Registration Dossier
Registration Dossier
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EC number: 240-539-0 | CAS number: 16484-77-8
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 study investigated whether MCPA could be detected in a soil to which the test material have 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. - 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
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- No. of replication treatments: Duplicate pots of each series were withdrawn weekly.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. - 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.
- 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, 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:
- not specified
- 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-methylphenolis 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.
- Endpoint:
- biodegradation in soil: simulation testing
- Type of information:
- other: Re-analysis of study data according to the latest guidelines.
- Adequacy of study:
- supporting study
- Study period:
- not applicable
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- secondary literature
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The report considers three degradation kinetics studies. The first is the degradation of the test material and metabolites in four aerobic soils (Schocken 1997), the second is the degradation of the test material and metabolites due to photolysis in soil (Connor 1996a) and the third is the degradation of the test material and its metabolites due to photolysis in water (Connor 1996b).
This study summary specifically relates to the degradation kinetics study to re-assess the degradation of the test material and metabolites in four aerobic soils, as reported in Schocken (1997), using FOCUS (2006, 2014). SFO kinetics were used in the first instance (most appropriate for use in environmental fate modelling), with FOMC and DFOP used to determine trigger values for additional studies where biphasic kinetics were observed. CAKE 3.1 was used to perform the kinetics fitting procedure, with the OLS optimiser. The IRLS optimiser was used where confidence limits with the initial OLS fit were unreliable (predominantly for metabolites). - GLP compliance:
- no
- Remarks:
- Summary of data
- Test type:
- other: Kinetic analysis
- Oxygen conditions:
- aerobic
- Soil No.:
- #1
- DT50:
- 4.2 d
- Type:
- other: DT50 as a trigger for further studies
- Soil No.:
- #1
- DT50:
- 4.4 d
- Type:
- other: DT50 for modelling
- Transformation products:
- yes
- Remarks:
- PCOC
- No.:
- #1
- Conclusions:
- In the FIFRA soil from the study by Schocken (1997) the data-set was best described by FOMC kinetics, whilst the three other data-sets from BBA soils were described well by SFO kinetics. On the basis of the analysis, the most appropriate values for the critical degradation end-points of the test material in aerobic soils are:
DT50 as a trigger for further studies: 4.2 days
DT90 as a trigger for further studies: 16.2 days
DT50 for modelling: 4.4 days
To calculate the kinetic end-points for the metabolite PCOC, the information on the parent was used as well as the information on the metabolite. The SFO kinetics showed a good fit to the data from two of the four soil data-sets in the study by Schocken (1997). The other two soil data-sets (FIFRA and BBA 2) either showed no decline in PCOC during the study or limited number of data-points and so it was not possible to obtain a reliable kinetics fit for PCOC from these data-sets. However, PCOC is a metabolite of other substances too, so to get the end-points for this metabolite the whole PCOC data-set could be consulted. On the basis of the analysis, and relying on the visual fits as well as the statistical criteria, conservative estimates of the critical degradation end-points of PCOC in aerobic soils are:
DT50 as a trigger for further studies: 10.2 days
DT90 as a trigger for further studies: 34.1 days
DT50 for modelling: 10.2 days
Conversion ratio of test material to PCOC in soil 1.9 % - Executive summary:
The study concerns a degradation kinetics study to re-assess the degradation of the test material and metabolites in four aerobic soils, as reported in Schocken (1997), using FOCUS (2006, 2014). SFO kinetics were used in the first instance (most appropriate for use in environmental fate modelling), with FOMC and DFOP used to determine trigger values for additional studies where biphasic kinetics were observed. CAKE 3.1 was used to perform the kinetics fitting procedure, with the OLS optimiser. The IRLS optimiser was used where confidence limits with the initial OLS fit were unreliable (predominantly for metabolites).
In the FIFRA soil from the study by Schocken (1997) the data-set was best described by FOMC kinetics, whilst the three other data-sets from BBA soils were described well by SFO kinetics. On the basis of the analysis, the most appropriate values for the critical degradation end-points of the test material in aerobic soils are:
DT50 as a trigger for further studies: 4.2 days
DT90 as a trigger for further studies: 16.2 days
DT50 for modelling: 4.4 days
To calculate the kinetic end-points for the metabolite PCOC, the information on the parent was used as well as the information on the metabolite. The SFO kinetics showed a good fit to the data from two of the four soil data-sets in the study by Schocken (1997). The other two soil data-sets (FIFRA and BBA 2) either showed no decline in PCOC during the study or limited number of data-points and so it was not possible to obtain a reliable kinetics fit for PCOC from these data-sets. However, PCOC is a metabolite of other substances too, so to get the end-points for this metabolite the whole PCOC data-set could be consulted. On the basis of the analysis, and relying on the visual fits as well as the statistical criteria, conservative estimates of the critical degradation end-points of PCOC in aerobic soils are:
DT50 as a trigger for further studies: 10.2 days
DT90 as a trigger for further studies: 34.1 days
DT50 for modelling: 10.2 days
Conversion ratio of test material to PCOC in soil 1.9 %
- 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
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Degradation and adsorption of 14C-ring-labelled test material was determined in three different soil types under different environmental conditions. Degradation was also determined in subsurface soil.
- GLP compliance:
- no
- Test type:
- laboratory
- Radiolabelling:
- yes
- Oxygen conditions:
- aerobic
- Soil classification:
- not specified
- Year:
- 1 993
- Soil no.:
- #1
- Soil type:
- sandy loam
- % Clay:
- 15.8
- % Silt:
- 18.4
- % Sand:
- 63.4
- % Org. C:
- 2.4
- pH:
- 6.9
- Soil no.:
- #2
- Soil type:
- sandy loam
- % Clay:
- 13.2
- % Silt:
- 28.6
- % Sand:
- 55.5
- % Org. C:
- 2.6
- pH:
- 6.7
- Soil no.:
- #3
- Soil type:
- other: Coarse sandy soil: 0 - 25 cm depth
- % Clay:
- 4
- % Silt:
- 6.6
- % Sand:
- 86.9
- % Org. C:
- 2.4
- pH:
- 6.6
- Soil no.:
- #4
- Soil type:
- other: Coarse sandy soil: 25 - 50 cm depth
- % Clay:
- 3.1
- % Silt:
- 3.4
- % Sand:
- 93.2
- % Org. C:
- 1
- pH:
- 6.8
- Soil no.:
- #5
- Soil type:
- other: Coarse sandy soil: 50 - 75 cm depth
- % Clay:
- 3.1
- % Silt:
- 2
- % Sand:
- 94.5
- % Org. C:
- 0.5
- pH:
- 6.5
- Soil no.:
- #6
- Soil type:
- other: Coarse sandy soil: 75 - 100 cm depth
- % Clay:
- 5.1
- % Silt:
- 2.3
- % Sand:
- 92.3
- % Org. C:
- 0.3
- pH:
- 5.9
- Details on soil characteristics:
- SOIL COLLECTION AND STORAGE
- Geographic location: Soils were from three Danish Experimental Farms (Flakkebjerg, Roskilde and Jyndevad). The soils were grown with rotational crops including cereals.
- Sampling depth: Subsoil was from undisturbed cores of coarse sandy soil sampled 33 - 66 and 66 - 99 cm below soil surface (Jyndevad).
- Storage conditions: Stored at 5 °C with about 25 % of total water holding capacity (WHC) until use. Subsoil was kept in stainless steel tubes at 5 °C.
- Storage length: Storage for less than one month.
- Soil preparation: After sampling, the surface soils were sieved (< 2 mm).
PROPERTIES OF THE SOILS (in addition to defined fields)
- Moisture at 1/3 atm (%):
Flakkebjerg: Water holding capacity, % of wet soil: 31
Roskilde: Water holding capacity, % of wet soil: 31
Jyndevad: Water holding capacity, % of wet soil: 29 - Soil No.:
- #1
- Duration:
- 227 d
- Soil No.:
- #2
- Duration:
- 227 d
- Soil No.:
- #3
- Duration:
- 227 d
- Soil No.:
- #1
- Initial conc.:
- 2 mg/kg soil d.w.
- Based on:
- test mat.
- Soil No.:
- #2
- Initial conc.:
- 2 mg/kg soil d.w.
- Based on:
- test mat.
- Soil No.:
- #2
- Initial conc.:
- 0.2 mg/kg soil d.w.
- Based on:
- test mat.
- Soil No.:
- #3
- Initial conc.:
- 2 mg/kg soil d.w.
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- radiochem. meas.
- Soil No.:
- #1
- Temp.:
- 20 °C
- Humidity:
- 50 % WHC
- Microbial biomass:
- Non-sterile
- Soil No.:
- #2
- Temp.:
- 5, 10 and 20 °C
- Humidity:
- 25, 50 or 200 % WHC
- Microbial biomass:
- Non-sterile
- Soil No.:
- #3
- Temp.:
- 20 °C
- Humidity:
- 50 % WHC
- Microbial biomass:
- Sterile and non-sterile
- Details on experimental conditions:
- EXPERIMENTAL DESIGN
- Soil condition: Sterile soil for the degradation experiment was obtained by autoclaving for 1 h at 120 °C on three consecutive days. The flasks were mounted with sterile filters on in- and outlets to remain sterile during incubation. After incubation sterility was controlled by transferring material to slants of nutrient agar (normal and 1/10 cone.) and V8 agar and to nutrient broth (normal and 1/10 cone.). The tubes were examined for growth after 3 weeks of incubation at 24 °C.
- Details of traps for CO2 and organic volatile, if any: 14C evolved as CO2 and as other compounds released from the soil containing 14C-test material was trapped by passing a slow stream (5 - 10 mL/min) of CO2-free atmospheric air through 100 mL Erlenmeyer flasks containing 50 g of surface soil. In the case of subsoil cores 380 g of soil were incubated in stainless steel tubes (i.d. 48 mm, length 150 mm). Air was passed through the cores twice a week for 3 h. The air was bubbled through 3 test tube absorbers, one with 1,2-ethanediol (ethyleneglycol) and two with 0.1 N KOH. In order to enhance absorption of CO2 the air was passed through scintered glass in one of the KOH absorbers and through glass beads in the others.
Test material application
- Application method: In surface soil 14C-test material was added to small soil samples dissolved in diethylether which was allowed to evaporate before the soil sample was mixed into the major soil portion. To the undisturbed cores of subsoil the test material was added in water solution injected with a long needle (three and four replicates in surface and subsurface soils, respectively).
- Is the co-solvent evaporated: Diethylether was allowed to evaporate.
SAMPLING DETAILS
Extraction of 14C-test material from soil was performed 4 times by sonication in H2O+Ca(OH)2 and centrifugation.
The combined extracts were extracted with dichloromethane (methylenechloride) pH < 1. The extract was evaporated to dryness in N2 and dissolved in 1 mL of mobile phase (methanol + tetrabutylammonia hydrogensulphate 67: 33) pH 5.4. After HPLC on ODS-2 the eluate was collected in fractions of 10 drops for counting of radioactivity and determination of test material. Recovery of freshly added test material was 89 - 94 %. Residual activity in extracted soil was determined by combustion in an induction furnace (Leco Corporation) in oxygen flow (0.2 g finely ground soil was added to 0.8 g iron and tin accelerator). 14CO2 evolved after combustion of soil was trapped in ethanolamine which was added to methanol and counted in toluene scintillant with PPO and POPOP (1 litre added to 4 g and 0.1 g, respectively).
14C was determined by Liquid Scintillation Counter. Radioactivity in KOH, 1,2-ethanediol, aqueous extracts, dichloromethane and methanol/TBAS was counted in Aqua Lyte at a rate of 1:5, 1:20 1:5, 1:20 and 1:20, respectively. - Soil No.:
- #1
- DT50:
- 3 d
- Type:
- not specified
- Temp.:
- 20 °C
- Remarks on result:
- other: Flakkebjerg S.L., 50 % WHC, 2.0 mg/kg
- Soil No.:
- #2
- DT50:
- 3 d
- Type:
- not specified
- Temp.:
- 20 °C
- Remarks on result:
- other: Roskilde S.L., 50 % WHC, 2.0 mg/kg
- Soil No.:
- #2
- DT50:
- 12 d
- Type:
- not specified
- Temp.:
- 10 °C
- Remarks on result:
- other: Roskilde S.L., 50 % WHC, 2.0 mg/kg
- Soil No.:
- #2
- DT50:
- 20 d
- Type:
- not specified
- Temp.:
- 5 °C
- Remarks on result:
- other: Roskilde S.L., 50 % WHC, 2.0 mg/kg
- Soil No.:
- #2
- DT50:
- 10 d
- Type:
- not specified
- Temp.:
- 20 °C
- Remarks on result:
- other: Roskilde S.L., 25 % WHC, 2.0 mg/kg
- Soil No.:
- #2
- DT50:
- 15 d
- Type:
- not specified
- Temp.:
- 15 °C
- Remarks on result:
- other: Roskilde S.L., 200 % WHC, 2.0 mg/kg
- Soil No.:
- #2
- DT50:
- 1.3 d
- Type:
- not specified
- Temp.:
- 20 °C
- Remarks on result:
- other: Roskilde S.L., 50 % WHC, 0.2 mg/kg
- Soil No.:
- #3
- DT50:
- 4 d
- Type:
- not specified
- Temp.:
- 20 °C
- Remarks on result:
- other: Jyndevad C.S, 50 % WHC, 2.0 mg/kg
- Transformation products:
- not specified
- Evaporation of parent compound:
- not specified
- Volatile metabolites:
- not specified
- Residues:
- not specified
- Details on results:
- - Correlation between 14CO2 evolution and degradation of 14C-test material:
In two experiments the correlation between the amounts of 14C evolved as CO2 and the corresponding amounts of test material decomposed, was shown by determination of 14C-test material degradation in soil (Jyndevad).
The concentration of the test material decreased to about 70 % before more than some few percent of 14C was evolved as CO2. This is due to formation of degradation products before evolution of the ring-labelled 14C.
Half of the test material had disappeared when about 12 % 14C was recovered as CO2 (this was after about 5 days in these experiments) and less than 10 % of added test material was left in the soil when 50 % 14C was evolved in CO2.
- Influence of temperature:
After 20 days of incubation about 10 % 14C was evolved at 5 °C against 40 % at 10 °C and above 60 % at 20 °C. After 50 % 14C was evolved as 14CO2 the 14C evolution was very slow since most test material was already decomposed.
Residual 14C was considereed built into soil microorganisms and in other organic compounds from where it is only slowly released. On the assumption that the half-life time is when 12 % 14C is evolved in CO2, the half-life times of the test material at 20, 10 and 5 °C were considered to be 3, 12 and 20 days, respectively. Regression analysis of these data showed that the degradation rate increases by a factor 3.6 for each 10 °C temperature increment between 5 and 20 °C. The influence of temperature on the test material-degradation is, thus, comparable to the influence found for several other pesticides.
