumulation of these metals.     

Conservation Strategies for Species Affected by Apparent Competition

Apparent competition is an indirect interaction between 2 or more prey species through a shared predator, and it is increasingly recognized as a mechanism of the decline and extinction of many species. Through case studies, we evaluated the effectiveness of 4 management strategies for species affected by apparent competition: predator control, reduction in the abundances of alternate prey, simultaneous control of predators and alternate prey, and no active management of predators or alternate prey. Solely reducing predator abundances rapidly increased abundances of alternate and rare prey, but observed increases are likely short‐lived due to fast increases in predator abundance following the cessation of control efforts. Substantial reductions of an abundant alternate prey resulted in increased predation on endangered huemul (Hippocamelus bisulcus) deer in Chilean Patagonia, which highlights potential risks associated with solely reducing alternate prey species. Simultaneous removal of predators and alternate prey increased survival of island foxes (Urocyon littoralis) in California (U.S.A.) above a threshold required for population recovery. In the absence of active management, populations of rare woodland caribou (Rangifer tarandus caribou) continued to decline in British Columbia, Canada. On the basis of the cases we examined, we suggest the simultaneous control of predators and alternate prey is the management strategy most likely to increase abundances and probabilities of persistence of rare prey over the long term. Knowing the mechanisms driving changes in species’ abundances before implementing any management intervention is critical. We suggest scientists can best contribute to the conservation of species affected by apparent competition by clearly communicating the biological and demographic forces at play to policy makers responsible for the implementation of proposed management actions. Estrategias de Conservación para Especies Afectadas por Competencia Aparente     

Assessing the Risk of Ships Striking Large Whales in Marine Spatial Planning

Marine spatial planning provides a comprehensive framework for managing multiple uses of the marine environment and has the potential to minimize environmental impacts and reduce conflicts among users. Spatially explicit assessments of the risks to key marine species from human activities are a requirement of marine spatial planning. We assessed the risk of ships striking humpback (Megaptera novaeangliae), blue (Balaenoptera musculus), and fin (Balaenoptera physalus) whales in alternative shipping routes derived from patterns of shipping traffic off Southern California (U.S.A.). Specifically, we developed whale‐habitat models and assumed ship‐strike risk for the alternative shipping routes was proportional to the number of whales predicted by the models to occur within each route. This definition of risk assumes all ships travel within a single route. We also calculated risk assuming ships travel via multiple routes. We estimated the potential for conflict between shipping and other uses (military training and fishing) due to overlap with the routes. We also estimated the overlap between shipping routes and protected areas. The route with the lowest risk for humpback whales had the highest risk for fin whales and vice versa. Risk to both species may be ameliorated by creating a new route south of the northern Channel Islands and spreading traffic between this new route and the existing route in the Santa Barbara Channel. Creating a longer route may reduce the overlap between shipping and other uses by concentrating shipping traffic. Blue whales are distributed more evenly across our study area than humpback and fin whales; thus, risk could not be ameliorated by concentrating shipping traffic in any of the routes we considered. Reducing ship‐strike risk for blue whales may be necessary because our estimate of the potential number of strikes suggests that they are likely to exceed allowable levels of anthropogenic impacts established under U.S. laws. Evaluación del Riesgo de Colisiones de Barcos y Ballenas en la Planificación Marina Espacial     

Effects of Dam‐Induced Landscape Fragmentation on Amazonian Ant–Plant Mutualistic Networks

Mutualistic networks are critical to biological diversity maintenance; however, their structures and functionality may be threatened by a swiftly changing world. In the Amazon, the increasing number of dams poses a large threat to biological diversity because they greatly alter and fragment the surrounding landscape. Tight coevolutionary interactions typical of tropical forests, such as the ant–myrmecophyte mutualism, where the myrmecophyte plants provide domatia nesting space to their symbiotic ants, may be jeopardized by the landscape changes caused by dams. We analyzed 31 ant–myrmecophyte mutualistic networks in undisturbed and disturbed sites surrounding Balbina, the largest Central Amazonian dam. We tested how ant–myrmecophyte networks differ among dam‐induced islands, lake edges, and undisturbed forests in terms of species richness, composition, structure, and robustness (number of species remaining in the network after partner extinctions). We also tested how landscape configuration in terms of area, isolation, shape, and neighborhood alters the structure of the ant–myrmecophyte networks on islands. Ant–myrmecophytic networks were highly compartmentalized in undisturbed forests, and the compartments had few strongly connected mutualistic partners. In contrast, networks at lake edges and on islands were not compartmentalized and were negatively affected by island area and isolation in terms of species richness, density, and composition. Habitat loss and fragmentation led to coextinction cascades that contributed to the elimination of entire ant–plant compartments. Furthermore, many myrmecophytic plants in disturbed sites lost their mutualistic ant partners or were colonized by opportunistic, nonspecialized ants. Robustness of ant–myrmecophyte networks on islands was lower than robustness near lake edges and in undisturbed forest and was particularly susceptible to the extinction of plants. Beyond the immediate habitat loss caused by the building of large dams in Amazonia, persistent edge effects and habitat fragmentation associated with dams had large negative effects on animal–plant mutualistic networks. Efectos de la Fragmentación del Paisaje Inducida por Presas sobre Redes Mutualistas Hormiga‐Planta Amazónicas