Preparation of antibodies and development of an enzyme immunoassay for determination of atrazine in environmental samples

An indirect competitive enzyme-linked immunosorbent assay (ELISA) has been developed and optimized for atrazine determination in soil at different depths (0-10, 10-20, and 20-30 cm) before and after 48 h of application, corn shoot and cow milk samples collected from Dina farm, Egypt. This assay was based on a specific polyclonal antibodies (PAb) raised by immunizing New Zealand rabbits with an immunogen prepared by coupling 3-{4-(ethylamino)-6-(isopropylamino)-1,3,5-triazine-2-yl} thiopropanoic acid to bovine serum albumin (BSA) via N-hydroxysuccinimide (NHS) active ester method. The sensitivity (estimated as IC?? value) was 17.5 μg mL?1 with a detection limit of 0.1 ng mL?1. The maximum atrazine concentration was found in soil especially in the deepest layer (325 and 890 μg kg?1 before and after application, respectively). Atrazine concentration in corn shoot was 333.28, μg kg?1 dry plant, while there was no detectable amount in milk. All samples screened by ELISA were validated by gas chromatography mass spectrometer procedure (GC/MS). Good correlation was achieved between the two methods (r = 0.997 for soil and 0.9814 for plant). This study demonstrates the utility and convenience of the simple, practical and cost-effective ELISA method in the laboratory for analysis of environmental samples. The method is ideal for the rapid screening of large numbers of samples in laboratories where access to GC/MS facilities, is limited or lacking.     

Development of a sensitive,competitive, indirect ELISA for the detection of fumonisin B1 in corn originating from Anhui province,China

Fumonisin B₁ (FB₁) is a secondary metabolite produced by Fusarium verticillioides or Fusarium proliferatum, which present in food and feed. It causes hazardous effects on human and animal health. A monoclonal antibody (mAb) against FB₁ was produced and a simple, reliable and sensitive, competitive, indirect enzyme-linked immunosorbent assay (ci-ELISA) for detection of FB₁ was developed and the experiment conditions were optimized. The coating concentration of FB₁-ovalbumin (FB₁-OVA) was 500 ng mL^?1, the action concentrations of anti-FB₁ mAb and goat anti-mouse IgG were 1.28 × 10⁴ and 1:5000, respectively. The 50% inhibitory concentration (IC₅₀) was 11 ng mL^?1, with a detectable range of 1.25–250 ng mL^?1, and a limit of determination (LOD) of 1.15 ng mL^?1. The cross-reactivity (CR) of the antibody against fumonisin B₂ (FB₂) was 60.4, and <1% against deoxynivalenol (DON), aflatoxin B₁ (AFB₁), ochratoxin A (OTA) or zearalenone (ZEN). In spiked samples (250 ng g^?1, 500 ng g^?1, 1000 ng g^?1), the mean recoveries ranged from 86.7 ± 5% to 102 ± 4%, and the coefficient of variation (CV) ranged from 3% to 10%. A survey of 96 corn samples from Bozhou, Fuyang, Bengbu, and Hefei, in Anhui province, China, was performed. Frequencies of FB₁ contamination were 83.3%, 95.8%, 20.8% and 91.7%, and the mean concentrations of positive samples were 0.702 μg kg^?1, 0.883 μg kg^?1, 0.074 μg kg^?1, and 0.276 μg kg^?1, respectively. The results of this study suggest that the ci-ELISA developed in this study can be used to identify FB₁ in corn, furthermore, further study is needed to investigate FB₁ contamination in food and feed to prevent its harmful health effects.     

Determination of the insecticide diflubenzuron in mushrooms by kinetic method and high-performance liquid chromatographic method

In the present study, a new sensitive and simple kinetic-spectrophotometric method for the determination of the insecticide diflubenzuron [1-(4-chlorophenyl)-3-(2,6-diflubenzoil)urea] is proposed. The method is based on the inhibited effect of diflubenzuron on the oxidation of sulphanilic acid (SA) by hydrogen peroxide in phosphate buffer in presence Cu(II) ion. Diflubenzuron was determined with linear calibration graph in the interval from 0.31 to 3.1 μg mL^?1 and from 3.1 to 31.0 μg mL^?1. The optimized conditions yielded a theoretical detection limit of 0.18 μg mL^?1corresponding to 0.036 mg Kg^?1mushroom sample based on the 3S_b criterion. The RSD is 5.03–1.83 % and 2.81–0.71 % for the concentration interval of diflubenzuron 0.31–3.1 μg mL^?1and 3.1–31.0 μg mL^?1, respectively. The reaction was followed spectrophotometrically at 370 nm. The kinetic parameters of the reaction are reported, and the rate equations are suggested. The developed procedure was successfully applied to the rapid determination of diflubenzuron in spiked mushroom samples of different mushroom species. The HPLC method was used like a comparative method to verify results.     

