Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 230-711-3 | CAS number: 7287-19-6
Biodegradation in soil
Administrative data
Link to relevant study record(s)
- Endpoint:
- biodegradation in soil: simulation testing
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 28 Jun 2000 to 16 Nov 2001
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with national standard methods
- Qualifier:
- according to guideline
- Guideline:
- other: BBA-Guideline Part IV, 4-1 ("Verbleib von Pflanzenschutzmitteln im Boden: Abbau, Umwandlung und Metabolismus").
- Version / remarks:
- December 1986
- GLP compliance:
- yes (incl. QA statement)
- Test type:
- laboratory
- Radiolabelling:
- yes
- Oxygen conditions:
- aerobic
- Soil classification:
- USDA (US Department of Agriculture)
- Year:
- 2 000
- Soil no.:
- #1
- Soil type:
- loam
- % Clay:
- 11.36
- % Silt:
- 50.46
- % Sand:
- 38.18
- % Org. C:
- 2.35
- pH:
- 7.23
- CEC:
- 15.7 other: mmole/z/100 g
- Bulk density (g/cm³):
- 0.95
- Details on soil characteristics:
- This study was carried out with one test soil. Freshly collected soil was used. The storage period of the soil before start of the study was less than two months. During storage, it was taken care that no anaerobic conditions occurred. For the first two days July 26 - July 28, 2000), the soil was stored at 20°C in the dark). Later, deviating from the study protocol, the soil was stored at the intended temperatures for the study instead of storing the total amount at 20°C After July 28, 2000, one part was stored at 10°C, the other part at 20°C.
Soil (loam)
- Name (supplier): „Gartenacker“ Batch 5/00*
- Site of collection: Les Barges, CH-1896 Vouvry, Switzerland*
- Time of collection: May 2000*
- Agricultural use of soils: none*
- Last year’s crop: not available*
- Depth of soil collection: 0 - 20 cm*
- Arrival at lab: July 26, 2000
- Storage conditions at the lab: 30 ± 2 % of max water holding capacity, for 20 °C study at 20 ± 2 °C, for 10 °C study a t 1 0 ± 2 °C aerobic, dark
- Storage period under these conditions at the lab: July 28, 2000- August 10, 2000
- pH-value (KCI): 7.23*
- CaCO3: 8.17 %*
- Max. water holding capacity (g H2O/100g dry weight): 67.9
- Organic carbon: 2.35 %*
- Total nitrogen: 0.25 %*
- Cation exchange capacity: 15.7 mmole/z*/100 g
- Microbial biomass (µg C/100g soil), test start: 565.5**
- Microbial biomass (µg C/100g), at the end of the test: Experiment at 10 °C: 583.9**, Experiment at 20 °C: 518.3**
- Clay (< 2 pm): 11.36*
- Silt (2- 50 pm): 50.46*
- Sand (50 - 2000 pm): 38.18*
- Bulk density: 0.95 g/cm3
(*Information given by the sponsor, ** Determined by: Bayerische Landesanstalt fur Bodenkultur und Pflanzenbau, Munchen)
- Preparation of soil for experiment: passed through 2 mm sieve; moisture content adjusted to 30% of maximum water holding capacity before application (40 % after application); storage at 22°C / 80% relative air humidity for 5 days. - Soil No.:
- #1
- Duration:
- >= 120 - <= 179 d
- Soil No.:
- #1
- Initial conc.:
- 3.3 mg/kg soil d.w.
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- CO2 evolution
- radiochem. meas.
- Soil No.:
- #1
- Temp.:
- 10 °C
- Humidity:
- 40 % of the maximum water capacity of the soil
- Microbial biomass:
- 583.9 µg C/100g soil
- Soil No.:
- #1
- Temp.:
- 20 °C
- Humidity:
- 40 % of the maximum water capacity of the soil
- Microbial biomass:
- 518.3 µg C/100g soil
- Details on experimental conditions:
- APPLICATION OF THE TEST SUBSTANCE
The amount of active ingredient to be applied was calculated as follows.
- Assumed Penetration Depth: 5 cm
- Treated Soil Volume: 500 m3/ha
- Specific Mass of Soil: 1.5 t/m3 (= 7501/500 m3)
- Prospective Amount to be Used: 2500 g active ingredient/ha = 2500 g/7501= 2500 mg/750 kg soil = approx. 3.3 mg/kg = 167 µg/50 g soil
- The total amount of purified radiolabelled active ingredient delivered was dissolved in approx. 30 mL acetone and transferred into another vial. The concentration of the test substance was chosen in such a way that not more than 0.5 g acetone/50 g soil (< 1 %) was used for application.
- Spec Radioact. of Test Substance: 2.02 kBq/µg
- Target Radioact. for Application: 337 kBq / 167 µg (amount for 50 g soil)
- Target Volume for Application: 337 kBq /0.492 mL test substance in acetone
- Actual Amount Applied: 351.8 kBq (= 174 µg = 104% of target amount)
- Before applying the active ingredient, the soil moisture was adjusted so that the soil moisture after
application of the test substance solution as given above was 40 % of the maximum water holding
capacity.
TEST PROCEDURE
- 50 g of the sieved soil were transferred into 300 mL Erlenmeyer flasks. After application of the test substance, the CO2-traps were mounted on top of the flasks. One half of the flasks was stored at 10°C ± 2°C, the other half at 20°C + 2°C in the dark. In order to assure that the moisture content of the soils remained constant, the moisture was controlled by weighing untreated control flasks at least once each week throughout the test period. For this procedure, the flasks had to be opened. Since the untreated control flasks did not contain radioactively labelled carbon, the gas phase needed no special treatment before opening the flasks. If the moisture content of the soil dropped below 36 % of maximum water capacity, bidestilled water was added to the soil surface by introducing a syringe through the septum of the side openings. For this reason the CCV traps need not be removed regularly for replacement of soil water. Traps were only exchanged if condensed water accumulated in the traps. Before opening the flasks for sampling, air was pressed into the side openings in order to trap 14CO2 eventually present in the gas phase. Two flasks (= duplicate determination) were opened at each of the following sampling time points: For the soil degradation at 10°C: 0.25 h, 7, 14, 21, 28, 56, 90, 120 and 179 days after application of the test substance, For the soil degradation at 20°C: 0.25 h, 7, 14, 21, 28, 56, 90 and 120 days after application of the test substance
SOIL EXTRACTION
- Two specimens were taken for each soil at each sampling date (complete double determination). The soil specimens were extracted using two to five steps:
- First step: 3 extractions with methanol/water (80/20, v/v)
- Second step: 3 extractions with redistilled water. Each of these extractions was carried out for 15 minutes in an ultrasonic bath (< 40°C) and by stirring the soil several times during sonification. Liquid and solid phase were separated by centrifugation (10 minutes, 2000 g). For determination of the radioactivity, aliquots of each extraction step were combined. For all specimens containing sufficient radioactivity, up to 1 mL of the extracts were used directly for thin-layer chromatography.
