Excessive use of pesticides and herbicides is a major environmental and health concern worldwide. Atrazine, a synthetic triazine herbicide commonly used to control grassy and broadleaf weeds in crops, is a major pollutant of soil and water ecosystems. Atrazine modifies the growth, enzymatic processes and photosynthesis in plants. Atrazine exerts mutagenicity, genotoxicity, defective cell division, erroneous lipid synthesis and hormonal imbalance in aquatic fauna and nontarget animals. It has threatened the sustainability of agricultural soils due to detrimental effects on resident soil microbial communities. The detection of atrazine in soil and reservoir sites is usually made by IR spectroscopy, ELISA, HPLC, UPLC, LC–MS and GC–MS techniques. HPLC/LC–MS and GC–MS techniques are considered the most effective tools, having detection limits up to ppb levels in different matrices. Biodegradation of atrazine by microbial species is increasingly being recognized as an eco-friendly, economically feasible and sustainable bioremediation strategy. This review presents the toxicity, analytical techniques, abiotic degradation and microbial metabolism of atrazine.

Toxicity, degradation and analysis of the herbicide atrazine

Biodegradation of xenobiotic compounds is examined with s-triazines as an example and with biological treatment of wastewater containing s-triazines as an aim. s-Triazines have been termed recalcitrant, but examination of the literature indicates that a potential for biodegradation exists. Nitrogen-limited enrichment cultures yield organisms able to degrade by-products of the industrial synthesis of s-triazine herbicides as sources of nitrogen. The choice of inoculum for these enrichments is important and often allows for successful enrichment after simple batch culture, but organisms containing several degradative reactions could be obtained only after selection in extended culture. Routine, specific determinations of all s-triazines (by HPLC) were essential throughout the work. Molar growth yields show complete mass balances for the utilization of s-triazines. Kinetic experiments indicate that specific degradation rates of s-triazines in growing cells are about 0.4 mkat/kg of protein. Characterised biochemical pathways consist of a series of hydrolytic cleavages of chloro-, amino- and alkylamino-groups from the s-triazine ring. Pathways converge to cyanuric acid, which is subject to hydrolytic ring cleavage to CO2 and NH4+ via hydrolysis of biuret and urea. Our cultures degraded all significant s-triazines in real wastewater. But the system was not practicable because the specific activities of some enzymes were too low, because of inhibition by salt in the wastewater and because expensive carbon sources were necessary. Improved planning for enrichment cultures is seen to be necessary and this depends on adequate knowledge of the chemistry of the wastes.

http://kops.uni-konstanz.de/bitstream/handle/123456789/6885/Biodegradation_of_s_triazine.pdf?sequence=1

Biodegration of s-triazine xenobiotics

Pseudomonas sp. strain ADP metabolizes atrazine to carbon dioxide and ammonia via the intermediate hydroxyatrazine. The genetic potential to produce hydroxyatrazine was previously attributed to a 1.9-kb AvaI DNA fragment from strain ADP (M. L. de Souza, L. P. Wackett, K. L. Boundy-Mills, R. T. Mandelbaum, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:3373-3378, 1995). In this study, sequence analysis of the 1.9-kb AvaI fragment indicated that a single open reading frame, atzA, encoded an activity transforming atrazine to hydroxyatrazine. The open reading frame for the chlorohydrolase was determined by sequencing to be 1,419 nucleotides and encodes a 473-amino-acid protein with a predicted subunit molecular weight of 52,421. The deduced amino acid sequence matched the first 10 amino acids determined by protein microsequencing. The protein AtzA was purified to homogeneity by ammonium sulfate precipitation and anion-exchange chromatography. The subunit and holoenzyme molecular weights were 60,000 and 245,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography, respectively. The purified enzyme in H2(18)O yielded [18O]hydroxyatrazine, indicating that AtzA is a chlorohydrolase and not an oxygenase. The most related protein sequence in GenBank was that of TrzA, 41% identity, from Rhodococcus corallinus NRRL B-15444R. TrzA catalyzes the deamination of melamine and the dechlorination of deethylatrazine and desisopropylatrazine but is not active with atrazine. AtzA catalyzes the dechlorination of atrazine, simazine, and desethylatrazine but is not active with melamine, terbutylazine, or desethyldesisopropylatrazine. Our results indicate that AtzA is a novel atrazine-dechlorinating enzyme with fairly restricted substrate specificity and contributes to the microbial hydrolysis of atrazine to hydroxyatrazine in soils and groundwater.

Atrazine chlorohydrolase from Pseudomonas sp. strain ADP: gene sequence, enzyme purification, and protein characterization.