Development of in-house ELISA for detection of chloramphenicol in bovine milk with subsequent confirmatory analysis by LC-MS/MS

This study was undertaken to develop and validate direct competitive ELISA for the determination of chloramphenicol residues in bovine milk. Antisera and an enzyme-tracer for chloramphenicol were prepared and used to develop an ELISA with inhibition concentrations, IC₂₀ and IC₅₀, of 0.09 and 0.44 ng mL^?1, respectively. Milk samples were spiked with standards equivalent to 0, 0.2, 0.3, 0.5, 1.0 &; 1.5 ng mL^?1 and extracted in methanol. The mean recoveries were found to be 73–100% with coefficient of variance 7–11%. The decision limit (CCα) and detection capability (CCβ) were calculated as 0.10 and 0.12 ng mL^?1, respectively. The results were found comparable with the commercial ELISA, having recoveries of 87 to 100%, CCα 0.09 ng mL^?1 and CCβ 0.12 ng mL^?1. As per Commission Decision 2002/657/EC, in-house ELISA was further validated by using LC-MS/MS. Mass spectral acquisition was done by using electrospray ionization in the negative ion mode applying single reaction monitoring of the diagnostic transition reaction for CAP (m/z 152, 194 and 257). The calibration curve showed good linearity in concentrations from 0.025 to 1.6 ng mL^?1 with correction coefficient 0.9902. The mean recoveries were found to be 88 to 100%. The CCα was calculated as 0.057 ng mL^?1 and CCβ 0.10 ng mL^?1. Since CCα and CCβ are less than half of the MRPL (0.15 ng mL^?1), the test was found suitable for screening and quantification of CAP residues in bovine milk samples. Results of surveillance studies indicated that out of 31 analyzed milk samples, 12.9% samples were found with CAP residues but only 3.2% samples were declared positive with maximum concentration 0.31 ng mL^?1, slightly above the MRPL.     

Assessment of the ecological security of immobilized enzyme remediation process with biological indicators of soil health

This study used the enzymes extracted from an atrazine-degrading strain, Arthrobacter sp. DNS10, which had been immobilized by sodium alginate to rehabilitate atrazine-polluted soil. Meanwhile, a range of biological indices were selected to assess the ecological health of contaminated soils and the ecological security of this bioremediation method. The results showed that there was no atrazine detected in soil samples after 28 days in EN?+?AT (the soil containing atrazine and immobilized enzyme) treatment. However, the residual atrazine concentration of the sample in AT (the soil containing atrazine only) treatment was about 5.02?±?0.93 mg?kg^?1. These results suggest that the immobilized enzyme exhibits an excellent ability in atrazine degradation. Furthermore, the immobilized enzyme could relieve soil microbial biomass carbon and soil microbial respiration intensity to 772.33?±?34.93 mg?C?kg^?1 and 5.01?±?0.17 mg?CO₂?g^?1?soil?h^?1, respectively. The results of the polymerase chain reaction–degeneration gradient gel electrophoresis experiment indicated that the immobilized enzyme also could make the Shannon–Wiener index and evenness index of the soil sample increase from 1.02 and 0.74 to 1.51 and 0.84, respectively. These results indicated that the immobilized enzymes not only could relieve the impact from atrazine on the soil, but also revealed that the immobilized enzymes did no significant harm on the soil ecological health.     