The test material was decomposed by soil microorganisms and the 14C-evolution also showed exponentially increasing degradation indicating proliferation of decomposers. By plotting daily evolution rates on an 1n scale against time elucidates lag phase and exponential phase at the three temperatures. Exponentially increasing evolution from the first day at 10 and 20 °C (14C-evolution as determined every 8 h for the first 48 h). At 5 °C there was a lag phase with slow (co-metabolic) degradation for 12 - 14 days before the exponential degradation starts.
The slope of the curves can elucidate the doubling time (Td) of the 14CO2 evolution by Td = In 2/slope. Doubling times from 1.4 days at 20 °C to 6.9 days at 5 °C indicate the temperature dependent growth of the decomposing microorganisms to be an important factor for the correlation between temperature and degradation rate. Since there is an increasing degradation rate with time, the first half-life time will always be the longest. The doubling times are relatively close to results for the test material where Td was found to be 0.7, 1.9 and 4.1 at 21, 10 and 6 °C, respectively (Helweg, 1987).
After 96 days of incubation the soils were extracted and residual 14C in the extracted soil determined by combustion. At all temperatures the concentration of the test material was below the limit of detection (< 6 % 14C in the extract, corresponding to < 0.1 mg/kg of test material).
Between 26 and 34 % 14C was not extractable and total recovery of 14C was 100, 90 and 103 at 20, 10 and 5 °C respectively.
- Influence of water content:
To show the influence of flooding and of drying out of soil on the degradation of the test material, evolution of 14C from the test material (2 mg/kg) in soil (Roskilde) with water added to 25 % WHC, 50 % WHC and 200 % WHC (flooded).
It appears that degradation was delayed in the flooded soil; on the other hand, fast degradation occured after 14 days and after 30 - 40 days all test material seemed to be decomposed. At the low water content (25 % WHC) degradation was initially slower than at 50 % WHC. The very slow degradation after 20 days was due to the fact that the soil was allowed to dry out. Addition of evaporated water to 25 % of WHC on day 60 increased degradation rate again.
- Influence of soil type, concentration and sterilisation:
The degradation of 14C-test material (2 mg/kg) was determined in three different soil types (Flakkebjerg, Roskilde and Jyndevad) in Roskilde soil also at 0.2 mg/kg and in Jyndevad soil also in sterilised soil.
During the first 2 days at 2 mg/kg the degradation was slowest in soil Jyndevad. After 12 days about 60 % 14C was evolved as CO2 from all 3 soils.
The degradation of 0.2 mg/kg in soil Roskilde was relatively faster during the first two days than the degradation of 2 mg/kg but after 12 days of incubation this difference was also eliminated. In sterile soil no evolution of 14C was observed and about 50 % of added 14C could be extracted and almost all 14C in the extract was identified as the test material by HPLC. About 35 % of the added 14C could not be extracted from the soil, possibly strongly adsorbed onto soil particles. All three replicates remained sterile during the experiment since the sterility test showed no growth on any of the substrates tested.
- Degradation in subsurface soil:
Since the test material has a low Koc-value it is possible that leaching of the compound may occur. Especially, there is a risk with heavy rain just after application or after late autumn use where the soil temperature is low and where there is constant infiltration of water through the soil.
The degradation of 14C-test material was determined in the coarse sandy soil profile from Jyndevad sampled at depths of 0 - 33, 33 - 66 and 66 - 99 cm. Test material (0.05 mg/kg) was added to all samples (four replicates) and incubated at 10 °C and water added to 50 % of WHC.
From surface soil about 30 % 14C is evolved in CO2 within 20 days after which the evolution decreased as a sign of the remaining 14C being now built into stable organic compounds in the soil. In subsurface soils the degradation was much slower and 30 % 14C was evolved after about 170 and 140 days at 33 - 66 and 66 – 99 cm, respectively. 14C-evolution still proceeded up to 227 days of incubation in subsurface soils.
If the half-life time is when 12 % 14C is evolved as CO2 the time for half of the test material to be decomposed is 7, 70 and 34 days in the soil layers 0 - 33, 33 - 66 and 66 - 99 cm, respectively.
After 227 days of incubation the soils were extracted. Test material in the extracts was determined by HPLC and the remaining radioactivity in the extracted soils was measured by combustion.
In the subsoil the variation in CO2 evolution from one replicate to another is very big as appears from the standard deviations (S.D.). The reason probably is that these are individual samples and have not, as for the top soil, have been mixed before addition of the test material. The addition of the test material to the subsoil cores by injection of an aqueous solution may also give an uneven distribution which may be another cause of variation.
The results also show that more test material (4 – 15 %) remains in the 33- to 66-cm layer than in the 66- to 99-cm layer. There is no determination of soil biomass or content of nutrients which possibly could explain the faster degradation in the deepest soil layer. - Conclusions:
- Under the conditions of the study estimated half-life times in the three surface soils were 3, 3 and 4 days, respectively at 20 °C. When the degradation was determined at 20, 10 and 5 °C the estimated half-life was 3, 12 and 20 days, respectively. The degradation rate of the test material increased initially with a doubling time of 1.4, 4.9 and 6.9 days, respectively at 20, 10 and 5 °C but with a 14-day lag-phase at 5 °C. In dry and in flooded soil (25 and 200 % of total water holding capacity) estimated half-lives were 10 and 15 days, respectively. At 0.2 mg/kg the half-life was 1.3 days compared to the 3 days at 2 mg/kg. In sterile soil no degradation was observed. In soil sampled at 0 - 33, 33 - 66 and 66 - 99 cm depth the estimated half-life time of the test material (0.05 mg/kg) was 7, 70 and 34 days, respectively at 10 °C.
- Executive summary:
Degradation of 14C-ring-labelled test material was determined in three different soil types under different environmental conditions. Degradation was also determined in subsurface soil. The correlation between test material disappearance at 2 mg/kg and 14CO2-evolution showed that when 12 % 14C was evolved as 14CO2, half of the test material was decomposed. Estimated half-life times in the three surface soils were 3, 3 and 4 days, respectively at 20 °C. When the degradation was determined at 20, 10 and 5 °C the estimated half-life was 3, 12 and 20 days, respectively. The degradation rate of the test material increased initially with a doubling time of 1.4, 4.9 and 6.9 days, respectively at 20, 10 and 5 °C but with a 14-day lag-phase at 5 °C. In dry and in flooded soil (25 and 200 % of total water holding capacity) estimated half-lives were 10 and 15 days, respectively. At 0.2 mg/kg the half-life was 1.3 days compared to the 3 days at 2 mg/kg. In sterile soil no degradation was observed. In soil sampled at 0 - 33, 33 - 66 and 66 - 99 cm depth the estimated half-life time of the test material (0.05 mg/kg) was 7, 70 and 34 days, respectively at 10 °C.
- 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:
- no guideline followed
- Principles of method if other than guideline:
- The test material, as well as its racemic substance, were 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.
- 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, as well as its racemic substance, were 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: 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: simulation testing
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Justification for type of information:
- Please see the read-across justification report in IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across source
- Endpoint:
- biodegradation in soil: simulation testing
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 07 July 1995 to 10 May 1996
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Guideline for pilot soil metabolsim study was not specified.
Prior to column leaching, a pilot soil metabolism study was conducted at an application rate of 2.4 lbs a.i./acre, representing the highest seasonal application rate of test material on turf. Based on chromatographic (HPLC-RAM) analysis of soil extracts and the assumption of first-order kinetics, soil half-lives of the test material were calculated in two different sandy loam soils, a sand, and a clay, at an incubation temperature of 25 ± 1 °C. - GLP compliance:
- yes
- Test type:
- laboratory
- Specific details on test material used for the study:
- Test concentrations were adjusted for purity of the test material and are reported as milligrams of test material per Litre of solution (mg/L).
The non-radiolabelled test material was used to isotopically dilute the radiolabelled test material as well as to serve as an analytical reference standard.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Preparation of the test material primary stock solution, purification of the radiolabelled test material and radiopurity analysis: The primary stock solution was prepared by transferring the entire contents of the received vial of [14C]test material via repetitive rinsing with acetonitrile to a final volume of 60 mL. The stock solution was analysed by liquid scintillation counting (LSC) and determined to have a concentration of 0.142 mg/mL (70 660 dpm/μL) based on a specific activity of 224.86 μCi/mg (499 189 dpm/μg; 48.3 mCi/mmol; 8.32 MBq/mg). HPLC-RAM analysis revealed, however, that the [14C]test material had a radiopurity of 94.5 %. Since the determined radiopurity was below 95 %, the [14C]test material primary stock solution was reduced to approximately 1 mL under nitrogen and purified by semi-preparative HPLC. The entire amount of impure [14C]test material was injected (in approximate 200 μL portions) onto a semi-preparative C18 HPLC column and the eluting region, corresponding to test material, was collected and pooled. Most of the acetonitrile (from the mobile phase) was removed under nitrogen, and 30 mL of reagent water was added to the remaining acidic water. After adjusting the pH to approximately 2 with acetic acid, the purified [14C]test material was partitioned with 3 x 50 mL of methylene chloride. The methylene chloride was then removed with nitrogen and the sample was redissolved in 50 mL of acetonitrile. Three 5-μL aliquots of the purified stock solution were profiled by HPLC-RAM, establishing a radiopurity of 100 %. The purified [14C]test material primary stock solution was used to prepare stock solutions for testing. This stock solution was analysed by LSC to have a mean measured concentration of 0.131 mg/mL (total of 6.53 mg [14C]test material), based on the radioactivity and the specific activity.
- Isotopic dilution, determination of specific activity and radiopurity of the [14C]test material Primary Stock Solution: The [14C]test material primary stock solution (43 mL containing 5.633 mg of [14C]test material) was combined with 16.881 mg of non-radiolabelled test material. The mean measured concentration of the isotopically diluted primary stock solution was determined to be 0.527 mg/mL, based on an HPLC-UV assay. The specific activity was determined to be 116 952 dpm/μg (11.3 mCi/mmol), based on a combination of LSC (to quantitate radioactivity), HPLC-UV (to determine the concentration of test material using a calibration curve) and HPLC-RAM (to determine radiopurity). Radiopurity was determined to be 100 %. - Radiolabelling:
- yes
- Oxygen conditions:
- aerobic
- Soil classification:
- not specified
- Year:
- 1 995
- Soil no.:
- #1
- Soil type:
- sandy loam
- % Clay:
- 7
- % Silt:
- 27
- % Sand:
- 66
- % Org. C:
- 0.9
- pH:
- 7.4
- CEC:
- 14.4 meq/100 g soil d.w.
- Bulk density (g/cm³):
- 1.28
- Soil no.:
- #2
- Soil type:
- sandy loam
- % Clay:
- 8
- % Silt:
- 36
- % Sand:
- 56
- % Org. C:
- 3.5
- pH:
- 6.3
- CEC:
- 11.9 meq/100 g soil d.w.
- Bulk density (g/cm³):
- 0.94
- Soil no.:
- #3
- Soil type:
- sand
- % Clay:
- 6
- % Silt:
- 2
- % Sand:
- 92
- % Org. C:
- 1.3
- pH:
- 6.7
- CEC:
- 4.3 meq/100 g soil d.w.
- Bulk density (g/cm³):
- 1.35
- Soil no.:
- #4
- Soil type:
- clay
- % Clay:
- 50
- % Silt:
- 34
- % Sand:
- 16
- % Org. C:
- 1.6
- pH:
- 7
- CEC:
- 34.9 meq/100 g soil d.w.
- Bulk density (g/cm³):
- 1.09
- Details on soil characteristics:
- SOIL COLLECTION AND STORAGE
Soils were obtained from various agricultural sites within the United States and were intended to represent both light and heavy-textured soils. The sand soil was collected from a site in Macon, Georgia and was received at the Test Facility on 8 June 1995. A sandy loam soil (designated Sandy Loam A) was collected from a site in Timmerman, Washington from the top 2 to 9 inches soil profile and was received at the Test Facility on 9 May 1995. Reportedly, the Washington soil had not received pesticide applications for 3 years prior to collection. Another sandy loam soil (designated as Sandy Loam B) was collected (on 5 June 1995) from a field in Hood River County, Oregon from a depth of 0 to 9 inches and was received at the Test Facility on 7 June 1995. Although an initial non-GLP soil characterisation of the Oregon soil indicated a soil texture corresponding to a silt loam, a subsequent characterisation conducted under GLP regulations indicated that this soil corresponded to a sandy loam. The latter characterisation was used. Reportedly, the Oregon soil had not been treated with agricultural chemicals or fertilizers within 5 years of its collection. A (Sharkey) clay soil was collected on 6 June 1995 from a farm near Watson, Arkansas and was received at the Test Facility on 7 June 1995. The clay soil had been left fallow for the previous ten years.
Prior to use, soils were stored at approximately 4 °C. Before use, the soils were sieved through a 2-mm stainless steel mesh to provide a uniform particle size as specified in the protocol. - Soil No.:
- #1
- Duration:
- 12 d
- Soil No.:
- #2
- Duration:
- 12 d
- Soil No.:
- #3
- Duration:
- 12 d
- Soil No.:
- #4
- Duration:
- 12 d
- Soil No.:
- #1
- Initial conc.:
- 1.16 other: µg/g (ppm)
- Soil No.:
- #2
- Initial conc.:
- 1.16 other: µg/g (ppm)
- Soil No.:
- #3
- Initial conc.:
- 1.16 other: µg/g (ppm)
- Soil No.:
- #4
- Initial conc.:
- 1.16 other: µg/g (ppm)
- Parameter followed for biodegradation estimation:
- radiochem. meas.
- Soil No.:
- #1
- Temp.:
- 25 ± 2 °C
- Soil No.:
- #2
- Temp.:
- 25 ± 2 °C
- Soil No.:
- #3
- Temp.:
- 25 ± 2 °C
- Soil No.:
- #4
- Temp.:
- 25 ± 2 °C
- Details on experimental conditions:
- A preliminary aerobic soil metabolism study was performed prior to conducting soil column leaching of aged residues to estimate the approximate half-life of the radiolabeled test material in each of the four soils.
