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Registration Dossier
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EC number: 230-386-8 | CAS number: 7085-19-0
Biodegradation in soil
Administrative data
Link to relevant study record(s)
- Endpoint:
- biodegradation in soil, other
- 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:
- The degradation of the test material in soil under laboratory conditions was studied using enantioselective high-resolution gas chromatography/mass spectrometry.
- GLP compliance:
- not specified
- Test type:
- laboratory
- Oxygen conditions:
- aerobic
- Soil classification:
- not specified
- Soil no.:
- #1
- Soil type:
- sandy loam
- % Org. C:
- 1.6
- pH:
- 7
- Details on soil characteristics:
- SOIL COLLECTION AND STORAGE
- Geographic location: Garden soil was taken from a plot near the research station in Wädenswil.
- Pesticide use history at the collection site: Phenoxy herbicides have never been used at the site.
- Storage conditions: The soil was then kept in a porous clay pot until used.
- Storage length: Used within a few days.
- Soil preparation: A portion of a few kilograms of soil was carefully air dried, 1 d at room temperature, and then sieved through 10 and 4 mm sieves.
PROPERTIES OF THE SOILS
- Moisture: The water content of the soil was determined at ≈ 18 %. - Soil No.:
- #1
- Duration:
- 35 d
- Soil No.:
- #1
- Initial conc.:
- 1 mg/kg soil d.w.
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- test mat. analysis
- Soil No.:
- #1
- Temp.:
- 20 – 23 °C
- Details on experimental conditions:
- EXPERIMENTAL DESIGN
- Soil condition: Air dried
- Soil (g/replicate): Portions of 400 g of the 18 % soil.
- Control conditions: A portion of the soil was sterilised by γ-irradiation from a commercial ^50Co source with a total dose of 25 kGy (24 h exposure). The controls were carried out in sterilised soil with incubation periods up to 16 d. Periodically 10.0 g samples were removed and placed into 20 mL glass vials for analysis; duplicate samples immediately after fortification and mixing and single samples periodically thereafter. Blank determinations of the soil prior to fortification revealed no phenoxy acids or dicamba present (Detection limit < 0.01 ppm). The herbicide concentrations of ≈ 1 ppm per compound enantiomer are within the range expected from field applications (≈ 1 kg/ha).
- Test apparatus: Experiments were carried out in 750 mL wide mouth clear glass jars covered with aluminium foil and lid.
- Details of traps for CO2 and organic volatile, if any: None specified.
Test material application
- Volume of test solution used/treatment: 400 μg of the test material dissolved in 10 mL water.
- Application method: Portions of 400 g of the 18 % soil were placed in a jar and fortified with solution containing 400 μg of each substance dissolved in 10 mL water, yielding a final water content of ≈ 20 % in soil (fortification level 1 ppm). The soil was carefully mixed.
Experimental conditions
- Moisture maintenance method: Covered to exclude losses by evaporation.
- Continuous darkness: No. Incubated with normal daylight.
OXYGEN CONDITIONS
- Methods used to create the an/aerobic conditions: The jars were opened for short time every day for aeration.
SAMPLING DETAILS
- Sampling method for soil samples: Prior to extraction, 10 μg of clofibric acid in 100 μL of methanol were added and after vigorous shaking the samples were centrifuged (4 000 rpm for 10 min). The clear supernatant was removed, added to 10 mL of distilled water and acidified with dilute H2SO4 to pH ≈2. The phenoxyacids and dicamba were re-extracted from this aqueous solution and two 3 mL portions of methylene chloride. The combined methylene chloride extract was then reduced in volume to ≈ 1 mL using a faint stream of nitrogen. The extracts were generally opaque due to the presence of small amounts of water. A few drops of methanol were then added to get clear solutions and methylation was effected by adding an ethereal solution of diazomethane until a clear yellow colour persisted. After 15 min standing for reaction at rt, the samples were concentrated, dried with a few milligrams of anhydrous Na2SO4 and then made to a volume of 5 mL with n-hexane. A 1 μL aliquot was used for analysis.
- Sample storage before analysis: The 10.0 g samples collected were kept at 4 °C until extracted and cleaned up. - Key result
- Soil No.:
- #1
- % Degr.:
- >= 90
- Parameter:
- test mat. analysis
- Sampling time:
- 35 d
- Transformation products:
- not measured
- Evaporation of parent compound:
- not measured
- Volatile metabolites:
- not measured
- Residues:
- not measured
- Details on results:
- The test material was readily degraded in soils to levels ≤ 10 % of the initial concentration after 22 - 35 days of incubation. The data showed linearity in an initial phase (< 16 d) but later showed some trend towards faster rates.
In the second, more rapid phase (> 16 d) the rates were significantly higher with a half-life of ≈ 4 d.
The data for the test material shows a continuous decrease of the concentration to levels < 1 – 3 % of the initial concentrations after 22 days of incubation, however with a less pronounced two-phase kinetic.
The concentration of the test material increased from initial values of 0.7 % to a maxima of 10 % after 8 – 9 days respectively and then decreased again.
The degradation observed can be chemically and / or biologically mediated. In order to distinguish this, the test material was incubated in sterilised soil. Chemical degradation in sterilised soil is expectedly non-enantioselective. The knet values were 2.5 – 4 times lower than those in non-sterilised soil. There was no increase in the concentrations of the inverted isomers. These data indicate that degradation was primarily biologically mediated. - Conclusions:
- Under the conditions of the study, the test material was determined was readily degraded in soils to levels ≤ 10 % of the initial concentration after 22 - 35 days of incubation. Degradation was primarily biologically mediated.
- Executive summary:
The degradation of the test material in soil under laboratory conditions was studied using enantioselective high-resolution gas chromatography/mass spectrometry. Garden soil (sandy load); 1.6 % organic carbon; pH 7.0) was taken from a plot near the research station in Wädenswil where phenoxy herbicides have never been used.
A portion of a few kilograms of soil was carefully air dried (1 d at room temperature, and then sieved through 10 and 4 mm sieves. The water content of the soil was determined at ≈ 18 %. The soil was then kept in a porous clay pot until used within a few days. A portion of the soil was sterilised by γ-irradiation from a commercial ^50Co source with a total dose of 25 kGy (24 h exposure).
The test material was readily degraded in soils to levels ≤ 10 % of the initial concentration after 22 - 35 days of incubation. The data showed linearity in an initial phase (< 16 d) but later showed some trend towards faster rates.
In the second, more rapid phase (> 16 d) the rates were significantly higher with a half-life of ≈ 4 d.
The data for the test material shows a continuous decrease of the concentration to levels < 1 – 3 % of the initial concentrations after 22 days of incubation, however with a less pronounced two-phase kinetic.
The concentration of the test material increased from initial values of 0.7 % to a maxima of 10 % after 8 – 9 days respectively and then decreased again.
The degradation observed can be chemically and / or biologically mediated. In order to distinguish this, the test material was incubated in sterilised soil.
Chemical degradation in sterilised soil is expectedly non-enantioselective. The knet values were 2.5 – 4 times lower than those in non-sterilised soil. There was no increase in the concentrations of the inverted isomers. These data indicate that degradation was primarily biologically mediated.
Under the conditions of the study, the test material was determined was readily degraded in soils to levels ≤ 10 % of the initial concentration after 22 - 35 days of incubation. Degradation was primarily biologically mediated.
- Endpoint:
- biodegradation in soil: simulation testing
- 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:
- according to guideline
- Guideline:
- other: draft G2 CTB/Steungroep M, Subgroep IB
- Version / remarks:
- August 1983
- GLP compliance:
- no
- Test type:
- laboratory
- Radiolabelling:
- yes
- Oxygen conditions:
- aerobic
- Soil classification:
- other: Classification according to the Netherlands
- Soil no.:
- #1
- Soil type:
- silty clay
- pH:
- 7.4
- Soil no.:
- #2
- Soil type:
- other: sandy peat
- pH:
- 7.4
- Soil no.:
- #3
- Soil type:
- other: loamy fine sand
- pH:
- 7.8
- Details on soil characteristics:
- SOIL COLLECTION AND STORAGE
- Geographic location: The water and sediment for the model system were collected from ditches in the eastern part of the Netherlands (De Graafschap): Duiven, Holocene river deposit (soil 1); Winterwijk, Holocene peat (soil 2); Wehl, Eolian pleistocene sand (soil 3).
- Storage conditions: The mixture was kept aerated at room temperature.
- Storage length: The test was started ca. 10 days after the samples had been taken from the field. During this period the samples were acclimatised to the laboratory test conditions.
- Soil preparation: The slurry was sieved (2 mm width) to remove coarse organic material such as leaves, branches, snail shells, etc.
PROPERTIES OF THE SOILS
- Total dry matter, g/L:
Soil 1, clay: 15.7
Soil 2, peat: 5.2
Soil 3, sand: 13.6
- Organic dry matter, g/L: The content of dry organic matter was diluted to about 1 g/L, resulting in varying contents of total dry matter.
Soil 1, clay: 1.9
Soil 2, peat: 1.1
Soil 3, sand: 0.8 - Soil No.:
- #1
- Duration:
- 3 mo
- Soil No.:
- #2
- Duration:
- 3 mo
- Soil No.:
- #3
- Duration:
- 3 mo
- Soil No.:
- #1
- Initial conc.:
- 0.94 other: mg/L
- Based on:
- test mat.
- Soil No.:
- #2
- Initial conc.:
- 0.94 other: mg/L
- Based on:
- test mat.
- Soil No.:
- #3
- Initial conc.:
- 0.94 other: mg/L
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- CO2 evolution
- Soil No.:
- #1
- Temp.:
- 17 to 20 °C
- Soil No.:
- #2
- Temp.:
- 17 to 20 °C
- Soil No.:
- #3
- Temp.:
- 17 to 20 °C
- Details on experimental conditions:
- EXPERIMENTAL DESIGN
- No. of replication controls, if used: For each soil one control bottle without test material was included.
- No. of replication treatments: The degradation of the test material was tested in three soils in triplicate.
- Test apparatus: The test system consisted of a 280 mL dark glass bottle filled with 50 mL of the water and sediment mixture.
- Details of traps for CO2 and organic volatile, if any: The CO2 evolving from the system was trapped in a wash-bottle filled with 7 mL ethanolamine (Baker, reagent grade). A correction was made for the slight increase of the volume of the ethanolamine due to moisture carried over by the CO2 into the ethanolamine. This increase was at the most 3 % of the total volume per week.
Test material application
- The test material was dissolved in water under alkaline conditions (pH 11). The stock solution contained approximately 50 mg/L.
Experimental conditions
- Moisture maintenance method: The system was continuously aerated by CO2-free moist air at a rate of 1 mL/min. As a consequence, the gas volume of the bottle was replaced about 7 times per day.
SAMPLING DETAILS
- Sampling intervals: The residual activity in the water and sediment was assessed at the end of the test period.
- Sampling method for soil samples: The radioactivity was counted with a Packard Tri-carb Liquid Scintillation Spectrometer, Model 3320.
Method of collection of CO2 and volatile organic compounds:
- The 14-CO2 traps of the test system were replaced periodically, after l, 3, 5, 7, 9 and 13 weeks. For the determination of the amount of 14-CO2 evolving from the system, a 1 mL aliquot of ethanolamine was diluted 1:10 in water and 1 mL of this solution was added to 9 mL of scintillant (Packard Insta Gel).
- The residual activity in the water and sediment was assessed at the end of the test period. For the measurement of the activity in the waterphase, 1 mL of the supernatant was added to the scintillant. To check the presence of dissolved CO2 or other volatile metabolites, the supernatant was acidified and aerated and the residual activity was measured again.
- The activity in the sediment was determined by total combustion. 0.5 mL samples of a well mixed water and sediment suspension were combusted at 1200 °C in the Packard Tri-carb Sample Oxidizer (306). CO2 was captured in 9 mL of Carbosorb (Packard), after which 11 mL of Permafluor (Packard) was added. The recovery of the Oxidizer was 97.1 percent. - Key result
- Soil No.:
- #1
- DT50:
- 12 d
- Type:
- other: 50 % recovery of 14-CO2
- Temp.:
- >= 17 - <= 20 °C
- Key result
- Soil No.:
- #2
- DT50:
- 18 d
- Type:
- other: 50 % recovery of 14-CO2
- Temp.:
- >= 17 - <= 20 °C
- Key result
- Soil No.:
- #3
- DT50:
- 44 d
- Type:
- other: 50 % recovery of 14-CO2
- Temp.:
- >= 17 - <= 20 °C
- Transformation products:
- yes
- No.:
- #1
- Details on transformation products:
- The major metabolic product for the test material is CO2.
- Evaporation of parent compound:
- not measured
- Volatile metabolites:
- not measured
- Residues:
- no
- Remarks:
- No appreciable residues in the water.
- Details on results:
- TEST CONDITIONS
- Moisture, temperature and other experimental conditions maintained throughout the study: Yes.
- Anomalies or problems encountered (if yes): The pH of the water in the test bottles was similar to that of the control. However, in a few test bottles the pH had risen to a pH-value high above normal. These systems were contaminated by NaOH at the end of the test. This resulted in an extraction of the sediment and a brown colour of the water. The total residual activity was not affected, but the activity in the water was higher than in the replicates. Data from these bottles were not used in the calculation of the water residues.
RECOVERY
- The average recovery of the test material was consistently low.
- The data for the triplicates showed that the reproducibility of the test systems is high: The difference between extreme replicates is mostly less than 4 %.
RATE OF DEGRADATION
- The initial rate of degradation varies slightly, depending on the origin of the water and sediment. The rate of degradation in the test model from the sandy soil is somewhat lower than those in the clay and peat mixtures. The half-life time of the test material, calculated as the time necessary for the recovery as CO2 of 50 % of the amount added, varies accordingly.
