mecoprop-P (ISO); (R)-2-(4-chloro-2-methylphenoxy)propionic acid

Additional information on environmental fate and behaviour

Administrative data

Endpoint:

additional information on environmental fate and behaviour

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

additional information on environmental fate and behaviour

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

A consortium of three bacteria was isolated from top soil through their capacity to utilise the chlorinated, aromatic herbicide test material as a single growth substrate.
The paper describes a novel laboratory study of test material degradation by both pure and mixed microbial cultures. This is an indicator of the potential of microorganisms to degrade the test material in situ. This work also includes an investigation of the ability of a microbial community to distinguish between the (±) stereoisomers of the test material. Finally, the ability of the consortium to degrade structurally related compounds that are often introduced into the environment as mixed formulations with the test material, to maximise weed control, was reported on.

GLP compliance:

not specified

Type of study / information:

Study of test material degradation by both pure and mixed microbial cultures

An inoculum taken from garden soil was incubated in batch culture using test material as the sole source of carbon and energy. Progressive enrichment resulted in the isolation of a consortium of three bacteria capable of growth on racemic test material. The consortium comprised Alcaligenes denitrificans, Pseudomonas glycinea and Pseudomonas marginalis. The organisms formed a tight association and because of their similar nutritional characteristics were difficult to separate. Growth of the consortium was monitored on the racemic herbicide and both the (R)-(+)- and (S)-(-)-enantiomers in independent experiments using an equivalent amount of total carbon for comparative purposes. During growth on the racemate, onset of chloride ion release occurred after a lag phase of 8 h. Degradation of the aryl moiety as monitored by the stoichiometric release of chloride ions took 37 h to complete.

The final accumulated chloride ion concentration equalled half the maximum calculated value for the complete degradation of the supplied racemic test material. Growth of the consortium supported by the (R)-(+)-enantiomer followed a similar time course, although in this case the final chloride ion concentration equalled the theoretical maximum present from the added herbicide. When growth on the (R)-(+)-enantiomer and racemate of test material had ceased, the maximum culture absorbance was 0.95 and 0.62 respectively, which corresponded to 0.63 and 0.37 g/L wet weight biomass. In both types of media, there was no significant difference between the maximum specific growth rate and release of chloride ions (P = 0.167) and the maximum population phase was reached at the time chloride ion release was completed. There was no growth of the consortium on the (S)-(-)-enantiomer and no subsequent release of chloride ions. Autoclaved, uninoculated growth media containing test material was used as a chloride ion release control to demonstrate there was no abiotic transformation of the herbicide.

Spectrophotometric scans of culture supernatants taken from both types of media were determined with time over the range 200 - 500 nm. During growth and chloride ion release, there was a concomitant decrease in the size of the 279 nm peak, probably demonstrating the disruption of the conjugated aryl moiety in the primary test material structure. During growth on the (R)-(+)-enantiomer, the decrease in A279 corresponded to a reduction in concentration from 1 to 0.03 g/L. This represents a 97 % reduction in the initial herbicide concentration as compared with only a 55 % decrease in test material concentration during growth on the racemate. The absorption maximum of media containing the racemate and the single isomer both remained at 279 nm until after 14 h of exponential growth when it shifted to 286 and 291 nm, respectively. After 35 - 40 h into the growth cycle, there was a noticeable decrease in UV absorption of the culture supernatant formulated with the (R)-(+)-enantiomer. The racemic carbon source showed no equivalent change and the residual test material in this culture remained stable on extended incubation. The decrease in the size of the 279 nm peak during incubation of the consortium on the (S)-(-)-enantiomer was negligible confirming chloride ion and culture absorbance analyses that it could not support growth.

The three-member community was also able to grow on the other herbicides as sole carbon and energy sources.