- Experimental set-up: The set-up consisted of individual glass 250-mL Erlenmeyer flasks fitted with glass Dreschel caps having inlet and outlet ports to facilitate aeration and collection of volatile degradates. Forced aeration was provided at a flow rate of 15 to 20 mL/min for approximately 30 minutes daily by using a vacuum pump to pull air through the soil flasks. Individual flasks were connected to trapping trains intended to capture volatile degradates during the incubation. The trapping trains contained trapping vials in the following order: One trap containing polyurethane foam plugs for the capture of volatile organics, one empty backflow trap, one trap containing 15 mL of ethylene glycol, also for the capture of volatile organics, one empty backflow trap, two traps containing 15 mL of 10 % potassium hydroxide for the capture of 14CO2 and other acidic volatiles, and an empty backflow trap.
- Test Procedure. Approximately 50 g (dry weight) of each soil type (moisturised to 75 % of field moisture capacity [FMC]) was added to seven Erlenmeyer flasks (250-mL). The flasks were fortified with 110 μL of the 0.527 mg/mL [14C]test material primary stock solution resulting in a soil concentration of 1.16 μg/g (ppm), equivalent to the highest seasonal application rate for turf (two applications/season at 1.2 lb/acre distributed within the upper 6-inch soil profile). The test material was evenly distributed throughout the soil by mixing with a stainless steel spatula. Solvent was allowed to evaporate from the soil samples for approximately 30 to 60 minutes in a fume hood.
The flasks were wrapped in aluminum foil to prevent exposure to light and connected to individual trapping trains. They were maintained in an environmental chamber at a temperature of 25 ± 2 °C. Temperatures were monitored using a minimum/maximum thermometer, which was recorded and reset daily. - Soil No.:
- #1
- DT50:
- 7.5 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 25 °C
- Soil No.:
- #2
- DT50:
- 2.5 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 25 °C
- Soil No.:
- #3
- DT50:
- 10.5 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 25 °C
- Soil No.:
- #4
- DT50:
- 8.2 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 25 °C
- Transformation products:
- not specified
- Evaporation of parent compound:
- not specified
- Volatile metabolites:
- not specified
- Residues:
- not specified
- Details on results:
- A pilot soil metabolism study was performed to estimate the approximate half-life of the test material in two different sandy loam soils, a clay soil and a sand soil. First-order half-lives were calculated to be 7.5 and 2.5 days for sandy loam A and sandy loam B, respectively; 8.2 days for the clay soil, and 10.5 days for the sand soil.
- Conclusions:
- A pilot soil metabolism study was performed to estimate the approximate half-life of the test material in two different sandy loam soils, a clay soil and a sand soil. First-order half-lives were calculated to be 7.5 and 2.5 days for sandy loam A and sandy loam B, respectively; 8.2 days for the clay soil, and 10.5 days for the sand soil at 25 ± 1 °C.
- Executive summary:
Prior to column leaching, a pilot soil metabolism study was conducted at an application rate of 2.4 lbs a.i./acre, representing the highest seasonal application rate of test material on turf. Based on chromatographic (HPLC-RAM) analysis of soil extracts and the assumption of first-order kinetics, soil half-lives of the test material were calculated in two different sandy loam soils, a sand, and a clay, at an incubation temperature of 25 ± 1 °C.
Under the conditions of the pilot study, first-order half-lives were calculated to be 7.5 and 2.5 days for sandy loam A and sandy loam B, respectively; 8.2 days for the clay soil, and 10.5 days for the sand soil.
- Endpoint:
- biodegradation in soil, other
- Remarks:
- AEROBIC SOIL METABOLISM
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 01 August 1995 to 14 March 1996
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: EPA/FIFRA Pesticide Assessment Guidelines, Subdivision N Chemistry: Environmental Fate Series §162-1 Aerobic Soil Metabolism Studies
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: BBA guidelines, Part IV, 4-1, Persistence of Plant Protection Products in the Soil Degradation, Transformation, and Metabolism
- Deviations:
- no
- Principles of method if other than guideline:
- An objective of this study was to determine the kinetics of degradation of the test material as well as to establish the patterns of formation and decline of major metabolites in a sandy loam soil collected in the United States. The degradation of the test material was further studied in three soils obtained from Germany in order to more fully characterise test material degradation in a variety of soil types. The fate of the test material in the three German soils also fulfils the German regulatory requirement for agrochemical soil metabolism (BBA Guideline Part IV, 4-1). Throughout this robust study summary, reference will be made either to the FIFRA study or the BBA study.
The FIFRA study terminated at Day 191 rather than after one year as intended since both the test material degradation kinetics and the formation and decline of major degradates had been well established by Day 191. - GLP compliance:
- yes
- Test type:
- laboratory
- Radiolabelling:
- yes
- Oxygen conditions:
- aerobic
- Soil classification:
- other: Characterised by Agvise Laboratories, Northwood, North Dakota
- Year:
- 1 995
- Soil no.:
- #1
- Soil type:
- other: Sandy loam (FIFRA)
- % Clay:
- 7
- % Silt:
- 27
- % Sand:
- 66
- pH:
- 7.4
- CEC:
- 14.4 meq/100 g soil d.w.
- Bulk density (g/cm³):
- 1.28
- Soil no.:
- #2
- Soil type:
- other: Sand (Speyer 2.1,BBA)
- % Clay:
- 5
- % Silt:
- 1
- % Sand:
- 94
- pH:
- 6.9
- CEC:
- 4.1 meq/100 g soil d.w.
- Bulk density (g/cm³):
- 1.38
- Soil no.:
- #3
- Soil type:
- other: Loamy sand (Speyer 2.2, BBA)
- % Clay:
- 7
- % Silt:
- 7
- % Sand:
- 86
- pH:
- 6
- CEC:
- 9 meq/100 g soil d.w.
- Bulk density (g/cm³):
- 1.04
- Soil no.:
- #4
- Soil type:
- other: Sandy loam (Speyer 2.3, BBA)
- % Clay:
- 13
- % Silt:
- 17
- % Sand:
- 70
- pH:
- 7.4
- CEC:
- 8.9 meq/100 g soil d.w.
- Bulk density (g/cm³):
- 1.17
- Details on soil characteristics:
- SOIL COLLECTION AND STORAGE
FIFRA SANDY LOAM
- Geographic location: Timmerman coarse sandy loam soil collected from Washington state.
- Pesticide use history at the collection site: The area where the soil was collected received no pesticide application during the previous three years.
- Storage conditions: Upon arrival at the testing facility the soil was stored refrigerated at approximately 7 °C.
- Soil preparation: Before testing, the soil was sieved through a 2-mm stainless steel mesh screen to provide a uniform particle size. Sieved soil was stored at approximately 17 °C. Prior to test initiation the soil remained well above 25 % field moisture capacity (FMC, 12.5 %) and did not require remoisturisation.
BBA SOILS
- Geographic location: Germany
- Pesticide use history at the collection site: Soils were collected from an area that had received no pesticide application during the previous three years.
- Storage conditions: Upon arrival at the testing facility the soil was stored refrigerated at approximately 7 °C.
- Soil preparation: Before testing, all three soils were sieved through a 2-mm stainless steel mesh screen to provide a uniform particle size. The sieved soils were stored at approximately 17 °C. Prior to test initiation, the soils remained well above 25 % FMC (7.1 % for sand, 13.7 % for loamy sand and 19.1 % for sandy loam) and did not require remoisturisation.
PROPERTIES OF THE SOILS (in addition to defined fields)
FIFRA SANDY LOAM
- Moisture at 1/3 atm: 12.5 %
- Organic matter: 0.9 %
- Total nitrogen: 0.051 %
- Base saturation cation data: Calcium 58.9 %, Magnesium 19.0 %, Sodium 0.7 %, Potassium 5.9 %, Hydrogen 15.5 %
BBA SAND
- Moisture at 1/3 atm: 7.1 %
- Organic matter: 0.8 %
- Total nitrogen: 0.040 %
- Base saturation cation data: Calcium 61.4 %, Magnesium 6.1 %, Sodium 3.5 %, Potassium 5.4 %, Hydrogen 23.6 %
BBA LOAMY SAND
- Moisture at 1/3 atm: 13.7 %
- Organic matter: 3.5 %
- Total nitrogen: 0.166 %
- Base saturation cation data: Calcium 61.4 %, Magnesium 5.6 %, Sodium 2.4 %, Potassium 1.2 %, Hydrogen 29.5 %
BBA SANDY LOAM
- Moisture at 1/3 atm: 19.1 %
- Organic matter: 0.9 %
- Total nitrogen: 0.089 %
- Base saturation cation data: Calcium 67.2 %, Magnesium 9.3 %, Sodium 2.0 %, Potassium 6.1 %, Hydrogen 15.2 % - Soil No.:
- #1
- Duration:
- 191 d
- Soil No.:
- #2
- Duration:
- 100 d
- Soil No.:
- #3
- Duration:
- 100 d
- Soil No.:
- #4
- Duration:
- 100 d
- Soil No.:
- #1
- Initial conc.:
- 1.16 ppm
- Based on:
- act. ingr.
- Soil No.:
- #2
- Initial conc.:
- 1.16 ppm
- Based on:
- act. ingr.
- Soil No.:
- #3
- Initial conc.:
- 1.16 ppm
- Based on:
- act. ingr.
- Soil No.:
- #4
- Initial conc.:
- 1.16 ppm
- Based on:
- act. ingr.
- Parameter followed for biodegradation estimation:
- radiochem. meas.
- Soil No.:
- #1
- Temp.:
- 20 ± 2 °C
- Microbial biomass:
- 29 mg C/100 g soil (Day -4)
- Soil No.:
- #2
- Temp.:
- 20 ± 2 °C
- Microbial biomass:
- 14 mg C/100 g soil (day -1)
- Soil No.:
- #3
- Temp.:
- 20 ± 2 °C
- Microbial biomass:
- 42 mg C/100 g soil (Day 1)
- Soil No.:
- #4
- Temp.:
- 20 ± 2 °C
- Microbial biomass:
- 20 mg C/100g soil (Day 2)
- Details on experimental conditions:
- EXPERIMENTAL DESIGN
The incubation vessels for the aerobic metabolism study were 250-mL Erlenmeyer flasks fitted with glass Dreschel caps having inlet and outlet ports for air exchange. Each test vessel was autoclaved (Magnaclave, Model MC) for 30 minutes to minimise introduction of air-borne spores or propagules before the addition of soil to the test vessel.
Individual test vessels and traps were connected via silicone tubing using gang valves as splitters. Each trap was a scintillation vial capped with an open-top cap and a Teflon®-coated septum.
For the FIFRA study the trapping train consisted of 1) two foam plugs in succession for the trapping of organic volatile compounds, 2) an empty trap (overflow), 3) a sulfuric acid (0.5 M) trap for the collection of basic volatiles, 4) an empty trap, 5) two 10 % potassium hydroxide traps for the collection of carbon dioxide, and 6) an empty trap.
Traps in the trapping train for the BBA study consisted of 1) two foam plugs in succession for the trapping of organic compounds, 2) an empty trap (overflow), 3) an ethylene glycol trap for the collection of organic compounds, 4) an empty trap, 5) two 10 % potassium hydroxide traps for the collection of carbon dioxide, and 6) an empty trap.
Aeration and sweeping of the head space (via vacuum pump) of the FIFRA test vessels was done for 30 minutes each day; aeration for the BBA study was done under a constant purge. A flow rate (2 mL/min), for both studies, was established which allowed for the collection of volatiles, but did not dry out the soils too quickly. Carbon dioxide from the incoming air was scrubbed with a 10 % potassium hydroxide solution so as not to saturate the potassium hydroxide trap solutions. Incoming air was rehydrated after scrubbing using sterile reagent water contained in a 250-mL Erlenmeyer flask and pulled through a 0.2-μm Acrodisc®. Check valves were placed before and after the trapping trains to prevent backflow.
Temperature for each study was measured using a min-max thermometer placed in 250-mL Erlenmeyer flasks covered with parafilm and filled with reagent water.
TEST PROCEDURE
FIFRA STUDY
Approximately 50 g (dry weight) of soil was added to a total of 36 Erlenmeyer flasks (250-mL). Soils were at 78.4 % of field moisture capacity (FMC) when added to the flasks and no water was added prior to dosing. Soils were dosed with 110 μL of dosing solution resulting in a soil concentration of 1.16 mg/kg (ppm). This concentration approximated the maximum seasonal application rate of test material based on soil incorporation to a depth of 6 inches, assuming a soil bulk density of 1.5 g/cm^3 and no interception by plant material or thatch, i.e., bare earth application. It also provided sufficient test material to establish degradation kinetics as well as degradate formation and decline curves.
After dosing, flasks were wrapped in aluminium foil to prevent exposure to light and placed in an environmental chamber. Temperatures were monitored daily using a minimum/maximum thermometer and maintained at 20 ± 2 °C. Flasks were connected in parallel to a series of trapping trains, which were used to trap volatile degradates during intermittent purging. Soil remoisturisation was performed gravimetrically on a weekly basis to maintain soil moisture at 75 ± 10 % of FMC. Vessels requiring the addition of water received sterile reagent grade water. Vessels were weighed prior to soil addition (to establish a vessel tare weight), after addition, after water addition and at the time of sampling.
Sampling intervals occurred on Days 0, 1, 3, 6, 14, 30, 64, 91, 128 and 191. Sampling order was determined by a random number generating computer program (i.e. Symphony®). Duplicate incubation flasks were analysed at each sampling interval. Samples were removed from the test system after purging for thirty minutes with CO2-free air. Contents of the flasks were removed and placed into 200-mL centrifuge tubes. Soil extraction was performed three times using 100 mL of acetonitrile/NANOpure® water/glacial acetic acid (80:20:2.5, v:v:v). Centrifuge tubes were secured to a shaker table and shaken at 250 revolution per minute (rpm) for fifteen minutes. Tubes were then centrifuged at 1000 rpm for 20 minutes. Extracts were combined and the volumes measured. Triplicate aliquots (100 or 500 μL) were removed for radioassay by LSC.
Since the three acetonitrile/water/acetic acid extractions of Day 30 soil sample replicates removed only 11.6 to 12.1 % of the applied radioactivity, an additional extraction was carried out using 100 mL of 2N sodium hydroxide. Samples were placed on a shaker table and shaken for 15 minutes at 250 rpm. Samples were centrifuged at 1000 rpm for 20 minutes and triplicate 500-μL aliquots were quantified by LSC.
Days 0, 1, and 3 were analysed by HPLC-RAM by direct injection of the combined soil extract. Because less radioactivity was extractable as incubation time increased, extracts from sampling Days 6, 14, 30, 64, 91 and 191 were concentrated from approximately 3 to 5-fold under a gentle stream of nitrogen. Soil extracts from the day 128 replicate samples were partitioned with ethyl acetate after removal of acetonitrile by rotary evaporation. The ethyl acetate fractions were dried with anhydrous sodium sulphate and solvent was removed by a combination of rotary evaporation and a gentle stream of nitrogen and reconstituted in 2.0 mL of acetonitrile prior to HPLC-RAM profiling. The sodium hydroxide extracts from Day 30 replicate samples were also profiled by HPLC-RAM.