- For the test material, it takes 12, 18 and 44 days to recover 50 % as 14-CO2 in the clay, peat and sand models, respectively.
MINERALISATION
- % of applied radioactivity present as CO2 at end of study: 96.7 %, 78.4 % and 92.2 % in clay, peat and sand respectively (expressed as percentages of the initial amounts (100 % = 1 mg/L). - Conclusions:
- Under the conditions of this study, degradation of the test material starts slowly, but after three weeks the major part of 14-CO2 was recovered. Additionally, although significant water residues are found in the sand system, the test material proved to be easily biodegradable in the clay and peat systems.
- Executive summary:
The rate of degradation of the test material was studied in an aquatic test system, comparable with conditions in Dutch ditches. The degradation was measured by the evolution of 14-C labelled carbon dioxide from ring-labelled test material.
The test material was shown to be mineralised completely after three months in the aquatic test systems in clay, peat and sandy soils, with no appreciable residues in the water. The degradation in the sandy soil system was somewhat slower and in the case of the test material some residual radioactivity was found. In general, the test system performed well, giving reproducible results and good recoveries of the labelled material.Under the conditions of this study, degradation of the test material starts slowly, but after three weeks the major part of 14-CO2 was recovered. Additionally, although significant water residues are found in the sand system, the test material proved to be easily biodegradable in the clay and peat systems.
- Endpoint:
- biodegradation in soil: simulation testing
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- not reported
- 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:
- The persistence of the test material (along with two other chlorinated herbicides) was investigated at the 2 µg/g level, under laboratory conditions, in three Saskatchewan soils at 85 % of their field capacity moistures and 20 ± 1 °C. Following extraction of the soils with aqueous acidic acetonitrile, the methylated extracts were analysed gas chromatographically for remaining herbicides.
- GLP compliance:
- not specified
- Test type:
- laboratory
- Radiolabelling:
- no
- Oxygen conditions:
- aerobic
- Soil classification:
- not specified
- Year:
- 1 980
- Soil no.:
- #1
- Soil type:
- clay loam
- Soil no.:
- #2
- Soil type:
- other: heavy clay
- Soil no.:
- #3
- Soil type:
- sandy loam
- Details on soil characteristics:
- SOIL COLLECTION AND STORAGE
- Geographic location: Saskatchewan (Canada)
- Pesticide use history at the collection site: Not specified; the herbicides under investigation are extensively used on the Canadian Prairies.
- Collection, preparation and storage procedures: Samples of the clay loam, heavy clay and sandy loam were collected in an air-dried state from the 0 - 5 cm soil horizon during October 1980 and sieved through a 2 mm screen. The soil was then stored in boxes at laboratory temperature for 4 weeks before use.
The soils used in the present study were collected from the same location as those used in an earlier comparative study involving di- and tri-chlorinated phenoxyalkanoic acid herbicides. Further details on the soil composition are not given in this paper. Additionally, the herbicide concentration, soil moisture and incubation temperature parameters in the two studies were identical. - Soil No.:
- #1
- Duration:
- 21 d
- Soil No.:
- #2
- Duration:
- 21 d
- Soil No.:
- #3
- Duration:
- 21 d
- Soil No.:
- #1
- Initial conc.:
- 2 other: µg/g moist soil
- Based on:
- test mat.
- Soil No.:
- #2
- Initial conc.:
- 2 other: µg/g moist soil
- Based on:
- test mat.
- Soil No.:
- #3
- Initial conc.:
- 2 other: µg/g moist soil
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- test mat. analysis
- Details on experimental conditions:
- The persistence study was carried out at a concentration of 2.0 µg/g; a concentration of 0.2 µg/g was also used to investigate the recovery of the test material form the soils.
METHOD
Samples (50 g) of all three soil types at 15 (air dried) and 85 % of their field capacity moistures were weighed into 175-mL Styrofoam cartons which were then capped with plastic lids and incubated in the dark at 20 ± 1 °C for 7 days to allow equilibration. The moisture levels were monitored, by weighing, every second day and distilled water added if necessary, with thorough mixing. After equilibration, cartons were treated with aliquots of the test material solution (40 µL, 100 µg) to give herbicide concentrations of 2.0 µg/g based on moist soil. This rate is equivalent to a field rate of 1 kg/ha, assuming incorporation in the field to a depth of 5 cm. All soils were thoroughly mixed to distribute the chemicals evenly. The cartons were re-capped and re-incubated in the dark at 20 ± 1 °C, water being added, with stirring, every second day to maintain the moisture content.
Duplicate samples from each treatment at the higher moisture regime were extracted and analysed for herbicides remaining after 7, 14 or 21 days, whilst duplicate soil samples at the 15 % moisture level were analysed only at the end of the breakdown study. - Soil No.:
- #1
- DT50:
- 9 d
- Type:
- not specified
- Temp.:
- 20 °C
- Remarks on result:
- other: Clay loam, 2.0 µg/g, 85 % of field capacity
- Soil No.:
- #2
- DT50:
- 8 d
- Type:
- not specified
- Temp.:
- 20 °C
- Remarks on result:
- other: Heavy clay, 2.0 µg/g, 85 % of field capacity
- Soil No.:
- #3
- DT50:
- 7 d
- Type:
- not specified
- Temp.:
- 20 °C
- Remarks on result:
- other: Sandy loam, 2.0 µg/g, 85 % of field capacity
- Transformation products:
- not specified
- Details on results:
- - Test material recovery
Recoveries of test material from the different soils were good, and reproducible. When 2.0 µg/g of test material was added, the recovery in clay loam, heavy clay and sandy loam was 100, 96 and 96 %, respectively. When 0.2 µg/g of test material was added, the recovery in clay loam, heavy clay and sandy loam was 99, 95 and 95 %, respectively.
- Persistence studies
The results from the persistence studies indicate that in the moist soils breakdown of the test material was rapid. In contrast, losses from air-dried soils was minimal. This lack of degradation in the air-dried soils suggests that herbicidal losses in the moist soils were due to biological processes, rather than inefficient extraction techniques.
Half-lives for the test material in the various soils were calculated from the graphs obtained by plotting the logarithm of percentage chemical remaining against incubation time. For the test material, the half-life in clay loam, heavy clay and sandy loam was determined to be 9, 8 and 7 days, respectively.
It can therefore be concluded that the test material, as a commonly used phenoxyalkanoic acid herbicide, appears to be degraded rapidly in all three Saskatchewan soils described. - Conclusions:
- The test material, as a commonly used phenoxyalkanoic acid herbicide, appears to be degraded rapidly in all three Saskatchewan soils described.
- Executive summary:
The persistence of the test material was investigated at the 2 µg/g level, under laboratory conditions, in three Saskatchewan soils at 85 % of their field capacity moistures and 20 ± 1 °C. Following extraction of the soils with aqueous acidic acetonitrile, the methylated extracts were analysed gas chromatographically for remaining herbicides.
The results from the persistence studies indicate that in the moist soils breakdown of the test material was rapid. In contrast, losses from air-dried soils was minimal. This lack of degradation in the air-dried soils suggests that herbicidal losses in the moist soils were due to biological processes, rather than inefficient extraction techniques.
Half-lives for the test material in the various soils were calculated from the graphs obtained by plotting the logarithm of percentage chemical remaining against incubation time. For the test material, the half-life in clay loam, heavy clay and sandy loam was determined to be 9, 8 and 7 days, respectively.
It can therefore be concluded that the test material, as a commonly used phenoxyalkanoic acid herbicide, appears to be degraded rapidly in all three Saskatchewan soils described.
- Endpoint:
- biodegradation in soil, other
- 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:
- The study investigated whether MCPA could be detected in a soil to which the test material has been added. Gas chromatography and bioassay were used to follow the degradation of the test material.
- GLP compliance:
- no
- Test type:
- laboratory
- Radiolabelling:
- no
- Oxygen conditions:
- aerobic
- Soil classification:
- not specified
- Year:
- 1 982
- Soil no.:
- #1
- Soil type:
- other: Not specified
- % Clay:
- 65
- % Silt:
- 32
- % Sand:
- 3
- % Org. C:
- 3.8
- pH:
- 6
- Details on soil characteristics:
- SOIL COLLECTION AND STORAGE
- Geographic location: Kotkaniemi, Ojakkala, Finland.
- Pesticide use history at the collection site: The soil was taken from a field not under cultivation.
- Soil preparation: The soil samples were air dried and sieved through < 2 mesh. The water holding capacity (WHC) of the soil was improved by adding 10 % (w/w) unfertilised peat, which lowered the soil pH to 5.75.
PROPERTIES OF THE SOILS
- Moisture at 1/3 atm (%): Distilled water was added to bring the soil moisture to 60 % of the WHC.
- Carbon: 3.8 %
- Sand: 3 %
- Silt: 32 %
- Clay: 65 % - Soil No.:
- #1
- Duration:
- 1 wk
- Soil No.:
- #1
- Initial conc.:
- 5 mg/kg soil d.w.
- Based on:
- test mat.
- Soil No.:
- #1
- Initial conc.:
- 15 mg/kg soil d.w.
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- test mat. analysis
- Soil No.:
- #1
- Temp.:
- 22 °C
- Humidity:
- Not specified
- Microbial biomass:
- Not specified
- Details on experimental conditions:
- EXPERIMENTAL DESIGN
Gas chromatograph and a bioassay were used to follow the degradation of the test material.
The formulated herbicide was thoroughly mixed with the soil at the concentrations of 5 mg and 15 mg test material/ kg soil. The soils were filled in earthen pots (125 mm i.d. in portions of ca. 400 g). The pots were kept in a glasshouse (22 °C).
- No. of replication treatments: Duplicate pots of each series were withdrawn weekly.
- Moisture maintenance method: The moisture content of the soil was kept between 50 and 70 % of the field capacity by adding distilled water to correct for evaporation loss.
- Sample storage before analysis: The soils were air dried and stored at 4 °C until analysed.
Soil that was treated with test material was periodically analysed for the presence of the theoretical metabolite, MCPA.
Bioassay:
- The method was based on the inhibition of root elongation of test plants. The test was performed in Petri dishes (90 mm i.d.).
- No. of replication treatments: Soil was filled in the dishes in triplicate as such or diluted 1: 10 with untreated soil.
- Twenty alfalfa seeds were sown in each dish. The dishes were sealed and incubated in a glasshouse (22 °C) for one week. The root length of the alfalfa seedlings were measured and the average root length for each sample (60 seeds) was expressed as percent of a control (alfalfa seedling grown in untreated soil). For the evaluation of the test material concentration in the samples, known amounts were added to soil and the assay performed. - Soil No.:
- #1
- Remarks on result:
- other: Not specified
- Remarks on result:
- other: Not specified
- Soil No.:
- #1
- Remarks on result:
- not measured/tested
- Transformation products:
- no
- Details on transformation products:
- The soil was periodically analysed for the presence of a degradation product, however it was not detected in any sample of the soil treated with the test material.
- Evaporation of parent compound:
- not measured
- Volatile metabolites:
- not measured
- Residues:
- no
- Details on results:
- The test material degraded fairly rapidly both in soil treated with 5 mg and in the soil with 15 mg test material/ kg soil. However the study was made under conditions that favoured microbial degradation.
MCPA was not detected in any sample taken from the soils that had been treated with the test material. This does not mean that MCPA was not produced as a degradation product, it might also be that degradation rate for this hypothetical intermediate was higher than for the parent product. It is reasonable however to assume that the degradation of the test material may proceed by the same mechanism as other related herbicides by an initial cleavage of the side chain to produce the corresponding phenol.
The degradation curves for the test material obtained by gas chromatography and by bioassay were very similar. The showed a correlation coefficient of 0.958, indicating that the two methods gave almost the same results. - Conclusions:
- Under the conditions of the study the test material degraded fairly rapidly both in soil treated with 5 mg and in the soil with 15 mg test material/ kg soil. However the study was made under conditions that favoured microbial degradation. The soil was periodically analysed for the presence of a degradation product, however it was not detected in any sample of the soil treated with the test material.
- Executive summary:
Gas chromatography was used in an investigation of the degradation of the test material in field soil under glasshouse conditions.
Under the conditions of the study the test material degraded fairly rapidly both in soil treated with 5 mg and in the soil with 15 mg test material/ kg soil. However the study was made under conditions that favoured microbial degradation. The soil was periodically analysed for the presence of a degradation product, however it was not detected in any sample of the soil treated with the test material.
- Endpoint:
- biodegradation in soil: simulation testing
- Type of information:
- experimental study
- Adequacy of study:
- key 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:
- The test material was incubated in three calcareous soils at 15 °C and 80 % of field capacity to try to elucidate its behaviour in soil and compare the dissipation rates when racemic and enantiopure compounds are used. Quantitation was made by HPLC and the R/S ratio by GC-MS.
- GLP compliance:
- not specified
- Test type:
- laboratory
- Radiolabelling:
- no
- Oxygen conditions:
- aerobic
- Soil classification:
- not specified
- Soil no.:
- #1
- Soil type:
- silt loam
- % Clay:
- 7.9
- % Silt:
- 61.4
- % Sand:
- 30.7
- % Org. C:
- 2.1
- pH:
- 8.2
- CEC:
- 10.4 other: cmol/kg
- Soil no.:
- #2
- Soil type:
- sandy loam
- % Clay:
- 0
- % Silt:
- 32.9
- % Sand:
- 67.6
- % Org. C:
- 1.5
- pH:
- 7.5
- CEC:
- 6.4 other: cmol/kg
- Soil no.:
- #3
- Soil type:
- clay loam
- % Clay:
- 32.7
- % Silt:
- 45.3
- % Sand:
- 22
- % Org. C:
- 1.4
- pH:
- 8.1
- CEC:
- 22.4 other: cmol/kg
- Details on soil characteristics:
- SOIL COLLECTION AND STORAGE
- Geographic location: Three calcareous soils, silt and sandy loam soils (Typic xerofluvents), and clay loam soil (Aquic xero fluvent) from the 'Vega de Granada' (SE, Spain) were selected.