- Third step: Soxhlet extraction using acetonitrile/water (80/20, v/v). This extraction was carried out approximately for 16 hours and was carried out routinely starting with 14 days after application. Quantities extracted before this time were marginal. The extracts of the first and second step were analysed separately by liquid scintillation counting. The sum of the results of step 1+2 and step 3 are reported separately. After extraction, aliquots of the soil were dried during 3-4 days at 40°C for soil combustion. Extraction using harsh conditions were applied only for the 120 d sampling date for the experiments at both temperatures. The following harsh conditions were used only for the 120 days samples which were considered as representative samples.
- Fourth step: A second reflux extraction using acetonitrile/0.1 N HCI (80/20, v/v)
- Fifth step: Extraction using 0.5 N NaOH (soluble fraction contains the fulvic acid fraction, the insoluble fraction corresponds to the humic acid fraction).
SOIL COMBUSTION
In order to determine the non-extracted residues in the soil, the soil samples were combusted. The 14CO2 generated during combustion was collected in Carbosorb E. The radioactivity of the combusted samples was analysed by liquid scintillation counting.
DETERMINATION OF 14CO2 IN THE TRAPS
The 14CO2-traps were removed from the test flasks and stored separately in plastic bags at -20°C until measurement. For measurement, the quartz wool was transferred into a 100 mL Erlenmeyer flask. Organic components present in the wool were extracted twice using approx. 45 mL ethyl acetate each time. For analysis, approximately 2 mL were analysed by liquid scintillation counting. In this study, no sample contained radioactivity above the background signal. The soda lime of each trap was combined and transferred into a gas tight flask containing a magnetic stirring rod. The flask was placed on a magnetic stirrer and the stirrer was started. Approx. 55 mL HCI (32 %) were added. 14CO2 trapped as carbonate was regenerated at this pH value. Fine bubbles of nitrogen were pressed into the measuring system. This procedure was carried out first for 30 minutes at room temperature, followed by 60 minutes at 60-70°C. 14CO2 was collected quantitatively in Carbosorb E (in two consecutive collecting traps). An aliquot of this liquid was analysed by liquid scintillation counting. - Soil No.:
- #1
- % Recovery:
- 95.9
- Remarks on result:
- other: for the experiment at 10°C
- Soil No.:
- #1
- % Recovery:
- 95
- Remarks on result:
- other: for the experiment at 20°C
- Key result
- Soil No.:
- #1
- DT50:
- 33.5 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 20 °C
- Soil No.:
- #1
- DT50:
- 85.6 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 10 °C
- Transformation products:
- not specified
- Details on transformation products:
- FORMATION AND DECLINE OF METABOLITES
In some cases, metabolites could be identified by comparing the Rf-values of authentical analytical standards with the Rf-values of the TL-chromatograms of the extracts. These tables contain data on identifiable peaks of all chromatograms. Column 4 with unidentified peaks is calculated by the difference of the sum of columns 2 , 5 and 6 to 100 %. Most unidentified signals were much more polar than the test substance. Unpolar compounds comprised less than 1 % ROI and are therefore not mentioned separately. The following degradation products used as analytical standards were found: M4 + M15 (could not be separated using the methods given above) and M2. The maximum concentrations of M4 + M15 were 0 7 % of applied radioactivity at 10°C and 0.6 % at 20°C. This compound occurred as an intermediate reaching the maximum concentration 28 days after application at both temperatures. Thereafter, it declined to <0.3 % at 10°C after 179 days and disappeared completely at 20°C after 120 days. The concentration of M2 was calculated using the peak found in solvent system 2 (corresponding to M4+M2+M15) and subtracting the results for M4 + M15 found using solvent system 1. Rather high amounts of M2 were found in the soil extracts during the study. The highest concentrations of this compound were found at the end of the study: 24.5 % of applied radioactivity at 10°C after 179 days and 34.0 % of applied radioactivity at 20°C after 120 days. Also some small amounts of either of the three products M11, M16 and M13 were found. These three products could not be separated using the analytical method described. However, these three compounds reached only a maximum of 1.1 %, 2.1 %, 3.7 % and 5 3 % of applied radioactivity at 20°C after 28, 56, 90 and 120 days of soil degradation at 20°C. - Evaporation of parent compound:
- no
- Volatile metabolites:
- no
- Residues:
- yes
- Details on results:
- RESULTS OF THE RADIOACTIVITY MEASUREMENTS
- Mass Balance: Table 1 in “Any other information on results incl tables” shows the results of the radioactivity measurements. All data refer to the applied radioactivity of 351.8 kBq / 50 g soil (dry mass). The average mass balance was 95.9 % for the experiment at 10°C and 95.0 % for the study at 20°C. The variation coefficient was ±2.3 % at both temperatures.
- Distribution of Radioactivity: The detailed results are shown in Table 1 in “Any other information on results incl tables”. In the experiment carried out at 10°C, the total radioactivity which could be released by cold extraction (aqueous methanol and water) and Soxhlet extraction (aqueous acetonitrile) amounted to 93.5 % at day 0 It decreased slowly to 73.0 % after 179 days of incubation. For the investigation at 20°C, the extractable residues were 92.9 % at the day of application and declined to 60.0 % until day 120. Harsh extraction using acetonitrile - 0.1N hydrochloric acid (80/20) and 0.5N sodium hydroxide (extraction steps 4+5) released only traces of radiocarbon (< 1.1 % of applied). During the same time, the non-extractable residues in the 10°C investigation increased steadily from 3.6 % at day 0 and finally reached 21 % at day 179. The non-extractable residues in the 20°C study augmented from 2.8 % to 32.6 % after 120 days. The final end product of degradation, CO2, was up to 0.7 % (10°C) and 2.4 % (20°C) at study termination.