In closed culture, the proportion of the community formed by each member varied with the age of the culture. Typically, a culture had the following composition after the completion of test material degradation: A. denitrificans (2.2 x 10^9 cfu/mL), P. marginalis (2.4 x 10^7 cfu/mL) and P. glycinea (2.6 x 10^5 cfu/mL). The consortium remained able to degrade test material after twenty passages through nutrient broth. After this period of non-selective culture, growth on test material required 72 h for completion and there was a slight increase in the lag phase. Samples were periodically removed to test for pure culture degradation of test material. Initially, none of the isolates was able to degrade test material in pure culture or in combination with any other single member of the consortium. However, after five months of sub-culturing and after approximately 80 days of chemostat growth, A. denitrificans acquired the complete capacity to degrade the test material as a single substrate. The isolate from the batch culture was chosen for further study. It formed recognisable colonies on test material selective agar. Growth, expressed as an increase in culture absorbance, occurred after a lag phase of 5 h and was associated with a drop in pH from 7.1 to 6.4. Release of chloride ions was initiated 12 h into the growth cycle. The doubling time of the organism during the exponential growth phase in batch culture was 6.5 h. There was no significant difference between growth rates of the consortium and pure culture (P = 0.184).

In chemostat culture with (R)-(+)-test material at a growth limiting concentration of 0.56 g of carbon/L and a dilution rate of 0.024/h, the three-member community was maintained as a stable association for over two months of continuous growth. After an initial fall in cfu, possibly attributed to wash-out of the inoculum, all three isolates stabilised with A. denitrificans remaining the dominant organism in the culture. The absorbance of the culture was constant at 0. 74 ± 0.04, a value that corresponded to approximately 90 % breakdown of the herbicide enantiomer in batch culture. After this period of growth in the chemostat, A. denitrificans was able to grow independently on racemic and (R)-(+)-test material as the sole carbon and energy source. The other two bacteria were not washed out of the chemostat but remained as a tight consortium.

Conclusions:

The culture exclusively degraded the (R)-(+)-isomer of the test material while the (S)-(-)-enantiomer remained unaffected. Initially, no single member of the bacterial consortium was able to degrade the test material as a pure culture but after prolonged incubation, A. denitrificans was able to grow on the herbicide as the sole source of carbon and energy.

Executive summary:

A consortium of three bacteria was isolated from top soil through their capacity to utilise the chlorinated, aromatic herbicide test material as a single growth substrate.

This paper describes a novel laboratory study of test material degradation by both pure and mixed microbial cultures. This is an indicator of the potential of microorganisms to degrade the test material in situ. This work also includes an investigation of the ability of a microbial community to distinguish between the (±) stereoisomers of the test material.

The culture exclusively degraded the (R)-(+)-isomer of the test material while the (S)-(-)-enantiomer remained unaffected. Initially, no single member of the bacterial consortium was able to degrade the test material as a pure culture but after prolonged incubation, A. denitrificans was able to grow on the herbicide as the sole source of carbon and energy.

Data source

Reference

Reference Type:

publication

Title:

Biodegradation of the chlorophenoxy herbicide (R)-(+)-mecoprop by Alcaligenes denitrificans

Author:

Tett VA, Willetts AJ and Lappin-Scott HM

Year:

1997

Bibliographic source:

Biodegradation 8: 43-52

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

An Alcaligenes denitrificans strain capable of utilising the herbicide test material as a sole carbon source was isolated from soil and cultured in liquid medium. Crude cell extracts of the bacterium were utilised in spectrophotometric assays to elucidate a biochemical pathway for the degradation of the test material.

GLP compliance:

not specified

Type of study / information:

Bacterial degradation and elucidation of the breakdown pathway of the test material

Test material

Test material information

Constituent 1

Reference substance name:

(R)-2-(4-chloro-2-methylphenoxy)propionic acid

EC Number:

240-539-0

EC Name:

(R)-2-(4-chloro-2-methylphenoxy)propionic acid

Cas Number:

16484-77-8

Molecular formula:

C10H11ClO3

IUPAC Name:

(R)-2-(4-chloro-2-methylphenoxy)propionic acid

Details on test material:

- Supplied by: Pennine Chemical Services Ltd. (Huddersfield, West Yorkshire, UK)

Results and discussion

Any other information on results incl. tables

The sequence of changes with time in the UV spectra of culture supernatants of A. denitrificans while growing on (R)-(+)-test material may be summarised as follows:

- In the first 24 h of growth there was a decrease in A279 indicating the disappearance of the test material;

- At 32 h there was a further decrease in A279 accompanied by a shift in maximum absorption (λmax) towards 260 nm, probably representing the accumulation of an intermediate;

- At 46 h, there was a decline in absorbance at 260 nm, the λmax occurring at 230 nm, this is more evident at 48 h.