Quantitation of non-extractable radioactivity (soil-bound residues) was accomplished by oxidative combustion analysis using a sample oxidiser. Prior to combustion, soils were placed in a fume hood and allowed to air-dry. After drying the soils, soils were homogenised using a mortar and pestle. Dried soils were analysed by removing approximate 100-mg aliquots in triplicate and placing them in burn cones for combustion analysis. Soil efficiency combustions were also performed at the time of analysis for bound residues.
Foam plugs and sulfuric acid traps were only quantified for radioactivity at sampling intervals. Quantification of radioactivity in potassium hydroxide traps occurred at each sampling interval and periodically throughout the study by sampling and replacing the potassium hydroxide traps. Foam plugs were extracted with two 5 mL portions of methanol by vortexing samples for several minutes with solvent, removing the solvent and mixing duplicate 1.0 mL aliquots with Monophase® cocktail for LSC analysis. Sulfuric acid traps and potassium hydroxide traps were analysed by measuring the volumes and removing duplicate 1.0 mL (sulfuric acid traps) or 0.5 mL aliquots (potassium hydroxide traps). Scintillation cocktails used for analysis included Monophase® and lnstagel® for the potassium hydroxide and sulfuric acid traps, respectively.
Overflow traps were also analysed by LSC.
- Confirmation of Trapped 14CO2 by Barium Precipitation
Trapped 14CO2 in the potassium hydroxide trapping solution for Day 30 samples was confirmed by barium precipitation. A composite was made of the Day 1, 3, 6, 14, 17, 23 and 30 traps. A 5 mL volume of the potassium hydroxide trapping solution composite and 5.0 mL of a saturated barium hydroxide (Ba(OH)2) solution were combined in an LSC vial. The contents of the tube were swirled and the precipitate was allowed to settle for 30 minutes. The precipitate was removed by suction filtration. Volume of the supernatant was recorded and duplicate aliquots (0.5 mL) were removed for quantification by LSC analysis. Radioactivity in the precipitate was quantified by combustion analysis using a sample oxidiser.
- Fractionation of Soil-Bound Radioactive Residues
By Day 3 of the FIFRA study, soil-bound radioactive residues were significant (i.e. > than 10 % of applied radioactivity). Therefore, extracted-soil replicates from Days 64 and 191 of the study were analysed by a humic and fulvic acid / humin fractionation procedure. Day 64 soil-bound residues accounted for an average of 50.2 % of the applied radioactivity and represented a substantial contribution at an intermediate point in the incubation. Day 191 samples were also analysed by the fractionation scheme in order to characterise bound residues at the termination of incubation. Approximately 20 g of the extracted soil (dry weight) was added to a centrifuge tube. Sodium hydroxide (100 mL of a 0.1 N solution) and 0.1 g of calcium chloride (to prevent the formation of fines) were added to the tube. After placement of the centrifuge tube on a shaker table at 250 rpm overnight, the centrifuge tube was centrifuged at 1 500 rpm for 30 minutes. The supernatant was decanted, its volume measured with a graduated cylinder and its pH determined. The pH of the supernatant was adjusted to approximately 1 by the addition of concentrated HCI and the centrifuge tube stored in a refrigerator overnight. The resulting precipitate was removed by suction filtration and quantified by combustion analysis (humic acid fraction). Quantification of the remaining supernatant (fulvic acid fraction) was done by triplicate LSC analysis. Extracted soil (humin fraction) was placed in a fume hood to dry and was quantified by combustion. Efficiency combustions were also performed during the analysis of the humin fraction.
BBA STUDY
For each of the three soil types, sand (Speyer 2.1), loamy sand (Speyer 2.2) and sandy loam (Speyer 2.3), 22 test vessels were prepared for a total of 66 test vessels. Approximately 50 g (dry weight) of soil was added to each Erlenmeyer flask (250 mL). Before dosing, reagent water was added to each incubation flask to hydrate the soils to 75 % of FMC. Soils were dosed with 110 μL of dosing solution resulting in a soil concentration of 1.16 mg/kg (ppm), identical to the procedure followed in the FIFRA study.
After dosing, flasks were wrapped in aluminium foil to prevent exposure to light and placed in an environmental chamber. Temperatures were monitored daily using a minimum/maximum thermometer and maintained at 20 ± 2 °C. Flasks were connected in parallel to a series of trapping trains, which were used to trap volatile degradates during purging. Soil remoisturisation was performed gravimetrically on a weekly basis to maintain soil moisture at 75 ± 10 % of FMC. Vessels requiring the addition of water received sterile reagent grade water. Vessels were weighed prior to soil addition (to establish a vessel tare weight), after addition, after water addition and at the time of sampling.
Sampling intervals occurred on Days 0, 1, 3, 7, 16, 36, 71 and 100. Duplicate incubation flasks for each soil type were analysed at each sampling interval. Samples were removed from the test system and the contents of the flasks were removed and placed into 200 mL centrifuge tubes. Soil extraction was performed three times using 100 mL of acetonitrile/NANOpure® water/glacial acetic acid (80:20:2.5, v:v:v). Centrifuge tubes were secured to a shaker table and shaken at 250 revolution per minute (rpm) for fifteen minutes. Tubes were then centrifuged at 1 000 rpm for 20 minutes. Extracts were combined and the volumes measured. Triplicate aliquots (100 or 500 μL) were removed for radioassay by LSC.
Days 0, 1, 3, 7 and 16 were analysed by HPLC by direct injection of the combined soil extract. Concentration of extracts from 10 to 3 mL (under a nitrogen stream) on sampling Days 36, 71 and 100 was necessary to provide sufficient radioactivity for HP LC-RAM profiling. Soil extracts from days 16 and 36 were also partitioned with ethyl acetate after removal of acetonitrile by rotary evaporation. The ethyl acetate fractions were dried with sodium sulphate, the solvent removed by rotary evaporation and reconstituted in 2.0 mL of acetonitrile prior to HPLC-RAM analysis.
Quantitation of non-extractable radioactive residues (soil-bound residues) was accomplished by combustion. Prior to combustion, soils were placed in a fume hood and allowed to dry. After drying, the soils were homogenised using a mortar and pestle. Dried soils were analysed by removing triplicate 100 mg aliquots and placing them in burn cones for combustion analysis. Soil efficiency combustions were performed at the time of analysis for bound residues.
Foam plugs and ethylene glycol traps were only quantified for radioactivity at sampling intervals. Quantification of radioactivity (by LSC) in potassium hydroxide traps occurred at each sampling interval and periodically throughout the study after replacing the potassium hydroxide traps and performing radioanalysis on the potassium hydroxide. Foam plugs were extracted with two 5 mL portions of methanol by vortexing samples for several minutes with methanol, removing the solvent and mixing duplicate 1.0 mL aliquots with Monophase® cocktail for LSC analysis. Ethylene glycol traps and potassium hydroxide traps were analysed by measuring the volumes and placing duplicate 1.0 mL (ethylene glycol traps) or 0.5 mL aliquots (potassium hydroxide traps) in Monophase® scintillation cocktail. Overflow traps were also analysed by LSC.
MICROBIAL BIOMASS DETERMINATION
- Plate Counts
Soil samples (1 g) were extracted with 99 mL of sterile reagent water and the extract was serially diluted from 10^-1 to 10^-7. One hundred microlitres of each dilution was plated onto semi-selective media (nutrient agar plus soil extract media [for determining total heterotrophic bacteria from soil] actinomycete media [for the enrichment and enumeration of actinomycetes] and peptone dextrose media [for determining total fungal colonies from soils]). Plates were incubated at a temperature of 25 ± 2 °C for approximately 2 (bacterial counts) or 3 days (actinomycete and fungal counts) prior to counting colonies.
- Substrate-Induced Respirometry (SIR).
The metabolically active microbial community in the soils used for both the FIFRA and BBA studies was estimated based on the respirometric method of Anderson and Domsch (1978). Twenty-five-gram (dry weight) aliquots of soil, at an approximate moisture content of 60 % of FMC, were fortified with the lowest glucose concentration which would give the maximum initial respirometric response and evolved CO2 was measured in a Micro-Oxymax Respirometer (Columbus Instruments) for at least 12 hours at 22 ± 1 °C. Total biomass carbon in the soil was measured using the following expression:
x = 40.4 y + 0.37
where:
x = Microbial biomass (mg C per unit dry weight soil)
y = Maximum initial rate of respiration (mL of CO2 per 100 grams of soil per hour)
- Fumigation-Extraction
Microbial biomass in the soil used for the FIFRA study (Day 225, representing test termination) was also estimated based on the fumigation-extraction method (Brookes et al., 1990; Brooks et al., 1987; Jenkinson and Powlson, 1976). Four 20 g (dry weight) aliquots of soil were fumigated with deuterated chloroform for 24 hours at 25 ± 1 °C and then extracted with 200 mL of 0.5 M potassium sulphate for 30 minutes, diluted into sodium hexametaphosphate and acidified to below pH 2 with phosphoric acid. Aliquots, 10 mL (1 mL injections), were used for total organic carbon analysis using a Dohrmann DC-80 TOC Analyser. Total organic carbon content in four 20 g (dry weight) aliquots of soils not fumigated (controls) was also determined and the mean value subtracted from the fumigated soil to determine the microbial carbon contribution of the soil. The expression describing the calculation is provided below:
Bc = 2.22 Ec
Where:
Ec= (organic C extracted by K2SO4 from fumigated soil) - (organic C extracted by K2SO4 from non-fumigated soil)
Bc = Biomass carbon
CALCULATIONS
During this study, calculations were performed using the following formulae:
- Applied radioactivity (dpm per test vessel) = (dpm in aliquot of dosing solution / aliquot size (mL)) x total volume of dose (mL)
Where:
dpm in aliquot of dosing solution = mean of 5 aliquots pre-dose and 5 aliquots post-dose
- Percent of applied radioactivity in soil extract at a given sampling interval = [((dpm in measured aliquot / aliquot size (mL)) x soil extract volume (mL)) / applied radioactivity]
- Determining volatile production (cumulative) = [(mean dpm in measured aliquot (Day 1) / size of aliquot (mL)) x measured volume of trap (mL)] + [(mean dpm in measured aliquot (Day 3) / size of aliquot (mL)) x measured volume of trap (mL)] + etc.
- Determining percent test material remaining (for kinetic analysis) at a given sampling interval = (% of applied radioactivity in soil extract at time t x % of HPLC profile contributed by test material) / 100
Half-life determination (FIFRA study)
At each sampling interval, the residual percentage of test material (relative to the applied dose) was determined in duplicate by HPLC-RAM analysis. The degradation rate constant (k) and half-life (t½) of the test material were calculated by plotting the natural logarithm of the residual percentage of test material versus time using linear regression analysis. A biphasic analysis was done by performing linear regression analysis on the Day 0 to Day 30 samples and also on the Day 30 to Day 191 samples. The degradation rate constant was calculated from the following equation based on apparent first-order kinetics:
Ln(test material at time t [relative to applied dose ]) = kt
where:
k = Rate constant (day^-1)
t = Time (days)
The linear regression of Ln (test material % at a given sampling interval [relative to the applied dose]) versus time was determined with the slope of the regression line equal to k, the kinetic rate constant.
The half-life (t½) in days was calculated from the following equation:
t½ = Ln 2 / k - Soil No.:
- #1
- % Recovery:
- 90
- Soil No.:
- #2
- Remarks on result:
- other: 101 %
- Soil No.:
- #3
- Remarks on result:
- other: 102 %
- Soil No.:
- #4
- % Recovery:
- 99.5
- Key result
- Soil No.:
- #1
- DT50:
- 30.1 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 20 °C
- Key result
- Soil No.:
- #2
- DT50:
- 8 d
- Type:
- zero order
- Temp.:
- 20 °C
- Key result
- Soil No.:
- #3
- DT50:
- 5 d
- Type:
- zero order
- Temp.:
- 20 °C
- Key result
- Soil No.:
- #4
- DT50:
- 7 d
- Type:
- zero order
- Temp.:
- 20 °C
- Transformation products:
- yes
- No.:
- #1
- No.:
- #2
- Details on transformation products:
- IDENTIFICATION OF DEGRADATES
- FIFRA Study
14CO2 was the major metabolite of the test material; few metabolites were detected in the soil extracts. Up to and including Day 6, 100 % of the radioactive distribution of the soil extracts was parent, test material. As the study continued, some minor metabolites accounting for less than 3 % of the applied radioactivity at any one time point were detected by HPLC. One metabolite, 4-chloro-2-methylphenol (also known as parachloro-ortho-cresol, PCOC), reached a maximum of 1.85 % applied radioactivity on Day 128. This metabolite was tentatively identified based on comparison of its HPLC retention time to an authentic reference standard.
An unidentified polar metabolite (designated Unknown A), with a retention time of approximately 3.5 minutes reached a maximum of 2.40 % of the applied radioactivity on Day 30 of the study. A second unknown metabolite (designated Unknown B), with a retention time of approximately 32 minutes was detected at several sampling intervals but did not account for more than 1 % of the applied dose. A third unknown metabolite (designated unknown C), less polar than the test material, with an HPLC retention time of approximately 44 minutes, reached a maximum value of 2.56 % of applied radioactivity on Day 191 of the study. Since the three unknown metabolites were considered minor, further efforts at identification were not pursued.
Since the three acetonitrile/water/acetic acid extractions of Day 30 soil sample replicates removed only 11.6 to 12.1 % of the applied radioactivity, an additional extraction was carried out using 100 mL of 2N sodium hydroxide in an attempt to remove more of the soil-bound radioactive residues. As indicated, minimal quantities of the test material were detected (0.34 and 1.12 % of the applied dose). Most of the extractable radioactivity corresponded to compounds more polar than the test material. Because the base extraction was sufficiently harsh to remove non-extractable residues as well as potentially creating artifacts, subsequent samples from later sampling intervals were not subjected to this treatment. Also, because of the potential for artifact production, the nature of the residues in the sodium hydroxide extracts were not quantified except for small amounts of the test material removed.
- BBA Study
As was found during the FIFRA study, the major metabolite of the test material produced in all three soils during the BBA study was 14CO2. Minor metabolites in the soil extracts were more polar than the test material. Formation of 4-chloro-2-methylphenol reached a maximum of 2.54 % of applied radioactivity on Day 16 in the sand (Speyer 2.1), 1.96 % on Day 3 in the loamy sand (Speyer 2.2) and 0.99 % on Day 16 in the sandy loam (Speyer 2.3). Maximum formation of an unknown polar metabolite (Unknown A) with a retention time of approximately 3.5 minutes represented 2.39 % on Day 36 in the sand (Speyer 2.1), 2.89 % on Day 36 in the loamy sand (Speyer 2.2) and 2.65 % on Day 100 in the sandy loam (Speyer 2.3).
