Differential bioavailability of soil-sorbed naphthalene to two bacterial species.

Prediction of the fate of hydrophobic organic contaminants in soils is complicated by the competing processes of sorption and biodegradation. To test the hypothesis that sorbed naphthalene is unavailable to degradative microorganisms, we developed a simple kinetic method to examine the rates and extents of naphthalene degradation in soil-free and soil-containing systems in a comparison of two bacterial species. The method is predicated on the first-order dependence of the initial mineralization rate on the naphthalene concentration when the latter is below the Michaelis-Menten half-saturation constant (Km) for naphthalene for the organism under study. Rates and extents of mineralization were estimated by nonlinear regression analysis of data by using both a simple first-order model and a three-parameter, coupled degradation-desorption model described for the first time here. Bioavailability assays with two bacterial species (Pseudomonas putida ATCC 17484 and a gram-negative soil isolate, designated NP-Alk) gave dramatically different results. For NP-Alk, sorption limited both the rate and extent of naphthalene mineralization, in accordance with values predicted on the basis of the equilibrium aqueous-phase naphthalene concentrations. For strain 17484, both the rates and extents of naphthalene mineralization exceeded the predicted values and resulted in enhanced rates of naphthalene desorption from the soils. We conclude that there are important organism-specific properties which make generalizations regarding the bioavailability of sorbed substrates inappropriate.     

Differential bioavailability of soil-sorbed naphthalene to two bacterial species.

Prediction of the fate of hydrophobic organic contaminants in soils is complicated by the competing processes of sorption and biodegradation. To test the hypothesis that sorbed naphthalene is unavailable to degradative microorganisms, we developed a simple kinetic method to examine the rates and extents of naphthalene degradation in soil-free and soil-containing systems in a comparison of two bacterial species. The method is predicated on the first-order dependence of the initial mineralization rate on the naphthalene concentration when the latter is below the Michaelis-Menten half-saturation constant (Km) for naphthalene for the organism under study. Rates and extents of mineralization were estimated by nonlinear regression analysis of data by using both a simple first-order model and a three-parameter, coupled degradation-desorption model described for the first time here. Bioavailability assays with two bacterial species (Pseudomonas putida ATCC 17484 and a gram-negative soil isolate, designated NP-Alk) gave dramatically different results. For NP-Alk, sorption limited both the rate and extent of naphthalene mineralization, in accordance with values predicted on the basis of the equilibrium aqueous-phase naphthalene concentrations. For strain 17484, both the rates and extents of naphthalene mineralization exceeded the predicted values and resulted in enhanced rates of naphthalene desorption from the soils. We conclude that there are important organism-specific properties which make generalizations regarding the bioavailability of sorbed substrates inappropriate.     

Ageing of atrazine in manure amended soils assessed by bioavailability to Pseudomonas sp. strain ADP

Animal manure is applied to agricultural land in areas of high livestock production. In the present study, we evaluated ageing of atrazine in two topsoils with and without addition of manure and in one subsoil. Ageing was assessed as the bioavailability of atrazine to the atrazine mineralizing bacteria Pseudomonas sp. strain ADP. Throughout an ageing period of 90 days bioavailability was investigated at days 1, 10, 32, 60 and 90, where ~10⁸ cells g^?1 of the ADP strain was inoculated to the ¹⁴C-atrazine exposed soil and ¹⁴CO₂ was collected over 7 days as a measure of mineralized atrazine. Even though the bioavailable residue decreased in all of the three soils as time proceeded, we found that ageing occurred faster in the topsoils rich in organic carbon than in subsoil. For one topsoil rich in organic carbon content, Simmelkær, we observed a higher degree of ageing when treated with manure. Contrarily, sorption experiments showed less sorption to Simmelkær treated with manure than the untreated soil indicating that sorption processes are not the only mechanisms of ageing. The other topsoil low in organic carbon content, Ringe, showed no significant difference in ageing between the manure-treated and untreated soil. The present study illustrates that not simply the organic carbon content influences adsorption and ageing of atrazine in soil but the origin and composition of organic matter plays an important role.     

