Triazines in the aquatic systems of the Eastern Chinese Rivers Liao-He and Yangtse

The results of a one-year monitoring program on the two Eastern Chinese River systems, i.e. the Liao-He and the Yangtse, with special emphasis on the presence of triazine herbicides are presented. Sediment, suspended solids and water samples from both rivers were analyzed. Additionally, recovery experiments on the SPE-in-field-enrichment procedure and the extraction methods were performed. The samples were measured by gas chromatography coupled with mass spectrometry, electron capture detection and a newly developed mu-plasma atomic emission detector. A typical result of a one-year monitoring was obtained in case of the Liao-He: During winter, at low water period, low triazine values were found. A similar situation was found in early spring. Highest concentrations of atrazine up to 1600 ng/l were found in late spring in the water samples. Maximum concentrations of atrazine, simazine, propazine, simetryn and prometryn were observed in this season as a result of the actual use of triazines. Finally, after the high water period in autumn the triazine concentrations decreased. Additionally, atrazine adsorbed on sediment (up to 2.8 ng/g) and suspended solids was determined (up to 8 ng/l) during late spring sampling. Therefore, the logarithm of the organic carbon based sorption coefficient of atrazine could be calculated. Low levels of atrazine were measured in the water of Yangtse (up to 18.3 ng/l). The concentrations from all sampling points and sampling stations of a particular sampling date were similar, which indicates a homogeneous distribution of this herbicide. Due to the high discharge rate of up to 79,000 m3/s in case of the Yangtse a considerable mass transport of up to 57.5 kg per day atrazine may take place, even at concentrations below the European drinking water limit of 100 ng/l.     

Comparison of in-house-developed ELISA with HPLC techniques for the analysis of atrazine residues

In-house developed ELISA was standardized to monitor atrazine residues in different environmental samples. The standard curve was linear, indicating an increase in log concentration with decrease in absorbance (%B/B(0)=1.075-0.042 Log C; r= -0.966). The middle of the test was at 75 ng/L and the lowest detection limit at 4 ng/L. ELISA significantly correlated with the high performance liquid chromatography (HPLC) (r=0.990). Internal validation showed good accuracy and precision. Maximum atrazine residues were present in Jehlum River water/sediments and maize/sugarcane plant roots. Most of the food samples were found to be contaminated. ELISA required less clean-up steps than HPLC, but showed matrix effect in soil/colored extracts.     

Comparison of in-house-developed ELISA with HPLC techniques for the analysis of atrazine residues

In-house developed ELISA was standardized to monitor atrazine residues in different environmental samples. The standard curve was linear, indicating an increase in log concentration with decrease in absorbance (%B/B₀ = 1.075–0.042 Log C; r = ?0.966). The middle of the test was at 75 ng/L and the lowest detection limit at 4 ng/L. ELISA significantly correlated with the high performance liquid chromatography (HPLC) (r = 0.990). Internal validation showed good accuracy and precision. Maximum atrazine residues were present in Jehlum River water/sediments and maize/sugarcane plant roots. Most of the food samples were found to be contaminated. ELISA required less clean-up steps than HPLC, but showed matrix effect in soil/colored extracts.     

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.     

Enzyme-immunoassay techniques for the detection of atrazine in water samples: Evaluation of a commercial tube based assay

Environmental immunoassays can help lower the operating costs and improve the effectiveness of residue laboratories. The present study assesses the ability of a commercially available enzyme immunoassay (EIA) to detect triazine herbicides in water. The tube based EIA could detect atrazine in lake and river water with detection limits of 62 pg/mL and 180 pg/mL respectively. The assay's ability to quantify atrazine in a set of 124 water samples taken from many parts of Canada was compared with a reference method that used gas chromatographic separation combined with a nitrogen phosphorous detector (GC-NPD) (R=0.919). A 71 % reduction in analytical load was achieved at a threshold concentration of 1 ng/mL. There were 2.4 % false negative and 0.8 % false positive results associated with that load reduction. The variability of the assay control parameters was generally within two standard deviations of the mean response for 65 assays. The EIA for atrazine is recommended for use as a screening technique and as an inexpensive way to monitor triazine levels in waters that are known to be contaminated with those herbicides.     