- Soil preparation: The soils were air dried and passed through a 2-mm sieve. The three calcareous soils were also amended with 10 % of peat and mixed homogeneously.
- Organic matter: The exogen organic matter used was a neutro-alkaline peat from 'El Padul' (Granada, Spain). It contains 78.5 % of organic matter. - Soil No.:
- #1
- Duration:
- 84 d
- Soil No.:
- #2
- Duration:
- 84 d
- Soil No.:
- #3
- Duration:
- 84 d
- Soil No.:
- #1
- Initial conc.:
- 0.5 other: µg/g soil d.w.
- Based on:
- test mat.
- Soil No.:
- #2
- Initial conc.:
- 0.5 other: µg/g soil d.w.
- Based on:
- test mat.
- Soil No.:
- #3
- Initial conc.:
- 0.5 other: µg/g soil d.w.
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- test mat. analysis
- Soil No.:
- #1
- Temp.:
- 15 °C
- Humidity:
- The mixture was rehumidified at 80 % of the field capacity.
- Microbial biomass:
- N/A
- Soil No.:
- #2
- Temp.:
- 15 °C
- Humidity:
- The mixture was rehumidified at 80 % of the field capacity.
- Microbial biomass:
- N/A
- Soil No.:
- #3
- Temp.:
- 15 °C
- Humidity:
- The mixture was rehumidified at 80 % of the field capacity.
- Microbial biomass:
- N/A
- Details on experimental conditions:
- PERSISTENCE STUDIES
Fresh soil samples (400 g) were preincubated at 50 % of their field capacity for 2 weeks. Then, the racemic test material was added at a concentration of 1 µg/g of dry soil (similar to the field dosage) and the mixture rehumidified at 80 % of the field capacity.
When the test material was applied, the amount used was halved, which corresponds to the relative amount of an enantiomer in the racemic mixture. Seven duplicates of 20 g of each soil (oven dry weight basis) were incubated in a thermostatic chamber at 15 °C in 80-mL bottles covered with aluminium foil in which a small hole was made. After an incubation period of 0, 1, 3, 7, 21, 42 and 84 days, the samples were frozen at -15 °C until they could be analysed. Samples were extracted for analysis by HPLC and GC-MS.
EXTRACTION AND ANALYSIS
The incubated flasks were extracted twice by shaking end-over-end, first with 50 mL and then with 25 mL of methanol-water-glacial acetic acid (49:49:2) for 1 h. The soil extracts were centrifuged at 4 000 rpm for 20 min, and the combined supernatants acidified with approximately 5 mL of concentrated HCI to a pH of 1 - 2. Phenoxyalkanoic acids present were recovered by extraction with 2 x 50 mL portions of dichloromethane. The combined extracts were evaporated under reduced pressure and brought to dryness under a stream of dry nitrogen. The evaporated extracts were dissolved in 2 mL of water for the HPLC and GC-MS analysis. Recoveries from the extraction process in the unamended and amended soil samples were 85 ± 4 and 79 ± 5 %, respectively, by HPLC. The R/S ratio values were determined by GC-MS after derivatisation to their corresponding methyl esters and in many samples confirmed by capillary zone electrophoresis. In general, the results obtained by these two techniques were in good agreement. - Soil No.:
- #1
- DT50:
- 4 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 15 °C
- Soil No.:
- #2
- DT50:
- 7 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 15 °C
- Soil No.:
- #3
- DT50:
- 40 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 15 °C
- Transformation products:
- not measured
- Evaporation of parent compound:
- not measured
- Volatile metabolites:
- not measured
- Residues:
- not measured
- Details on results:
- DISSIPATION OF RACEMIC TEST MATERIAL
The rates of disappearance of the racemic test material were greater in the sandy and silt loam soils than in the clay loam soil. Half-lives change significantly from 15 or 11 days in the silt and sandy soils to 50 or 62 days in the clay loam soil.
The kinetic data calculated for the enantiomeric forms show an enantioselective transformation in the soils. The rate of disappearance of the S-form; in the clay loam soil was more than two times greater than for the R form. In the silt and sandy loam soils, the greater rate of dissipation corresponds to the R-form. The occurrence of such selectivity implies the mediation of bacterial, enzymatic or other biological entities. Abiotic processes are not enantioselective.
The observed differences in the persistence in the three soils may be attributed to their textural composition. In the clay loam soil, porosity and ventilation must be lower than in silt and sandy loam soils. Therefore, if dissipation takes place by aerobic degradation in the clay loam soil it might be less favoured than in the others.
In agreement with the enantioselective dissipation observed from the kinetic data, the R/S values during the dissipation of the racemic test material during the first 3 days of incubations remain close to one. After 3 days there is a decrease in the silt and sandy soils, which indicates a faster degradation of the R-enantiomer. In the clay loam soil, the R/S ratio up to 42 days of incubation remains close to one, but at 84 days this ratio increases approximately three times, which indicates a slower degradation of the R-enantiomer.
DISSIPATION OF ENANTIOPURE TEST MATERIAL IN CALCAREOUS SOILS
Kinetic data from an exponential equation of first-order reaction provide similar results to those observed for the incubation of the racemic compound, where the persistence of the active R-enantiomer in the clay loam soil is approximately five times longer than in the silt and sandy loam soils. The R-forms' peristence is approximately two times lower when they are incubated alone than when they are incubated as racemic compounds.
As can be observed for the R/S ratio values during the dissipation of the test material in the three soils, there is a quick but small inversion reaction of the R-form to the inactive S-form from the beginning. The R/S values decrease throughout the whole incubation time, mainly due to a dissipation of the R-form and, in a small quantity, to the above mentioned inversion reaction of the R- to the S-form. This explanation is based on the small quantity of the Sform determined at the different incubation times (from 0.01 to 0.07 mg/kg of soil). The amounts of the R- and S-forms are too low to be measured after 21 days in silt loam soil but still great enough in clay loam soil to be measured at 84 days.
INFLUENCE OF PEAT AMENDMENT ON THE DISSIPATION OF RACEMIC TEST MATERIAL
- Influence of reaction kinetics: In the silt loam soil, the presence of exogen organic matter does not cause any significant difference in the rate of transformation of racemic test material.
In the sandy loam soil amended with peat, the rate of disappearance decreases, showing higher increments in the half-lives than those observed in the silt loam soil. The greatest increases in the half-lives correspond to the S-form and the smallest to the R-form. The persistence of racemic test material is very short to short. This decrease in the dissipation rate of racemic test material in the silt and sandy loam soils amended with peat might be explained by a reduction in the availability of the molecules in the amended soils due to an enhancement of their sorption capacities.
The dissipation of racemic test material in the clay loam soil added with peat is signicantly different in comparison with the other two soils. The addition of peat means a great reduction in the persistence of the R-form, more or less an increase in the persistence of the S-form, and consequently a considerable decrease in the persistence of the R,S-form. Thus, the persistence in amended clay loam soil changes from moderate to short.
The peat effect in the dissipation of these molecules in the clay loam soil could be related to an increase in the microbiological activity and/or to a change in the soil structure, since the degree of aggregation, in this soil with fine particles, could affect the oxygen diffusion and then the persistence of the racemic substance.
- Influence of ER
The addition of peat to soil modifies the ER-evolution in the clay loam soil, while in the silt and sandy loam soils the R/S ratio values are similar to that observed in the unamended soils. Consequently, the exogen organic matter, in this clay loam soil, seems to have a clear influence on the enantioselective degradation of racemic test material and allows for an estimation that the dissipation is mainly related to a biological transformation.
Compared to the low percentage of organic matter in the raw soils, the addition of 10 % peat plays an important role in the degradation of the racemic test material. Nevertheless, this importance is related to the soil type. - Conclusions:
- It can be concluded that dissipation rates of racemic and enantiomeric forms are influenced by the soil properties.
The enantiomers were found to degrade at different rates in three different soils. The Renantiomer degraded faster in silt and sandy loam soils, while in the clay loam soil the opposite occured. The R-enantiomer was partially converted into its S-enatiomer. The addition of organic matter (peat) to the soils changed the degradation rates of the enantiomers, even modifying the enatioselectivity in the clay loam soil.
Biological degradation is the most probable cause of the enantioselective disappearance of the racemic test material. It is known that the enantiocomposition of a chemical can only be changed by a biological process. - Executive summary:
The test material and its enantiopure R-form was incubated in three calcareous soils at 15 °C and 80 % of their field capacity to try to elucidate their behaviour in soil and compare the dissipation rates when racemic and enantiopure compounds are used. Quantitation was made by HPLC and the R/S ratio by GC-MS.
Under the conditions of the study, the inactive S-enantiomer from the racemic form persisted longer than the R-form in silt and sandy loam soils, but for shorter time in the clay loam soil. The pure R-enantiomer, after incubation in soil, was partially converted into its S-form. In all cases, the dissipation of racemic and pure enatiomeric forms was lower in the clay loam soil than in the silt and sandy loam soils. The R-forms' peristence, in the three soils, was approximately two times lower when it was incubated alone than when it was incubated as a racemic compound. When peat was added, the persistence of the racemic substance in the silt and sandy loam soils increased, while in the clay loam soil it decreased. Besides, in the clay loam soil, the enantiomeric ratio (ER) changes from its S-preferential degradation to a preferential degradation of its R-form, so an increase in the persistence of the inactive S-form occurs.
- Endpoint:
- biodegradation in soil, other
- Remarks:
- Aerobic and anaerobic soil metabolism
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 22 April 1987 to 20 October 1987
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: Environmental Protection Agency Pesticide Assessment Guidelines, Subdivision N, Sections 162-1 and 162-2.
- Deviations:
- no
- GLP compliance:
- not specified
- Test type:
- laboratory
- Radiolabelling:
- yes
- Remarks:
- Ring-labelled [14C]test material
- Oxygen conditions:
- aerobic/anaerobic
- Soil classification:
- not specified
- Year:
- 1 987
- Soil no.:
- #1
- Soil type:
- sandy loam
- % Clay:
- 13
- % Silt:
- 36
- % Sand:
- 51
- % Org. C:
- 2.4
- pH:
- 5.6
- CEC:
- 5 meq/100 g soil d.w.
- Bulk density (g/cm³):
- 1.15
- % Moisture content:
- 30.7
- Details on soil characteristics:
- SOIL COLLECTION AND STORAGE
- Geographic location: Dane County, Wisconsin.
- Soil preparation: 2 mm sieved - Soil No.:
- #1
- Duration:
- 181 d
- Soil No.:
- #1
- Initial conc.:
- 9.98 other: μg/g
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- CO2 evolution
- Soil No.:
- #1
- Temp.:
- 25 °C
- Details on experimental conditions:
- 1. PRELIMINARY EXPERIMENTS:
A preliminary study was conducted to investigate extraction and analytical procedures for the test material under the test conditions. Preliminary study findings were used to establish the experimental parameters for the definitive study.
Approximately 17 g (dry-weight equivalent) of 2-mm sieved soil was placed in each of two glass containers. The two samples (Nos.1 and 2) were fortified with 200 µL of a fortification solution of 14C-test material in MeOH at a level of 1.31 x 10^7 dpm or 12.3 µg/g (test material/ soil sample). The moisture level of each sample was adjusted to 75 % FMC by adding H2O. Sample Nos. 1 and 2 were extracted with acetonitrile (ACN): acetic acid (HOAc) (99:1) and MeOH: HOAc (99:1), respectively. The radioactivity in the extracts and the extracted soil (oxidised by combustion) was quantitated by LSC. The radioactivity in the extracts was investigated by TLC using four different solvent systems.
Two additional samples (Nos. 3 and 4) were prepared. Approximately 20 g (dry-weight equivalent) of 2-mm sieved soil was added to each of two glass containers. The samples were fortified with 200 µL of a fortification solution (14C-test material in MeOH) at a level of 1.31 x 10^7 dpm or 10.6 µg/g (test material/soil sample). Water (approximately 23 or 29 g), through which nitrogen had been bubbled, was added to each fortified sample. The samples were centrifuged, and the H2O layer was decanted into a separate container. The H2O layer for Sample No. 3 was acidified to a pH of approximately 1 to 2 with phosphoric acid (H3PO4) and extracted with methyl-t-butyl ether. For Sample No. 4, the H2O layer was extracted with dichloromethane (DCM): hydrochloric acid (HCl) (99:1). The soil from each sample was extracted with ACN: HOAc (99:1). The sample extracts and the extracted soil and H2O layers were analysed for radioactivity content.
2. EXPERIMENTAL DESIGN
- Soil condition:
Aerobic Incubation Phase: Air dried (moisture content of weighed soil approximately 17 %.
- Soil (g/replicate):
Aerobic Incubation Phase: 20 dry weight equivalent of 2 mm sieved soil.
- Test apparatus (Type/material/volume):
Aerobic Incubation Phase: The samples were placed in a sealed glass chamber.
