Chapter 16
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Integrated Chemical and Biological Remediation of Atrazine-Contaminated Aqueous Wastes
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S. M. Arnold, W. J. Hickey, R. F. Harris, and R. E. Talaat 1
Soil Science Department, University of Wisconsin—Madison, Madison, WI 53706 Corning Hazleton, Inc., Madison, WI 53707 2
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Treatment of [2,4,6- C]atrazine with Fenton's reagent (FR) produced seven major dechlorinated, dealkylated, and/or partially oxidized products identified by high-performance liquid chromatography megaflow electrospray tandem mass spectrometry. The best FR mixture, 2.69 mM (1:1) FeSO :H O , completely degraded atrazine (140 μΜ) in≤30s,primarily to two dealkylated, chlorinated products: 23% diaminochloro-s-triazine and 28% deisopropylatrazine amide. About 55% chloride release indicated dehalogenated s-triazines accounted for the balance or products. Rhodococcus corallinus degraded these chlorinated products in≤10minutes and converted 47% of [2,4,6C]atrazine to CO in 7 d. R. corallinus combined with Pseudomonas sp. strain D increased CO production to 73%. When applied to a pesticide rinse water containing atrazine, cyanazine, alachlor, metolachlor, and EPTC,≥99%of the pesticides were degraded with 12.2 mM FR. Subsequent treatment with R. corallinus and Pseudomonas sp. strain D degraded all chlorinated s-triazine intermediates and released 70% 14CO from an [2,4,6- C]atrazine tracer in 10 d. Collectively, these studies have demonstrated that the integrated approach has potential as an on-site treatment for pesticide rinse water. In this study, Fenton's reagent (FR) in combination with Rhodococcus corallinus and/or Pseudomonas sp. strain D were used to treat atrazine alone and in mixed pesticide wastes. Fenton's reagent (Fe and H 0 ) generates hydroxyl radical (HO*), a powerful non-specific oxidant (/). 4
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Fe + H 0 -> FE + HO* + HO 2
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©1998 American Chemical Society In Triazine Herbicides: Risk Assessment; Ballantine, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.
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178 Previous research has focused on degrading s-triazine herbicides using HO* generated by ozone (2-6), T1O2/UV light (7-9), H2O2/UV light (70), photodecomposition of Fe(OH) (77), and Fenton's reagent (FR) (12-13). The main advantage of Fenton's reagent (FR) over other HO" systems is its simplicity: the chemicals are commonly available and inexpensive, and there is no need for special equipment like U V lamps, complex reaction vessels, T1O2 particles, or ozone generators. Because of its simplicity, FR has the potential for widespread use in treating atrazine wastes. The drawback of treating s-triazines with HO*generating systems is that one or more stable chlorinated products commonly accumulate. Thus, treatment is not complete in a remediation context because the toxicity of chlorinated s-triazine products may be as great as that of the parent compound (14). Microbial processes are often well-suited for degradation of single pesticides, but may be limited in treating pesticide mixtures because of the limitations of most enzymes in attacking structurally diverse chemicals. Moreover, wastes such as pesticide rinse water generated during field applications may contain a variety of pesticides, formulating agents, surfactants, emulsifiers and fertilizers, which may inhibit microbial growth (75). Thus, chemical pretreatment can eliminate microbial inhibitors and breakdown target pesticides into common substrates for bacterial degradation. We hypothesized that the collective catabolic activities of R. corallinus (contains an inducible hydrolase capable of dechlorination and deamination of partially dealkylated s-triazines) and Pseudomonas sp. strain D (metabolizes a broader spectrum of dechlorinated, partially dealkylated s-triazines degradation products) could be used to efficientiy degrade the stable chlorinated end-products generated by FR treatment of atrazine.
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Materials and Methods Chemicals. The Glossary of this volume lists the common names and chemical names of s-triazines and pesticides described in this study. Atrazine (99% pure), cyanazine (99%), alachlor (99%), and EPTC (98%) were purchased from Chem Service (West Chester, PA). [2,4,6- C]Atrazine (19.4 μ α / m g , 97%), desethylatrazine (99%), desisopropylatrazine (98%), chaminochloro-s-triazine (90%), desethylhydroxyatrazine (98%), ammeline (95%), desisopropylhydroxyatrazine (97%), cyanazine (96%), and metolachlor (98%) were provided by Ciba-Geigy Corporation (Greensboro, NC). Pesticide rinse water, collected from a commercial pesticide application facility in Dane County, WI, was a yellowish liquid (pH 8.1) in which the following pesticides were detected: atrazine (131 μΜ), cyanazine (132 μΜ), EPTC (159 μΜ), metolachlor (209 μΜ), and alachlor (98 μΜ). The pesticide rinse water contained 235 mg/L total organic carbon; Fe, Mg, Μη, P, S, Zn were detected at 0.01, 0.55, 0.06, 44, 193, and 0.63 mg/L, respectively. Other heavy metals were not detected (76). Suspended solids were removed by centrifugation (10,000 rpm; 14,336 χ g) with a Sorvall Szent-Gyorgyi and Blum continuous flow-through system. 14
In Triazine Herbicides: Risk Assessment; Ballantine, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.