VOLATILE DEGRADATES
- FIFRA Study
Volatile degradates captured in the potassium hydroxide trapping solution accounted for an average of 37.8 % of the applied radioactivity at Day 191 of the study (test termination). Trapped 14CO2 in the form of HCO3^- and CO3^-2 was confirmed by barium precipitation from an aliquot of a composite of potassium hydroxide traps (Days 1, 3, 6, 14, 17, 23 and 30; latter samples were not used since volatiles already accounted for an average of 25.7 % of the applied radioactivity at Day 30). The precipitated Ba14CO3 was quantified by combustion analysis to account for an average of 79.4 % of the radioactivity present in the composite potassium hydroxide traps prior to barium addition. No radioactivity was detected in the supernatant. Thus, all of the volatiles trapped in the potassium hydroxide solutions were presumed to represent 14CO2. - Evaporation of parent compound:
- no
- Volatile metabolites:
- yes
- Details on results:
- SOIL VIABILITY
- FIFRA Study
Four days prior to dosing with the test material, the microbial viability of the sandy loam soil used for testing was determined by both standard plate counts and substrate-induced respirometry. Initial microbial biomass of the soil was determined to be 29 mg C/100 g soil by substrate-induced respirometry.
Four days after test termination (Day 195), microbial carbon was determined to be 7 mg C/100 g soil. A Day 225 sample was analysed by the fumigation-extraction method to confirm the Day 195 results. This alternative method for quantitatively establishing microbial biomass in soils provided a mean value of 3.7 mg microbial C/100 g soil. Plate counts enumerating bacteria, actinomycetes and fungi were somewhat diminished at Day 225 (representing test termination) relative to test initiation but were consistent with the substrate-induced respirometry and fumigation-extraction results.
- BBA Study
The microbial viability of the three German soils (Speyer 2.1, sand; Speyer 2.2, loamy sand; and Speyer 2.3, sandy loam) used in the 100-day study was determined a few days prior to test material dosing and at test termination (Day 104). Little change in microbial carbon was observed Speyer 2.1 and Speyer 2.3 over the 100-day incubation period. Speyer 2.2 displayed a marked drop in microbial activity (approximately half) over the 100-day study. The changes observed by SIR were paralleled in the plate counts.
DISTRIBUTION OF RADIOACTIVE RESIDUES AND MATERIAL BALANCE
- FIFRA Study
The material balance ranged from 71.4 to 105 % over the course of the study and averaged 90.0 %. Extractable residues started at 103 % (mean of two replicates) and were 4.58 % on Day 191, test termination. Bound residues (soil-bound, non-extractable radioactivity) were an average of 0.62 % of applied radioactivity at test initiation and accounted for 44.4 % of applied radioactivity at test termination. Bound residues (including base-extractable radioactivity) had a maximum average of 56.9 % on Day 30 and decreased towards the end of the study. Volatiles (predominantly CO2) represented an average of 1.18 % of applied radioactivity at the first sampling interval (Day 1) and increased to 37.8 % of applied radioactivity at test termination.
Based on the pattern of formation and decline of bound residues, it is postulated that mineralisation (formation of 14CO2 ) of the test material resulted from the slow microbial turnover of soil-bound residues.
Somewhat low material balance during the latter part of the study (averaging 78.1 to 86.8 % from Day 64 to Day 191) probably resulted from the loss of volatiles in the early part of the incubation when the majority of the volatiles were being produced. During the BBA study, average material balance was over 89 % throughout the 100-day incubation and the amount of volatiles produced was higher than in the FIFRA study. Purging of the volatiles during the FIFRA study was intermittent, 30 minutes per day. For the BBA study, a constant purge was maintained to prevent the loss of volatiles. The deficit in material balance for the FIFRA study is therefore probably the result of loss of volatiles due to intermittent purging and does not adversely affect the integrity of the study.
BBA Study
Material balance ranged from 76. 7 to 110 % of applied radioactivity for the sand (Speyer 2.1), 95. 7 to 108 % for the loamy sand (Speyer 2.2) and from 87.1 to 107 % for the sandy loam (Speyer 2.3). Material balances averaged 101, 102, and 99.5 % for the sand (Speyer 2.1), loamy sand (Speyer 2.2), and sandy loam (Speyer 2.3) soils, respectively. For the sand (Speyer 2.1), extractable residues represented an average of 102 % of the applied dose at Day 0 and accounted for 3.68 % of the dose on Day 100, test termination. For the loamy sand (Speyer 2.2), extractable residues went from an average of 102 to 5.14 % during the course of the study. Extractable residues went from an average of 103 to 4.03 % in the sandy loam (Speyer 2.3) during the 100-day study. Bound residues represented an average of 0.60 % of applied radioactivity at test initiation, reached 59.8 % on Day 36, and decreased to 43.1 % of applied radioactivity at test termination for the sand (Speyer 2.1). Bound residues in the loamy sand (Speyer 2.2) went from an average of 1.27 % of applied radioactivity at test initiation to 50.3 % on Day 71 and accounted for 44.0 % at the end of the 100-day study. Bound residues in the sandy loam (Speyer 2.3) were an average of 1.23 % of applied radioactivity at test initiation, increased to 60.6 % on Day 36, and were 51.2 % at Day 100. Volatiles (predominantly CO2) accounted for averages of 50.3, 49.6, and 42.3% of applied radioactivity at test termination in the sand (Speyer 2.1), loamy sand (Speyer 2.2) and sandy loam (Speyer 2.3) soils, respectively. Based on the formation and decline of bound residues throughout the 100-day incubations, it is postulated that CO2, in part, results from the slow, microbially mediated turnover of soil-bound residues.
The material balances and amount of volatiles produced during the BBA study were higher than for the FIFRA study. It is suspected that this was a result of the volatiles being collected by a constant purge in the BBA study compared to an intermittent purge during the FIFRA study.
TEST MATERIAL TIME COURSE
- FIFRA Study
At test termination (Day 191), test material accounted for only 1.60 % of the applied dose (average of two replicate samples). The extractable radioactivity and amount of test material in the soil extracts decreased significantly during the course of the study with the majority of the radioactive residues being either strongly associated with the soil (non-extractables) or mineralised to 14CO2. Since the vast majority of test material had been degraded and the pattern of formation and decline of major degradates had already been established, the study was terminated at Day 191.
- BBA Study
By the end of the study, test material accounted for only averages of 1.25, 2.58, and 1.16 % of the applied dose in the sand (Speyer 2.1), loamy sand (Speyer 2.2) and sandy loam (Speyer 2.3) soils, respectively. As with the FIFRA study, extractable radioactivity and amount of test material in the soil extracts decreased significantly during the 100 days of the BBA study while volatiles increased throughout.
KINETIC ANALYSIS
- FIFRA Study
The initial plot of the Ln (% test material) versus time (days) over the 191-day incubation suggested a biphasic kinetic pattern where degradation of test material proceeded much more rapidly in the initial phase. Nevertheless, the first-order half-life (based on data from Day 0 through 191) was calculated to be 30.1 days with a degradation rate constant of 0.023 days^-1 and a coefficient of determination of 0.768. The half-life of the test material was also calculated using a biphasic analysis. Between Day 0 and Day 30 the half-life for the test material was calculated to be 8.89 days. The degradation rate constant was 0.078 days^-1 and the coefficient of determination for the linear regression was calculated to be 0.998 which demonstrated an excellent fit of the data. From Day 30 to Day 191 the half-life of the test material was calculated to be 53.3 days. The degradation rate constant was 0.013 days^-1 and the coefficient of determination was calculated to be 0.444. These results demonstrate that the test material is readily degraded under aerobic conditions.
- BBA Study
Based on zero-order kinetics, the time required to achieve 50 % and 90 % degradation (DT50 and DT90) of the test material was estimated for each of the three soil types. DT50s were 8, 5 and 7 days for the sand (Speyer 2.1), loamy sand (Speyer 2.2) and sandy loam (Speyer 2.3) soils, respectively. DT90s were 18, 35 and 24 days for the sand (Speyer 2.1), loamy sand (Speyer 2.2) and sandy loam (Speyer 2.3) soils, respectively. These results indicate that the test material is readily degraded under aerobic conditions.
SOIL-BOUND RADIOACTIVE RESIDUES
Soil-bound residues from Day 64 and Day 191 sampling intervals from the FIFRA study were characterised by a humic acid/fulvic acid/humin fractionation scheme. Day 64 replicates were chosen for characterisation since they represented intermediate as well as substantial values during the incubation period; Day 191 replicates were chosen because they represented the final sampling interval of the study. The distribution of radioactivity between humic acid, fulvic acid and humin were somewhat similar at both sampling intervals. Any differences most likely reflect the continued metabolism of test material degradates during the incubation period.
TEMPERATURE
- FIFRA Study
Temperatures for the FIFRA study (Day 0 to 191) ranged from 17.6 to 27.7 °C with a mean minimum ± standard deviation of 20.0 ± 1.17 °c and a mean maximum ± standard deviation of 21.2 ± 1.25 °C.
- BBA Study
The temperature range for the BBA study was from 18.8 to 23.0 °C with a mean minimum ± standard deviation of 20.6 ±1.0 °C and a mean maximum ± standard deviation of 21.0 ± 0.8 °C. - Conclusions:
- Based on results from the FIFRA and SBA studies, the test material was found to degrade relatively rapidly in a variety of soils. Its degradative pathway involves conversion to a transient, intermediate degradate, 4-chloro-2-methylphenol, subsequent incorporation into soil bound residues, and gradual mineralisation to CO2. Thus, the test material or its degradates should not persist in soil.
- Executive summary:
During the course of this study, two separate aerobic soil metabolism studies were conducted (at 20 ± 2 °C) under GLP conditions The first study, using a sandy loam soil collected from agricultural land in the United States (Washington State) and designated the FIFRA study, was performed according to the Pesticide Assessment Guidelines, Subdivision N Chemistry: FIFRA Guideline 162-1. The second study was conducted with three soils obtained from Germany and carried out according to the BBA IV-4-1 Guidelines (designated the BBA study). The German soils included a sand (Speyer 2.1), a loamy sand (Speyer 2.2) and a sandy loam (Speyer 2.3).
The test material was applied at an application rate of 1.16 μg/g (parts per million [ppm]), which is equivalent to 2.4 lbs a.i./acre and represented the maximum seasonal application rate of the herbicide.
Several days prior to study initiation, all four soils used in these studies were determined to be microbially active based on substrate-induced respirometry and plate count methodologies. Soil microbial activity was also established shortly after test termination, again by substrate-induced respirometry and plate counts, as well as by fumigation-extraction (for the FIFRA study only). Microbial activity in the three soils used for the BBA study was essentially constant throughout the 100-day incubation period. In contrast, microbial activity in the sandy loam soil used for the FIFRA study diminished somewhat over the 191-day incubation period, probably reflecting the experimental difficulty of maintaining viable microbial populations in laboratory-style soil metabolism studies incubated over relatively long periods of time.
The FIFRA study was terminated on Day 191 of the incubation period although it had originally been intended to be a one-year study. Since data points needed to accurately calculate the kinetics of the test material degradation were obtained and the patterns of formation and decline of significant degradates were also well established, the study was terminated. The half-life of the test material was calculated to be 30.1 days over the 191-day incubation period, based on first-order kinetics. Since test material degradation appeared to follow biphasic kinetics, first-order half-lives were also calculated for Days 0 to 30 (8.89 days) and for Days 30 to 191 (53.3 days). During the 191-day incubation period, extractables from the soil markedly decreased to a mean of 11.9 % of the applied dose at Day 30 and were 4.58 % of the applied at Day 191. Soil-bound residues, in contrast, steadily increased to a maximum average of 56.9 % (mean of two replicates) at Day 30 and decreased somewhat (mean of 44.4 %) at Day 191. Volatiles, comprised predominantly of 14CO2 (based on barium hydroxide precipitation), accounted for a mean of 37.8 % of the applied dose at Day 191. Material balance ranged from 71.4 to 105 % in individual samples during the 191-day incubation with a mean ± standard deviation of 90.0 ± 8.6 %.
No individual metabolite in soil extracts from the FIFRA study exceeded 3 % of the applied dose. Based on chromatographic (HPLC) comparison to the authentic reference standard, a minor metabolite was identified as 4-chloro-2-methylphenol. This metabolite never exceeded 3 % of the applied radioactivity and appeared only as a transient metabolite in the metabolic pathway leading to the mineralisation of the test material. There was no build-up or accumulation of 4-chloro-2-methylphenol in any phase of the study. Three other metabolites, two more polar (Metabolite A and Metabolite B) and one less polar than the test material (Metabolite C) were detected but not identified due to their minimal contributions relative to the applied dose.
The degradation kinetics of the test material observed in the BBA study was quite similar to the FIFRA study. The time required to reach 50 % degradation (DT50) was estimated to be approximately 8, 5 and 7 days for Speyer 2.1, Speyer 2.2 and Speyer 2.3 respectively, based on zero-order kinetic plots (compared to a 9-day first-order half-life of the test material calculated from the FIFRA study over the first 30 days of incubation). Additionally, the time required to reach 90 % degradation (DT90) was estimated to be approximately 18, 35 and 24 days in Speyer 2.1, Speyer 2.2 and Speyer 2.3, respectively.
The distribution of radioactive residues in the BBA study was also similar to the FIFRA study. For all three German soils, extractable radioactive residues were initially quantitatively removable at Day 0 yet accounted for only 3 to 5 % of the applied dose at test termination (Day 100). Volatiles were substantial, accounting for 42 to 50 % of the applied dose. Bound residues were also significant, representing 43 to 51 % of the applied dose. Material balance for individual samples during the 100-day incubations in the three German soils ranged from 76.7 to 110 %, 95.7 to 104 % and 87.1 to 107 % in Speyer 2.1, 2.2 and 2.3, respectively. The mean ± standard deviation material balances were calculated to be 101.1 ± 7.8 %, 101.6 ± 3.0 % and 99.5 ± 5.3 %, respectively.
No individual metabolite in soil extracts from the BBA study exceeded 3 % of the applied dose. Based on chromatographic (HPLC) comparison to the authentic reference standard, a minor metabolite was identified as 4-chloro-2-methyl phenol. One other metabolite (Metabolite A) was detected but not identified due to its minimal contribution relative to the applied dose.