DEGRADATION OF THE SUBSTANCE
Table 2 - Table 3 in “Any other information on results incl tables” show the results of thin layer chromatographic measurements with respect to the degradation of the parent compound. All results refer to the applied radioactivity of 351.8 kBq / 50 g soil (dry mass). The average results of solvent systems 1+2 (last column of Table 2 - Table 3 in “Any other information on results incl tables”) were used for calculation of the half-lives and DT-90 values (see Table 4 in “Any other information on results incl tables”). In the experiment conducted at 10°C, the initial concentration of the substance declined from 89.7% of the applied radiocarbon to 22.5 % after incubation for 179 days When incubated at 20°, the parent active ingredient degraded from 89.7 % at day 0 to 8.2 % at day 120. Using best fit (1st order kinetic), the following half-lives and DT-90 values were calculated.
RESULTS OF HALF-LIVES AND DT-90 CALCULATIONS
Table 4 in “Any other information on results incl tables” shows the results of half-life and DT-90 calculations. For both degradation conditions in the Gartenacker soil, the best fit was found to be a 1st order kinetic (exponential function). - Conclusions:
- The half life of the substance was found to be 85.6 days at 10°C and 33.5 days at 20°C.
- Executive summary:
The soil degradation of the radiolabelled test substance was determined at 10°C and 20°C according to the BBA-Guideline Part IV, 4-1 and in compliance with GLP criteria. For this purpose, one agriculturally used loam soil containing 2.35 % organic carbon was used. The test substance was applied to 50 g of each of these soils. The application rate was174 µg a.i./ 50 g soil, which was slightly higher (104 %) than the intended use of 2500 g active ingredient / ha on the field. The soil degradation study was carried out at 10°C and at 20°C over a period of 120 d (20°C) and 179 d (10°C) after the application. During this period, the soil moisture was adjusted to 40 % of the maximum water capacity of the soil. Sampling dates were: 0.01 d, 7 d, 14 d, 28 d, 56 d, 90 d and 120 d for both temperatures. An additional sample was taken for the 10°C experiment after 179 d. At each of these sampling dates, the amount of extractable radioactivity in soil, the amount of non-extractable residues in soil and the amount of 14CO2 generated were determined. Additionally, the extracts of the samples were analysed using two thin-layer chromatographic methods to find out which portion of the radioactivity was the parent compound. The following results were obtained. The average mass balance was 95.9 % for the experiment at 10°C and 95.0 % for the experiment at 20°C. The variation coefficient was +2.3 % at both temperatures. The extractable residues in the experiment at 10°C decreased from initial values of 93.5 % to 73.0 % after 179 days of incubation. The corresponding values in the study at 20°C declined from 92.9 % to 58.9 % at day 120. The parent compound was degraded from 89.7 % of applied to 22.5 % after 179 d at 10°C. At 20°C, the parent compound was degraded from 89.7 % of applied to 8.2 % after 120 d. Within the same period, the concentration of the major degradation product increased to 24.5 % of applied radioactivity after 179 days at 10°C and to 34.0 % after 120 d at 20°C The amount of non-extractable residues increased from 3 - 4 % at the beginning to 21.0 % at 10°C on day 179 and 32.6 % at 20°C on day 120 (in each case the end of the investigations). The final end product of degradation, CO2, was up to 0.7 % (10°C) and 2.4 % (20°C) at study termination. Beside the parent compound two other major metabolites were found: M4 + M15 (could not be separated using the methods given above) and M2. The maximum concentrations of M4 + M15 were 0.7 % of applied radioactivity at 10°C and 0.6 % at 20°C. These compounds occurred as intermediates reaching the maximum concentration 28 d after application at both temperatures and declined to <0.3 % at study termination. The highest concentrations of M2 were found at the end of the study: 24.5 % of applied radioactivity after 179 d at 10°C and 34.0 % of applied radioactivity after 120 d at 20°C. Using a first order kinetic model for the calculation of the half-life (DT-50, disappearance time of 50 %) and the DT-90 (disappearance time of 90 %), the following results were obtained:
Half-life
DT-90
Loam, Gartenacker, 10°C
85.6 days
284 days
Loam, Gartenacker, 20°C
33.5 days
111 days
- Endpoint:
- biodegradation in soil: simulation testing
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 01 Jul 1996 to 29 Jun 1998
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with national standard methods
- Qualifier:
- according to guideline
- Guideline:
- other: BBA-Guideline Part IV, 4-1 ("Verbleib von Pflanzenschutzmitteln im Boden: Abbau, Umwandlung und Metabolismus").
- Version / remarks:
- December 1986
- GLP compliance:
- yes
- Test type:
- laboratory
- Radiolabelling:
- yes
- Oxygen conditions:
- aerobic/anaerobic
- Soil classification:
- USDA (US Department of Agriculture)
- Year:
- 1 998
- Soil no.:
- #1
- Soil type:
- silt loam
- % Clay:
- 8.2
- % Silt:
- 55.8
- % Sand:
- 36
- % Org. C:
- 2.2
- pH:
- 7.25
- CEC:
- 14.9 meq/100 g soil d.w.
- Bulk density (g/cm³):
- 1.02
- Soil no.:
- #2
- Soil type:
- loamy sand
- % Clay:
- 2.9
- % Silt:
- 22.7
- % Sand:
- 74.4
- % Org. C:
- 1.1
- pH:
- 7.6
- CEC:
- 6.5 meq/100 g soil d.w.
- Bulk density (g/cm³):
- 1.3
- Soil no.:
- #3
- Soil type:
- loamy sand
- % Clay:
- 7.3
- % Silt:
- 12.1
- % Sand:
- 80.6
- % Org. C:
- 2.2
- pH:
- 5.65
- CEC:
- 12 meq/100 g soil d.w.