Assessment of atrazine decontamination by epiphytic root bacteria isolated from emergent hydrophytes

Aim of the study was to identify atrazine remediating bacteria that can potentially succeed in situ where they encounter varied environmental conditions. Three epiphytic root bacteria, genus Pseudomonas and Arthrobacter, were isolated from rhizoplanes of hydrophytes Acorus calamus, Typha latifolia, and Phragmites karka. Potential of these strains to decontaminate environmentally relevant concentrations of atrazine was determined in liquid atrazine medium (LAM) and Luria-Bertani (LB) medium at varying pH and temperature. There was an increase in decontamination by the strains with time upon exposure to 2.5 to 10 mg l^?1 atrazine over a period of 15 days, notably, in both minimal and nutrient-rich media. Growth in terms of O.D.₆₀₀ and biomass determined during the same period also showed a corresponding surge. Pseudomonas sp. strain AACB mitigated atrazine in a wide range of pH (5 to 8). Pseudomonas sp. strains AACB and TTLB decontaminated >?62% atrazine at 10 °C. All the strains exhibited plant growth–promoting traits in vitro, reported for the first time in the presence of atrazine. Strain AACB exhibits the novel trait of atrazine decontamination under harsh environmental conditions mimicked in lab. Strains isolated in the present study promise success in in situ remediation. Bioreactors and water treatment plants can be designed comprising the hydrophytes and the strains inoculated into their rhizospheres to improve efficacy of the treatment. They can be used to study plant-bacterium mutualistic symbiosis or other interactions occurring during atrazine mitigation.     

Assessment of the bacterial community structure in a Brazilian clay soil treated with atrazine

In the present paper, the bacterial communities in two soils, one from an agricultural sugarcane cropped field and the other from an unperturbed soil with similar geopedological characteristics, were characterized using the Fluorescence In Situ Hybridization (FISH) method. FISH consists of in situ identification of bacteria using fluorescent labeled 16S rRNA targeted oligonucleotide probes visualizable under epifluorescence microscope. In the cultivated soil, in line with agricultural practice, the pre-emergence herbicide atrazine had been regularly applied each year at a concentration of 5 L/ha. The Shannon Diversity and Evenness Indices were also calculated using the phylogenetic data obtained from the FISH analysis. Although, at the sampling time (6 months after soil atrazine treatment), no residual herbicide concentration was found, the overall bacterial community results show a lower diversity and evenness in the agricultural soil than in the unperturbed one, demonstrating how microbiological indicators are sensitive to anthropogenic disturbance. In the natural soil, the dominant groups were α-Proteobacteria, β-Proteobacteria, and γ-Proteobacteria (representing more than 50 % of the bacteria), but in the agricultural soil, their abundance decreased significantly and represented just 31 % of the bacteria domain.     

Assessment of bioavailability of heavy metals using lux modified constructs of Pseudomonas fluorescens

The bioluminescence response of a genetically modified ( lux -marked) bacterium to potentially toxic elements (PTEs) was monitored using an in vitro assay. Washed cells of Pseudomonas fiuorescens were added to solutions containing various concentrations of metal salts. Bioluminescence, involving either plasmid or chromosomally encoded lux genes, declined as the metal concentration increased. The plasmid marked construct was significantly more sensitive to all metals except Cr. The order of metal sensitivity was found to be Cu = Zn > Cd > Cr > Ni for the chromosomally marked construct and Cu = Zn > Cd > Ni > Cr for the plasmid marked construct. The very sensitive response of lux -marked terrestrial bacteria to PTEs identified the potential for a rapid and flexible ecotoxicity assay for assessing the pollution of soil or fresh water environments.     

Assessment of selenium bioavailability from naturally produced high-selenium soy foods in selenium-deficient rats

We assessed the bioavailability of selenium (Se) from a protein isolate and tofu (bean curd) prepared from naturally produced high-Se soybeans. The Se concentrations of the soybeans, the protein isolate and tofu were 5.2 ± 0.2, 11.4 ± 0.1 and 7.4 ± 0.1 mg/kg, respectively. Male weanling Sprague–Dawley rats were depleted of Se by feeding them a 30% Torula yeast-based diet (4.1 μg Se/kg) for 56 days, and then they were replenished with Se for an additional 50 days by feeding them the same diet containing 14, 24 or 30 μg Se/kg from the protein isolate or 13, 23 or 31 μg Se/kg from tofu, respectively. l-Selenomethionine (SeMet) was used as a reference. Selenium bioavailability was determined on the basis of the restoration of Se-dependent enzyme activities and tissue Se concentrations in Se-depleted rats, comparing those responses for the protein isolate and tofu to those for SeMet by using a slope-ratio method. Dietary supplementation with the protein isolate or tofu resulted in linear or log-linear, dose-dependent increases in glutathione peroxidase activities in blood and liver and in thioredoxin reductase activity in liver. Furthermore, supplementation with the protein isolate or tofu resulted in linear or log-linear, dose-dependent increases in the Se concentrations of plasma, liver, muscle and kidneys. These results indicated an overall bioavailability of approximately 101% for Se from the protein isolate and 94% from tofu, relative to SeMet. We conclude that Se from naturally produced high-Se soybeans is highly bioavailable in this model and that high-Se soybeans may be a good dietary source of Se.     