- Details of traps for CO2 and organic volatile, if any:
Aerobic Incubation Phase: The sealed chamber was connected to two traps in series for the collection of volatile components; Trap 1 contained ethylene glycol and Trap 2 contained 2-ethoxyethanol: ethanolamine (1:1). An H2O trap was installed after the traps for volatile components to collect solvent vapours; the H2O from this trap was not analysed. A continuous airflow from the chamber to the traps was created by vacuum. The air entering the incubation system was passed through H2O in the chamber bottom at a rate of 35 to 70 mL/minute (except Day 181, where the flow rate pulsed between 0 and 160 mL/minute) to create a humid, aerobic environment. The samples were maintained in a dark, temperature-controlled room at a temperature range of 23.3 ° to 25.6 °C. Water was added to the samples periodically throughout the study to maintain the initial moisture level of the soil (75 % FMC).
Anaerobic Incubation Phase: After 30 days of aerobic incubation, four samples were removed from the incubation apparatus and flooded with H2O to a height of 3 cm above the soil surface. The samples were placed in a sealed glass chamber connected to two traps in series for the collection of volatile components. Trap 1 contained ethylene glycol and Trap 2 contained 2-ethoxyethanol: ethanolamine (1:1). An H2O trap was installed after the traps for volatile components to collect solvent vapors; the H2O from this trap was not analysed. Nitrogen was introduced into the chamber through H2O in the chamber bottom to create a humid, anaerobic environment (air rather than nitrogen was inadvertently introduced into the incubation chamber for the first 7 days after the flooded samples were placed in the chamber). The chamber was kept in a dark, temperature-controlled room at a temperature range of 24.2 ° to 25.6 °C.
Test material application
- Sample Fortification:
Aerobic Incubation Phase: The samples were each fortified with 200 µL of a fortification solution of 14C-test material in MeOH at a level of 9 960.0 dpm or 9.98 µg/g (test material/ soil sample). The concentration and homogeneity of the radioactivity in the fortification solution and the amount of radioactivity applied to the samples were determined by LSC of pre-, mid-, and post-fortification aliquots of the fortification solution. After fortification, nitrogen was gently blown on the soil surface to evaporate the MeOH from the fortification solution. The moisture level of each sample was adjusted to 75 % FMC, and the final weight of the samples was recorded.
3. SAMPLING DETAILS
- Sampling intervals:
Aerobic Incubation Phase: Two samples were analysed for radioactivity at Day 0. Two samples were removed from test conditions at Days 3, 7, 14, 21, 30, 61, 91, and 181. The order of sample removal was determined by a computer randomisation program. The traps were sampled at Days 3, 7, 14, 21, 30, 61, 75, 91, 149, and 181. The total volume of each trapping medium was measured, and duplicate 1.0 mL aliquots were collected for analysis. The traps were emptied and refilled with fresh trapping media.
Anaerobic Incubation Phase: The two samples collected and analysed after 30 days of aerobic incubation were considered to be the Day 0 samples for the anaerobic phase of the study. Two samples were removed from test conditions after 31 and 61 days of anaerobic incubation (61 and 91 days of aerobic/anaerobic incubation, respectively). The order of sample collection was determined by a computer randomisation program. The traps were sampled at Days 31 and 61 (anaerobic incubation time). The total volume of each trapping medium was measured, and duplicate 1.0 mL aliquots were collected for analysis. The traps were emptied and refilled with fresh trapping media.
- Sampling method for soil samples:
Aerobic Incubation Phase: Approximately 40 mL of ACN:HOAc (99:1) was added to the sample container and the soil/ solvent mixture was stirred on a magnetic stir plate for 15 minutes. The soil/ solvent mixture was centrifuged for approximately 15 minutes at 2 000 rpm. The supernatant was decanted. The above procedure was repeated, and the supernatants were combined. The solvent remaining in the soil was evaporated using a gentle stream of nitrogen.
- Method of collection of CO2 and volatile organic compounds:
Aerobic Incubation Phase:
Sample Analysis - ACN: HOAc (99:1) Extract: The radioactivity in the combined ACN: HOAc (99:1) extract was quantitated. Duplicate aliquots (approximately 0.1 to 0.2 g, accurately weighed) of the extracts were analysed by LSC. The distribution of radioactivity for the extracts was determined by TLC. A 100-µL aliquot of the extracts for Days 0, 3, 7, 14, 21, and 30 was applied to a silica gel TLC plate 2 cm from the plate bottom along with a 10-µL aliquot of the non-radiolabelled standard solution of the test material, or 2-chloro-2- methylphenol, or both (only non-radiolabelled test material was applied on Day 21). The standards were applied either on the same TLC plate strip as the extract or on an adjacent strip. The TLC plate was developed in ACN: H2O: ammonium hydroxide (NH4OH) (80:18:2) until the solvent front migrated 18 cm from the plate bottom. The plate was removed from the development tank and air-dried at room temperature. The location of the non-radiolabelled standards was determined by fluorescent quenching under ultraviolet (UV) light. The plate was analysed by a radioactivity scanner and/or autoradiography to determine the location and quantity of the radioactivity.
Sample Analysis - Extracted Soil: The radioactivity in the extracted soil was quantitated. Duplicate aliquots (approximately 0.1 to 0.2 g, accurately weighed) of the soil were oxidised by combustion for 3 minutes. The resulting 14CO2 was trapped in Carbo-Sorb:Perma-Fluor V (1:1) and analysed by LSC.
Sample Analysis - Traps for Volatile Components: The radioactivity content in the traps was quantitated. Duplicate aliquots (1.0 mL) of the trapping medium were analysed in 8 mL of H2O and 10 mL of scintillation cocktail by LSC.
Anaerobic Incubation Phase:
Sample Extraction - H2O Layer: Samples were centrifuged at 2 000 rpm for approximately 10 minutes. The H2O layer was decanted into a separatory funnel and extracted with approximately 40 mL of DCM:HCl (99:1). The organic fraction was drained into a tared container. The extraction procedure was repeated, and the organic fractions were combined. The extracted H2O layer was placed in a separate tared container.
Sample Extraction – Soil: Approximately 40 mL of ACN:HOAc (99:1) was added to the soil in the sample container and the soil/ solvent mixture was stirred on a magnetic stir plate for 15 minutes. The soil/ solvent mixture was centrifuged for approximately 10 minutes at 2 000 rpm. The supernatant was decanted. The above procedure was repeated and the supernatants were combined. The solvent remaining in the soil was evaporated using a gentle stream of nitrogen.
Sample Analysis - DCM:HCl (99:1) Extract of H2O Layer: The radioactivity in the combined DCM:HCl (99:1) extract was quantitated. Duplicate aliquots (approximately 0.2 to 0.3 g, accurately weighed) of the extracts were analysed by LSC.
The distribution of radioactivity for the extracts was determined by TLC. A 200 mL aliquot of the extracts was applied to a silica gel TLC plate along with a 10 μL aliquot of non-radiolabelled test material. The TLC plate was developed in ACN: H2O: NH4OH (80:18:2) and analysed as described in the Sample Analysis - ACN:HOAc (99:1) Extract section for the aerobic phase of the study.
Sample Analysis - Extracted H2O Layer: The radioactivity in duplicate aliquots (approximately 0.2 g, accurately weighed) of the H2O layer after extraction was quantitated by LSC.
Sample Analysis - ACN:HOAc (99:1) Extract of Soil: The radioactivity in duplicate aliquots (approximately 0.1 to 0.2 g, accurately weighed) of the extracts was quantitated by LSC. The distribution of radioactivity for the extracts was determined by TLC as described in the aerobic phase of the study.
Sample Analysis - Extracted Soil: The radioactivity in duplicate aliquots (approximately 0.1 to 0.2 g, accurately weighed) of the extracted soil was quantitated. The aliquots were oxidised by combustion, and the resulting 14CO2 was trapped in Carbo-Sorb:Perma-Fluor V (1 :1) and analysed by LSC.
Sample Analysis - Traps for Volatile Components: The radioactivity in duplicate aliquots (1.00 mL) of the trapping media was quantitated by LSC.
Validation of the Soil Oxidation Procedure: The efficiency of the oxidation procedure was determined. Triplicate aliquots (approximately 0.2, accurately weighed) of the soil were fortified with 3 590 or 44 833 dpm (mean of duplicate aliquots of the fortification solution) of 14C-test material and oxidised by combustion. The resulting 14CO2 was trapped in Carbo-Sorb:Perma-Fluor V (1:1) and quantitated by LSC. The recovered radioactivity (dpm) was divided by the applied radioactivity (dpm) and multiplied by 100 to determine the percentage of recovery. Since the mean recovery values were higher than 95 % (99.3 % and 96.3 % for the low and high radioactivity levels, respectively), radioactivity values for extracted soil were not corrected for oxidation efficiency.
Characterisation of Radioactivity in the ACN:HOAc (99:1) Extracted Soil and Trapping Medium
- Extracted Soil: The radioactivity was characterised in the extracted soil from two samples incubated under aerobic conditions (Day 67) and from two samples incubated under aerobic/ anaerobic conditions (Day 61 of aerobic/ anaerobic incubation or Day 31 of anaerobic incubation). Duplicate aliquots (approximately 5 g, accurately weighed) of the ACN:HOAc (99:1) extracted soil were extracted in a Soxhlet apparatus using MeOH for approximately 12 hours. The radioactivity in duplicate aliquots of the MeOH extracts (approximately 0.7 to 0.8 g, accurately weighed) was quantitated by LSC.
The MeOH-extracted soil aliquots were transferred to tared glass containers. Twenty or 30 mL of 0.5 N sodium hydroxide (NaOH) was added to the containers, and the soil/ NaOH mixtures were stirred overnight on a magnetic stir plate and then centrifuged for 10 minutes at 2 000 rpm. The supernatants were decanted. Five millilitres of H2O was added to the soil and stirred for 30 minutes on a magnetic stir plate. The soil/ H2O mixtures were centrifuged for 10 minutes at 2 000 rpm. The supernatant for the sample was decanted and combined with the above supernatants.
The pH of the combined supernatants was adjusted to approximately 1 with concentrated HCl, causing a precipitate (humic acid) to form. After centrifugation, the supernatants (fulvic acid) were decanted. The humic acid fractions were dissolved in 5 mL of 0.5N NaOH. The radioactivity in the fulvic and humic acid fractions was quantitated by LSC.
- Trapping Medium for Volatile Components: The trapping medium, 2-ethoxyethanol: ethanolamine (1:1), for the Day 30 sampling interval was analysed for the presence of 14CO2. Six aliquots (1.0 mL) were placed in separate scintillation vials. Nine millilitres of H2O was added to each of six vials, and the vials were mixed on a vortex mixer. Three vials were placed on a magnetic stir plate. While stirring, the pH of the vial contents was adjusted to approximately 1 to 2 with concentrated HCl. The headspace in each vial was flushed with nitrogen, and the vials were capped. After acidification, the contents of each vial were divided equally between three vials. The radioactivity in the acidified and nonacidified aliquots was quantitated by LSC. The LSC results of the acidified and nonacidified aliquots were compared; the loss of radioactivity from the acidification process presumably represented 14CO2 evolution.
Calculations
- Percentage of Applied Radioactivity in Sample Matrices: Calculations of the applied radioactivity in the sample matrices were performed using Symphony® Version 1.2 software on an IBM personal (or 100 % IBM compatible) computer.
- Distribution of Radioactivity Applied to the TLC Plate: The distribution of radioactivity on TLC plates of sample extracts was determined from the quantitation of radioactivity peaks by a radioactivity scanner.
- Distribution of Radioactivity in Sample Extracts Expressed as Radioactivity: Applied to the Sample The fraction of applied radioactivity recovered from each sample extract was multiplied by the fraction of radioactivity detected for each peak identified by the radioactivity scanner. The resulting values were expressed as percentages.
- Degradation Half-Life of 14C-Test Material under Aerobic Conditions: The degradation of half-life of 14 C-test material in the samples was calculated by linear regression analysis of the natural log percentage of 14C-test material with time. Calculations were performed using Symphony Version 1.2 software on an IBM personal (or 100 % IBM compatible) computer.
t½ = -(1n 2 / k)
Where:
t½ = Half-life of 14 C-test material
1n 2 = Natural log of two (0.693)
k = Degradation rate constant of 14 C-test material (day^-1)
Residue levels or concentrations of the test material in soil during the reported experimental period were used for estimating the half-life in soil (DT50) as well as the time period required for the degradation of 75 % of the initial residues present (DT75).
The calculation was performed by a validated software program on a PC (Datamini Multiflex 486 DX2, Models for describing the decline of pesticide residues PC Evaluation Programs, Version 2.0 (IVA)) using the 'best fit' option.
Residue levels or concentrations of the test material in soil during the reported experimental period were used for estimating the half-life in soil (DT50). The calculation was performed by a validated software program on a PC (Datamini Multiflex 48 6 DX2) using the 'best fit' as well as the '1st order' option.
Residue levels or concentrations of the test material in soil during the reported experimental period were used for estimating the half-life in soil (DT50). The calculation was performed by a validated software program (Models for describing the decline of pesticide residues)PC Evaluation Programs, Version 2.0 (IVA)) on a PC (Datamini Multiflex 486 DX2) using the 'best fit' as well as the '1st order' option. - Soil No.:
- #1
- DT50:
- 13.26 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 25 °C
- Remarks on result:
- other: St. dev. not reported.
- Soil No.:
- #1
- DT50:
- 17.89 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 25 °C
- Remarks on result:
- other: St. dev. not reported.
- Soil No.:
- #1
- DT50:
- 65.12 d
- Type:
- second order
- Temp.:
- 25 °C
- Remarks on result:
- other: St. dev. not reported.
- Transformation products:
- no
- Details on transformation products:
- The major products of aerobic metabolism were 14CO2 and soil-bound residues. No additional degradation of 14C-test material or formation of products was observed in aerobic samples that were subsequently made anaerobic by flooding them with H2O.