- Bulk density (g/cm³):
- 1.09
- Soil no.:
- #4
- Soil type:
- sandy loam
- % Clay:
- 4.4
- % Silt:
- 31.8
- % Sand:
- 63.8
- % Org. C:
- 1.3
- pH:
- 7.5
- CEC:
- 8.2 meq/100 g soil d.w.
- Bulk density (g/cm³):
- 1.18
- Details on soil characteristics:
- - The study was carried out with four different soil types.
- Soil Types: 1) Gartenacker (for aerobic and anaerobic metabolism study), 2) Pappelacker 2 (for aerobic rate study), 3) Speyer 2.2 (for aerobic rate study), 4) Weide (for aerobic rate study)
- Sources: 1) Les Barges, Vouvry / VS, Switzerland., 2) Les Barges, Vouvry / VS, Switzerland., 3) LUFA, Speyer, Germany., 4) Les Barges, Vouvry / VS, Switzerland. The soils were stored under a grass cover in the greenhouse until use.
- Specifications: Soil parameters, e.g. organic carbon (%), pH, cation exchange capacity (CEC), Ntota|, CaCO3, particle size, maximum water holding capacity (MWC), field capacity (FC), and microbial biomass were determined. The corresponding data are given in Table 1 in “Any other information on materials and methods incl tables”. The water of the anaerobic samples was specified for pH, oxygen content and redox potential. Extra samples with vehicle only were set-up to follow these properties.
- Soil Preparation: Before use the soils were sieved through a 2 mm sieve. After treatment the soil moisture was adjusted to 40 % of the max. water holding capacity. - Soil No.:
- #1
- Duration:
- 351 d
- Soil No.:
- #2
- Duration:
- 168 d
- Soil No.:
- #3
- Duration:
- 168 d
- Soil No.:
- #4
- Duration:
- 168 d
- Initial conc.:
- 2.5 kg/ha d.w.
- Based on:
- test mat.
- Remarks:
- for all soils
- Parameter followed for biodegradation estimation:
- CO2 evolution
- radiochem. meas.
- Temp.:
- 20 ± 2 °C
- Humidity:
- 40 % of the max. water holding capacity.
- Microbial biomass:
- 18.6 - 23.5 mg microbial carbon / 100 g soil.
- Details on experimental conditions:
- EXPERIMENTAL DESIGN
- Apparatus: a) Metabolism study: Soil samples (aerobic and anaerobic) were incubated in 1L Steilbrust flasks connected to an absorption bottle containing 50 ml ethylene glycol and two absorption bottles each containing 50 ml 2N NaOH to pass through the effluent air or nitrogen, b) Rate study: Soil samples (aerobic) were incubated in 200 ml Erlenmeyer flasks connected to an absorption bottle containing 50 mL ethylene glycol and two absorption bottles each containing 50 mL 2N NaOH to pass through the effluent air. General: The aerobic samples were stored under ventilation with moistened air (flow-rate: 10 - 60 ml / min) in incubation chambers. The anaerobic samples were waterlogged with about 200 ml water and ventilated with nitrogen twice per day each for 30 min. Prior to treatment the anaerobic samples were equilibrated at 20 °C for about 30 days.
- Sample Size: a) Metabolism study: 200 g soil (dry weight) b) Rate study: 75 g soil (dry weight). For each biomass determination 660 g soil (dry weight) were used. Thus, three flasks each with about 220 g soil were incubated for each biomass analysis. The samples received the equivalent amount of vehicle (acetone) as the treated samples.
- Number of Samples: a) Metabolism study: Single samples were set up, each equipped with a trapping system. b) Rate study: Soil samples were incubated in Erlenmeyer flasks (75 g samples)
connected in one line equipped with a trapping system.
- Moisture Content: The soil moisture was set to 40 % of the maximum water holding capacity (MWC). The soil moisture was checked and adjusted, if necessary, every week during the first month and thereafter in intervals of about two weeks.
- Microbial Biomass: The microbial biomass of the soils was determined at the beginning of the study and after 120 d of incubation (Table 1 in “Any other information on materials and methods incl tables”).
- Temperature: Soil samples were incubated in the dark at 20 ± 2 °C. The temperature was monitored during incubation.
- Concentration: The compound was individually added to the soil samples at a quantity corresponding to the maximum recommended field rate of 2.5 kg a.i. / ha or 3.3 mg / kg (based on the soils dry weight). The test substance was added as acetone solution to the soil, whereby not more than 0.5 ml acetone / 100 g dry soil were used. After treatment the soil moisture content was adjusted to 40 % of its MWC.
PREPARATION OF THE TEST SUBSTANCE
The substance (18.59 mg labelled and 15.75 mg not labelled) was dissolved in acetone. Thereafter, three dilutions in acetone were prepared and analysed for amount of radioactivity, radiochemical purity and amount of G 34161. Based on the analytical results, the amount of the test substance was calculated to be 34.35 mg / 5 ml acetone. For treatment, individual aliquots of the acetone solution (0.0427 - 0.1455 mL for each sample) were placed into vials (Stock Solutions I) and kept deep frozen until application.
TREATMENT OF SOIL SAMPLES
Mean amounts actually applied are given in Table 2 in “Any other information on materials and methods incl tables”. The individual aliquots of Stock Solution I were evenly applied to the surface of 200 and 75 g soil by means of a Hamilton syringe. Thereafter, the test substance was incorporated into the soil and its moisture content adjusted to 40 % MWC by adding de-ionised water. For the anaerobic samples the application aliquots of Stock Solution I were applied to the water surface.
INCUBATION
- Metabolism flasks were placed into an incubation chamber and connected to the other parts of the metabolism apparatus. Thereafter, the aerobic systems were continuously ventilated with moistened air at a flow rate of 20 - 60 mL / min. The anaerobic samples were ventilated with nitrogen twice per day each for 30 min. Incubation was carried out at 20 ± 2 °C in the dark. During the incubation period the temperature in the incubation chambers was automatically controlled.