Atrazine, simazine, and propazine are closely related herbicides that can be used for weed control. The degradation of s‐triazines in soil, water, and sediments has received considerable attention. In this review, the literature related to degradation of atrazine and related s‐triazines is reviewed. Particular emphasis is directed toward those substances formed along the degradative pathways and to the biodegradation aspects of mineralization. The s‐triazines are unusual in that the carbon and nitrogen ring contains no available electrons for aerobic biodegradation. The only sources of energy for aerobic biodegradation of atrazine are found on the ethyl and isopropy I groups. There is some evidence to support the concept that dealkylation of s‐triazines occurs to support the bioenergetic needs of microorganisms. Biodegradation studies with simazine or atrazine as the carbon and energy source have generally shown biodegradation of the alkyl side chains with evolution of radioactive carbon dioxide from chai...

Degradation of atrazine and related s‐triazines

Strain YAYA6 was isolated from a mixed microbial community that was growing on atrazine as a sole carbon source and formed quantitative amounts of chloride and nitrate. This strain was identified as a member of the true pseudomonad group (RNA group I) and was given the designation DMS 93-99. The growth yield when atrazine was the sole carbon and nitrogen source was 80 g (dry weight) of cells per mol of atrazine, and the cell doubling time was around 11 h. Approximately 20% of [U-ring 14C]atrazine was mineralized during primary degradation of atrazine. After atrazine disappeared from the culture supernatant, mineralization continued until the level of mineralization was more than 50%. Under different experimental conditions 10% of the atrazine supplied initially was converted to cyanuric acid and < 1% was converted to other s-triazines after prolonged incubation. Degradation proceeded via dechlorination and N-dealkylation. Atrazine was degraded until the concentration was circa 0.1 milligrams/liter. We obtained evidence showing that strain YAYA6 has specific uptake mechanisms for atrazine but less specific degradation mechanisms for s-triazines.

Mineralization of the herbicide atrazine as a carbon source by a Pseudomonas strain.

The metabolism of atrazine herbicide by Nocardia, a soil bacterium previously shown to degrade the herbicide to 4-amino-2-chloro-1,3,5-triazine, has been studied. The primary route of the metabolism appeared to be N-dealkylation and was similar to that found in several fungi. The data suggested that the isolated dealkylated metabolites were precursors of 4-amino-2-chloro-1,3,5-triazine. The supposed metabolites 4-alkylamino-2-chloro-1,3,5-triazines were synthesized but there was not direct proof of their involvement in the degradation process.

Atrazine Metabolism by Nocardia: Elucidation of Initial Pathway and Synthesis of Potential Metabolites

The degradation of herbicide atrazine (2-chloro-4-ethylamino-6-isopropylamino-1, 3, 5-triazine) by a soil bacterium is reported. The bacterium involved is a species of Nocardia, which utilizes the atrazine as the sole source of carbon and nitrogen. A new metabolite, 4-amino-2-chloro-1, 3, 5-triazine, of the degradation of atrazine in the presence of glucose has been identified. The results further substantiated that atrazine can be degraded by soil microorganisms and indicated that deamination can also occur, as well as dealkylation. 4-Amino-2-chloro-1,3,5-triazine did not show phytotoxic activity to oat (Avena sativa L.), demonstrating that deamination insures detoxification.

4-Amino-2-chloro-1,3,5-triazine: a new metabolite of atrazine by a soil bacterium.

The purpose of this paper is to elucidate the final degradative pathways of atrazine (I) by a Nocardia strain through experiments on the degradation of its metabolite, 4-amino-2-chloro-6-isopropylamino-1,3,5-triazine (IIa). This compound, in the bacterial medium, was transformed into several products identified as 2-chloro-4,6-diamino-1,3,5-triazine (IIc), 4-amino-2-hydroxy-6-isopropylamino-1,3,5-triazine (VIII), 4-amino-1,2-dihydro-1,3,5-triazin-2-one (IV) and dicyanodi-amidine (VII). The formation of (IIc) was attributed to a microbial N-dealkylation, while the detection of (IV) and (VII) was attributed to a chemical degradation of the intermediate 4-amino-2-chloro-1,3,5-triazine (III). Compound (III) undergoes rapid hydrolysis and does not accumulate in the culture medium. On the basis of the microbial and chemical results, we propose a degradative pathway for atrazine.

M. C. Giardina

Papers

Atrazine Metabolism by Nocardia: Elucidation of Initial Pathway and Synthesis of Potential Metabolites

4-Amino-2-chloro-1,3,5-triazine: a new metabolite of atrazine by a soil bacterium.

Chemical and Biological Degradation of Primary Metabolites of Atrazine by a Nocardia Strain