- Evaporation of parent compound:
- not measured
- Volatile metabolites:
- not specified
- Residues:
- not specified
- Details on results:
- PRELIMINARY STUDY
The recovery of applied radioactivity from soil fortified with 14C-test material and extracted three times with ACN:HOAc (99:1) was 94.8 %; 2.4 % of the radioactivity was non-extractable.
The recovery of applied radioactivity from fortified soil extracted three times with MeOH:HOAc (99:1) was 93.8 %; 3.1 % of the radioactivity was non-extractable.
The recoveries of applied radioactivity from two fortified soil samples that were flooded with H2O, were 109.8 % and 112.2 %. The amount of applied radioactivity recovered after three extractions of the H2O layer (acidified to pH 1 to 2 with H3PO4) with methyl-t-butyl ether was 46.0 %; three extractions with DCM:HCl (99:1) recovered 59.2 %. The remaining radioactivity was recovered from the soil by extraction and combustion. In each case, the first extraction was sufficient to recover more than 98 % of the radioactivity present in the H2O layer. The Rf value of test material applied to a TLC plate developed in a solvent system of ACN: H2O: NH4OH (80:18:2) was approximately 0.50.
DEFINTIVE STUDY
- Aerobic Incubation Phase
Material Balance of Radioactivity in Sample Matrices: The radioactivity in the ACN:HOAc (99:1) extract declined from a mean of 104.3 % at Day 0 to 2.4 % at Day 61 and did not change significantly from Days 91 to 181 (2.2 % and 1 .7 %, respectively). Correspondingly, the radioactivity remaining in the extracted soil (soil-bound residues) increased from 4.5 % at Day 0 to 50 % at Day 61, and then declined to 39.2 % and 30.3 % at Days 91 and 181, respectively. The amount of radioactivity in the ethylene glycol trap was 0.2 % or less at any sample interval. The amount of radioactivity detected in the 2-ethoxyethanol:ethanolamine (1:1) trap (determined to be evolved 14CO2) increased from 1.7 % at Day 3 to 47.4 % at Day 61, and reached a maximum of 58.1 % at Day 181. The overall recovery of radioactivity (material balance) for the samples ranged from 108.8 % (Day 0) to 90.3 % (Day 181).
Degradation of 14C-Test Material and Formation of Products: The major component in the ACN:HOAc extract was 14C-test material. The amount of 14C-test material declined from a mean of 102.1 % (Day 0) to 20.1 % (Day 30) and then declined to 2.4 % or less at subsequent sample intervals. Since the total radioactivity in the ACN:HOAc extracts at Days 61, 91, and 181 was 2.4 % or less, no TLC analysis of these extracts was conducted. A minor product, labelled as Peak 1 (mean of 3.5 % of applied radioactivity), was initially observed at Day 21 and declined to a mean value of 0.9 % at Day 30. Cochromatography of non-radiolabelled test material and the radiolabelled peak in the TLC scan was observed. When incubated in soil under aerobic conditions, the major metabolism products of 14C-test material were 14CO2 and soil-bound residues.
Half-Life of 14C-Test Material: The calculated half-life of 14C-test material when incubated in soil under aerobic conditions was determined using linear regression analysis. The half-life of 14C-test material was 13.3 days (y-intercept 4.65, rate constant -0.052, and correlation coefficient -0.988).
- Anaerobic Incubation Phase
Material Balance of Radioactivity in Sample Matrices: The amount of radioactivity recovered in the ACN:HOAc (99:1) extract of soil declined from 22.3 % (anaerobic Day 0) to 8.0 % (anaerobic Day 61). The amount of radioactivity recovered in the DCM:HCl (99:1) extract of the H2O layer was relatively constant at 15.3 % and 17.1 % at anaerobic Days 31 and 61, respectively. Therefore, flooding the soil samples with H2O caused a distribution of radioactivity into the H2O layer. However, the combined radioactivity in the organic extracts of H2O and soil (24.4 % and 25.1 % at anaerobic Days 31 and 61, respectively), was not significantly different from the anaerobic Day 0 value (22.3 %). Approximately 1 % of the applied 14C was not organosoluble and remained in the H2O layer. The soil-bound radioactivity in the anaerobic samples, 37.6 % and 40.5 % (anaerobic Days 31 and 61, respectively), showed no marked differences from the anaerobic Day 0 value (44.1 %). No radioactivity was detected in the ethylene glycol trap. The amount of evolved 14CO2 during anaerobic incubation was small because the anaerobic Day 61 value (32.1 %) showed no significant increase over the anaerobic Day 0 value (30.8 %). The overall material balance for the anaerobic samples was 94.5 % and 98.8 % for anaerobic Days 31 and 61, respectively.
Degradation of 14C-Test Material: A minor product, Peak 1, was observed only in the ACN:HOAc extracts of soil samples; the amount of this product did not exceed 1.1 % of the applied radioactivity. Cochromatography of non-radiolabelled test material and the radiolabelled peak in the TLC scan was observed. No decline in the concentration of 14C-test material or changes in the concentration of Peak 1 was observed in the anaerobic samples.
Half-Life of 14C-Test Material: No degradation of 14C-test material occurred in soil samples that had been incubated for 30 days aerobically and subsequently flooded and incubated under anaerobic conditions. Therefore, no half-life of 14C-test material under anaerobic incubation conditions could be calculated.
Comparison of Aerobic and Anaerobic Incubation
Degradation of 14C-test material: After 61 days of aerobic incubation conditions, approximately 97 % or more of the 14C-test material applied to soil was converted to 14CO2 or soil-bound residues. After 30 days of aerobic incubation, approximately 20 % of the applied radioactivity remained as 14c- test material. At Day 30, four aerobic samples were flooded. The concentration of 14C-test material in the flooded samples remained unchanged during the 61 days of anaerobic incubation. In the same time period under aerobic conditions, 14C-test material decreased from 20 % to less than 3 %. Therefore, flooding the soil inhibited the degradation of the test material.
Formation of Products: At aerobic Day 30/ anaerobic Day 0, the percent values of 14CO2 and soil-bound residues were 30.8 % and 44.0 %, respectively. These values showed no marked increase during the subsequent 61 days of anaerobic incubation. In the same time period, under aerobic conditions the 14CO2 increased to 52.4 %; under aerobic conditions the soil-bound residues increased to 50.01 at Day 61 and then decreased to 39.2 % at Day 91. Aerobic metabolism products were not formed to any great extent when the soil samples were flooded.
Half-Life of 14C-Test Material: A half-life of 13.3 days was calculated for 14C-test material under aerobic incubation conditions. No half-life could be calculated for 14C-test material under anaerobic incubation conditions.
Characterisation of Major Products
Characterisation of Bound Residues: Selected samples from the aerobic and anaerobic phases of the study were extracted with MeOH using a Soxhlet apparatus. The MeOH-extracted soils were further extracted with NaOH to determine the distribution of radioactivity in the fulvic acid, humic acid, and humin fractions of the soil organic matter. The percentage of soil-bound (non-extractable) radioactivity present before the Soxhlet extraction of these soil samples is also included for comparison. Approximately between 2 % to 3 % of the applied radioactivity was recovered from Soxhlet extraction of soil. No further characterisation of the MeOH-soluble radioactivity was attempted. The radioactivity remaining in the Soxhlet-extracted soil was somewhat evenly distributed between fulvic acid, humic acid, and humin fractions of the soil organic matter.
Characterisation of Radioactivity in Trapping Medium: Approximately 89 % of the radioactivity trapped in the 2-ethoxyethanol:ethanolamine trap was lost upon acidification. These data suggest that the radioactivity trapped was 14CO2.
Evaluation of the study results by DT50 (disappearance time 50) and DT75 (disappearance time 75) data: No DT90 value could be estimated using the model for describing the decline of pesticide residues, PC Evaluation Programs, Version 2.0 (IVA).
Residue levels or concentrations of the test material in soil during the reported experimental period were used for estimating the half-life in soil (DT50) using the "best fit" as well as the "1st order" option. The degradation can be described by a first order kinetic (resulting in a DT50 = 17 .89 d) and a square root second order model (best fit option, resulting in a DT50 = 65.12 d). Due to the nature of the data, no DT90 value could be estimated.
Further analysis using the 'best fit' as well as the '1st order' option was conducted. The degradation can be described by a first order kinetic (resulting in a DT50 = 13.26 d) which is also the best fit option. Due to the nature of the data, no DT90 value could be estimated. - Conclusions:
- Under aerobic conditions, 14C-test material degraded and decreased from a mean of 102.1 % (Day 0) to 20.1 % (Day 30), and then to less than approximately 3 % on subsequent sampling intervals. The calculated half-life was 13.3 days. The major products observed were 14CO2 (maximum of 58.1 % of applied radioactivity, Day 181) and soil-bound residues (maximum of 50.0 %, Day 61). No degradation of 14C-test material was observed in soil samples that were flooded with water after 30 days of aerobic incubation and were maintained under anaerobic conditions. The formation of 14CO2 or soil-bound residues was inhibited by anaerobic incubation conditions; therefore, the half-life of 14C-test material under anaerobic incubation conditions could not be calculated. Characterisation of soil-bound residues indicated that less than 3 % of the radioactivity was released by Soxhlet extraction with methanol. The bulk of the radioactivity was distributed into the fulvic acid, humic acid, and humin fractions of the soil organic matter.
- Executive summary:
The soil metabolism of the test material was studied under aerobic incubation conditions and subsequent aerobic/anaerobic incubation conditions using a Fox sandy loam soil (Dane County, Wisconsin). The study was conducted in accordance with the Environmental Protection Agency Pesticide Assessment Guidelines, Subdivision N, Sections 162-1 and 162-2.
Soil samples were fortified with 14C-test material at 9.98 ppm (µg/g) and placed in a glass chamber maintained at 25 °C ± 2 ° in a dark, temperature-controlled room. The glass chamber was connected to traps containing ethylene glycol and 2-ethoxyethanol:ethanolamine (1: 1) for the collection of organic volatiles and carbon dioxide (CO2), respectively. Air was passed through the system continuously to maintain aerobic conditions. Duplicate samples were collected for analysis on Days 0, 3, 7, 14, 21, 30, 61, 91, and 181 of aerobic incubation. On Day 30, four aerobic samples were flooded with water and incubated similarly, except that nitrogen was passed through the system.
Duplicate anaerobic samples were collected for analysis 31 and 61 days after flooding. Under aerobic conditions, 14C-test material degraded and decreased from a mean of 102.1 % (Day 0) to 20.1 % (Day 30), and then to less than approximately 3 % on subsequent sampling intervals. The calculated half-life was 13.3 days. The major products observed were 14CO2 (maximum of 58.1 % of applied radioactivity, Day 181) and soil-bound residues (maximum of 50.0 %, Day 61). A minor product (Peak 1; maximum of 3.5 %, Day 21) was also observed. No degradation of 14C-test material was observed in soil samples that were flooded with water after 30 days of aerobic incubation and were maintained under anaerobic conditions. The formation of 14CO2 or soil-bound residues was inhibited by anaerobic incubation conditions; therefore, the half-life of 14C-test material under anaerobic incubation conditions could not be calculated. Characterisation of soil-bound residues indicated that less than 3 % of the radioactivity was released by Soxhlet extraction with methanol. The bulk of the radioactivity was distributed into the fulvic acid, humic acid, and humin fractions of the soil organic matter.
- Endpoint:
- biodegradation in soil, other
- 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:
- The degradation of ring-labelled [14C]test material was investigated in three soil types to determine whether 4-chloro-2-methylphenol could be isolated as a degradation product.
- GLP compliance:
- no
- Test type:
- laboratory
- Radiolabelling:
- yes
- Remarks:
- Ring-labelled [14C]test material
- Oxygen conditions:
- aerobic
- Soil classification:
- not specified
- Year:
- 1 983
- Soil no.:
- #1
- Soil type:
- clay
- Soil no.:
- #2
- Soil type:
- clay loam
- Soil no.:
- #3
- Soil type:
- sandy loam
- Details on soil characteristics:
- The composition and physical characteristics of the clay, clay loam and sandy loam in this study have been described in other studies.
- Soil No.:
- #1
- Duration:
- 20 d
- Soil No.:
- #2
- Duration:
- 20 d
- Soil No.:
- #3
- Duration:
- 20 d
- Soil No.:
- #1
- Initial conc.:
- 50 other: µL, 50 µg herbicide
- Based on:
- test mat.
- Soil No.:
- #2
- Initial conc.:
- 50 other: µL, 50 µg herbicide
- Based on:
- test mat.
- Soil No.:
- #3
- Initial conc.:
- 50 other: µL, 50 µg herbicide
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- radiochem. meas.
- Soil No.:
- #1
- Temp.:
- 20 °C
- Soil No.:
- #2
- Temp.:
- 20 °C
- Soil No.:
- #3
- Temp.:
- 20 °C
- Details on experimental conditions:
- EXPERIMENTAL DESIGN
- Soil preincubation conditions: Duplicate samples (50 g) of the three soil types at 85 % of their field capacity moisture levels were incubated in the dark for 7 days at 20 °C in Bartha and Pramer flasks before being treated with the [14C]-test material solution (50 µL, 50 µg herbicide).
Because the boiling point of this phenol has been reported to be 222 – 225 °C at 760 mm, the experiments were conducted in Bartha and Pramer flasks to reduce the volatility losses of degradation products during the soil incubation period.
After treatment the flasks were incubated in the dark at 20 °C for 20 days before extraction and analysis.
- No. of replication treatments: Duplicate
- Test apparatus: Bartha and Pramer flasks.