Application of an enzyme-linked immunosorbent assay for the detection of clenbuterol residues in swine urine and feeds

The present study outlines applications of an enzyme-linked immunosorbent assay (ELISA) for the analysis of clenbuterol residues. Antisera were raised from rabbits immunized with diazotized clenbuterol-bovine serum albumin (BSA) conjugate. The assay was specific to clenbuterol with a half-maximum inhibition concentration (IC(50)) of 1.8 ng/mL and 2.5 ng/mL in blank swine urine and phosphate buffer solution, respectively. The assay had high cross-reactivity (86%) with mabuterol, but low with other adrenergic agonists and antagonists. The average recovery of clenbuterol, as measured with the ELISA, ranged from 90% to 112% in swine urine samples and from 86% to 95% in feeds, respectively. This new assay was compared with commercial ELISA test kits. An excellent correlation (r(2) = 0.98) between the two methods and satisfactory recoveries suggest that the new assay can be suitable for the determination of clenbuterol residues in real samples. The assay was used to analyze clenbuterol residues in 103 swine urine samples and 68 feed samples collected from northern China. Approximately 50% of the urine samples and 25% of the feed samples analyzed were found positive (concentration of clenbuterol > or = 1 ppb). The results indicate that clenbuterol was misused in some of the areas surveyed.     

Application of an enzyme-linked immunosorbent assay for the detection of clenbuterol residues in swine urine and feeds

The present study outlines applications of an enzyme-linked immunosorbent assay (ELISA) for the analysis of clenbuterol residues. Antisera were raised from rabbits immunized with diazotized clenbuterol-bovine serum albumin (BSA) conjugate. The assay was specific to clenbuterol with a half-maximum inhibition concentration (IC₅₀) of 1.8 ng/mL and 2.5 ng/mL in blank swine urine and phosphate buffer solution, respectively. The assay had high cross-reactivity (86%) with mabuterol, but low with other adrenergic agonists and antagonists. The average recovery of clenbuterol, as measured with the ELISA, ranged from 90% to 112% in swine urine samples and from 86% to 95% in feeds, respectively. This new assay was compared with commercial ELISA test kits. An excellent correlation (r ² = 0.98) between the two methods and satisfactory recoveries suggest that the new assay can be suitable for the determination of clenbuterol residues in real samples. The assay was used to analyze clenbuterol residues in 103 swine urine samples and 68 feed samples collected from northern China. Approximately 50% of the urine samples and 25% of the feed samples analyzed were found positive (concentration of clenbuterol ≥ 1 ppb). The results indicate that clenbuterol was misused in some of the areas surveyed.     

Multiresidue screening methods for the determination of pesticides in tomatoes

The possibility of applying thin layer chromatography (TLC) detection for the analysis of pesticide residues in tomatoes was investigated. Samples of tomatoes that have never been treated with pesticide were fortified with atrazine, carbaryl, carbofuran, chloroxuron, diuron, dimethoate, imazalil, oxamyl and methamidophos. The samples were extracted, cleaned-up by gel permeation chromatography and then applied on silica gel plates. The pesticides were eluted with ethyl acetate and dichloromethane. Two eluting solvent systems were tested, one using the reagents o-toluidine + potassium iodite (o-TKI) and the other p- nitrobenzene fluoroborate (NBFB). After the development of the plates, the diameter of the spots was measured. The lowest minimum detection quantity (MDQ) for o-TKI system for atrazine was 12 ng. The highest was 125 ng for carbofuran. Using NBFB system, the lowest MDQ was 60 ng for carbaryl and the highest was 70 ng obtained for carbofuran. Considering the concentration of these pesticides in the spiked tomato samples, the minimum concentration was 1.1 ng/microL and 32.3 ng/microL for atrazine and carbofuran, respectively, by using o-TKI system. For NBFB system the minimum concentration reached was 3.5 ng/microL and 4.3 ng/microL for carbaryl and carbofuran, respectively. This study showed that TLC can be used for semi-quantitative analysis.     

Monitoring of atrazine in milk using a rapid tube-based ELISA and validation with HPLC

Although atrazine has been banned in the European Union since 2007 it still persists in soil from where it can enter the food chain. Milk-producing animals accumulate atrazine from contaminated feed and water and since large quantities of milk and milk products are consumed its quality should be constantly monitored. The objective of this investigation was to develop a simple tube ELISA procedure suitable for use in non-specialised laboratories and in the field. A polyclonal antibody raised in sheep and the hapten-gelatine conjugate was immobilised onto polystyrene tubes. This enables the colour produced to be read on a basic spectrophotometer. Milk samples were collected from three farms in different regions of Poland and diluted before immunoassay was performed. Samples were extracted with hexane-acetone for HPLC analysis. The amount of fat in the milk samples interferes with the dose response so it essential that the standards are prepared in the same samples matrix. A good correlation between 1% and 2% was found between the two methods in the analysis of real samples. However the ELISA procedure was more sensitive that the HPLC method since atrazine was detected in some samples by the ELISA but was not confirmed by the HPLC method. The study demonstrated that the simple antigen-coated tube assay provides a cost effective and valuable screening test that can be easily modified for direct use as a screening tool in the field.     