Residual tembotrione and atrazine in carrot

Carrot (Daucus carota L.) is a vegetable crop that is grown throughout the year across various regions of Brazil in rotation or in succession to other cultures. Herbicide residual effect has emerged as a concern, because of the possibility of carryover. Thus, the objective of this study was to evaluate the effect of tembotrione and atrazine residues – in mixture and isolated – on carrot planted in succession to corn. The experiment was designed in randomized blocks with five replications. Treatments consisted of tembotrione (50.4 g ha^?1), tembotrione (100.8 g ha^?1), tembotrione + atrazine (50.4 g ha^?1+ 2 L ha^?1), tembotrione + atrazine (100.8 g ha^?1+ 2 L ha^?1), and atrazine (2.00 L ha^?1) applied eight months before carrot seeding, plus a control treatment with no herbicide application. Investigated variables were shoot dry mass, productivity, and classification of carrot roots. The presence of atrazine and tembotrione decreased dry mass in the area, and only tembotrione reduced total root productivity. Thus, there is a carryover effect to tembotrione application that reduces the dry matter accumulation of shoot and total productivity, and an atrazine + tembotrione (100.8 g ha^?1) mixture reduces the total productivity after application of these herbicides to soil.     

Validation of a QuEChERS-based gas chromatographic method for analysis of pesticide residues in Cassia angustifolia (senna)

A simple multi-residue method based on modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) approach was established for the determination of 17 organochlorine (OC), 15 organophosphorous (OP) and 7 synthetic pyrethroid (SP) pesticides in an economically important medicinal plant of India, Senna (Cassia angustifolia), by gas chromatography coupled to electron capture and flame thermionic detectors (GC/ECD/FTD) and confirmation of residues was done on gas chromatograph coupled with mass spectrometry (GC-MS). The developed method was validated by testing the following parameters: linearity, limit of detection (LOD), limit of quantification (LOQ), matrix effect, accuracy–precision and measurement uncertainty; the validation study clearly demonstrated the suitability of the method for its intended application. All pesticides showed good linearity in the range 0.01–1.0 μg mL^?1 for OCs and OPs and 0.05–2.5 μg mL^?1 for SPs with correlation coefficients higher than 0.98. The method gave good recoveries for most of the pesticides (70–120%) with intra-day and inter-day precision < 20% in most of the cases. The limits of detection varied from 0.003 to 0.03 mg kg^?1, and the LOQs were determined as 0.01-0.049 mg kg^?1. The expanded uncertainties were <30%, which was distinctively less than a maximum default value of ±50%. The proposed method was successfully applied to determine pesticide residues in 12 commercial market samples obtained from different locations in India.     

Characterization of bacterial diversity in an atrazine degrading enrichment culture and degradation of atrazine,cyanuric acid and biuret in industrial wastewater

An enrichment culture was used to study atrazine degradation in mineral salt medium (MSM) (T1), MSM+soil extract (1:1, v/v) (T2) and soil extract (T3). Results suggested that enrichment culture required soil extract to degrade atrazine, as after second sequential transfer only partial atrazine degradation was observed in T1 treatment while atrazine was completely degraded in T2 and T3 treatments even after fourth transfer. Culture independent polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique confirmed selective enrichment of genus Bacillus along with Pseudomonas and Burkholderia. Degradation of atrazine/metabolites in the industrial wastewater was studied at different initial concentrations of the contaminants [wastewater-water (v/v) ratio: T1, 1:9; T2, 2:8; T3, 3:7; T4, 5:5 and T5, undiluted effluent]. The initial concentrations of atrazine, cyanuric acid and biuret ranged between 5.32 and 53.92 µg mL^?1, 265.6 and 1805.2 µg mL^?1 and 1.85 and 16.12 µg mL^?1, respectively. The enrichment culture was able to completely degrade atrazine, cyanuric acid and biuret up to T4 treatment, while no appreciable degradation of contaminants was observed in the undiluted effluent (T5). Inability of enrichment culture to degrade atrazine/metabolites might be due to high concentrations of cyanuric acid. Therefore, a separate study on cyanuric acid degradation suggested: (i) no appreciable cyanuric acid degradation with accumulation of an unidentified metabolite in the medium where cyanuric acid was supplemented as the sole source of carbon and nitrogen; (ii) partial cyanuric acid degradation with accumulation of unidentified metabolite in the medium containing additional nitrogen source; and (iii) complete cyanuric acid degradation in the medium supplemented with an additional carbon source. This unidentified metabolite observed during cyanuric acid degradation and also detected in the enrichment culture inoculated wastewater samples, however, was degraded up to T4 treatments and was persistent in the T5 treatment. Probably, accumulation of this metabolite inhibited atrazine/cyanuric acid degradation by the enrichment culture in undiluted wastewater.     