- Details of traps for CO2 and organic volatile, if any: 0.2 N sodium hydroxide solution (25 mL) was added to the side arm of the flasks to absorb [14C]-carbon dioxide evolved; the solution being replaced fresh every second day.
- Identity and concentration of co-solvent: The recovery of the test material from the three soils under test using aqueous acidic acetonitrile is over 95 %.
Experimental conditions
- Moisture maintenance method: The three soil types were at 85 % of their field capacity moisture levels.
- Continuous darkness: Yes
SAMPLING DETAILS
For extraction, the soil from each duplicate treatment flask was placed in a 250 mL glass-stoppered flask and shaken on a wrist-action shaker for 1 h with sufficient 20 % aqueous acetonitrile containing 2 % glacial acetic acid so that the total volume of extractant together with the water present in the soil was equivalent to 100 mL. Following centrifugation at 2 000 g for 4 minutes aliquots (5 mL) of the supernatant were assayed for radioactivity extracted. A further portion (25 mL) of the supernatant was added to 5 % aqueous sodium chloride solution (100 mL) containing N hydrochloric acid (2 mL) and vigorously shaken with dichloromethane (10 mL). The organic phase was dried over anhydrous sodium chloride and gently evaporated at room temperature to approximately 0.25 mL using a stream of dry nitrogen. The evaporated extracts were examined by thin-layer chromatographic and radiochemical techniques to detect and quantify the various [14C]-components present.
- Sampling method for soil samples: The radioactivity in the various solutions was measured by adding aliquots to scintillation solution (15 mL) consisting of an equivalent mixture of toluene and 2-methoxymethanol containing 0.4 % of PPO and 0.01 % of POPOP. Samples were counted on Packard TRICARB 300C scintillation spectrometer, with counting efficiencies being determined using an external [^226Ra] standard. - Remarks on result:
- not measured/tested
- Soil No.:
- #1
- % Degr.:
- > 70
- Parameter:
- radiochem. meas.
- Sampling time:
- 20 d
- Soil No.:
- #2
- % Degr.:
- > 70
- Parameter:
- radiochem. meas.
- Sampling time:
- 20 d
- Soil No.:
- #3
- % Degr.:
- > 70
- Parameter:
- radiochem. meas.
- Sampling time:
- 20 d
- Transformation products:
- yes
- No.:
- #1
- Details on transformation products:
- In addition to the [14C]-test material, small amounts of single radioactive compound were recovered from all soils whose Rf values in a four solvents were identical to those for 4-chloro-2-methylphenol. There was no trace of any compound with a higher Rf value that could be attributed to 4-chloro-2-methylanisole.
- Evaporation of parent compound:
- yes
- Volatile metabolites:
- not specified
- Residues:
- not specified
- Details on results:
- Breakdown of the ring-labelled [14C]test material was rapid in all soils, being over 70 % complete in 20 days. During this time, between 20 and 29 % of the soil-applied radioactivity was released as [14C]-carbon dioxide, indicating that fission of the aromatic nucleus was occurring.
Between 48 and 52 % of the total activity could be attributed to radioactively labelled compounds.
It was assumed that at least some of the remaining activity had been converted into soil organic matter, since this is known to occur with carbon dioxide or carbon-containing fragments formed by breakdown of herbicide being incorporated into the soil biomass.
Losses on [14C]-carbon dioxide and the [14C]-phenol could have occurred as a result of volatilisation from the Bartha and Pramer flasks during the daily sampling of the alkaline solution or during exchange of sodium hydroxide with fresh solution. Similar losses of the phenol could also have occurred during work up of the soil extracts, especially during the evaporation stage of the dichloromethane solutions with nitrogen.
Although mass spectral data are necessary to confirm the identity of the degradation product isolated, the TLC results strongly indicate that 4-chloro-2-methylphenol is formed in soils from the test material. There is no indication that the 4-chloro-2-methylphenol underwent methylation to the corresponding anisole. - Conclusions:
- Under the conditions of the study breakdown of the ring-labelled [14C]test material was rapid in all soils, being over 70 % complete in 20 days. During this time, between 20 and 29 % of the soil-applied radioactivity was released as [14C]-carbon dioxide, indicating that fission of the aromatic nucleus was occurring. In addition to the [14C]-test material, small amounts of single radioactive compound were recovered from all soils whose Rf values in a four solvents were identical to those for 4-chloro-2-methylphenol. There was no trace of any compound with a higher Rf value that could be attributed to 4-chloro-2-methylanisole. Between 48 and 52 % of the total activity could be attributed to radioactively labelled compounds.
- Executive summary:
The degradation of ring-labelled [14C]-test material was investigated in three soil types to determine whether 4-chloro-2-methylphenol could be isolated as a degradation product. Because the boiling point of this phenol has been reported to be 222 – 225 °C at 760 mm, the experiments were conducted in Bartha and Pramer flasks to reduce the volatility losses of degradation products during the soil incubation period.
Breakdown of the ring-labelled [14C]test material was rapid in all soils, being over 70 % complete in 20 days. During this time, between 20 and 29 % of the soil-applied radioactivity was released as [14C]-carbon dioxide, indicating that fission of the aromatic nucleus was occurring. In addition to the [14C]-test material, small amounts of single radioactive compound were recovered from all soils whose Rf values in a four solvents were identical to those for 4-chloro-2-methylphenol. There was no trace of any compound with a higher Rf value that could be attributed to 4-chloro-2-methylanisole. Between 48 and 52 % of the total activity could be attributed to radioactively labelled compounds.
It was assumed that at least some of the remaining activity had been converted into soil organic matter, since this is known to occur with carbon dioxide or carbon-containing fragments formed by breakdown of herbicide being incorporated into the soil biomass.
Losses on [14C]-carbon dioxide and the [14C]-phenol could have occurred as a result of volatilisation from the Bartha and Pramer flasks during the daily sampling of the alkaline solution or during exchange of sodium hydroxide with fresh solution. Similar losses of the phenol could also have occurred during work up of the soil extracts, especially during the evaporation stage of the dichloromethane solutions with nitrogen.
Although mass spectral data are necessary to confirm the identity of the degradation product isolated, the TLC results strongly indicate that 4-chloro-2-methylphenolis formed in soils from the test material. There is no indication that the 4-chloro-2-methylphenol underwent methylation to the corresponding anisole.
Referenceopen allclose all
Distribution and Recovery of 14-C Labelled Test Material After Three Months, Expressed as Percentages of the Initial Amounts (100 % = 1 mg/L). Artihmatic Mean of the Triplicates.
Herbicide |
Soil Type |
Air (CO2) |
Sediment + Water |
Water |
Recovery |
Test material |
Clay |
75.4 |
21.3b |
2.2a1 |
96.7 |
Peat |
62.7 |
15.8 |
1.7a1 |
78.4 |
|
Sand |
55.5 |
36.7 |
32.5 |
92.2 |
a1: Due to contamination by NaOH in some test bottles at the end of the test, the mean activity in the water is not in all cases based on three replicates: One test bottle only.
b: In one test bottle the degradation was delayed considerably, leaving high residues in the test system. In the other replicates the mean recovery of CO2 was 81.5 % and the mean residual activity in the water and the sediument was 15.7 %.
Breakdown of Test Material in Soils (2 µg/g) at 20 ± 1 °C and 85 % Field Capacity
Soil type |
Remaining (%)* |
Half-life (days) |
||
7 days |
14 days |
21 days |
||
Clay loam |
58 |
36 |
17 (90) |
9 ± 1 |
Heavy clay |
60 |
35 |
10 (90) |
8 ± 2 |
Sandy loam |
65 |
24 |
8 (87) |
7 ± 2 |
*Average of 2 replicates, corrected for appropriate recovery factor
Figures in parentheses represent percentage recovered from soils at 20 ± 1 °C and 15 % of field capacity moisture (air dried)
Kinetic Data for the Dissipation of the Test Materials in the Three Soils
Soil Type |
Test Material |
Half Life (Days) |
Kt x 10^3 (Days^-1) |
R* |
Silt loam |
Test material |
15 |
43 |
0.989 |
R-test material |
12 |
58 |
0.996 |
|
S-test material |
21 |
33 |
0.972 |
|
Sandy loam |
Test material |
11 |
63 |
0.980 |
R-test material |
8 |
86 |
0.992 |
|
S-test material |
12 |
56 |
0.994 |
|
Clay loam |
Test material |
50 |
14 |
0.990 |
R-test material |
77 |
9 |
0.957 |
|
S-test material |
32 |
22 |
0.956 |
* Significance at P < 0.01
R/S Ratio Values at Different Incubation Days for Racemic Test Material in the Three Soils
Incubation Time (Days) |
|||||||
Soil Type |
0 |
1 |
3 |
7 |
21 |
42 |
84 |
Silt loam |
0.92 |
0.94 |
0.92 |
0.89 |
0.58 |
nd |
nd |
Sandy loam |
1.08 |
1.16 |
1.15 |
0.71 |
0.6 |
nd |
nd |
Clay loam |
1.18 |
1.08 |
0.86 |
0.95 |
0.91 |
1.00 |
3.2 |
nd: Under detection limits.
Kinetic Data for the Dissipation of Test Material in the Three Soils
Soil Type |
Half Life (Days) |
Kt x 10^3 (Days^-1) |
R* |
Silt loam |
4 |
157 |
0.987 |
Sandy loam |
7 |
97 |
0.915 |
Clay loam |
40 |
17 |
0.997 |
* Significance at P < 0.01 in most cases.
R/S Ratio Values at Different Incubation Days for the Test Material in the Three Soils
Incubation Time (days) |
|||||
Soil Type |
0 |
3 |
7 |
21 |
84 |
Silt loam |
35.9 |
19.8 |
5.5 |
nd |
nd |
Sandy loam |
32.3 |
13.8 |
5.2 |
nd |
nd |
Clay loam |
47.6 |
22.9 |
13.2 |
9.5 |
4.7 |
nd: Under detection limits.
Individual Distribution of the Applied Radioactivity among the Matrices of the Samples Incubated under Aerobic Conditions
Sample Interval (Day) |
Sample Number |
Applied Radioactivity (%) |
||||
ACN:HOAc (99:1) Extract |
Extracted Soil |
Traps for Volatile Components |
Total |
|||
Ethylene Glycol |
2-Ethoxyethanol: Ethanolamine (1:1) |
|||||
0 |
A |
103.4 |
5.2 |
N/A |
N/A |
108.6 |
B |
105.2 |
3.7 |
108.9 |
|||
3 |
3 |
85.4 |
15.0 |
<0.1 |
1.7 |
102.1 |
21 |
84.7 |
12.8 |
992 |
|||
7 |
8 |
81.2 |
18.2 |
<0.1 |
3.8 |
103.2 |
5 |
76.0 |
22.9 |
102.7 |
|||
14 |
14 |
55.9 |
32.4 |
<0.1 |
11.2 |
99.5 |
1 |
56.4 |
35.6 |
103.2 |
|||
21 |
4 |
37.7 |
35.7 |
<0.1 |
20.4 |
93.8 |
13 |
49.1 |
36.4 |
105.9 |
|||
30 |
23 |
20.6 |
42.4 |
<0.1 |
30.8 |
93.8 |
10 |
23.9 |
45.5 |
100.2 |
|||
61 |
17 |
2.4 |
42.8 |
<0.1 |
47.4 |
92.6 |
16 |
2.4 |
57.1 |
106.9 |
|||
75 |
N/A |
N/A |
N/A |
0.2 |
50.0 |
N/A |
N/A |
N/A |
N/A |
N/A |
|||
91 |
11 |
2.6 |
35.5 |
0.2 |
52.4 |
90.7 |
18 |
1.8 |
42.9 |
97.3 |
|||
149 |
N/A |
N/A |
N/A |
0.2 |
58.0 |
N/A |
N/A |
N/A |
N/A |
N/A |
|||
181 |
12 |
2.1 |
29.2 |
0.2 |
58.1 |
89.6 |
24 |
1.3 |
31.4 |
91.0 |
|||
N/A: Not applicable
Mean Distribution of the Applied Radioactivity Among the Matrices of the Samples Incubated Under Aerobic Conditions*
Sample Interval (Day) |
Applied Radioactivity (%) |
||||
ACN:HOAc (99:1) Extract |
Extracted Soil |
Traps for Volatile Components |
Total** |
||
Ethylene Glycol |
2-Ethoxyethanol: Ethanolamine (1:1) |
||||
0 |
104.3 |
4.5 |
N/A |
N/A |
108.8 |
3 |
85.1 |
13.9 |
<0.1 |
1.7 |
100.7 |
7 |
78.6 |
20.6 |
<0.1 |
3.8 |
103.3 |
14 |
56.2 |
34.0 |
<0.1 |
11.2 |
101.4 |
21 |
43.4 |
36.1 |
<0.1 |
20.4 |
99.9 |
30 |
22.3 |
44.0 |
<0.1 |
30.8 |
97.2 |
61 |
2.4 |
50.0 |
<0.1 |
47.4 |
99.8 |
75 |
N/A |
N/A |
0.2 |
50.0 |
N/A |
91 |
2.2 |
39.2 |
0.2 |
52.0 |
94.0 |
149 |
N/A |
N/A |
0.2 |
58.0 |
N/A |
181 |
1.7 |
30.3 |
0.2 |
58.1 |
90.3 |
N/A: Not applicable
*: Mean of dup1icate values in table above.