A sensitive enzyme‐linked immunosorbent assay amplified by biotin–streptavidin system for detecting non‐steroidal anti‐inflammatory drug ketoprofen

A sensitive biotin–streptavidin‐amplified enzyme‐linked immunosorbent assay (BA‐ELISA) method was developed for detecting non‐steroidal anti‐inflammatory drug ketoprofen. Compared with traditional ELISA method, the sensitivity of proposed immunoassay was enhanced by the biotin–streptavidin system. Under the optimal condition, the median inhibitory concentration (IC₅₀) was 0.25 ng mL^?1, with minor cross‐reactivity to a number of structural analogs. This developed assay was successfully applied to detect the ketoprofen residues in different fish samples, and good recoveries (72.6–105.5%) were obtained. The results indicated that this immunoassay method could specifically detect trace ketoprofen residues and could be widely used for routine monitoring of food samples.     

Distribution and ecotoxicity of chlorotriazines in the Scheldt Estuary (B-Nl)

As part of the Endis-Risks project, the current study describes the occurrence of the chlorotriazine pesticides atrazine, simazine and terbutylazine in water, sediment and suspended matter in the Scheldt estuary (B-Nl) from 2002 to 2005 (3 samplings a year, 8 sampling points). Atrazine was found at the highest concentrations, varying from 10 to 736 ng/l in water and from 5 up to 10 ng/g in suspended matter. Simazine and terbutylazine were detected at lower concentrations. Traces of the targeted pesticides were also detected in sediments, but these were below the limit of quantification. As part of an ecotoxicological assessment, we studied the potential effect of atrazine on molting of Neomysis integer (Crustacea:Mysidacea), a resident invertebrate of the Scheldt Estuary and a proposed test organism for the evaluation of endocrine disruption. Following chronic exposure ( approximately 3 weeks), atrazine did not significantly affect mysid molting at environmentally relevant concentrations (up to 1 microg/l).     

Determination of trace triazine and chloroacetamide herbicides in tile-fed drainage ditch water using solid-phase microextraction coupled with GC-MS

Solid-phase microextraction coupled with gas chromatography-mass spectrometry (SPME-GC-MS) was used to analyze two triazine (atrazine and simazine) and three chloroacetamide herbicides (acetochlor, alachlor, and metolachlor) in water samples from a midwest US agricultural drainage ditch for two growing seasons. The effects of salt concentration, sample volume, extraction time, and injection time on extraction efficiency using a 100-mum polydimethylsiloxane-coated fiber were investigated. By optimizing these parameters, ditch water detection limits of 0.5 microgL(-1) simazine and 0.25 microgL(-1) atrazine, acetochlor, alachlor, and metolachlor were achieved. The optimum salt concentration was found to be 83% NaCl, while sample volume (10 or 20 mL) negligibly affected analyte peak areas. The optimum extraction time was 40 min, and the optimum injection time was 15 min. Results indicated that atrazine levels in the ditch water exceeded the US maximum contaminant level for drinking water 12% of the time, and atrazine was the most frequently detected among studied analytes.     

Evaluation of atrazine positive and false positive immunoassay detections in ground water

Abstract

False positive responses on an atrazine (6‐chloro‐N‐ethyl‐N'‐(l‐methylethyl)‐1, 3, 5‐triazine‐2, 4‐diamine) immunoassay kit were investigated to explain possible causes for these occurences. Ground water samples were evaluated with the immunoassay kit and positive responses (>0.20 μg L^‐1) were confirmed using gas chromatography/mass spectrometry (GC/MS). Non‐confirming samples (false positives) were analyzed for seven additional compounds on GC. Resulting GC/MS and GC analyses showed that 70% of the false positives could be attributed to two compounds. Prometon (6‐methoxy‐N,N'‐bis(l‐methylethyl)‐1, 3, 5‐triazine‐2, 4‐diamine) was responsible for the majority (64%) of the false positive responses The atrazine metabolite, deethylatrazine (2‐chloro‐4‐amino‐6‐isopropylamino‐1, 3, 5‐triazine), was responsible for the other 6% of the false positives measured. Unattributed false positives (30%) were probably due to an overestimation of pesticide concentrations in the kit's lower detection range.     