Toxicity of metalaxyl,azoxystrobin, dimethomorph,cymoxanil, zoxamide and mancozeb to Phytophthora infestans isolates from Serbia

A study of the in vitro sensitivity of 12 isolates of Phytophthora infestans to metalaxyl, azoxystrobin, dimethomorph, cymoxanil, zoxamide and mancozeb, was conducted. The isolates derived from infected potato leaves collected at eight different localities in Serbia during 2005–2007. The widest range of EC₅₀ values for mycelial growth of the isolates was recorded for metalaxyl. They varied from 0.3 to 3.9 μg mL^?1 and were higher than those expected in a susceptible population of P. infestans. The EC₅₀ values of the isolates were 0.16–0.30 μg mL^?1 for dimethomorph, 0.27–0.57 μg mL^?1 for cymoxanil, 0.0026–0.0049 μg mL^?1 for zoxamide and 2.9–5.0 μg mL^?1 for mancozeb. The results indicated that according to effective concentration (EC₅₀) the 12 isolates of P. infestans were sensitive to azoxystrobin (0.019–0.074 μg mL^?1), and intermediate resistant to metalaxyl, dimethomorph and cymoxanil. According to resistance factor, all P. infestans isolates were sensitive to dimethomorph, cymoxanil, mancozeb and zoxamide, 58.3% of isolates were sensitive to azoxystrobin and 50% to metalaxyl. Gout's scale indicated that 41.7% isolates were moderately sensitive to azoxystrobin and 50% to metalaxyl.     

Persistence of C-labeled atrazine and its residues in a field lysimeter soil after 22 years

Twenty-two years after the last application of ring-¹⁴C-labeled atrazine at customary rate (1.7 kg ha⁻¹) on an agriculturally used outdoor lysimeter, atrazine is still detectable by means of accelerated solvent extraction and LC-MS/MS analysis. Extractions of the 0-10 cm soil layer yielded 60% of the residual ¹⁴C-activity. The extracts contained atrazine (1.0 μg kg⁻¹) and 2-hydroxy-atrazine (42.5 μg kg⁻¹). Extractions of the material of the lowest layer 55-60 cm consisting of fine gravel yielded 93% of residual ¹⁴C-activity, of which 3.4 μg kg⁻¹ was detected as atrazine and 17.7 μg kg⁻¹ was 2-hydroxy-atrazine. The detection of atrazine in the lowest layer was of almost four times higher mass than in the upper soil layer. These findings highlight the fact that atrazine is unexpectedly persistent in soil. The overall persistence of atrazine in the environment might represent a potential risk for successive groundwater contamination by leaching even after 22 years of environmental exposure.     

Optimization and validation of enzyme-linked immunosorbent assay for the determination of endosulfan residues in food samples

In this study, an enzyme-linked immunosorbent assay (ELISA) was optimized and applied to the determination of endosulfan residues in 20 different kinds of food commodities including vegetables, dry fruits, tea and meat. The limit of detection (IC₁₅) was 0.8 μg kg^?1 and the sensitivity (IC₅₀) was 5.3 μg kg^?1. Three simple extraction methods were developed, including shaking on the rotary shaker at 250 r min^?1 overnight, shaking on the rotary shaker for 1 h and thoroughly mixing for 2 min. Methanol was used as the extraction solvent in this study. The extracts were diluted in 0.5% fish skin gelatin (FG) in phosphate-buffered saline (PBS) at various dilutions in order to remove the matrix interference. For cabbage (purple and green), asparagus, Japanese green, Chinese cabbage, scallion, garland chrysanthemum, spinach and garlic, the extracts were diluted 10-fold; for carrots and tea, the extracts were diluted 15-fold and 900-fold, respectively. The extracts of celery, adzuki beans and chestnuts, were diluted 20-fold to avoid the matrix interference; ginger, vegetable soybean and peanut extracts were diluted 100-fold; mutton and chicken extracts were diluted 10-fold and for eel, the dilution was 40-fold. Average recoveries were 63.13–125.61%. Validation was conducted by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The results of this study will be useful to the wide application of an ELISA for the rapid determination of pesticides in food samples.     