Biodegradation of atrazine under denitrifying conditions

Anaerobic biodegradation of atrazine by the bacterial isolate M91-3 was characterized with respect to mineralization, metabolite formation, and denitrification. The ability of the isolate to enhance atrazine biodegradation in anaerobic sediment slurries was also investigated. The organism utilized atrazine as its sole source of carbon and nitrogen under anoxic conditions in fixed-film (glass beads) batch column systems. Results of HPLC and TLC radiochromatography suggested that anaerobic biotransformation of atrazine by microbial isolate M91-3 involved hydroxyatrazine formation. Ring cleavage was demonstrated by ¹⁴CO₂ evolution. Denitrification was confirmed by detection of ¹⁵N₂ in headspace samples of K¹⁵NO₃-amended anaerobic liquid cultures. In aquatic sediments, mineralization of uniformly ring-labeled [¹⁴C]atrazine occurred in both M91-3-inoculated and uninoculated sediment. Inoculation of sediments with M91-3 did not significantly enhance anaerobic mineralization of atrazine as compared to uninoculated sediment, which suggests the presence of indigenous organisms capable of anaerobic atrazine biodegradation. Results of this study suggest that the use of M91-3 in a fixed-film bioreactor may have applications in the anaerobic removal of atrazine and nitrate from aqueous media. Received: 3 September 1997 / Received revision: 4 December 1997 / Accepted: 2 January 1998     

Biodegradation of atrazine in transgenic plants expressing a modified bacterial atrazine chlorohydrolase (atzA) gene

Atrazine is one of the most widely used herbicides in the USA. Atrazine chlorohydrolase (AtzA), the first enzyme in a six-step pathway leading to the mineralization of atrazine in Gram-negative soil bacteria, catalyses the hydrolytic dechlorination and detoxification of atrazine to hydroxyatrazine. In this study, we investigated the potential use of transgenic plants expressing atzA to take up, dechlorinate and detoxify atrazine. Alfalfa, Arabidopsis thaliana and tobacco were transformed with a modified bacterial atzA gene, p-atzA, under the control of the cassava vein mosaic virus promoter. All transgenic plant species actively expressed p-atzA and grew over a wide range of atrazine concentrations. Thin layer chromatography analyses indicated that in planta expression of p-atzA resulted in the production of hydroxyatrazine. Hydroponically grown transgenic tobacco and alfalfa dechlorinated atrazine to hydroxyatrazine in leaves, stems and roots. Moreover, p-atzA was found to be useful as a conditional-positive selection system to isolate alfalfa and Arabidopsis transformants following Agrobacterium-mediated transformation. Our work suggests that the in planta expression of p-atzA may be useful for the development of plants for the phytoremediation of atrazine-contaminated soils and soil water, and as a marker gene to select for the integration of exogenous DNA into the plant genome.     

Cadmium bioavailability

Cadmium slowly accumulates in the liver and kidney and has a long biological half-life, estimated to be 2-3 decades in the kidney. If the kidney cadmium concentration reaches a critically high level, proximal tubular damage results, which can be followed by severe bone mineral loss. There are only a few measurements of cadmium bioavailability in foods; however, the data are indicative of lower utilization from foods than from inorganic salts. In animal tissues cadmium is bound primarily to a heat-resistant small protein with a high cysteine content (metallothionein), whereas little is known about the form in which cadmium occurs in the edible parts of plants. Low intakes of many nutrients exacerbate the effects of cadmium and supplemental intakes are protective. Newborn and young animals absorb much higher quantities of cadmium than adults. There is some evidence in animals that females may be more adversely affected than males. Itai-itai disease, a painful disease with kidney damage and bone demineralization, occurred in elderly Japanese women who had borne several children and who were exposed to cadmium via food and drinking water. Inasmuch as cadmium in the U.S. food supply affords an estimated safety factor of only 4- to 15-fold, it is important to establish factors that affect the bioavailability of cadmium from foods.     

Electrochemical immunosensor for the detection of atrazine

An amperometric immunosensor for the detection of the herbicide atrazine has been developed. A redox polymer PVPOs(bpy)2Cl was co-immobilized with the specific antibody on the surface of the electrode by crosslinking with PEGDGE to form an electron-conducting hydrogel. In a competitive assay the occurrence of the antibody-antigen reaction on the surface of the sensing film was detected through the 'electrical wiring' of the redox centres of antigen-labelled horseradish peroxidase and the electrode surface in the presence of H2O2 at 0.1 V (vsAg/AgCl).     

Phytohormones in soil after atrazine application

The herbicide atrazine influences the content of phytohormones (auxins, gibberellins, cytokinins) in maize-cultivated soils. The effect is probably related to variations in the metabolism of hormones in soil microorganisms.     