SAMPLING
- Soil Samples: Aerobic soil samples were taken for analysis of parent molecule and number and amount of metabolites after 0, 7, 14, 28, 42, 56, 70, 98, 120, 168 (all soils) and 351 days of incubation (only Gartenacker soil). The anaerobic soil samples were taken for analysis after 0, 14, 42, 70 and 120 days of incubation. Due to a technical error positive redox potentials were recorded, although the oxygen concentration was at or below 0.1 mg/L, thus, clearly demonstrating anaerobic conditions. However, partial repetition of the experiment with new samples over 120 days showed negative redox potentials, thus, confirming the low oxygen concentration (0.1 - 0.2 mg/L) and anaerobic conditions. Soil samples were directly analysed.
- Microbial Biomass of Soil: Microbial biomass was determined prior to application and after 120 d of incubation.
- Absorption Solutions: The absorption solutions were exchanged in about weekly intervals during the first month and thereafter in intervals of about two weeks.
- Redox Potential, pH and Oxygen Concentration of Anaerobic Samples: Redox potential, pH and oxygen concentration in the aqueous layer of anaerobically incubated samples were determined in untreated reference flasks and in treated flasks at sampling by using commercially available electrodes. Prior to treatment the parameters were followed for about one month.
SAMPLE PREPARATION AND EXTRACTION PROCEDURE
- EXTRACTION OF SOIL SAMPLES
- Extraction at Room Temperature: The aerobic and anaerobic8 soil samples were transferred into 500 and 250 mL centrifuge tubes and extracted with about 400 and 200 mL acetone-water (80:20, v:v) under strong agitation (300 rpm) at 20 °C for 60 min. Thereafter, the suspensions were centrifuged at 2500 rpm for 10 minutes at 10 °C and the supernatant filtered into an adequate measuring cylinder. This extraction procedure was repeated three times under the same conditions. The volumes of all extracts were recorded and triplicate aliquots of 1 ml submitted to radioactivity measurements by liquid scintillation counting (LSC). Thereafter, the extracts were combined. Aliquots of the combined extracts were concentrated at 35 °C to a volume of 10.0 mL using a vacuum rotary evaporator. Recovery checks were performed for each step by taking aliquots and determining their radioactivity by LSC.
- Soxhlet Extraction: After extraction at room temperature the soil samples were extracted in a Soxhlet apparatus with acetone for 6 h. Soxhlet extracts containing less than 5 % of the radioactivity applied were not further analysed, due to the low amount of radioactive material present. After Soxhlet extraction selected soil samples were submitted to further harsh extraction procedures and organic matter fractionation.
- Harsh Extraction: Two subsequent extraction steps were performed: a) First harsh extraction step:
An aliquot (20 g) of the Soxhlet extracted soil was air dried, pulverised and thereafter suspended in 100 ml acetonitrile : water (4:1, v:v) and refluxed in a centrifuge tube (250 mL) for two hours. After centrifugation (2500 rpm, 10 min) the solvent was decanted, filtered through a fluted filter paper and the volume and radioactivity determined. b) Second harsh extraction step. The second extraction step was performed by adding 100 ml acetonitrile : 0.1 N HCI (9:1, v:v) to the same soil sample and refluxing it again in the centrifuge tube for two hours. After centrifugation (2500 rpm, 10 min) the solvent was decanted, filtered through a fluted filter paper and the volume and radioactivity determined.
- Organic Matter Fractionation: After the harsh extraction procedures the remaining soil sample (20 g) was mixed with 60 mL 0.5 N aqueous NaOH solution and extracted by shaking at room temperature for about 17 hours at 200 rpm followed by centrifugation at 2500 rpm for 10 min. To the decanted supernatant concentrated HCI was added until a pH < 1 was reached. The resulting suspension was centrifuged as described above, the supernatant decanted, the volume determined (fulvic acid fraction) and radioassayed by LSC. The remaining solid (humic acid fraction) was dissolved in 25 ml 0.5 N NaOH and radioassayed by LSC. The amount of the insoluble humin fraction was calculated by subtraction of the amounts of harsh extracts and alkali extract from the amount of non-extractables after Soxhlet extraction.
- DETERMINATION OF NON-EXTRACTABLES
- The extracted soil was air-dried, weighed, homogenised and the residual activity determined by combustion. For this purpose, aliquots of soil samples of up to approximately 1 g were combusted in an oxygen stream at about 800 °C with copper oxide as catalyst using a Robox II sample oxidiser. Radioactivity present in the absorption solution was measured by LSC.
- PREPARATION OF WATER SAMPLES
- Aliquots of 20 - 300 mL of the decanted water were taken and concentrated to a volume of 2 - 10 mL using a vacuum rotary evaporator. Recovery checks were performed for each step by taking aliquots and determining their radioactivity by LSC.
- ANALYSIS OF VOLATILE PRODUCTS
- Aliquots of the trapping solutions were measured in duplicate by LSC. For representative samples, the nature of radioactivity trapped in NaOH (14CO2) was proven. For this purpose, 1 mL of the NaOH-solution was mixed with 1 mL aqueous 1 M-BaCI2-solution. After centrifugation (5 min, 13000 rpm) 1 mL of the clear supernatant was measured by LSC.
- MICROBIAL BIOMASS OF THE SOIL
- The microbial biomass in the soil was determined by using the respiratory method of Anderson and Domsch. - Soil No.:
- #1
- % Recovery:
- 89.3
- Remarks on result:
- other: Aerobic system after 351 days incubation
- Soil No.:
- #1
- % Recovery:
- 101.5
- Remarks on result:
- other: Anaerobic system after 120 days incubation
- Soil No.:
- #2
- % Recovery:
- 101.2
- Remarks on result:
- other: Aerobic system after 168 days incubation
- Soil No.:
- #3
- % Recovery:
- 105.1
- Remarks on result:
- other: Aerobic system after 168 days incubation
- Soil No.:
- #4
- % Recovery:
- 93.2
- Remarks on result:
- other: Aerobic system after 168 days incubation
- Soil No.:
- #1
- % Degr.:
- 88
- Parameter:
- radiochem. meas.