Determination of atrazine in rainfall and surface water by enzyme immunoassay

Rainwater and surface water from four sites in Germany (Bavaria and Lower Saxony) were analyzed for atrazine by enzyme immunoassay from June 1990 until October 1992. The limit of quantification of the immunoassay was 0.02 μg/L with a middle of the test at 0.2 μg/L. About 60 % of the samples contained measurable amounts of atrazine. Seasonal trends were observed, with the highest concentration in the summer months of up to 4 μg/L for rainwater and up to 15 μg/L for surface waters. The highest concentrations were found in agricultural areas, while in the investigated national parks up to 0.56 μg/L could be detected in rain water. This points to long-range atmospheric transport from agricultural areas to pristine national parks. Samples from forest stands usually showed higher atrazine concentrations than samples from open fields. Deposition rates of 10 – 50 μg/m² · yr were observed in the national parks and 10–180 μg/m² · yr at the agricultural sites. Comparison of results obtained by enzyme immunoassay and GC/MS showed a good correlation of r = 0.95.     

Fate in soil of 14C-sulfadiazine residues contained in the manure of young pigs treated with a veterinary antibiotic

The fate of ¹⁴C-labeled sulfadiazine (¹⁴C-SDZ) residues was studied in time-course experiments for 218 days of incubation using two soils (A_p horizon of loamy sand, orthic luvisol; A_p horizon of silt loam, cambisol) amended with fresh and aged (6 months) ¹⁴C-manure [40 g kg^?1 of soil; 6.36 mg of sulfadiazine (SDZ) equivalents per kg of soil], which was derived from two shoats treated with ¹⁴C-SDZ. Mineralization of ¹⁴C-SDZ residues was below 2% after 218 days depending little on soil type. Portions of extractable ¹⁴C (ethanol-water, 9:1, v/v) decreased with time to 4–13% after 218 days of incubation with fresh and aged ¹⁴C-manure and both soils. Non-extractable residues were the main route of the fate of the ¹⁴C-SDZ residues (above 90% of total recovered ¹⁴C after 218 days). These residues were high immediately after amendment depending on soil type and aging of the ¹⁴C-manure, and were stable and not remobilized throughout 218 days of incubation. Bioavailable portions (extraction using CaCl₂ solution) also decreased with increasing incubation period (5–7% after 218 days). Due to thin-layer chromatography (TLC), 500 μg of ¹⁴C-SDZ per kg soil were found in the ethanol-water extracts immediately after amendment with fresh ¹⁴C-manure, and about 50 μg kg^?1 after 218 days. Bioavailable ¹⁴C-SDZ portions present in the CaCl₂ extracts were about 350 μg kg^?1 with amendment. Higher concentrations were initially detected with aged ¹⁴C-manure (ethanol-water extracts: 1,920 μg kg^?1; CaCl₂ extracts: 1,020 μg kg^?1), probably due to release of ¹⁴C-SDZ from bound forms during storage. Consistent results were obtained by extraction of the ¹⁴C-manure-soil samples with ethyl acetate; portions of N-acetylated SDZ were additionally determined. All soluble ¹⁴C-SDZ residues contained in ¹⁴C-manure contributed to the formation of non-extractable residues; a tendency for persistence or accumulation was not observed. SDZ's non-extractable soil residues were associated with the soluble HCl, fulvic acids and humic acids fractions, and the insoluble humin fraction. The majority of the non-extractable residues appeared to be due to stable covalent binding to soil organic matter.     

Fate of atrazine in a soil under different agronomic management practices

Agricultural management affects the movement of atrazine in soil and leaching to groundwater. The objective of this study was to determine atrazine adsorption in a soil after 20 years of atrazine application under agronomic management practices differing in tillage practice (conventional and zero tillage), residue management (with and without residue retention) and crop rotation (wheat-maize rotation and maize monoculture). Atrazine sorption was determined using batch and column experiments. In the batch experiment, the highest distribution coefficient K_d (1.1 L kg^?1) at 0–10 cm soil depth was observed under zero tillage, crop rotation and residue retention (conservation agriculture). The key factor in adsorption was soil organic matter content and type. This was confirmed in the column experiment, in which the highest K_d values were observed in treatments with residue retention, under either zero or conventional tillage (0.81 and 0.68 L kg^?1, respectively). Under zero tillage, the fact that there was no soil movement helped to increase the K_d. The increased soil organic matter content with conservation agriculture may be more important than preferential flow due to higher pore connectivity in the same system. The soil's capacity to adsorb 2-hydroxyatrazine (HA), an important atrazine metabolite, was more important than its capacity to adsorb atrazine, and was similar under all four management practices (K_d ranged from 30 to 40 L kg^?1). The HA adsorption was attributed to the type and amount of clay in the soil, which is unaffected by agronomic management. Soils under conservation agriculture had higher atrazine retention potential than soils under conventional tillage, the system that predominates in the study area.     