Anaerobic treatment of atrazine bearing wastewater.

Performance of mixed microbial anaerobic culture in treating synthetic waste-water with high Chemical Oxygen Demand (COD) and varying atrazine concentration was studied. Performance of hybrid reactors with wood charcoal as adsorbent, with a dose of 10 g/l and 40 g/l, along with the microbial mass was also studied. All the reactors were operated in sequential mode with Hydraulic Retention Time (HRT) of 5 days. In all the cases, COD removal after 5 days was found to be above 81%. Initial COD was above 1,000 mg/l. From a hybrid reactor COD removal after 2 days was observed to be 90%. Atrazine reduction after 5 days by microbial mass alone was 43.8%, 40% and 33.2% with an initial concentration of 0.5, 1.0 and 2.0 mg/l respectively. MLSS on all the cases were almost same. Increasing MLSS concentration by about 2 fold did not increase the atrazine removal efficiency significantly. Maximum atrazine removal was observed to be 64% from the hybrid reactor with 10 g/l of wood charcoal and 69.4% from the reactor with 40 g/l of wood charcoal. Atrazine removal from the hybrid reactors after 15 days were observed to be 35.7% and 38.7%, which showed that the higher dose of wood charcoal in hybrid reactor did not improve the atrazine removal efficiency significantly. Specific methanogenic activity test showed no inhibitory effect of atrazine on methane producing bacteria. The performance of anaerobic microorganisms in removing atrazine with no external carbon source and inorganic nitrogen source was studied in batch mode. With an initial concentration of 1.0 mg/l, reduction of atrazine by the anaerobic microorganisms in absence of external carbon source after 35 days was observed to be 61.8% where as in absence of external carbon and inorganic nitrogen source the reduction was only 44.2% after 150 days. Volatilization loss of atrazine was observed to be insignificant.     

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.     

Influence of humic substances on the photolysis of aqueous pesticide residues

The present work investigated the direct and indirect photolysis of pesticide residues (atrazine, imazaquin, iprodione), in aqueous solutions and under UV-visible radiation (280-480nm). Different kinds of humic substances (HS) were added to samples in order to evaluate their behaviour as possible photocatalysts and their effect on the photolysis of pesticides. The fulvic acids were purchased from the International Humic Substances Society, and they were added to samples in concentrations ranging from 1 to 150 mgl(-1). Titanium dioxide was used as the photocatalyst, in concentration ranging from 10 to 150 mgl(-1). Pesticides photolysis were measured by UV-visible absorption spectroscopy and differential pulse polarography with all used pesticides, reaching total degradation after 2h of irradiation, thus indicating a fast direct photolysis. Photocatalysis by TiO(2) could increase the pesticides photolysis rate up to 40%. This effect, however, was not observed for imazaquin photolysis. Again, except for imazaquin, HS presence showed a positive effect in increasing pesticide degradation, but only within specific concentration ranges (below 10mg l(-1) for iprodione and about 30mgl(-1) for atrazine). Above these ranges HS induce a decrease in the pesticides photolysis rate. Spin-trapping measurements by electronic paramagnetic resonance spectroscopy, using the spin-trap DMPO, showed that HS are able to photogenerate hydroxyl radicals, increasing the pesticides molecule degradation. However, the HS also react with the photogenerated hydroxyl radical, influencing the pesticide photolysis, leading to a decrease in the photolysis rate and causing it to be strongly dependent on the nature and concentration of residues in the water to be treated.     