- Sampling time:
- 351 d
- Soil No.:
- #2
- % Degr.:
- 58.3
- Parameter:
- radiochem. meas.
- Sampling time:
- 168 d
- Soil No.:
- #3
- % Degr.:
- 24.1
- Parameter:
- radiochem. meas.
- Sampling time:
- 168 d
- Soil No.:
- #4
- % Degr.:
- 59.3
- Parameter:
- radiochem. meas.
- Sampling time:
- 168 d
- Key result
- Soil No.:
- #1
- DT50:
- 26.4 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 20 °C
- Remarks on result:
- other: Aerobic system
- Key result
- Soil No.:
- #2
- DT50:
- 26.5 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 20 °C
- Remarks on result:
- other: Aerobic system
- Key result
- Soil No.:
- #3
- DT50:
- 121 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 20 °C
- Remarks on result:
- other: Aerobic system
- Key result
- Soil No.:
- #4
- DT50:
- 20.6 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 20 °C
- Remarks on result:
- other: Aerobic system
- Soil No.:
- #1
- DT50:
- 1 066 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 20 °C
- Remarks on result:
- other: Anaerobic system 1
- Soil No.:
- #1
- DT50:
- 1 035 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 20 °C
- Remarks on result:
- other: Anaerobic system repetition
- Transformation products:
- not specified
- Details on transformation products:
- RATES AND ROUTE OF FORMATION OF METABOLITES
- One minor, (M30), and three major metabolite fractions (M3, M2 and M29) were found in the soil extracts of Gartenacker, Pappelacker and Weide, whereas in the Speyer soil extracts only two major metabolite fractions (M2 and M3) and traces of M30 were detected. Under anaerobic conditions no significant amounts of metabolites were observed (< 2.1 %).
- Metabolite M3: Metabolite M3 built-up first and reached its highest amounts with 14.9 % in soil Gartenacker (day 28), 19.2 % in soil Pappelacker (day 28) and 22.6 % (0.91 mg parent equivalents/kg soil) in soil Weide (day 14). At the end of the study metabolite M3 had completely disappeared from these soils. In the soil Speyer 2.2 metabolite M3 reached a maximum of 4.7 % (0.22 mg/kg) after 14 days and then slowly decreased to 3.0 % at the end of the study (day 168).
- Metabolite M2: The formation of the second major metabolite M2 started after 14 days of incubation in all soils. Metabolite M2 reached its highest amounts with 19.7 % (0.96 mg parent equivalents/kg soil) in soil Gartenacker (day 98), 17.4 % in soil Pappelacker (day 120) and 18.5 % in soil Weide (day 98). At the end of the study metabolite M2 accounted for 0.8 % (351 d), 17.4 % (168 d) and 15.3 % (168 d) in the corresponding soils. In soil Speyer 2.2 metabolite M2 reached its maximum amount of 31.1 % (1.41 mg/kg) at the of the study (day 168).
- Metabolite M29: Metabolite M29 was first detected after 28 days in all soils. The maxima accounted for 14.7 % (Gartenacker, day 120), 16.1 % (Pappelacker, day 120) and 20.6 % (0.88 mg/kg, Weide, day 98). The corresponding amounts at the end of the study were 5.5 % (351 d), 14.3 % (168 d) and 14.7 % (168 d). Metabolite M29 was not detected in the soil Speyer 2.2.
- Metabolite M30: Metabolite M30 was first formed after 14 days in soils Gartenacker, Pappelacker and Weide. It reached maximum amounts of 5.6 % (0.27 mg/kg), 7.7 % (0.29 mg/kg) and 9.0 % (0.37 mg/kg) after 168 days in soils Gartenacker, Pappelacker and Weide, respectively, and dropped to 4.4 % (0.22 mg/kg) after 351 days in soil Gartenacker. In soil Speyer 2.2 metabolite M30 was only detected twice (day 120 and 168) in amounts < 1.8 % (0.08 mg/kg).
- Estimation of the Half-Lives of the Metabolites: Based on the amounts of metabolites found under aerobic conditions in the different soils after various time intervals the degradation rate constants and the half-lives were determined by applying exponential models. The results are summarised in Table 1 in “Any other information on results incl tables”. The half-life of metabolite M4 in the different soils was 11.9 - 19.3 days. For metabolite M2 half-lives of 74.8, 70.7 and 142.3 days were determined for soils Gartenacker, Pappelacker and Weide, respectively. The half-lives of metabolite M29 were calculated to be 140.9, 113.9 and 100.3 days in the respective soils.
- Nature of Metabolites: Metabolite M3: Metabolite M3 was slightly basic by nature, showing a migration of about 3.5 cm versus the cathode after high voltage electrophoresis (HVE) at pH 10. According to co-chromatography (TLC) with extracts of soil samples from a previous study metabolite M3 was identified. Co-chromatography (HPLC and TLC) showed, that metabolite M3 is not identical with M4 or M12. Metabolite M2: Metabolite M2 showed no significant migration after HVE at pH 10, indicating its neutral properties. In addition, an acidic impurity was observed, which migrated 8.4 cm versus the anode. HVE at pH 2, confirmed the neutral behaviour of M3, as no migration was observed under these conditions. However, M2 appeared to be unstable under strong acidic pH-conditions. The identity of M2 with the reference substance was proven, when submitted to cochromatography with the referenc esubstance, on 2d-TLC. In addition, this result was confirmed by mass-spectroscopy in the positive APCI-mode, where for the protonated molecule ion m/z = 212 amu (corresponding to a molecular mass of 211 amu) was found. This molecule ion and the fragmentation pattern of the daughter ion scans with the parent ion 212 were consistent with pattern of the reference compound. After methylation with diazomethane a molecule ion of m/z = 226 amu was detected, indicating M12. Metabolite M29: Metabolite M29 was acidic by nature, showing a migration of about 10.1 cm versus the anode after HVE at pH 10. The positive atmospheric pressure ionisation mass spectrum showed a protonated molecular ion at m/z 276 Dalton. MS/MS fragmentation indicated the elimination of SO3 followed by the elimination of two isopropyl groups. Thus M29 was identified. Metabolite M30: Metabolite M30 also had acidic properties, as it migrated about 12.1 cm versus the anode after HVE at pH 10. Metabolite M30 accounting for 1.8 % up to 9.0 % of the dose applied in soils Speyer and Weide, respectively, was identified in a previous study as a mixture of two metabolite fractions, one of them was identified. - Evaporation of parent compound:
- no
- Volatile metabolites:
- no
- Residues:
- yes
- Details on results:
- RECOVERY AND DISTRIBUTION PATTERN OF RADIOACTIVITY
- Recovery: The total individual recoveries of radioactivity from soils for all experimental parts ranged from 89.3 % to 110.8 %.