179 Fenton's Reagent Treatments. For treatment of atrazine, ratios (FeS0 :H 0 ) of 1:11, 1:100, and 2:1 were examined at concentrations from 0.1 to 25 mM. Aliquots of 0.05 ml to 1.5 ml of 50 mM FeS0 were mixed with 25 ml of 135 μΜ atrazine in 50-ml Erlenmeyer flasks; HO*production was initiated by adding 0.05 ml to 1.5 ml of 50 mM H 0 . For pesticide rinse water treatment, 0.15-2.0 ml of 50 mM FeS0 and 50 mM H 0 were mixed with 10 ml pesticide rinse water. The aluminum foil-wrapped flasks were incubated for 24 h on a rotary shaker at 200 rpm (25 ± 1°C). Treated atrazine and pesticide rinse water samples (0.5 ml) were mixed with methanol (0.5 ml) to quench the reaction, centrifuged (10 min; 3,200 rpm), and the supernatants analyzed by high-performance liquid chromatography (HPLC). Treated pesticide rinse water samples were also extracted and analyzed by gas chromatography (GC) as described later. 4
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Bacterial Degradation Experiments. Rhodococcus corallinus (NRRL B15444R) was a gift from Ciba-Geigy. Pseudomonas sp. strain D (NRRL B-12228) was obtained from the USDA, National Center for Agricultural Utilization Research, Peoria, IL. Cells were grown on cyanuric acid-glucose (CAG) medium that consisted of potassium phosphate buffer (10 mM, pH 7.3), M g S 0 (1 mM), glucose (10.0 mM), cyanuric acid (1.7 mM), and a trace element mixture (77). Cultures were harvested during their early stationary phase of growth 1 L C A G , washed four times with potassium phosphate buffer (130 mM, pH 7.4), and resuspended to an optical density (measured at 600 nm) of 2.5 in 80 ml potassium phosphate buffer. Duplicate incubations were established by adding 10 ml cell suspension to 10 ml FR-treated atrazine or pesticide rinse water. Duplicate controls had 10 ml of FR-treated solutions added to 10 ml of 130 mM potassium phosphate buffer. Reactions were terminated at selected intervals by immersing 1ml aliquots in dry ice baths. The samples were thawed by boiling for 10 min, clarified by centrifugation (10 min, 3200 rpm), and the supernatant analyzed by HPLC. 4
Mineralization Studies of Atrazine and its FR Products. FR-treated atrazine and/or pesticide rinse water spiked with [2,4,6- C]atrazine (0.026 to 0.046 μΟ/ητΙ in 2.0 ml) was added to a 25-ml serum bottle and crimp sealed with a Teflon-lined septum. Next, 2.0 ml of either R. corallinus or Pseudomonas sp. strain D, 1.0 ml of both cultures, or 2.0 ml of 130 mM buffer, was injected and the samples incubated on a rotary shaker (150 rpm) for 7-10 days. A l l treatments were incubated in duplicate. At selected times the flask headspaces were flushed for 1 hour to trap C 0 in carbon 14 cocktail (R. J. Harvey Instrument Co., Hillsdale, NJ) and volatile organic compounds in ethylene glycol monoethyl ether. After incubation, 1 ml was acidified with 0.5 ml 6 Ν H S 0 and flushed for 1 hour to release dissolved C 0 . Radioactivity was quantified using a Rackbeta model 1209 liquid scintillation counter (LKB-Wallac, San Francisco, CA). 14
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HPLC/UV Analysis for Atrazine and s-Triazine Transformation Products. Analysis for atrazine and s-triazine transformation products was done with a Hewlett-Packard (Palo Alto, C A , USA) Model 1050 HPLC equipped with a
In Triazine Herbicides: Risk Assessment; Ballantine, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.
Downloaded by NORTH CAROLINA STATE UNIV on October 1, 2012 | http://pubs.acs.org Publication Date: May 14, 1998 | doi: 10.1021/bk-1998-0683.ch016
180 variable-wavelength U V detector at 220 nm. Analytes were separated using a Hewlett-Packard ODS Hypersil C-18 reversed-phase column (200 mm χ 4.6 mm i.d.; 5 μπι mean particle diameter) at a flow-rate of 1 ml/min. The mobile phase, potassium phosphate (5 mM, pH 4.6)-acetonitrile, was run in a gradient from 5 to 89% acetonitrile in 18 min. The column was re-equilibrated at the starting conditions in a 10 min post-run. Five-point calibration curves were run for atrazine, desethylatrazine, desisopropylatrazine, diaminochloro-5-triazine, desethylhydroxyatrazine, desisopropylhydroxyatrazine, and ammeline, which had detection limits of 0.24, 0.24, 0.14, 0.29, 0.63, 0.39, and 0.77 μΜ, respectively. Atrazine amide, simazine amide, N-isopropylammelide, and hydroxyatrazine were quantified using a response factor, which is the ratio of the U V (220 nm) response to C activity in fraction-collected peaks. The s-triazine transformation products were identified by HPLC/Electrospray (ES)-MS/MS equipped with a radioactivity detector, and confirmatory analysis was done using high resolution-electron impact-mass spectrometry (HR-EI-MS) and GC/MS as described previously (18). Chloride ion was determined using an ion-specific electrode (12,19). 1 4
GC Analysis for Pesticides in Pesticide Rinse Water. Atrazine, cyanazine, metolachlor, alachlor, and EPTC were extracted from FR-treated pesticide rinse water by shaking for 2 min with 2 χ 25 ml dichloromethane and 1.25 g of NaCl. The organic phase was separated and then dried by filtering through 50 g anhydrous Na S0 , and concentrated to 5 ml by rotoevaporation. The aqueous phase was extracted with ethyl acetate and the organic phases were combined, rotoevaporated to approximately 2 ml, and dried with nitrogen. The residue was resuspended in 5 ml ethyl acetate, filtered with a 0.22-μιη nylon syringe filter, and analyzed by GC. Percent recoveries (all ±