Based on results from the FIFRA and SBA studies, the test material was found to degrade relatively rapidly in a variety of soils. Its degradative pathway involves conversion to a transient, intermediate degradate, 4-chloro-2-methylphenol, subsequent incorporation into soil bound residues, and gradual mineralisation to CO2. Thus, the test material or its degradates should not persist in soil.
Referenceopen allclose all
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.
Test Material Degradation Kinetics in Aerobic Soils
FIFRA Soil
SFO and FOMC passed the statistical requirements to be considered a good fit including t-test, confidence limits not including zero and X-error. The FOMC was shown to have a slightly lower X-error and was a better visual fit as the SFO fit under-estimated later time-points. Outliers were excluded from the analysis (the later data-points – due to the long study duration the microbial biomass had decreased) and the data was weighted (log-transformed) to assess the applicability of bi-phasic degradation kinetics to this data-set. FOMC once again gave a better visual fit. DFOP kinetics were fitted to compare with FOMC, however the confidence limits for k2 included zero and so FOMC is considered to be the best fit for this data-set.
- Kinetic model for modelling is SFO
- Kinetic model for trigger values is FOMC
- Uncorrected
o DT50 for use as trigger value 6.4 days
o DT90 for use as trigger value 38 days
o DT50 for use in modelling 7.6 days
- Corrected values (for moisture):
o DT50 for use as trigger value 4.4 days
o DT90 for use as trigger value 26.4 days
o DT50 for use in modelling 5.3 days
BBA Soil 1
SFO and FOMC kinetics produced a good visual fit to the data-points from BBA soil 1. SFO kinetics passed all statistical requirements, with an X-error of 10.5 %. FOMC kinetics passed with an X-error of 11.2 %, however the confidence limits of α and β both included zero. Therefore, SFO kinetics provide a reliable estimation of the degradation of the test material in this soil and DFOP was not performed as it was unnecessary.
- Kinetic model for modelling and trigger values is SFO
- Uncorrected values
o DT50 for use as trigger value 7.0 days
o DT90 for use as trigger value 23.1 days
o DT50 for use in modelling 7.0 days
- Corrected values (for moisture):
o DT50 for use as trigger value 3.9 days
o DT90 for use as trigger value 13.0 days
o DT50 for use in modelling 3.9 days
BBA Soil 2
SFO and FOMC kinetics produced a good visual fit to the data-points (with outlier removed) from BBA soil 2. SFO kinetics passed the t-test for the degradation constant k and was below the guidance threshold of 15 % for the X-error value, with an X-error of 14.4 %. FOMC kinetics failed both statistical tests, with an X-error or 15.2 % and the confidence limits of α and β both included zero. FOCUS guidance states that the use of the 15 % X-error is a guideline and in fact a fit may still be considered acceptable if the visual fit is good. SFO kinetics provide a reliable estimation of the degradation of the test material in this soil and DFOP was not performed as it was unnecessary.
- Kinetic model for modelling and trigger values is SFO
- Uncorrected values
o DT50 for use as trigger value 7.3 days
o DT90 for use as trigger value 24.2 days
o DT50 for use in modelling 7.3 days
- Corrected values (for moisture):
o DT50 for use as trigger value 4.4 days
o DT90 for use as trigger value 14.6 days
o DT50 for use in modelling 4.4 days
BBA Soil 3
SFO and FOMC kinetics produced a reasonable visual fit to the data-points from BBA soil 3, though both slightly under-estimated concentrations at later time-points. Both SFO and FOMC kinetics pass the statistical tests, including t-tests, confidence limits of parameters not including zero and X-error values under 15 %. SFO had a marginally lower X-error of 3.98 %, compared with that of FOMC of 4.24 %. The SFO model provides a better fit to the data and given that the DT90 value produced (19.9 days) is covered by the fit well (in the earlier time-points where the visual fit is good) supports the use of this SFO fit in determining the degradation kinetics of the test material in this soil.
- Kinetic model for modelling and trigger values is SFO
- Uncorrected values:
o DT50 for use as trigger value 6.0 days
o DT90 for use as trigger value 19.9 days
o DT50 for use in modelling 6.0 days
- Corrected values (for moisture):
o DT50 for use as trigger value 4.2 days
o DT90 for use as trigger value 13.8 days
o DT50 for use in modelling 4.2 days
PCOC Degradation Kinetics in Aerobic Soils
FIFRA Soil
In this data-set, no decline in PCOC concentrations is observed (the study was terminated before a decline was observable).
SFO + SFO kinetics for both parent and metabolite provided reasonable visual fits for the metabolite. However, the t-test failed on the metabolite degradation constant, the confidence limits of ffM included zero and the X-error for the metabolite was 29.6 % (in exceedance of the 15 % recommended by FOCUS 2006, 2015). FOMC + SFO kinetics for the parent and metabolite respectively once again provided a reasonable visual fit for the metabolite, though all statistical tests failed as for the SFO + SFO fit. A third fit, using FOMC + DFOP for the parent and metabolite respectively also provided a reasonable visual fit for the metabolite, though all statistics failed for this fitting also.
The results from these kinetic fits for PCOC are not supported statistically, with the X-error value exceeding 15 % and the t-test on the metabolite degradation rate exceeding the 10 % threshold. This is likely due to the early conclusion of the study that did not allow the observation of a decline in the concentration of the metabolite PCOC. Therefore, the results from this soil may not be used to determine the degradation kinetics of PCOC and default values should be used in modelling.
- Kinetic model for trigger values for further studies is: None
- Kinetic model for modelling is: None
- Uncorrected values:
o DT50 for use as trigger value: Unreliable
o DT90 for use as trigger value: Unreliable
o DT50 for use in modelling: 1 000 days (default)
o ffM for use in modelling: 1 (default)
BBA Soil 1
The OLS had difficulty in securing confidence limits for kinetics fitting to this soil data-set and therefore the IRLS optimiser was used as a refinement.
SFO + SFO kinetics for both parent and metabolite provided reasonable visual fits for the metabolite. The t-test passed on the metabolite degradation constant and the confidence limits of ffM did not include zero. The X-error for the metabolite was 47.7 % (in exceedance of the 15 % recommended by FOCUS 2006, 2014), though this can be considered acceptable given the good visual fit. FOMC + SFO kinetics for the parent and metabolite respectively once again provided a reasonable visual fit for the metabolite, and though the t-test passed for the metabolite degradation constant, confidence limits could not be determined for α and β. Therefore, the SFO + SFO kinetics fit can be considered to provide reliable values for the formation and degradation of the PCOC metabolite.
- Kinetic model for modelling and trigger values is SFO + SFO
- Uncorrected values:
o DT50 for use as trigger value is 10.9 days
o DT90 for use as trigger value is 36.2 days
o DT50 for use in modelling is 10.9 days
o ffM for use in modelling is 0.029
- Corrected values (moisture):
o DT50 for use as trigger value is 6.1 days
o DT90 for use as trigger value is 20.4 days
o DT50 for use in modelling is 6.1 days
o ffM for use in modelling is 0.029
BBA Soil 2
The OLS had difficulty in securing confidence limits for kinetics fitting to this soil data-set and therefore the IRLS optimiser was used as a refinement.
SFO + SFO kinetics for both parent and metabolite provided reasonable visual fits for the metabolite, however, the t-test fails on the metabolite degradation constant, the confidence limits of ffM did include zero and the X-error for the metabolite was 63.8 % (in exceedance of the 15 % recommended by FOCUS 2006, 2014). FOMC + SFO kinetics for the parent and metabolite respectively once again provided a reasonable visual fit for the metabolite, however all statistical tests failed with an X-error of 62.7 %. Neither fit can be considered reliable.
- Kinetic model for trigger values for further studies is: None
- Kinetic model for modelling is: None
- Uncorrected values:
o DT50 for use as trigger value: Unreliable
o DT90 for use as trigger value: Unreliable
o DT50 for use in modelling: 1 000 days (default)
o ffM for use in modelling: 1 (default)
BBA Soil 3
The OLS had difficulty in securing confidence limits for kinetics fitting to this soil data-set and therefore the IRLS optimiser was used as a refinement.
SFO + SFO kinetics for both parent and metabolite provided reasonable visual fits for the metabolite. The t-test passed on the metabolite degradation constant and the confidence limits of ffM did not include zero. The X-error for the metabolite was 66.5 % (in exceedance of the 15 % recommended by FOCUS 2006, 2014), though this can be considered acceptable given the reasonable visual fit. FOMC + SFO kinetics for the parent and metabolite respectively once again provided a reasonable visual fit for the metabolite, and though the t-test passed for the metabolite degradation constant, confidence limits could not be determined for α and β. Therefore, the SFO + SFO kinetics fit can be considered to provide reliable values for the formation and degradation of the PCOC metabolite.
- Kinetic model for modelling and trigger values is SFO + SFO
- Uncorrected values:
o DT50 for use as trigger value is 24.6 days
o DT90 for use as trigger value is 81.9 days
o DT50 for use in modelling is 24.6 days
o ffM for use in modelling is 0.009
- Corrected values (moisture):
o DT50 for use as trigger value is 17.1 days
o DT90 for use as trigger value is 56.9 days
o DT50 for use in modelling is 17.1 days
o ffM for use in modelling is 0.009
The calculated values for the test material and the metabolite PCOC for degradation in soil are shown below.
Summary of Fitted Parameters for the Decline of the Test Material and the Production and Decline of PCOC for All Soil Data-Sets.
Compound |
Kinetic Model |
Fitted Parameters |
FIFRA |
BBA 1 |
BBA 2 |
BBA 3 |
Test material |
SFO |
M0(%) |
98.698 |
107.1 |
102.6 |
103.9 |
k1(d^-1) |
0.091 |
0.100 |
0.095 |
0.116 |
||
FOMC |
M0(%) |
102.6 |
107.1 |
103.8 |
103.9 |
|
α |
1.6 |
8.2 x 10^5 |
3.54 |
5.5 x 10^7 |
||
β |
11.82 |
8.2 x 10^6 |
32.08 |
4.8 x 10^8 |
||
DFOP |
M0(%) |
100.2 |
Not required |
Not required |
Not required |
|
k1(d^-1) |
0.11 |
|||||
k2(d-1) |
< 0.001 |
|||||
g |
0.91 |
|||||
PCOC |
SFO + SFO |
M0(%) |
98.68 |
107.1 |
102.6 |
103.9 |
k1(d^-1) |
0.091 |
0.100 |
0.095 |
0.116 |
||
km(d^-1) |
0.001 |
0.063 |
0.785 |
0.028 |
||
ffM |
0.009 |
0.029 |
0.119 |
0.009 |
||
FOMC + SFO |
M0 (%) |
102.2 |
107.1 |
103.9 |
103.9 |
|
α |
1.82 |
666.4 |
3.02 |
980.6 |
||
β |
14.05 |
6.7 x 10^3 |
26.78 |
8.5 x 10^3 |
||
km(d^-1) |
< 0.001 |
0.063 |
0.813 |
0.028 |
||
ffM |
0.009 |
0.029 |
0.119 |
0.009 |
||
FOMC + DFOP |
M0(%) |
102.2 |
Not required |
Not required |
Not required |
|
α |
1.82 |
|||||
β |
14.05 |
|||||
k1(d^-1) |
0.086 |
|||||
k2(d^-1) |
< 0.001 |
|||||
g |
0.59 |
|||||
ffM |
0.014 |
The most appropriate optimisations for risk assessment (either trigger values for further studies, modelling end-points or both) are highlighted in bold.
Taken as a whole, this sequence of data-sets is very useful for the calculation of the appropriate kinetic parameters for the test material and PCOC in aerobic soils. Kinetics for the parent test material could be fitted to all four soils, whilst kinetics for the metabolite, PCOC, could be fitted to two out of the four soils. For the two data-sets that could not provide reliable kinetic fits for the PCOC metabolite this was because PCOC in the FIFRA soil showed no clear decline, whilst there were limited datapoints and therefore no statistical support for the fit in the BBA 2 soil. In these cases where reliable kinetics could not be fitted, FOCUS guidance (FOCUS 2006, 2015) recommends the use of a formation fraction of 1 and a default DT50 (e.g. 1 000 days). It is clear from the two successful fits to the other two soils that this would be overly conservative and therefore only the reliable kinetics fits for the PCOC metabolite were used in the final calculation of formation fraction or degradation rate of the metabolite PCOC in aerobic soils.
A summary of the key kinetic parameters for the test material in aerobic soils is shown below. The final aerobic soil DT50 for modelling is calculated to be 4.4 days, shorter than the value of 7.1 days suggested in the Review Report (EC 2003), which was based on the same data-set but which was not normalised nor produced with the benefit of the FOCUS kinetics guidance (FOCUS 2006, 2014). The main difference is due to the moisture correction to reference conditions pF2 used in the FOCUS models.
Summary of Fitting Parameters for the Decline of the Test Material for All Data-Sets in Aerobic Soils
Endpoint |
FIFRA |
BBA 1 |
BBA 2 |
BBA 3 |
Geometric Mean |
Moisture correction |
0.695 |
0.564 |
0.603 |
0.695 |
|
Temperature correction |
- |
- |
- |
- |
|
Kinetic model for use to trigger studies |
FOMC |
SFO |
SFO |
SFO |
|
DT50 trigger (uncorrected) (days) |
6.4 |
7.0 |
7.3 |
6.0 |
|
DT90 trigger (uncorrected) (days) |
38 |
23.1 |
24.2 |
19.9 |
|
DT50 trigger (corrected) (days) |
4.4 |
3.9 |
4.4 |
4.2 |
4.2 |
DT90 trigger (corrected) (days) |
26.4 |
13.0 |
14.6 |
13.8 |
16.2 |
Kinetic model for use in modelling |
SFO |
SFO |
SFO |
SFO |
|
DT50 modelling (uncorrected) (days) |
7.6 |
7.0 |
7.3 |
6.0 |
|
DT50 modelling (corrected) (days) |
5.3 |
3.9 |
4.4 |
4.2 |
4.4 |
A summary of the key kinetic parameters for PCOC in aerobic soils is shown below. The final DT50 for modelling is calculated to be 10.2 days, rather shorter than the value of 21 days suggested in the Review Report for PCOC (UNEP 1998). However, the UNEP (1998) value was based on the “realistic worst case concept” rather than geometric mean values from specific studies as reported here. The formation fraction of PCOC from the test material was 1.9 %, which is consistent with those reported in UNEP (1998) of 2 - 5 %. These values appear reasonable for use in modelling, given the visual and statistical support for their kinetics fit.
