The initial culture had an OD600 of 0. 10, and resuspended infresh buffertoan OD600 of1. 0. Metolachlor disappearance and metabolite formation were determined by HPLC analysis. Analyses were performed using a Waters high performing liquid chromatograph equipped with a C 18 5 m column and a UV photodioarray detector. For the detection of the metolachlor, the isocratic mobile phase consisted of water and acetonitrile. The flow rate was 1. 0 mL min, the column was operated at room temperature, and the injection volume was 50 L. Metolachlor was detected at 210 nm after approximately 5. 8 min. For the detection of the acetochlor, the isocratic mobile phase consisted of water and methanol at a flow rate of 1. 0 mL min, the column was operated at room temperature, and the injection volume was 25 L.
Acetochlor was detected at 200 nm after approximately 4. 8 min. For the LY294002 detection of the alachlor, the isocratic mobile phase consisted of water and acetonitrile at a flow rate of 1. 0 mL min, the column was operated at room temperature, and the injection volume was 25 L. Alachlor was detected at 205 nm after approximately 6. 5 min. Mineralization of the metolachlor and alachlor ring structures was determined in 250 mL biometer flasks containing 10 mL of 25 mM phos phate buffer. Bacteria and yeast cells obtained from cultures grown on metolachlor or alachlor were added to final concentrations of 10 9 or 10 cells mL, respectively. Metolachlor or alachlor was added to flasks to a final concentration of 50 g mL and metolachlor or alachlor was added to a final concentration of 3000 dpm mL.
A 7 mL vial LY294002 containing 5 mL of 0. 5 N NaOH was placed into the biometer flasks to quantify CO 2 released. The vials containing NaOH were removed and replaced at selected times during the incubation period. To determine CO 2, a 1 mL aliquot of the NaOH solution was mixed with 6 mL of Ecolite cocktail and radioactivity was quantified by using a Beckman model LS 6800 scintillation counter. Samples were held in the dark for 24 48 h prior to counting and were corrected for quenching. No chemiluminescence was observed. The buffer medium was analyzed for the presence of metolachlor or alachlor and potential metabolites by HPLC as described below. Mass Balance Determination. After the final sampling period, the solution in biometer flaskswas dried to a constantweight at 80 C for 24h.
Duplicate aliquots of the dried samples were weighed and mixed with an checkpoint kinase equal volume of powdered micro crystalline cellulose powder CF 11, and samples were oxidized for 1. 4 min using a model 306 sample oxidizer. The CO 2 evolved during combus tion process was trapped in Carbosorb solvent, mixed with Permafluor in a liquid scintillation vial, and quantified by using a Beckman model LS 6800 scintillation counter. The instrument combustion efficiency was determined before and after the combustion of each set of test samples. The efficiency of the oxidizer was calculated on the basis of the recovery of radioactivity from cellulose containing a known quantity of metolachlor or alachlor, and averaged 97. 0% during the course of the study. LC MS Analysis.
The concentration of metolachlor and its metabo lites in growth medium was determined by using HPLC and LC MS analyses. The HPLC analyses were done as described above. The LC MS analysisfor lossofparentcompoundmetolachlor was doneusing a Waters Alliance 2695 high performance liquid Neuronal Signaling chromatograph, coupled to an Applied Biosystems API 3200 LC MS MS. A Zorbax RX C8 column was used for separa tion. The column temperature was maintained at 40 C, and the mobile phase was a gradient starting with 95% water /5% acetonitrile, 95% A at 0 min, 95% A at 5 min, 50% A at 10 min, 3% A at 15 min, 3% A at 20 min, 95% A at 25 min, and 95% A at 30 min. The mobilephaseflowratewas 0. 2 mLmin, and thesampleinjectionvolume was 50 L. Samples were maintained at 10 C in the autosampler to minimize decomposition.
Tuning parameters were optimized by direct infusion. NSCLC All compounds were detected using LC DAD, and positive ionization or thermospray ESIt multiple reaction monitoring mode with the following mass spectrometer conditions: curtain gas interface, 30 psi, IS voltage, 4000 V, gas 1, 30 psi, gas 2, 30 psi, ion source temperature, 300 C, collision gas, medium, dwell time, 200 ms. DAD monitoring was done at 210 400 nm. growth was measured at 600 nm by using a DU 70 spectrophotometer. Data reported are mean values of two independent growth experiments carried out under identical condi tions. Fortheexperimentswithacetochlorandalachlor,MMmediumplus 0. 04% yeast extract was exclusively used. Herbicide Degradation. Exponential phase yeast cells grown in MM containing50 gmL herbicidewereharvestedbycentrifugationat10000g 622 J. Agric. Food Chem., Vol. 59, No. 2, 2011 Munoz et al.