In an attempt to elucidate the biodegradative pathway the authors postulated a pathway then undertook enzyme assays to test the hypothetical route. Crude enzyme extracts were produced from (R)-(+)-test material-grown A. denitrificans to assay for a range of enzymes. The first step in the biodegradation may involve a test material monooxygenase. However, spectrophotometric tests for this enzyme based on the oxidation of NAD(P)H failed to detect its presence, and evidence for this step was drawn from whole-cell methods using GLC analysis and respirometry.

Assays for activity of MCP were performed by monitoring the consumption of NADPH by test material­ grown cells. The maximum specific activity of the monooxygenase was 0.78 µmoles NADPH/min/mg protein. The reaction was specific for NADPH and there was no conversion of MCP to 5-chloro-3-methylcatechol in the presence of NADH. Similarly, there was no detectable monooxygenase activity in extracts obtained from cells grown on nutrient broth. The monooxygenase was found to be intracellular and not membrane-bound as the activity was almost exclusively present in the supernatant and not in the pellet of cellular debris.

Utilisation of catechol was monitored by the production of the corresponding muconic acid which absorbs maximally at 265 nm. The rate of change of optical density is directly proportional to the enzyme concentration within a 10-fold range of enzyme dilutions. Catechol dioxygenase activity toward a number of catechols was induced in cells of A. denitrificans grown on the test material, but not on cells grown on nutrient broth. The bacterium possessed an enzyme capable of utilising catechol, 3-methyl and 4-methylcatechol. The specific activity of catechol dioxygenase was calculated using the molar extinction coefficients of the tested substrates.

The spectrum of cis, cis-muconic acid has a λmax at 257 nm. Muconate cycloisomerase activity was assayed by following the decrease in absorbance at 260 nm without interference from the lactone at this wavelength. Crude cell extracts prepared from cells grown on both (R)-(+)-test material and benzoic acid utilised muconic acid in the presence of Mn (II). In order to demonstrate lactonising enzyme activity, the assay required very specific reaction conditions. Maximum absorption of 4-carboxymethylbut-2-en-4-olide occurs at 220 nm. Disappearance of the lactone was followed at 230 nm since absorbance of β-ketoadipate is negligible at this wavelength. 4-carboxymethyl but-2-en-4-olideutilisation was observed in cell extracts produced separately from cells grown on benzoate and test material.

The Gibbs reaction was used to detect the presence of phenol-containing compounds within the biodegradation pathway. Although reported to be specific for 4- alkoxy-substituted phenolic compounds, the test material, 4-chloro-2-methylphenol and muconate were all detected using the reaction giving respective Rf values of 0.47, 0.71 and 0.08. There was no positive Gibbs response of phenol formation observed when test material biodegradation was monitored in the presence of 2,2'-bipyridyl. Using diagnostic tests to determine ortho and meta cleavage, test material-grown A. denitrificans gave a negative test for meta cleavage but a positive Rothera reaction demonstrating that the degradation of the test material by A. denitrificans proceeds via ortho cleavage.

Oxygen uptake was measured and compared when (R)-(+)-test material grown cells were supplied with test material, MCP, catechol, 3-methyl and 4-methylcatechol. All substrates were rapidly oxidised without a lag phase. The rate of oxygen consumption during oxidation of each catechol was greater than that observed for oxidation of the test material or MCP.