Triazine and Metolachlor Herbicide Residues in Farm Areas of the Lower Fraser Valley,British Columbia,Canada

Crop soils, ditch sediments and water flowing from several Lower Fraser River (LFR) farm areas of British Columbia, Canada, to salmon tributary streams of that river were sampled in 2004–2005 to quantify for residues of triazine [atrazine, desethylatrazine (a transformation product of atrazine), propazine, and simazine] and metolachlor (a chloroacetamide) herbicides. Average concentrations [μg kg^?1 dry weight (d.w.)] of triazine (10,110) and metolachlor (8,910) herbicides detected in crop soils at the start (May 2004, 2005) of the growing season were about 17 and 6 times, respectively, higher than those found for both herbicide groups during (June–Sept, 2004, 2005) the growing season. In contrast, mean concentrations (μg L^?1) of triazines (0.092) and metolachlor (0.014) in permanent ditches adjacent to farms were about 7 and 28 times, respectively, lower at the start than during the growing season. Both herbicide groups in ditch sediments were detected only during the growing season at concentrations averaging about 315 μg kg^?1 d.w. The risk potential of these herbicides for non-target aquatic organisms inhabiting permanent farm ditches contiguous to tributary streams of the LFR during the growing season is evaluated and discussed.     

Concentration and dissipation of chlorantraniliprole and thiamethoxam residues in maize straw,maize, and soil

To study the dissipation rates and final residual levels of chlorantraniliprole and thiamethoxam in maize straw, maize, and soil, two independent field trials were conducted during the 2014 cropping season in Beijing and Anhui Provinces of China. A 40% wettable powder (20% chlorantraniliprole?+?20% thiamethoxam) was sprayed onto maize straw and soil at an application rate of 118 g of active ingredient per hectare (g a.i.ha^?1). The residual concentrations were determined by ultra-high-performance liquid chromatography–tandem mass spectrometry. The chlorantraniliprole half-lives in maize straw and soil were 9.0–10.8 and 9.5–21.7 days, respectively. The thiamethoxam half-lives in maize straw and soil were 8.4–9.8 and 4.3–11.7 days, respectively. The final residues of chlorantraniliprole and thiamethoxam in maize straw, maize, and soil were measured after the pesticides had been sprayed two and three times with an interval of 7 days using 1 and 1.5 times the recommended rate (72 g a.i. ha^?1 and 108 g a.i. ha^?1, respectively). Representative maize straw, maize, and soil samples were collected after the last treatment at pre-harvest intervals of 7, 14, and 28 days. The chlorantraniliprole residue was below 0.01 mg kg^?1 in maize, between 0.01 and 0.31 mg kg^?1 in maize straw, and between 0.03 and 1.91 mg kg^?1 in soil. The thiamethoxam residue concentrations in maize, maize straw, and soil were <0.01, <0.01, and 0.01–0.03 mg kg^?1, respectively. The final pesticide residues on maize were lower than the maximum residue limit (MRL) of 0.02 mg kg^?1 after a 14-day pre-harvest interval. Therefore, a dosage of 72 g a.i. ha^?1 was recommended, as it can be considered safe to human beings and animals.     

Simultaneous analysis of free and sulfo-conjugated steroid estrogens in artificial urine solution and agricultural soils by high-performance liquid chromatography

A simple and robust analytical method was developed to simultaneously detect and quantify 17β-estradiol (E2), estrone (E1), 17β-estradiol-3-sulphate (E2-3S), and estrone-3-sulphate (E1-3S) in aqueous solutions (calcium chloride and artificial urine solutions) and agricultural soils using high performance liquid chromatography and UV detection. The standards for all four compounds were linear in the range of 0.01 to 1.0 μg mL^?1 (n = 6) and 1.0 to 20 μg mL^?1 (n = 6), respectively, with correlation coefficients > 0.999. The on-column limits of detection at an injection volume of 50 μL and S/N (signal: noise) ratio of 3 were: 9.0 ng mL^?1, 10 ng mL^?1, 5.0 ng mL^?1, and 7.0 ng mL^?1 for E2-3S, E1-3S, E2 and E1, respectively. The limit of detection and quantification in artificial urine solution and CaCl₂ solution was 1.0 ng mL^?1 for all four compounds. Method detection limits for the compounds in the 3 soils ranged from 2 to 2.4 ng g^?1 (E2-3S and E1-3S), and 1.0 to 2.9 ng g^?1 (E2 and E1), respectively.     