Summary of Fitting Parameters for the Decline of PCOC for All Data-Sets in Aerobic Soils
Endpoint |
FIFRA |
BBA 1 |
BBA 2 |
BBA 3 |
Geometric Mean |
Moisture correction |
0.695 |
0.564 |
0.603 |
0.695 |
|
Temperature correction |
- |
- |
|||
Kinetic model for use to trigger studies |
- |
SFO + SFO |
- |
SFO + SFO |
|
DT50 trigger (uncorrected) (days) |
Unreliable |
10.9 |
Unreliable |
24.6 |
|
DT90 trigger (uncorrected) (days) |
36.2 |
81.9 |
|||
DT50 trigger (corrected) (days) |
6.1 |
17.1 |
10.2 |
||
DT90 trigger (corrected) (days) |
20.4 |
56.9 |
34.1 |
||
Kinetic model for use in modelling |
SFO + SFO |
SFO + SFO |
|||
DT50 modelling (uncorrected) (days) |
10.9 |
24.6 |
|||
DT50 modelling (corrected) (days) |
6.1 |
17.1 |
10.2 |
||
Formation Fraction |
0.029 |
0.009 |
0.019 (arithmetic mean) |
In the FIFRA soil from the study by Schocken (1997) the data-set was best described by FOMC kinetics, whilst the three other data-sets from BBA soils were described well by SFO kinetics. On the basis of the analysis, the most appropriate values for the critical degradation end-points of the test material in aerobic soils are:
DT50 as a trigger for further studies: 4.2 days
DT90 as a trigger for further studies: 16.2 days
DT50 for modelling: 4.4 days
To calculate the kinetic end-points for the metabolite PCOC, the information on the parent was used as well as the information on the metabolite. The SFO kinetics showed a good fit to the data from two of the four soil data-sets in the study by Schocken (1997). The other two soil data-sets (FIFRA and BBA 2) either showed no decline in PCOC during the study or limited number of data-points and so it was not possible to obtain a reliable kinetics fit for PCOC from these data-sets. However, PCOC is a metabolite of other substances too, so to get the end-points for this metabolite the whole PCOC data-set could be consulted. On the basis of the analysis, and relying on the visual fits as well as the statistical criteria, conservative estimates of the critical degradation end-points of PCOC in aerobic soils are:
DT50 as a trigger for further studies: 10.2 days
DT90 as a trigger for further studies: 34.1 days
DT50 for modelling: 10.2 days
Conversion ratio of test material to PCOC in soil 1.9 %
Period with exponentially increasing 14C-evolution and calculated doubling time (Td) for the 14CO2-evolution from soil supplied with 14C-test material (2 mg/kg) and incubated at 20, 10 and 5 °C.
Temperature (°C) |
Exponential Period (Days) |
Slope |
Doubling Time (Days) Td = 1n 2/slope |
20 |
0.3 – 5 |
0.48 |
1.4 |
10 |
0.3 - 17 |
0.14 |
4.9 |
5 |
14 - 29 |
0.1 |
6.9 |
Degradation of 14C-test material (2 mg/kg) after 96 days of incubation in soil (Roskilde) at 20, 10 and 5 °C (mean ± S.D. of three replicates).
Temperature (°C) |
% 14C in CO2 |
% 14C in Extract |
Test Material % of Added |
% 14C in Extracted Soil |
Recovery of 14C* |
20 |
71.9 ± 1.6 |
2.2 ± 0.6 |
ND |
25.6 ± 1.7 |
99.7 ± 1.7 |
10 |
58.4 ± 7.1 |
3.3 ± 0.1 |
ND |
28.7 ± 3.0 |
90.4 ± 10.2 |
5 |
63.8 ± 4.6 |
5.7 ± 0.4 |
ND |
33.8 ± 1.9 |
103.3 ± 6.8 |
ND, < 6 % of added 14C in extracts approx. ~ < 0.1 mg/kg of test material.
*14C evolved as CO2 + 14C in extracts + 14C in extracted soil.
Degradation of 14C-test material (2 mg/kg) in the 3 soil types (Flakkebjerg, Roskilde and Jyndevad). Roskilde soil at 2 and 0.2 mg/kg and Jyndevad in sterile and unsterile soil (where given, mean ± SD of three replicates).
Soil Type |
Conc. (mg/kg) |
Total Evolution of 14CO2% after Day |
%14C in Extract |
%14C in Extracted Soil |
Total Recovery of 14C* |
||
2 |
12 |
96 |
|||||
Flakkebjerg |
2 |
5.7 |
61.1 |
74.1 ± 5.7 |
1.7 ± 0.3 |
28.1 ± 0.3 |
103.9 ± 6.1 |
Roskilde |
2 |
5.8 |
59.8 |
71.9 ± 1.6 |
2.2 ± 0.6 |
25.6 ± 1.7 |
99.7 ± 1.7 |
Roskilde |
0.2 |
19.4 |
51.3 |
63.5 ± 8.8 |
3.1 ± 0.3 |
31.4 ± 5.9 |
98.0 ± 14.9 |
Jyndevad |
2 |
2.8 |
63.7 |
84.3 ± 7.2 |
2.8 ± 0.1 |
27.2 ± 1.9 |
114.2 ± 9.0 |
Jyndevad (Sterile) |
2 |
0 |
0 |
0 |
52.6 ± 4.04 |
35.4 ± 0.4 |
88.0 ± 3.7 |
* 14C evolved in CO2 + 14C extracted + 14C in extracted soil.
Degradation of 14C-test material (0.05 mg/kg ) in a soil profile (Jyndevad). Total 14C evolved in CO2 after 227 days, 14C recovered in extracts, % test material recovered by HPLC, 14C in extracted soil and total recovery of 14C (mean ± S.D. of four replicates).
Soil Depth (cm) |
% 14C in CO2 |
%14C in Extract |
Test Material % of Added |
% 14C in Extracted Soil |
Recovery of 14C* |
0 - 33 |
43.1 ± 6.3 |
3.1 ± 0.2 |
ND |
45.6 ± 1.5 |
91.8 ± 6.4 |
33 - 66 |
36.8 ± 11.2 |
15.0 ± 6.0 |
8.8 ± 4.8 |
39.5 ± 6.5 |
91.3 ± 8.4 |
66 - 99 |
35.6 ± 13.8 |
5.9 ± 1.0 |
0.7 ± 0.1 |
40.5 ± 2.9 |
81.9 ± 16.4 |
ND, < 6 % of added 14C in extracts approx. < 0.003 mg/kg of test material.
*14C evolved as CO2 + 14C in extracts + 14C in extracted soil.
Estimated half-life of test material in soil sampled at 0 - 33, 33 - 66 and 66 - 99 cm.
Soil Depth (cm) |
Estimated Half-Life Time (Days) |
0 - 33 |
7 |
33 – 66 |
70 |
66 - 99 |
34 |
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 |
R,S-test material |
15 |
43 |
0.989 |
test material |
12 |
58 |
0.996 |
|
S-test material |
21 |
33 |
0.972 |
|
Sandy loam |
R,S-test material |
11 |
63 |
0.980 |
test material |
8 |
86 |
0.992 |
|
S-test material |
12 |
56 |
0.994 |
|
Clay loam |
R,S-test material |
50 |
14 |
0.990 |
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.
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)
Summary of the Distribution of Radioactivity (Expressed as % of Applied Radioactivity)
Sampling Interval (Days) |
Soil Extracts* |
Volatiles† |
Bound Residues |
Total (Material Balance) |
Sandy Loam A (Soil # 1) |
||||
0 |
97.6 |
NA |
NA |
97.6 |
2 |
72.4 |
6.1 |
19.0 |
97.5 |
6 |
51.5 |
5.7 |
36.4 |
93.6 |
12 |
38.0 |
16.6 |
34.9 |
89.5 |
Sandy Loam B (Soil # 2) |
||||
0 |
86.4 |
NA |
NA |
86.4 |
2 |
42.5 |
4.7 |
36.3 |
83.5 |
6 |
14.7 |
10.7 |
56.4 |
81.8 |
12 |
7.5 |
22.9 |
44.4 |
74.8 |
Sand (Soil # 3) |
||||
0 |
97.0 |
NA |
NA |
97.0 |
2 |
90.1 |
2.3 |
10.1 |
102.5 |
6 |
69.6 |
3.9 |
13.7 |
87.2 |
12 |
49.7 |
21.5 |
21.0 |
92.2 |
Clay (Soil # 4) |
||||
0 |
191.5 |
NA |
NA |
191.5‡ |
2 |
86.5 |
1.7 |
11.4 |
99.6 |
6 |
65.8 |
7.3 |
19.4 |
92.5 |
12 |
42.6 |
16.3 |
26.2 |
85.1 |
NA: Not analysed
*
CH3CN/H2O/CH3COOH (80/ 20/ 2.5; v/v/v) extracts, combined.
† Greater than 97 % of volatile radioactivity was captured in the
potassium hydroxide trapping solutions. The remaining volatile
radioactivity (in summation not exceeding 1 % of the applied dose) was
captured by the polyurethane foam plugs and ethylene glycol solution.
‡ Sample thought to be dosed twice.
Time Course of Test Material Degradation
Sampling Interval (Days) |
Test Material Remaining (% of Applied) |
Sandy Loam A (Soil # 1) |
|
0 |
97.6 |
2 |
68.9 |
6 |
49.0 |
12 |
30.8 |
Sandy Loam B (Soil # 2) |
|
0 |
86.4 |
2 |
38.2 |
6 |
11.1 |
12 |
0.0 |
Sand (Soil # 3) |
|
0 |
97.0 |
2 |
86.9 |
6 |
63.5 |
12 |
44.4 |
Clay (Soil # 4) |
|
0 |
191.5* |
2 |
80.8 |
6 |
57.4 |
12 |
34.9 |
* Sample appears to have been inadvertently dosed twice.
Summary of the Distribution of Radioactive Residues (Material Balance) During the FIFRA Study (Mean Values)
Interval (day) |
% of Applied Dose |
||||
Soil Extraction |
NaOH Extraction* |
Soil Combustion |
Volatiles |
Total |
|
0 |
103 |
ND |
0.62 |
NA |
104 |
1 |
89.9 |
ND |
7.57 |
1.18 |
98.7 |
3 |
70.0 |
ND |
20.8 |
1.78 |
92.6 |
6 |
55.6 |
ND |
31.7 |
5.88 |
93.1 |
14 |
33.1 |
ND |
39.7 |
15.5 |
88.3 |
30 |
11.9 |
15.6 |
41.3 |
26.4 |
95.2 |
64 |
8.64 |
ND |
50.2 |
25.6 |
84.4 |
91 |
8.18 |
ND |
47.1 |
25.0 |
80.3 |
128 |
9.52 |
ND |
52.0 |
22.1 |
83.6 |
191 |
4.58 |
ND |
44.4 |
39.7 |
88.7 |
*Because of relatively low extractable (CH3CN:H2O:HOAc) radioactivity from Day 30 replicate samples, an additional 2N NaOH extraction was performed. This procedure was subsequently not continued for Days 64 through 191. For purposes of quantitation, the base-extractable radioactivity was considered part of the overall soil-bound residue.
ND = Not done
Summary of the Distribution of Radioactive Residues (Material Balance) from the Sand Soil (Speyer 2.1) During the BBA Study (Mean Values)
Interval (day) |
% of Applied Dose |
|||
Extraction |
Bound Residues |
Volatiles |
Total |
|
0 |
102 |
0.60 |
NA |
102 |
1 |
94.5 |
6.97 |
1.18 |
103 |
3 |
84.2 |
18.0 |
5.17 |
107 |
7 |
56.6 |
32.0 |
13.8 |
102 |
16 |
14.4 |
50.3 |
24.6 |
89.2 |
36 |
6.52 |
59.8 |
38.4 |
105 |
71 |
4.02 |
51.0 |
47.8 |
103 |
100 |
3.68 |
43.1 |
50.5 |
97.3 |
NA = Not analysed
Summary of the Distribution of Radioactive Residues (Material Balance) from the Loamy Sand Soil (Speyer 2.2) During the BBA Study (Mean Values)
Interval (day) |
% of Applied Dose |
|||
Extraction |
Bound Residues |
Volatiles |
Total |
|
0 |
102 |
1.27 |
NA |
103 |
1 |
87.9 |
10.3 |
1.88 |
100 |
3 |
72.9 |
23.6 |
7.34 |
104 |
7 |
40.8 |
40.3 |
20.2 |
101 |
16 |
25.7 |
48.4 |
29.3 |
103 |
36 |
12.7 |
47.5 |
39.8 |
100 |
71 |
6.27 |
50.3 |
46.4 |
103 |
100 |
5.14 |
44.0 |
49.6 |
98.7 |
NA = Not analysed
Summary of the Distribution of Radioactive Residues (Material Balance) from the Sandy Loam Soil (Speyer 2.3) During the BBA Study (Mean Values)
Interval (day) |
% of Applied Dose |
|||
Extraction |
Bound Residues |
Volatiles |
Total |
|
0 |
103 |
1.23 |
NA |
104 |
1 |
91.1 |
7.65 |
1.20 |
100 |
3 |
76.8 |
16.8 |
5.93 |
99.4 |
7 |
47.8 |
31.9 |
17.0 |
96.7 |
16 |
15.4 |
47.7 |
37.9 |
101 |
36 |
6.31 |
60.6 |
33.8 |
101 |
71 |
3.62 |
52.9 |
40.5 |
97.0 |
100 |
4.03 |
51.2 |
42.3 |
97.5 |
NA = Not analysed
Time Course of Test Material Degradation During the FIFRA Study (Mean Values)
Interval (day) |
%Radioactivity in Soil Extract* |
% Test Material from HPLC-RAM |
% Test Material |
0 |
103 |
100 |
103 |
1 |
89.9 |
100 |
89.9 |
3 |
70.0 |
100 |
70.0 |
6 |
55.6 |
98.1 |
54.5 |
14 |
33.1 |
96.1 |
31.7 |
30 |
27.5 |
NA |
9.11 |
64 |
8.64 |
59.3 |
5.22 |
91 |
8.18 |
51.9 |
4.89 |
128 |
9.52 |
51.7 |
5.66 |
191 |
4.58 |
33.2 |
1.60 |
*Based on applied dose
NA = Not applicable
Time Course of Test Material Loss Under Aerobic Conditions in the Sand Soil (Speyer 2.1) During the BBA Study (Mean Values)
Interval (day) |
% Radioactivity in Soil Extract* |
% Test Material from HPLC-RAM |
% Test Material |
0 |
102 |
100 |
102 |
1 |
94.5 |
100 |
94.5 |
3 |
84.2 |
99.5 |
83.7 |
7 |
56.6 |
97.5 |
55.2 |
16 |
14.4 |
84.2 |
12.1 |
36 |
6.52 |
52.9 |
3.45 |
71 |
4.02 |
44.3 |
1.78 |
100 |
3.68 |
34.1 |
1.25 |
*Based on applied dose
Time Course of Test Material Loss Under Aerobic Conditions in the Loamy Sand Soil (Speyer 2.2) During the BBA Study (Mean Values)
Interval (day) |
% Radioactivity in Soil Extract* |
% Test Material from HPLC-RAM |
% Test Material |
0 |
102 |
100 |
102 |
1 |
87.9 |
99.4 |
87.3 |
3 |
72.9 |
97.2 |
70.8 |
7 |
40.8 |
94.8 |
38.6 |
16 |
25.7 |
96.5 |
24.7 |
36 |
12.7 |
80.5 |
10.2 |
71 |
6.27 |
49.4 |
3.38 |
100 |
5.14 |
48.5 |
2.58 |
*Based on applied dose
Time Course of Test Material Loss Under Aerobic Conditions in the Sandy Loam Soil (Speyer 2.3) During the BBA Study (Mean Values)
Interval (day) |
% Radioactivity in Soil Extract* |
% Test Material from HPLC-RAM |
% Test Material |
0 |
103 |
100 |
103 |
1 |
91.1 |
100 |
91.1 |
3 |
76.8 |
100 |
76.8 |
7 |
47.8 |
97.8 |
46.7 |
16 |
15.4 |
94.0 |
14.5 |
36 |
6.31 |
57.2 |
3.60 |
71 |
3.62 |
49.2 |
1.77 |
100 |
4.03 |
27.9 |
1.16 |
*Based on applied dose
Characterisation of Bound Residues from the FIFRA Soil Metabolism Study (Humic Acid/Fulvic Acid/ Humin Fractionation)
Interval (day) |
Replicate |
% of Applied Dose |
||||
Humic Acid |
Fulvic Acid |
Humin |
Total |
Recovery |
||
64 |
A |
0.20 |
3.5 |
43.9 |
47.7 |
96.3 |
B |
0.10 |
4.0 |
44.2 |
48.3 |
94.9 |
|
191 |
A |
2.19 |
8.06 |
27.8 |
38.1 |
86.8 |
B |
3.26 |
6.75 |
27.9 |
37.9 |
88.1 |
|
Description of key information
Schocken (1997)
Based on results from the FIFRA and SBA studies, the test material was found to degrade relatively rapidly in a variety of soils. Its degradative pathway involves conversion to a transient, intermediate degradate, 4-chloro-2-methylphenol, subsequent incorporation into soil bound residues, and gradual mineralisation to CO2. Thus, the test material or its degradates should not persist in soil.