**: Sum of the mean values in this table.
Individual and Mean Distribution of Radioactivity Applied to the TLC Plates for the ACN:HOAc (99:1) Extract of the Samples Incubated under Aerobic Conditions*
Sample Interval (Day) |
Sample Number |
Radioactivity Applied to TLC Plate (%) |
|||
Test Material |
Peak 1 |
||||
Individual |
Mean |
Individual |
Mean |
||
0 |
A |
97.9 |
97.9 |
ND |
ND |
B |
97.9 |
ND |
|||
3 |
3 |
96.4 |
96.8 |
ND |
ND |
21 |
97.2 |
ND |
|||
7 |
8 |
98.6 |
98.2 |
ND |
ND |
5 |
97.7 |
ND |
|||
14 |
14 |
96.8 |
96.4 |
ND |
ND |
1 |
96.0 |
ND |
|||
21 |
4 |
94.1 |
87.8 |
ND |
7.1 |
13 |
81.4 |
14.2 |
|||
30 |
23 |
88.0 |
90.3 |
3.8 |
4.0 |
10 |
92.6 |
4.2 |
|||
*: TLC Plates were developed in ACN: H2O:NH4OH (80:18:2)
ND: Not detected
Individual and Mean Distribution of Radioactivity Expressed as the Percentage of Radioactivity Applied to the Samples for the ACN:HOAc (99:1) Extract of the Samples Incubated under Aerobic Conditions*
Sample Interval (Day) |
Sample Number |
Radioactivity Applied to TLC Plate (%) |
|||
Test Material |
Peak 1 |
||||
Individual |
Mean |
Individual |
Mean |
||
0 |
A |
101.2 |
102.1 |
ND |
ND |
B |
103.0 |
ND |
|||
3 |
3 |
82.3 |
82.3 |
ND |
ND |
21 |
82.3 |
ND |
|||
7 |
8 |
80.1 |
77.2 |
ND |
ND |
5 |
74.3 |
ND |
|||
14 |
14 |
54.1 |
54.1 |
ND |
ND |
1 |
54.1 |
ND |
|||
21 |
4 |
35.5 |
37.8 |
ND |
3.5 |
13 |
40.0 |
7.0 |
|||
30 |
23 |
18.1 |
20.1 |
0.8 |
0.9 |
10 |
22.1 |
1.0 |
|||
*: Percentage of radioactivity recovered from TLC plate multiplied by the applied radioactivity recovered from ACN:HOAc (99:1) extract of the sample divided by 100.
ND: Not detected
Data for Calculation of Degradation Half-Life of 14C-Test Material Measured in the ACN:HOAc (99:1) Extract of the Samples Incubated under Aerobic Conditions
Sample Interval (Day) |
Sample Number |
Test Material |
|
Applied Radioactivity (%) |
Natural Log Percent of Applied Radioactivity |
||
0 |
A |
101.2 |
4.617 |
B |
103.0 |
4.635 |
|
3 |
3 |
82.3 |
4.410 |
21 |
82.3 |
4.410 |
|
7 |
8 |
80.1 |
4.383 |
5 |
74.3 |
4.308 |
|
14 |
14 |
54.1 |
3.991 |
1 |
54.1 |
3.991 |
|
21 |
4 |
35.5 |
3.570 |
13 |
40.0 |
3.689 |
|
30 |
23 |
18.1 |
2.896 |
10 |
22.1 |
3.096 |
|
Individual Distribution of the Applied Radioactivity in the Matrices of the Samples Incubated under Aerobic/Anaerobic Conditions
Sample Interval (Day)* |
Sample Number |
Applied Radioactivity (%) |
||||||
Soil |
H2O Layer |
Traps for Volatile Components |
Total |
|||||
ACN: HOAc (99:1) Extract |
Extracted Soil |
DCM:HCl (99:1) Extract |
Extracted H2O Layer |
Ethylene Glycol |
2E:E** |
|||
30 [0] |
23 |
20.6 |
42.4 |
N/A |
N/A |
<0.1 |
30.8 |
93.8 |
10 |
23.9 |
45.5 |
N/A |
N/A |
100.2 |
|||
61 [31] |
19 |
9.4 |
39.3 |
15.1 |
0.9 |
<0.1 |
31.6 |
96.3 |
20 |
8.8 |
35.9 |
15.5 |
0.9 |
92.7 |
|||
91 [61] |
9 |
7.5 |
42.1 |
15.7 |
1.0 |
<0.1 |
32.1 |
98.4 |
15 |
8.4 |
38.9 |
18.5 |
1.1 |
99.0 |
|||
N/A: Not applicable
*: Sample interval expressed as the length of aerobic/ anaerobic and anaerobic incubation time; values in brackets [ ] represent anaerobic incubation time.
**: 2-Ethoxyethanol: ethanolamine (1:1)
Mean Distribution of the Applied Radioactivity in the Matrices of the Samples Incubated under Aerobic/Anaerobic Conditions*
Sample Interval (Day)* |
Applied Radioactivity (%) |
||||||
Soil |
H2O Layer |
Traps for Volatile Components |
Total†† |
||||
ACN: HOAc (99:1) Extract |
Extracted Soil |
DCM:HCl (99:1) Extract |
Extracted H2O Layer |
Ethylene Glycol |
2E:E† |
||
30 [0] |
22.3 |
44.0 |
N/A |
N/A |
<0.1 |
30.8 |
97.1 |
61 [31] |
9.1 |
37.6 |
15.3 |
0.9 |
<0.1 |
31.6 |
94.5 |
91 [61] |
8.0 |
40.5 |
17.1 |
1.1 |
<0.1 |
32.1 |
98.8 |
N/A: Not applicable
*Mean of duplicate values above.
**: Sample interval expressed as the length of aerobic/anaerobic and anaerobic incubation time; values in brackets [ ] represent anaerobic incubation time.
†:2E:E = 2-ethoxyethanol:ethanolamine (1:1).
††: Sum of the mean values in this table.
Individual and Mean Distribution of Radioactivity Applied to the TLC Plates for the DCM:HCl (99:1) Extract of the H2O Layer from the Samples Incubated under Aerobic/Anaerobic Conditions*
Sample Interval (Day)** |
Sample Number |
Radioactivity Applied to TLC Plate (%) |
|||
Test Material |
Peak 1 |
||||
Individual |
Mean |
Individual |
Mean |
||
30 [0] |
N/A |
N/A |
N/A |
N/A |
N/A |
N/A |
N/A |
N/A |
|||
61 [31] |
19 |
97.6 |
96.8 |
ND |
ND |
20 |
96.0 |
ND |
|||
91 [61] |
9 |
104.6 |
103.2 |
ND |
ND |
15 |
101.7 |
ND |
|||
N/A: Not applicable
ND: Not detected
*: TLC plates were developed in ACN: H2O: NH4OH (80:18:2)
**: Sample interval expressed as the length of aerobic/anaerobic and anaerobic incubation time; values in brackets [ ] represent anaerobic incubation time.
Individual and Mean Distribution of Radioactivity Applied to the TLC Plates for the ACN:H0Ac (99:1) Extract of the Soil from the Samples Incubated under Aerobic/Anaerobic Conditions*
Sample Interval (Day)** |
Sample Number |
Radioactivity Applied to TLC Plate (%) |
|||
Test Material |
Peak 1 |
||||
Individual |
Mean |
Individual |
Mean |
||
30 [0] |
23 |
88. |
90.3 |
3.8 |
4.0 |
10 |
92.6 |
4.2 |
|||
61 [31] |
19 |
77.2 |
79.3 |
11.6 |
11.7 |
20 |
81.3 |
11.7 |
|||
91 [61] |
9 |
84.5 |
83.6 |
15.0 |
13.7 |
15 |
82.7 |
12.4 |
|||
*: TLC plates were developed in ACN:H2O:NH4OH (80:18:2).
**: Sample interval expressed as the length of aerobic/anaerobic and anaerobic incubation time; values in brackets [ ] represent anaerobic incubation time.
Individual and Mean Distribution of Radioactivity Expressed as the Percentage of Radioactivity Applied to the Samples in the DCM:HCl (99:1) Extract of the H2O Layer from the Samples Incubated under Aerobic/Anaerobic Conditions*
Sample Interval (Day)** |
Sample Number |
Radioactivity Applied to Sample (%) |
|||
Test Material |
Peak 1 |
||||
Individual |
Mean |
Individual |
Mean |
||
30 [0] |
N/A |
N/A |
N/A |
N/A |
N/A |
N/A |
N/A |
N/A |
|||
61 [31] |
19 |
14.7 |
14.8 |
ND |
ND |
20 |
14.9 |
ND |
|||
91 [61] |
9 |
16.4 |
17.6 |
ND |
ND |
15 |
18.8 |
ND |
|||
N/A: Not applicable
ND: Not detected
*: Percentage of radioactivity recovered from TLC plate multiplied by the applied radioactivity recovered from the DCM:HCl (99:1) extract of the H2o layer divided by 100.
**: Sample interval expressed as the length of aerobic/anaerobic and anaerobic incubation time; values in brackets [ ] represent anaerobic incubation time.
Individual and Mean Distribution of Radioactivity Expressed as the Percentage of Radioactivity Applied to the Samples in the ACN:HOAc (99:1) Extract of the Soil from the Samples Incubated under Aerobic/Anaerobic Conditions*
Sample Interval (Day)** |
Sample Number |
Radioactivity Applied to Sample (%) |
|||
Test Material |
Peak 1 |
||||
Individual |
Mean |
Individual |
Mean |
||
30 [0] |
23 |
18.1 |
20.1 |
0.8 |
0.9 |
10 |
22.1 |
1.0 |
|||
61 [31] |
19 |
7.3 |
7.2 |
1.1 |
1.1 |
20 |
7.2 |
1.0 |
|||
91 [61] |
9 |
6.3 |
6.6 |
1.1 |
1.1 |
15 |
6.9 |
1.0 |
|||
*: Percentage of radioactivity recovered from TLC plate multiplied by the applied radioactivity recovered from the ACN:HOAc (99:1) extract of the soil divided by 100.
**: Sample interval expressed as the length of aerobic/anaerobic and anaerobic incubation time; values in brackets [ ] represent anaerobic incubation time.
Summary of the Mean Distribution of Radioactivity Expressed as the Percentage of Radioactivity Applied to the Samples for the Extracts of the H2O Layer and Soil of the Samples Incubated under Aerobic/Anaerobic Conditions
Sample Interval (Day)** |
Radioactivity Applied to Sample (%) |
|
14C-Test Material |
Peak 1 |
|
30 [0] |
20.1 |
0.9 |
61 [31] |
22.0 |
1.1 |
91 [61] |
24.2 |
1.1 |
*: Sum of mean values
**: Sample interval expressed as the length of aerobic/anaerobic and anaerobic incubation time; values in brackets [ ] represent anaerobic incubation time .
Individual Distribution of the Radioactivity among Fractions of the Extracted Soi1 from Selected Samples Incubated Under Aerobic and Aerobic/Anaerobic Conditions
Sample Interval (Day)* |
Sample Number |
Applied Radioactivity (%) |
||||
Extracted Soil† |
Extracted Soil Fractions |
|||||
MeOH Extract (Soxhlet) |
Fulvic Acid |
Humic Acid |
Humin†† |
|||
Aerobic Samples |
||||||
61 |
16 |
57.1 |
2.0 |
15.0 |
12.1 |
28.0 |
17 |
42.8 |
1.9 |
14.5 |
14.6 |
11.8 |
|
Aerobic/ Anaerobic Samples |
||||||
61 [31] |
19 |
39.3 |
1.7 |
12.9 |
10.5 |
14.2 |
20 |
35.9 |
2.6 |
11.0 |
10.1 |
12.2 |
|
*: Sample interval for the aerobic/anaerobic samples expressed as the length of aerobic/anaerobic and anaerobic incubation time; values in brackets [ ] represent anaerobic incubation time.
†: Values from the tables above for aerobic and anaerobic samples.
††: Percentage of applied radioactivity in the extracted soil minus the sum of the applied radioactivity in the MeOH extract, and fulvic and humic acid fractions.
Radiopurity and Stability of the Test Material as Determined by HLA
The radiopurity of 14C-test material was determined at the test facility by TLC analysis. An aliquot of 14C-test material in acetone and a non-radiolabelled test material standard in MeOH were applied to a silica-gel TLC plate. The TLC plate was developed in ACN:H2O:NH4OH (80:18:2) and analysed by a radioactivity scanner. The non-radiolabelled test material standard comigrated with 14C-test material. The radiopurity of 14C-test material was 96.9 %. As a measure of stability the radiopurity of 14C-test material in the fortification solution (14C-test material in MeOH) was determined by TLC analysis using the chromatographic conditions described above. Radiopurity was determined after completion of the sample fortification. The non-radiolabelled test material standard comigrated with 14C-test material. The radiopurity of 14C-test material in the fortification solution was 97.3 %. The radiopurity value (97.3 %) was compared with the Sponsor-determined radiopurity value (96.3 % by HPLC). The 14C-test material was stable (within 5 % of the Sponsor-determined radiopurity) in MeOH.
Degradation of Active Ingredient in Soil
t (d) |
R (%) |
0.0 |
101.2 |
0.0 |
103.0 |
3.0 |
82.3 |
3.0 |
82.3 |
7.0 |
80.1 |
7.0 |
74.3 |
14.0 |
54.1 |
14.0 |
54.1 |
21.0 |
35.5 |
21.0 |
40.0 |
30.0 |
18.1 |
30.0 |
22.1 |
t (d) = Sampling date in days
R (%) = % of active ingredient present (nominal concentration on day 0 = 100 %)
The degradation can be described by a first order kinetic (resulting in a DT50 = 13.26 d) which is also the best fit option.
Due to the nature of data, no DT90 value could be estimated.