- Extractability of aerobic soil samples: In the four soils the extractable radioactivity steadily decreased during incubation, reaching 12.0 % (Gartenacker), 41.7 % (Pappelacker), 75.9 % (Speyer 2.2) and 40.7 % (Weide) of the radioactivity applied. Accordingly, the non-extractable fraction increased to 46.3 %, 47.5 %, 28.4 % and 43.3 % of the dose applied for the corresponding soils.
- Harsh extraction: The harsh-extraction of two Gartenacker soil samples (120 and 168 days) with acetonitrile/water recovered 4.5 and 4.1 % of the radioactivity applied, respectively. Analysis of the extract of day 120 showed, that mainly M2 (3.4 % of the radioactivity applied) and several minor fractions < 0.3 % of the radioactivity applied were present. The second reflux extraction with acetonitrile/0.1 N HCI recovered only 2.0 % of the radioactivity applied for both samples. These fractions were not further analysed due to the low amount of radioactivity.
ORGANIC MATTER FRACTIONATION
After harsh extraction of the samples (Gartenacker 120 and 168 days; see above) the extraction with 0.5 N NaOH recovered amounts of 13.9 and 14.4 %, respectively, of the radioactivity applied (soluble fraction). In the soluble fraction the fulvic acid fraction and the humic acid fraction amounted to 9.1 % and 4.8 - 5.2 % of the radioactivity applied, respectively. The remaining bound residues (insoluble humin fraction) were calculated to be 22.6 and 24.2 % of the radioactivity applied for the corresponding Gartenacker soil samples. This distribution of radioactivity in the organic matter showed, that the major part of the non-extractable radioactivity (63.6 - 65.6 %) was associated with the less mobile soil organic fractions (humic acid fraction and humin fraction).
DISTRIBUTION OF RADIOACTIVITY IN THE ANAEROBIC SAMPLES
After application the amount of radioactivity in the water steadily decreased within 120 days, reaching 9.6 %. The extractable radioactivity from soil steadily increased to 89.9 % after 120 days of incubation. The non-extractable radioactivity remained with 2.0 % after 120 days of incubation on a very low level. The data for the repetition of the anaerobic part were similar after 120 days of incubation: 19.3 % of the applied radioactivity was still present in the water, 70.1 % was extractable from soil, and 2.5 % remained non-extractable.
VOLATILES
The 14CO2-evolution reached 30.9 % after 351 days with Gartenacker soil and 12.0 %, 0.9 % and 9.3 % after 168 days for Pappelacker, Speyer 2.2 and Weide soil, respectively. No significant amount of radioactivity was detected in the NaOH solution after precipitation of CO2 as BaCO3, proving the identity of the radioactivity with carbon dioxide. In anaerobically incubated soil (both experiments) only negligible amounts of 14CO2 and organic volatiles were found.
RATE OF DEGRADATION OF THE TEST SUBSTANCE
Based on the concentration of the parent molecule found in the different soils after various time intervalsthe degradation rate constants and the half-lives were determined by applying an exponential model. The results are summarised in Table 1 in “Any othe rinformation on results incl tables”. Under aerobic conditions the substance degraded in soil Gartenacker, Pappelacker, Speyer 2.2 and Weide with a half-life (DT50) of 26.4, 26.5, 121.0 and 20.6 days and a DT90-value of 87.7, 87.9, 401.8 and 68.5 days, respectively. Under anaerobic conditions the half-life for the substance in the total anaerobic system was > 1000 days for both experiments. - Conclusions:
- The substance was degraded in soil with half-lives of 20.6 to 121.0 days under aerobic conditions and with a half-life of > 1000 days under anaerobic conditions. Under aerobic conditions, major metabolites formed were M3, M2 and metabolite M29 (triazine sulfonic acid derivative) with half-lives of 11.9 - 19.3, 70.7 - 142.3 and 100.3 - 140.9 days, respectively. The three metabolites were of transient nature. Finally, between 0.9 % (Speyer 2.2) and 30.9 % (Gartenacker) of the applied substance was mineralised and 28.4 % to 47.5% was transformed into bound residues. Under anaerobic conditions no significant degradation of the substance was observed.
- Endpoint:
- biodegradation in soil: simulation testing
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 18 Sep 1987 to 29 Dec 1987
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: Pesticide Assessment Guidelines, Subdivision N, Section 162-2 of the Environmental Protection Agency
- GLP compliance:
- yes
- Test type:
- laboratory
- Radiolabelling:
- yes
- Oxygen conditions:
- aerobic/anaerobic
- Soil classification:
- USDA (US Department of Agriculture)
- Year:
- 1 987
- Soil no.:
- #1
- Soil type:
- sandy loam
- % Clay:
- 5
- % Silt:
- 35
- % Sand:
- 60
- % Org. C:
- 0.41
- pH:
- 4.6
- CEC:
- 3.4 meq/100 g soil d.w.
- Bulk density (g/cm³):
- 1.59
- Details on soil characteristics:
- The study was conducted using a California sandy loam soil (Citrus Heights, California). Before physical analysis, the soil was air-dried and passed through a 2-mm sieve. Physical analysis was performed by the University of Wisconsin-Extension Soil and Forage Laboratory. Field moisture capacity and bulk density were measured by HLA. Physical characteristics of the soil are summarized in Table 1 in “Any other information on materials and methods incl tables”. Microbial assays were conducted on the soil before study and initiation and presented in Table 2 in “Any other information on materials and methods incl tables”.