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: 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.
Supporting Study: Helweg (1993)
Under the conditions of the study estimated half-life times in the three surface soils were 3, 3 and 4 days, respectively at 20 °C. When the degradation was determined at 20, 10 and 5 °C the estimated half-life was 3, 12 and 20 days, respectively. The degradation rate of the test material increased initially with a doubling time of 1.4, 4.9 and 6.9 days, respectively at 20, 10 and 5 °C but with a 14-day lag-phase at 5 °C. In dry and in flooded soil (25 and 200 % of total water holding capacity) estimated half-lives were 10 and 15 days, respectively. At 0.2 mg/kg the half-life was 1.3 days compared to the 3 days at 2 mg/kg. In sterile soil no degradation was observed. In soil sampled at 0 - 33, 33 - 66 and 66 - 99 cm depth the estimated half-life time of the test material (0.05 mg/kg) was 7, 70 and 34 days, respectively at 10 °C.
Supporting Study: Müller & 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: Romero et al. (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: Hartley (1996)
A pilot soil metabolism study was performed to estimate the approximate half-life of the test material in two different sandy loam soils, a clay soil and a sand soil. First-order half-lives were calculated to be 7.5 and 2.5 days for sandy loam A and sandy loam B, respectively; 8.2 days for the clay soil, and 10.5 days for the sand soil, at an incubation temperature of 25 ± 1 °C.
Supporting Study: Hazlerigg & Garrett (2015)
In the FIFRA soil from the study by Schocken (1997) the data-set was best described by FOMC kinetics, whilst the three other data-sets from BBA soils were described well by SFO kinetics. On the basis of the analysis, the most appropriate values for the critical degradation end-points of the test material in aerobic soils are:
DT50 as a trigger for further studies: 4.2 days
DT90 as a trigger for further studies: 16.2 days
DT50 for modelling: 4.4 days
To calculate the kinetic end-points for the metabolite PCOC, the information on the parent was used as well as the information on the metabolite. The SFO kinetics showed a good fit to the data from two of the four soil data-sets in the study by Schocken (1997). The other two soil data-sets (FIFRA and BBA 2) either showed no decline in PCOC during the study or limited number of data-points and so it was not possible to obtain a reliable kinetics fit for PCOC from these data-sets. However, PCOC is a metabolite of other substances too, so to get the end-points for this metabolite the whole PCOC data-set could be consulted. On the basis of the analysis, and relying on the visual fits as well as the statistical criteria, conservative estimates of the critical degradation end-points of PCOC in aerobic soils are:
DT50 as a trigger for further studies: 10.2 days
DT90 as a trigger for further studies: 34.1 days
DT50 for modelling: 10.2 days
Conversion ratio of test material to PCOC in soil 1.9 %
Read-Across Substance: Smith & Hayden (1981)
The test material, as a commonly used phenoxyalkanoic acid herbicide, appears to be degraded rapidly in all three Saskatchewan soils described.
Key value for chemical safety assessment
- Half-life in soil:
- 30.1 d
- at the temperature of:
- 20 °C
Additional information
Schocken (1997)
During the course of this study, two separate aerobic soil metabolism studies were conducted (at 20 ± 2 °C) under GLP conditions The first study, using a sandy loam soil collected from agricultural land in the United States (Washington State) and designated the FIFRA study, was performed according to the Pesticide Assessment Guidelines, Subdivision N Chemistry: FIFRA Guideline 162-1. The second study was conducted with three soils obtained from Germany and carried out according to the BBA IV-4-1 Guidelines (designated the BBA study). The German soils included a sand (Speyer 2.1), a loamy sand (Speyer 2.2) and a sandy loam (Speyer 2.3). The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
The test material was applied at an application rate of 1.16 μg/g (parts per million [ppm]), which is equivalent to 2.4 lbs a.i./acre and represented the maximum seasonal application rate of the herbicide.
Several days prior to study initiation, all four soils used in these studies were determined to be microbially active based on substrate-induced respirometry and plate count methodologies. Soil microbial activity was also established shortly after test termination, again by substrate-induced respirometry and plate counts, as well as by fumigation-extraction (for the FIFRA study only). Microbial activity in the three soils used for the BBA study was essentially constant throughout the 100-day incubation period. In contrast, microbial activity in the sandy loam soil used for the FIFRA study diminished somewhat over the 191-day incubation period, probably reflecting the experimental difficulty of maintaining viable microbial populations in laboratory-style soil metabolism studies incubated over relatively long periods of time.
The FIFRA study was terminated on Day 191 of the incubation period although it had originally been intended to be a one-year study. Since data points needed to accurately calculate the kinetics of the test material degradation were obtained and the patterns of formation and decline of significant degradates were also well established, the study was terminated. The half-life of the test material was calculated to be 30.1 days over the 191-day incubation period, based on first-order kinetics. Since test material degradation appeared to follow biphasic kinetics, first-order half-lives were also calculated for Days 0 to 30 (8.89 days) and for Days 30 to 191 (53.3 days). During the 191-day incubation period, extractables from the soil markedly decreased to a mean of 11.9 % of the applied dose at Day 30 and were 4.58 % of the applied at Day 191. Soil-bound residues, in contrast, steadily increased to a maximum average of 56.9 % (mean of two replicates) at Day 30 and decreased somewhat (mean of 44.4 %) at Day 191. Volatiles, comprised predominantly of 14CO2 (based on barium hydroxide precipitation), accounted for a mean of 37.8 % of the applied dose at Day 191. Material balance ranged from 71.4 to 105 % in individual samples during the 191-day incubation with a mean ± standard deviation of 90.0 ± 8.6 %.
No individual metabolite in soil extracts from the FIFRA study exceeded 3 % of the applied dose. Based on chromatographic (HPLC) comparison to the authentic reference standard, a minor metabolite was identified as 4-chloro-2-methylphenol. This metabolite never exceeded 3 % of the applied radioactivity and appeared only as a transient metabolite in the metabolic pathway leading to the mineralisation of the test material. There was no build-up or accumulation of 4-chloro-2-methylphenol in any phase of the study. Three other metabolites, two more polar (Metabolite A and Metabolite B) and one less polar than the test material (Metabolite C) were detected but not identified due to their minimal contributions relative to the applied dose.
The degradation kinetics of the test material observed in the BBA study was quite similar to the FIFRA study. The time required to reach 50 % degradation (DT50) was estimated to be approximately 8, 5 and 7 days for Speyer 2.1, Speyer 2.2 and Speyer 2.3 respectively, based on zero-order kinetic plots (compared to a 9-day first-order half-life of the test material calculated from the FIFRA study over the first 30 days of incubation). Additionally, the time required to reach 90 % degradation (DT90) was estimated to be approximately 18, 35 and 24 days in Speyer 2.1, Speyer 2.2 and Speyer 2.3, respectively.
The distribution of radioactive residues in the BBA study was also similar to the FIFRA study. For all three German soils, extractable radioactive residues were initially quantitatively removable at Day 0 yet accounted for only 3 to 5 % of the applied dose at test termination (Day 100). Volatiles were substantial, accounting for 42 to 50 % of the applied dose. Bound residues were also significant, representing 43 to 51 % of the applied dose. Material balance for individual samples during the 100-day incubations in the three German soils ranged from 76.7 to 110 %, 95.7 to 104 % and 87.1 to 107 % in Speyer 2.1, 2.2 and 2.3, respectively. The mean ± standard deviation material balances were calculated to be 101.1 ± 7.8 %, 101.6 ± 3.0 % and 99.5 ± 5.3 %, respectively.
No individual metabolite in soil extracts from the BBA study exceeded 3 % of the applied dose. Based on chromatographic (HPLC) comparison to the authentic reference standard, a minor metabolite was identified as 4-chloro-2-methyl phenol. One other metabolite (Metabolite A) was detected but not identified due to its minimal contribution relative to the applied dose.
Based on results from the FIFRA and SBA studies, the test material was found to degrade relatively rapidly in a variety of soils. Its degradative pathway involves conversion to a transient, intermediate degradate, 4-chloro-2-methylphenol, subsequent incorporation into soil bound residues, and gradual mineralisation to CO2. Thus, the test material or its degradates should not persist in soil.
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: 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. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
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.
Supporting Study: Helweg (1993)
Degradation of 14C-ring-labelled test material was determined in three different soil types under different environmental conditions. Degradation was also determined in subsurface soil. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
The correlation between test material disappearance at 2 mg/kg and 14CO2-evolution showed that when 12 % 14C was evolved as 14CO2, half of the test material was decomposed. Estimated half-life times in the three surface soils were 3, 3 and 4 days, respectively at 20 °C. When the degradation was determined at 20, 10 and 5 °C the estimated half-life was 3, 12 and 20 days, respectively. The degradation rate of the test material increased initially with a doubling time of 1.4, 4.9 and 6.9 days, respectively at 20, 10 and 5 °C but with a 14-day lag-phase at 5 °C. In dry and in flooded soil (25 and 200 % of total water holding capacity) estimated half-lives were 10 and 15 days, respectively. At 0.2 mg/kg the half-life was 1.3 days compared to the 3 days at 2 mg/kg. In sterile soil no degradation was observed. In soil sampled at 0 - 33, 33 - 66 and 66 - 99 cm depth the estimated half-life time of the test material (0.05 mg/kg) was 7, 70 and 34 days, respectively at 10 °C.
Supporting Study: Müller & Buser (1997)
The degradation of the test material in soil under laboratory conditions was studied using enantioselective high-resolution gas chromatography/mass spectrometry. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
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.
Supporting Study: Romero et al. (2001)
The test material, as well as its racemic substance, were 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.
Supporting Study: Hartley (1996)
Prior to column leaching, a pilot soil metabolism study was conducted at an application rate of 2.4 lbs a.i./acre, representing the highest seasonal application rate of test material on turf. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
Based on chromatographic (HPLC-RAM) analysis of soil extracts and the assumption of first-order kinetics, soil half-lives of the test material were calculated in two different sandy loam soils, a sand, and a clay, at an incubation temperature of 25 ± 1 °C.
Under the conditions of the pilot study, first-order half-lives were calculated to be 7.5 and 2.5 days for sandy loam A and sandy loam B, respectively; 8.2 days for the clay soil, and 10.5 days for the sand soil.
Supporting Study: Hazlerigg & Garrett (2015)
The study concerns a degradation kinetics study to re-assess the degradation of the test material and metabolites in four aerobic soils, as reported in Schocken (1997), using FOCUS (2006, 2014). SFO kinetics were used in the first instance (most appropriate for use in environmental fate modelling), with FOMC and DFOP used to determine trigger values for additional studies where biphasic kinetics were observed. CAKE 3.1 was used to perform the kinetics fitting procedure, with the OLS optimiser. The IRLS optimiser was used where confidence limits with the initial OLS fit were unreliable (predominantly for metabolites). The study was awarded a reliability score of 4 in accordance with the criteria set forth by Klimisch et al. (1997).
In the FIFRA soil from the study by Schocken (1997) the data-set was best described by FOMC kinetics, whilst the three other data-sets from BBA soils were described well by SFO kinetics. On the basis of the analysis, the most appropriate values for the critical degradation end-points of the test material in aerobic soils are:
DT50 as a trigger for further studies: 4.2 days
DT90 as a trigger for further studies: 16.2 days
DT50 for modelling: 4.4 days
To calculate the kinetic end-points for the metabolite PCOC, the information on the parent was used as well as the information on the metabolite. The SFO kinetics showed a good fit to the data from two of the four soil data-sets in the study by Schocken (1997). The other two soil data-sets (FIFRA and BBA 2) either showed no decline in PCOC during the study or limited number of data-points and so it was not possible to obtain a reliable kinetics fit for PCOC from these data-sets. However, PCOC is a metabolite of other substances too, so to get the end-points for this metabolite the whole PCOC data-set could be consulted. On the basis of the analysis, and relying on the visual fits as well as the statistical criteria, conservative estimates of the critical degradation end-points of PCOC in aerobic soils are:
DT50 as a trigger for further studies: 10.2 days
DT90 as a trigger for further studies: 34.1 days
DT50 for modelling: 10.2 days
Conversion ratio of test material to PCOC in soil 1.9 %
Read-Across Substance: 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.
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