Diffuse atrazine pollution in German aquifers

Until its prohibition in Germany in 1991, atrazine was the mostfrequently applied herbicide in maize cultivation. Moreover, it was used in orchards andvineyards and as a total herbicide on non-cultivated grounds (railways, factory grounds).Later on, atrazine was substituted mainly by terbutylazine. Terbutylazine and terbutrynare the only s-triazines presently permitted in Germany. Nevertheless, atrazine and itsmetabolite desethylatrazine are by far the most abundant herbicides detected in near surfacegroundwater. This might be due to wash-outs from the pools of atrazine and its metabolitesfrom the soil into the groundwater or continuing illegal applications. Samples takenfrom maize fields in 1994 showed that 6.2% of 471 fields tested were treated with atrazinedespite the prohibition of its use. Nevertheless, the overall trend is in fact a slow decreasein atrazine concentrations where it is detected in groundwater and, simultaneously often aslight increase in desethylatrazine concentrations. But this is not the case forall sampling points, and increasing concentrations in several aquifers are observedas well. Factors governing the adsorption, degradation, persistence and the possible transferinto the aquifer and the current situation concerning atrazine occurrence in German aquiferswill be discussed.     

Interaction of the herbicide atrazine with model membranes. II: Effect of atrazine on fusion of phospholipid vesicles

The effect of atrazine on Ca2+ induced fusion of cardiolipin(CL) and phosphatidylserine (PS) vesicles is studied by Tb3+/dipicolinic acid fluorescence and turbidity measurements. The interaction of herbicide with CL and PS membranes is studied by DPH fluorescence polarization. At low concentrations the pesticide partially inhibits fusion, especially in CL vesicles. Higher concentrations of atrazine decrease inhibition of fusion in CL, while fusion is slightly increased in PS. The Ca2(+)-induced increase of turbidity is not affected by atrazine in both PS and CL aggregation experiments. DPH polarization measurements show a perturbation only of the membrane hydrophobic core of PS, in presence of Ca2+. It is hypothesized that this biphasic effect shown by low and high atrazine concentrations on Ca2(+)-induced fusion of vesicles is due to a different localization of the pesticide in the membrane.     

Use of atrazine sensitive leguminous plants as biological indicators to evaluate the atrazine degradation efficiency of a bacterial inoculum

Atrazine sensitive leguminous plants were grown in a soil spiked with atrazine and augmented with an atrazine-degrading bacterium, Arthrobacter sp. strain MCM B-436, to ascertain its degradative efficiency. Germination and survival of plants was correlated with atrazine removal from soil. This experiment was carried out at laboratory as well as field level, showing consistent results. This bioindicator approach serves as an efficient measure for atrazine removal and could be easily adapted to determine atrazine degradation efficiency of other microbial strains.     

Assessment of bioavailability and risk of iron in phytomedicines Aconitum carmichaeli and Paeonia lactiflora

Since n-octanol, a long-chain alkanol, presents a configuration similar to that of carbohydrates and lipids, the lipophilicity and absorptivity of organic medicines may be assessed from their distribution coefficients between octanol and water. n-octanol is used as a model of biomembrane. This strategy has been used in order to define the species of iron in two phytomedicines and to study the distribution of iron in decoctions of phytomedicines in the stomach and the intestine. The concentrations of iron in the original herbal materials and in n-octanol- and water-soluble species were determined by flame atomic spectrometry following mixed acid digestion. The acidities of digestive and absorptive juices (saliva, gastric juice, bile and intestine), the phytomedical composition and the compatibility of phytomedicines, i.e., the combination ratio of single phytomedicines, greatly affected the iron complexing ligands and determined the species and bioavailability of iron. The concentration of octanol-soluble iron could be used for the bioavailability assessment of iron in phytomedicines. The dosage of iron in phytomedicines could be designed according to the level of the octanol-soluble iron and the reference daily intakes for iron (18 mg/d). Risk assessment could be examined through the comparison between the level of the octanol-soluble iron and the tolerable upper intake level for iron (45 mg iron/d).     

Assessment of heavy metal bioavailability using Escherichia coli zntAp::lux and copAp::lux-based biosensors

To determine the amount of metals detectable by bacteria, two plasmids were constructed in which the metal-inducible zntA and copA promoters from Escherichia coli were fused to a promoterless Vibrio fischeri luxCDABE operon. The luminescence response of E. coli bearing these constructs was studied as a function of the concentration of several heavy metals and was shown to be influenced by cell growth phase. The zntAp::lux fusion is induced mainly by salts of cadmium, lead, mercury and zinc, with significant induction by other metal ions, whereas the specificity of copA induction is restricted to copper and silver. In optimized assay conditions, metals could be detected at threshold concentrations ranging from nanomolar to micromolar, with maximal induction observed after only 60-100 min incubation. The ability of these biosensor strains to distinguish bioavailable quantities of metals in a sample makes them good candidates as useful tools to monitor metal contamination in environmental samples.