GLC analysis was used to follow the breakdown of (R)-(+)-test material and to detect and identify specific intermediates of the test material biodegradation pathway. Results from GC-MS confirmed that MCP was an intermediate soon after the cells were exposed to the test material. Other intermediates were identified within the culture supernatants, occurring in minor quantities throughout growth of the bacterium and identified by their retention times against equivalent chemically-synthesised reference standards as follows: 5-chloro-3-methylcatechol RT 11.54 min, 2-methyl-4-carboxymethylene but-2-en-4-olide (lactone) RT 9.76 min. Upon disappearance of MCP after approximately 200 h growth on the test material, the formation of a more hydrophobic peak with a retention time of 5.8 - 5.9 min was observed. The compound increased steadily in the supernatants and was still present after 900 h. This peak could not be identified by GLC or GC-MS. After 900 h, a total amount of 1 mg of the test material remained at a 93 % extraction efficiency. The muconic acid was not detected in the extracts from A. denitrificans and the chemically-synthesised standard was not able to dissolve in the ethyl acetate extraction solvent. Muconic acid was considered too polar for the extraction and partitioning characteristics required in the gas chromatographic procedure to isolate the test material.

Specific Activities of Catechol Dioxygenase on Three Catechol Analogues of 5 -Chloro-3-methylcatechol.

Substrate	Wavelength λmax (nm)	Molar extinction coefficient (x 10³ M)	Specific activity (units/ min/ mg protein)
Catechol	274	2.59	0.07
3-methylcatechol	272	1.73	0.23
4-methylcatechol	278	2.91	0.26

The extinction coefficients were measured at the wavelengths cited in 50 mM phosphate buffer (pH 7.0). The protein concentration of the crude cell extract was 12.0 mg/mL. One enzyme unit is defined as that amount of enzyme that catalyses the formation of 1 µmol of the product or the disappearance of 1 µmol of the substrate per minute under the assay conditions

 

Spectrophotometric Investigation of the Effect of Substrate Concentration on the Specific Activity of the Muconate Lactonising and Delactonising Enzymes of (R)-(+)-Test Material-grown A. denitrificans.

[cis, cis-muconic acid] (µM)	Specific activity (µmols cis, cis-muconic acid utilised/min/mg protein)	[4-carboxymethyl- but-2-en-4-olide] (µM)	Specific activity (µmols lactone utilised/ min/ mg protein)
0.0	0	0.0	0
0.03	0.016	0.13	0.08
0.09	0.017	0.25	0.15
0.18	0.029	0.38	0.27
0.27	0.033	0.63	0.40
0.36	0.034	1.38	0.55

The assays were conducted at 260 and 230 nm, respectively, with crude cell extracts containing 12.4 mg/mL protein and started by the addition of substrate. The data represent the change in specific activity as a function of concentration of cis, cis-muconic acid and 4-carboxymethylbut-2-en-4-olide, structural analogues of 4-chloro-2-methyl muconate and 2-methyl-4-carboxymethylene but-2-en-4-olide

 

Applicant's summary and conclusion

Conclusions:

GC-MS analysis provided direct evidence for the biotransformation of the test material to the transient metabolite 4-chloro-2-methylphenol (MCP). No NADPH-dependent activity was observed during this reaction. Pyruvate was verified as the second product derived from the aliphatic side chain of the test material. MCP was subsequently transformed to a substituted catechol by an NADPH-dependent monooxygenase.

Executive summary:

An Alcaligenes denitrificans strain capable of utilising the herbicide (R)-(+)-test material as a sole carbon source was isolated from soil and cultured in liquid medium. Crude cell extracts of the bacterium were utilised in spectrophotometric assays to elucidate a biochemical pathway for degradation of the test material. Results indicated a reaction sequence analogous to the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D). GC-MS analysis provided direct evidence for the biotransformation of the test material to the transient metabolite 4-chloro-2-methylphenol (MCP). No NADPH-dependent activity was observed during this reaction. Pyruvate was verified as the second product derived from the aliphatic side chain of the test material. MCP was subsequently transformed to a substituted catechol by an NADPH-dependent monooxygenase. When grown on the test material, A. denitrificans was adapted to oxidise catechol and its 4- and 3-methylated derivatives indicating the broad substrate specificity of catechol dioxygenase. The microorganism was demonstrated to adopt the ortho mechanism of aromatic cleavage which resulted in the formation of 2-methyl-4-carboxymethylene but-2-en-4-olide, a reaction intermediate of the β-ketoadipate pathway.