ANAEROBIC TREATMENT OF ATRAZINE BEARING WASTEWATER

Performance of mixed microbial anaerobic culture in treating synthetic wastewater with high Chemical Oxygen Demand (COD) and varying atrazine concentration was studied. Performance of hybrid reactors with wood charcoal as adsorbent, with a dose of 10 g/l and 40 g/l, along with the microbial mass was also studied. All the reactors were operated in sequential mode with Hydraulic Retention Time (HRT) of 5 days. In all the cases, COD removal after 5 days was found to be above 81%. Initial COD was above 1000 mg/l. From a hybrid reactor COD removal after 2 days was observed to be 90%. Atrazine reduction after 5 days by microbial mass alone was 43.8%, 40% and 33.2% with an initial concentration of 0.5, 1.0 and 2.0 mg/l respectively. MLSS on all the cases were almost same. Increasing MLSS concentration by about 2 fold did not increase the atrazine removal efficiency significantly. Maximum atrazine removal was observed to be 64% from the hybrid reactor with 10 g/l of wood charcoal and 69.4% from the reactor with 40 g/l of wood charcoal. Atrazine removal from the hybrid reactors after 15 days were observed to be 35.7% and 38.7%, which showed that the higher dose of wood charcoal in hybrid reactor did not improve the atrazine removal efficiency significantly. Specific methanogenic activity test showed no inhibitory effect of atrazine on methane producing bacteria. The performance of anaerobic microorganisms in removing atrazine with no external carbon source and inorganic nitrogen source was studied in batch mode. With an initial concentration of 1.0 mg/l, reduction of atrazine by the anaerobic microorganisms in absence of external carbon source after 35 days was observed to be 61.8% where as in absence of external carbon and inorganic nitrogen source the reduction was only 44.2% after 150 days. Volatilization loss of atrazine was observed to be insignificant.     

Behaviour and biodegradation of sulfonamides (p-TSA, o-TSA, BSA) during drinking water treatment

Three sulfonamides -para-toluenesulfonamide (p-TSA), ortho-toluenesulfonamide (o-TSA) and benzenesulfonamide (BSA) - have recently been detected in groundwater within a catchment area of one drinking water treatment plant (DWTP), which is located downstream of a former sewage farm. The degradation pathways of p-TSA, o-TSA and BSA were investigated during drinking water treatment with incubation experiments and an experimental filter. Incubation experiments showed that p-TSA is removed during the treatment by microbiological processes. Removal of p-TSA is performed by adapted microorganisms only present in polluted groundwater. The elimination in an experimental filter of 1.6m length applying filtration velocities from 2 to 6 m h(-1) was approximately 93% of p-TSA. The microbial degradation rates in the incubation experiment were approximately 0.029 microg l(-1) h(-1) (zero order reaction). In the experimental filter, the reaction rate constants were around 0.0063 s(-1) for all filtration velocities (1st order reaction). Drinking water treatment does not reduce the concentration of o-TSA and BSA under conditions encountered in Berlin. p-TSA, o-TSA and BSA were only measured in the low microg l(-1) concentrations range in the purified water.     

Localization of atrazine non-extractable (bound) residues in soil size fractions

Three different soils were incubated under field conditions with ¹⁴C-ring labelled atrazine. After six months, the soils were exhaustively extracted with methanol and sonicated in water. The dispersed material was then fractionated by sieving, sedimentation and centrifugation, and each fraction was separated into humin, fulvic and humic acids. In all soils, the well humified organic matter and the atrazine residues were mainly located in the 20-2 and 2-0.2 μm fractions. There was a very large concentration of bound residues in the coarsest fractions, especially in the 200-50 μm fraction. These could be related to the active degradation of coarse plant residues, or to bioconcentration by soil actinomycetes and fungi.     

Identification and significance of phenazone drugs and their metabolites in ground- and drinking water

Residues of three phenazone-type pharmaceuticals have been identified in routine analyses of groundwater samples from selected areas in the north-western districts of Berlin, Germany. Phenazone, propiphenazone, and dimethylaminophenazone have been detected in some wells at concentrations up to the low microg/l-level. Additionally, three phenazone-type metabolites namely 1-acetyl-1-methyl-2-dimethyl-oxamoyl-2-phenylhydrazide (AMDOPH), 1-acetyl-1-methyl-2-phenylhydrazide, and dimethyloxalamide acid-(N'-methyl-N-phenyl)-hydrazide have also been identified in these groundwater samples. The residues are suspected to originate from former production spills of a pharmaceutical plant located in a city north of Berlin. It was observed that with the exception of AMDOPH all other residues were efficiently removed during conventional drinking water treatment. The drug metabolite AMDOPH deriving from dimethylaminophenazone residues was found at concentrations of 0.9 microg/l in finished drinking water. However, a following study on the toxicological relevance of the AMDOPH residues has shown that there is no toxicological harm for humans at the low concentrations of AMDOPH observed in Berlin drinking water.