Degradation of Active Ingredient in Soil (1st Order, Best Fit)
t(d) |
R (%) |
0.0 |
101.2 |
0.0 |
103.0 |
3.0 |
82.3 |
3.0 |
82.3 |
7.0 |
80.1 |
7.0 |
74.3 |
t(d) = Sampling date in days
R (%) = % of active ingredient present on Day 0 = 100 %
Degradation of Active Ingredient in Soil (1stOrder, Best Fit)
t(d) |
R (%) |
0.0 |
101.2 |
0.0 |
103.0 |
3.0 |
82.3 |
3.0 |
82.3 |
7.0 |
80.1 |
7.0 |
74.3 |
14.0 |
54.1 |
14.0 |
54.1 |
21.0 |
35.5 |
21.0 |
40.0 |
30.0 |
18.1 |
30.0 |
22.1 |
t(d) = Sampling date in days
R (%) = % of active ingredient present on Day 0 = 100 %
Radioactivity Recovered from Soils Treated with 1 µg/g[ring-U-^14C]-Test Material Following Incubation at 20 °C for 20 Days.
Soil |
% Applied Radioactivity Extracted as* |
|||
Test Material |
4-Chloro-2-methylphenol |
CO2 |
Total |
|
Clay |
30 |
2 |
20 |
52 |
Clay loam |
21 |
3 |
26 |
5 |
Sandy loam |
16 |
3 |
29 |
48 |
* Average from two replicates.
Description of key information
Key Study: Romero (2001)
It can be concluded that dissipation rates of racemic and enantiomeric forms are influenced by the soil properties.
The enantiomers were found to degrade at different rates in three different soils. The Renantiomer degraded faster in silt and sandy loam soils, while in the clay loam soil the opposite occured. The R-enantiomer was partially converted into its S-enatiomer. The addition of organic matter (peat) to the soils changed the degradation rates of the enantiomers, even modifying the enatioselectivity in the clay loam soil.
Biological degradation is the most probable cause of the enantioselective disappearance of the racemic test material. It is known that the enantiocomposition of a chemical can only be changed by a biological process.
Supporting Study: Smith & Hayden (1981)
The test material, as a commonly used phenoxyalkanoic acid herbicide, appears to be degraded rapidly in all three Saskatchewan soils described.
Supporting Study: Lindholm et al. (1982)
Under the conditions of the study the test material degraded fairly rapidly both in soil treated with 5 mg and in the soil with 15 mg test material/ kg soil. However the study was made under conditions that favoured microbial degradation. The soil was periodically analysed for the presence of a degradation product, however it was not detected in any sample of the soil treated with the test material.
Supporting Study: Balk (1985)
Under the conditions of this study, degradation of the test material starts slowly, but after three weeks the major part of 14-CO2 was recovered. Additionally, although significant water residues are found in the sand system, the test material proved to be easily biodegradable in the clay and peat systems.
Supporting Study: Müller and Buser (1997)
Under the conditions of the study, the test material was determined was readily degraded in soils to levels ≤ 10 % of the initial concentration after 22 - 35 days of incubation. Degradation was primarily biologically mediated.
Supporting Study: Saxena (1988)
Under aerobic conditions, 14C-test material degraded and decreased from a mean of 102.1 % (Day 0) to 20.1 % (Day 30), and then to less than approximately 3 % on subsequent sampling intervals. The calculated half-life was 13.3 days. The major products observed were 14CO2 (maximum of 58.1 % of applied radioactivity, Day 181) and soil-bound residues (maximum of 50.0 %, Day 61). No degradation of 14C-test material was observed in soil samples that were flooded with water after 30 days of aerobic incubation and were maintained under anaerobic conditions. The formation of 14CO2 or soil-bound residues was inhibited by anaerobic incubation conditions; therefore, the half-life of 14C-test material under anaerobic incubation conditions could not be calculated. Characterisation of soil-bound residues indicated that less than 3 % of the radioactivity was released by Soxhlet extraction with methanol. The bulk of the radioactivity was distributed into the fulvic acid, humic acid, and humin fractions of the soil organic matter.
Supporting Study: Smith (1985)
Under the conditions of the study breakdown of the ring-labelled [14C]test material was rapid in all soils, being over 70 % complete in 20 days. During this time, between 20 and 29 % of the soil-applied radioactivity was released as [14C]-carbon dioxide, indicating that fission of the aromatic nucleus was occurring. In addition to the [14C]-test material, small amounts of single radioactive compound were recovered from all soils whose Rf values in a four solvents were identical to those for 4-chloro-2-methylphenol. There was no trace of any compound with a higher Rf value that could be attributed to 4-chloro-2-methylanisole. Between 48 and 52 % of the total activity could be attributed to radioactively labelled compounds.
Key value for chemical safety assessment
- Half-life in soil:
- 40 d
- at the temperature of:
- 15 °C
Additional information
Key Study: Romero (2001)
The test material and its enantiopure R-form was incubated in three calcareous soils at 15 °C and 80 % of their field capacity to try to elucidate their behaviour in soil and compare the dissipation rates when racemic and enantiopure compounds are used. Quantitation was made by HPLC and the R/S ratio by GC-MS. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
Under the conditions of the study, the inactive S-enantiomer from the racemic form persisted longer than the R-form in silt and sandy loam soils, but for shorter time in the clay loam soil. The pure R-enantiomer, after incubation in soil, was partially converted into its S-form. In all cases, the dissipation of racemic and pure enatiomeric forms was lower in the clay loam soil than in the silt and sandy loam soils. The R-forms' peristence, in the three soils, was approximately two times lower when it was incubated alone than when it was incubated as a racemic compound. When peat was added, the persistence of the racemic substance in the silt and sandy loam soils increased, while in the clay loam soil it decreased. Besides, in the clay loam soil, the enantiomeric ratio (ER) changes from its S-preferential degradation to a preferential degradation of its R-form, so an increase in the persistence of the inactive S-form occurs.
Supprting Study: Smith & Hayden (1981)
The persistence of the test material was investigated at the 2 µg/g level, under laboratory conditions, in three Saskatchewan soils at 85 % of their field capacity moistures and 20 ± 1 °C. Following extraction of the soils with aqueous acidic acetonitrile, the methylated extracts were analysed gas chromatographically for remaining herbicides. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
The results from the persistence studies indicate that in the moist soils breakdown of the test material was rapid. In contrast, losses from air-dried soils was minimal. This lack of degradation in the air-dried soils suggests that herbicidal losses in the moist soils were due to biological processes, rather than inefficient extraction techniques.
Half-lives for the test material in the various soils were calculated from the graphs obtained by plotting the logarithm of percentage chemical remaining against incubation time. For the test material, the half-life in clay loam, heavy clay and sandy loam was determined to be 9, 8 and 7 days, respectively.
It can therefore be concluded that the test material, as a commonly used phenoxyalkanoic acid herbicide, appears to be degraded rapidly in all three Saskatchewan soils described.
Supporting Study: Lindholm et al. (1982)
Gas chromatography was used in an investigation of the degradation of the test material in field soil under glasshouse conditions. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
Under the conditions of the study the test material degraded fairly rapidly both in soil treated with 5 mg and in the soil with 15 mg test material/ kg soil. However the study was made under conditions that favoured microbial degradation. The soil was periodically analysed for the presence of a degradation product, however it was not detected in any sample of the soil treated with the test material.
Supporting Study: Balk (1985)
The rate of degradation of the test material was studied in an aquatic test system, comparable with conditions in Dutch ditches. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
The degradation was measured by the evolution of 14-C labelled carbon
dioxide from ring-labelled test material.
The test material was shown to be mineralised completely after three
months in the aquatic test systems in clay, peat and sandy soils, with
no appreciable residues in the water. The degradation in the sandy soil
system was somewhat slower and in the case of the test material some
residual radioactivity was found. In general, the test system performed
well, giving reproducible results and good recoveries of the labelled
material.
Under the conditions of this study, degradation of the test material starts slowly, but after three weeks the major part of 14-CO2 was recovered. Additionally, although significant water residues are found in the sand system, the test material proved to be easily biodegradable in the clay and peat systems.
Supporting Study: Müller and Buser (1997)
The degradation of the test material in soil under laboratory conditions was studied using enantioselective high-resolution gas chromatography/mass spectrometry. Garden soil (sandy load); 1.6 % organic carbon; pH 7.0) was taken from a plot near the research station in Wädenswil where phenoxy herbicides have never been used. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
A portion of a few kilograms of soil was carefully air dried (1 d at
room temperature, and then sieved through 10 and 4 mm sieves. The water
content of the soil was determined at ≈ 18 %. The soil was then kept in
a porous clay pot until used within a few days. A portion of the soil
was sterilised by γ-irradiation from a commercial ^50Co source with a
total dose of 25 kGy (24 h exposure).
The test material was readily degraded in soils to levels ≤ 10 % of the
initial concentration after 22 - 35 days of incubation. The data showed
linearity in an initial phase (< 16 d) but later showed some trend
towards faster rates.
In the second, more rapid phase (> 16 d) the rates were significantly
higher with a half-life of ≈ 4 d.
The data for the test material shows a continuous decrease of the
concentration to levels < 1 – 3 % of the initial concentrations after 22
days of incubation, however with a less pronounced two-phase kinetic.
The concentration of the test material increased from initial values of
0.7 % to a maxima of 10 % after 8 – 9 days respectively and then
decreased again.
The degradation observed can be chemically and / or biologically
mediated. In order to distinguish this, the test material was incubated
in sterilised soil.
Chemical degradation in sterilised soil is expectedly
non-enantioselective. The knet values were 2.5 – 4 times lower than
those in non-sterilised soil. There was no increase in the
concentrations of the inverted isomers. These data indicate that
degradation was primarily biologically mediated.
Under the conditions of the study, the test material was determined was
readily degraded in soils to levels ≤ 10 % of the initial concentration
after 22 - 35 days of incubation. Degradation was primarily biologically
mediated.
Supporting Study: Saxena (1988)
The soil metabolism of the test material was studied under aerobic incubation conditions and subsequent aerobic/anaerobic incubation conditions using a Fox sandy loam soil (Dane County, Wisconsin). The study was conducted in accordance with the Environmental Protection Agency Pesticide Assessment Guidelines, Subdivision N, Sections 162-1 and 162-2. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
Soil samples were fortified with 14C-test material at 9.98 ppm (µg/g) and placed in a glass chamber maintained at 25 °C ± 2 ° in a dark, temperature-controlled room. The glass chamber was connected to traps containing ethylene glycol and 2-ethoxyethanol:ethanolamine (1: 1) for the collection of organic volatiles and carbon dioxide (CO2), respectively. Air was passed through the system continuously to maintain aerobic conditions. Duplicate samples were collected for analysis on Days 0, 3, 7, 14, 21, 30, 61, 91, and 181 of aerobic incubation. On Day 30, four aerobic samples were flooded with water and incubated similarly, except that nitrogen was passed through the system.
Duplicate anaerobic samples were collected for analysis 31 and 61 days after flooding. Under aerobic conditions, 14C-test material degraded and decreased from a mean of 102.1 % (Day 0) to 20.1 % (Day 30), and then to less than approximately 3 % on subsequent sampling intervals. The calculated half-life was 13.3 days. The major products observed were 14CO2 (maximum of 58.1 % of applied radioactivity, Day 181) and soil-bound residues (maximum of 50.0 %, Day 61). A minor product (Peak 1; maximum of 3.5 %, Day 21) was also observed. No degradation of 14C-test material was observed in soil samples that were flooded with water after 30 days of aerobic incubation and were maintained under anaerobic conditions. The formation of 14CO2 or soil-bound residues was inhibited by anaerobic incubation conditions; therefore, the half-life of 14C-test material under anaerobic incubation conditions could not be calculated. Characterisation of soil-bound residues indicated that less than 3 % of the radioactivity was released by Soxhlet extraction with methanol. The bulk of the radioactivity was distributed into the fulvic acid, humic acid, and humin fractions of the soil organic matter.
Supporting Study: Smith (1985)
The degradation of ring-labelled [14C]-test material was investigated in three soil types to determine whether 4-chloro-2-methylphenol could be isolated as a degradation product. Because the boiling point of this phenol has been reported to be 222 – 225 °C at 760 mm, the experiments were conducted in Bartha and Pramer flasks to reduce the volatility losses of degradation products during the soil incubation period. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
Breakdown of the ring-labelled [14C]test material was rapid in all soils, being over 70 % complete in 20 days. During this time, between 20 and 29 % of the soil-applied radioactivity was released as [14C]-carbon dioxide, indicating that fission of the aromatic nucleus was occurring. In addition to the [14C]-test material, small amounts of single radioactive compound were recovered from all soils whose Rf values in a four solvents were identical to those for 4-chloro-2-methylphenol. There was no trace of any compound with a higher Rf value that could be attributed to 4-chloro-2-methylanisole. Between 48 and 52 % of the total activity could be attributed to radioactively labelled compounds.
It was assumed that at least some of the remaining activity had been converted into soil organic matter, since this is known to occur with carbon dioxide or carbon-containing fragments formed by breakdown of herbicide being incorporated into the soil biomass.
Losses on [14C]-carbon dioxide and the [14C]-phenol could have occurred as a result of volatilisation from the Bartha and Pramer flasks during the daily sampling of the alkaline solution or during exchange of sodium hydroxide with fresh solution. Similar losses of the phenol could also have occurred during work up of the soil extracts, especially during the evaporation stage of the dichloromethane solutions with nitrogen.
Although mass spectral data are necessary to confirm the identity of the degradation product isolated, the TLC results strongly indicate that 4-chloro-2-methylphenolis formed in soils from the test material. There is no indication that the 4-chloro-2-methylphenol underwent methylation to the corresponding anisole.
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