Behavior of atrazine in soils of tropical zone

Abstract

Movement and degradation of ¹⁴C‐atrazine (2‐chloro 4‐(ethylamino)‐6‐(isopropylamino)‐s‐triazine, was studied in undisturbed soil columns (0.50m length × 0.10m diameter) of Gley Humic and Deep Red Latosol from a maize crop region of Sao Paulo state, Brazil. Atrazine residues were largely confined to the 0–20cm layers over a 12 month period Atrazine degraded to the dealkylated metabolites deisopropylatrazine and deethylatrazine, but the major metabolite was hydroxyatrazine, mainly in the Gley Humic soil. Activity detected in the leachate was equivalent to an atrazine concentration of 0.08 to 0.11μg/1.

The persistence of ¹⁴C‐atrazine in a maize‐bean crop rotation was evaluated in lysimeters, using Gley Humic and Deep Red Latosol soils. Uptake of the radiocarbon by maize plants after 14‐days growth was equivalent to a herbicide concentration of 3.9μg/g fresh tissue and was similar in both soils. High atrazine degradation to hydroxyatrazine was detected by tic of maize extracts. After maize harvest, when beans were sown the Gley Humic soil contained an atrazine concentration of 0.29 μg/g soil and the Deep Red Latosol, 0.13 μg/g soil in the 0–30 cm layer. Activity detected in bean plants corresponded to a herbicide concentration of 0.26 (Gley Humic soil) and 0.32μg/g fresh tissue (Deep Red Latossol) after 14 days growth and 0.43 (Gley Humic soil) and 0.50 μg/g fresh tissue (Deep Red Latossol) after 97 days growth. Traces of activity equivalent to 0.06 and 0.02μg/g fresh tissue were detected in bean seeds at harvest. Non‐extractable (bound) residues in the soils at 235 days accounted for 66.6 to 75% (Gley Humic soil and Deep Red Latossol) of the total residual activity.     

Development of indirect competitive ELISA using egg yolk-derived immunoglobulin (IgY) for the detection of Gentamicin residues

Gentamicin (Gent) is an aminoglycoside antibiotic being used in livestock sector. Gent residues could cause some genetic disorders by nonsense mutations. This study aimed to develop IgY-based ELISA for the detection of Gent in animal products. Gent was conjugated with Bovine serum albumin (BSA) by carbodiimide method for further immunization in the laying chickens. PEG-6000 extraction method was employed to extract IgY from the egg yolk. The titer of anti-Gent-IgY attained the peak of 1:256,000 after the 5^th booster immunization. Checkerboard titration confirmed that, anti-Gent IgY in 1:2,000 dilution could give an Optical Density (OD) 1.0 at 2 µg mL^?1 of Gent-OVA coating concentration. IgY-based indirect competitive ELISA (Ic-ELISA) showed that, the IC₅₀ value of anti-Gent IgY was 2.69 ng mL^?1 and regression curve equation was y = ?16.27x + 56.97 (R² = 0.95, n = 3), confirming that, the detection limit (LOD, IC₁₀ value) was 0.01 ng mL^?1. Recoveries from fresh milk, pork and chicken samples were ranged from 69.82% to 94.32%, with relative standard deviation lower than 10.88%. Our results suggested that generated anti-Gent IgY antibodies can be used in routine screening analysis of Gent residues in food samples.     

Biodegradation of phthalate esters in polluted soil by using organic amendment

This study investigated the biodegradation of the phthalate esters (PAEs) di-n-butyl phthalate (DBP) and di-(2-ethyl hexyl) phthalate (DEHP) in sludge and sludge-amended soil. DBP (100 mg kg^?1) and DEHP (100 mg kg^?1) were added to sewage sludge, which was subsequently added to soil. The results showed that sewage sludge can degrade PAEs and the addition of sewage sludge to soil enhanced PAE degradation. Sludge samples were separated into fractions with various particle size ranges, which spanned 0.1–0.45 μm to 500–2000 μm. The sludge fractions with smaller particle sizes demonstrated higher PAE degradation rates. However, when the different sludge fractions were added to soil, particle size had no significant effect on the rate of PAE degradation. The results from this study showed that microbial strains F4 (Rhodococcus sp.) and F8 (Microbacterium sp.) were constantly dominant in the mixtures of soil and sludge.