- Soil No.:
- #1
- Duration:
- 102 d
- Soil No.:
- #1
- Initial conc.:
- 0.6 mg/kg soil d.w.
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- CO2 evolution
- radiochem. meas.
- Soil No.:
- #1
- Temp.:
- 24.4 ºC to 25.0 ºC
- Humidity:
- 75% FMC
- Details on experimental conditions:
- PRELIMINARY STUDY
- A preliminary study was conducted to investigate extraction and analytical procedures for the test material under the test conditions. Preliminary study findings were used to establish the experimental parameters for the definitive study. Approximately 20 g (dry-weight equivalent) of 2-mm sieved soil was placed in each of two glass containers. The two samples (Nos. A and B) were fortified with 210 µL of a fortification solution of the radiolabelled test substance in methanol (MeOH) at a radioactivity level of 1.30E+07 dpm/sample and a test material concentration of 10.0 µg/g of soil. The moisture level of each sample was adjusted to 75% field moisture capacity (FMC) by adding water. The samples were extracted twice with MeOH:H2O (80:20); the extracts were combined for each sample. The radioactivity in the combined extracts and extracted soil (oxidized by combustion) was quantitated by liquid scintillation counting (LSC). Cochromatography of each combined extract and nonradiolabeled test substance was conducted on a silica gel TLC plate developed in isopropanol:H2O (98:2). The radioactivity distribution on the plate was analyzed by a radioactivity scanner. An Rf value was also determined for each of the nonradiolabeled standards. Nonradiolabeled standards were located by fluorescent quenching under UV light.
DEFINITIVE STUDY - AEROBIC INCUBATION PHASE
- Sample Preparation: Eighteen test samples were prepared in separate glass containers. Each sample consisted of approximately 20 g (dry-weight equivalent) of 2-mm sieved soil (moisture content, approximately 6%). The sample containers were labeled with the study number, unique sample number, and test conditions.
- Sample Fortification: The samples were each fortified with 210 of a solution of the radiolabelled test substance in MeOH at a radioactivity level of 1.38E+07 dpm/sample and a test material concentration of 10.6 µg/g of soil. The concentration and homogeneity of the radioactivity in the fortification solution were determined by LSC analysis of pre- and post-fortification aliquots of the solution. The radioactivity concentration of each of the aliquots was within 3% of the mean concentration (65,600 dpm/µL). Because the differences from the mean were less than 5%, the radioactivity was considered to be homogeneously dispersed in the fortification solution. After fortification, the sample containers were capped and gently shaken by hand to mix the contents. The moisture level of each sample was adjusted to 75% FMC, and the final weight of the samples was recorded.
- Sample Test Conditions: The samples were placed in a sealed glass chamber. The sealed chamber was connected to two traps in series for the collection of volatile components; Trap 1 contained ethylene glycol and Trap 2 contained 2-ethoxyethanol:ethanolamine (1:1). A water trap was installed after the traps for volatile components to collect solvent vapors; the water from this trap was not analyzed. A continuous airflow from the chamber to the traps was created by vacuum. The air entering the incubation system was passed through water in the chamber bottom at a rate of 62 to 76 mL/minute to create a humid, aerobic environment. The samples were maintained in a dark, temperature controlled room at 24.4 ºC to 25.0 ºC. Samples were weighed periodically throughout the study to ensure that the initial moisture level of the soil (75% FMC) was maintained.
- Sample Collection: Two samples (Sample Nos. C and D) were not placed under test conditions. These samples (Day 0) were analyzed for radioactivity level and distribution approximately 0.25 hours after fortification. Two samples were removed from test conditions at Days 15, 31, 67, and 102. The order of sample removal was determined by a computer randomization program. The traps for volatile components were sampled at Days 15, 31, 67, and 102. The total volume of each trapping medium was measured, and duplicate 1.0-mL aliquots were collected for analysis. The traps were emptied and refilled with fresh trapping media.
- Sample Extraction: Approximately 20 mL of MeOH:H2O (80:20) was added to the sample container and the soil/solvent mixture was stirred on a magnetic stir plate for 30 minutes. The soil/solvent mixture was centrifuged for approximately 10 minutes at 2,200 rpm. The supernatant was transferred to a tared container. The above procedure was repeated using 15 mL of MeOH:H2O (80:20) with an extraction time of 15 minutes. The supernatants from the two extractions were combined.
- Sample Analysis - MeOH:H2O (80:20) Extract: The radioactivity in the combined MeOH:H2O (80:20) extract was quantitated. Duplicate aliquots (weight values in Appendix C-l) of the extracts were analyzed by LSC. The distribution of radioactivity for the extracts was determined by TLC. A 20-ni aliquot of a nonradiolabeled test substance standard was applied to a silica gel TLC plate 2 cm from the plate bottom and overlaid with a 50 µL aliquot of the MeOH:H2O (80:20) extract. The TLC plates were predeveloped to approximately 0.5 cm above the origin in MeOH for Day 31, and in acetonitrile (ACN) for Days 67 and 102. The TLC plates were then developed in isopropanol:H2O (98:2) until the solvent front migrated 18 cm from the plate bottom. The plate was removed from the development tank and air-dried at room temperature. The location of the nonradiolabeled standards was determined by fluorescent quenching under UV light. The plate was analyzed by a linear analyzer and/or autoradiography to determine the location of the radioactivity. The relative quantity of the radioactivity in the radioactive peaks was determined by a linear analyzer.
- Sample Analysis - Extracted Soil: The level of radioactivity remaining in duplicate aliquots of the extracted soil was measured by LSC after oxidation. The weight values of the soil aliquots were analyzed.
- Sample Analysis - Traps for Volatile Components: The radioactivity in the traps was quantitated. Duplicate aliquots (1.0 mL) of the trapping medium were analyzed in 9 mL of H2O and 10 mL of scintillation cocktail by LSC.
DEFINITIVE STUDY - ANAEROBIC INCUBATION PHASE
- Sample Test Conditions: After 31 days of aerobic incubation, six samples were removed from the incubation apparatus. Because of the low organic matter content of the soil (0.7%) , 0.2 g of dextrose was added to each sample. The soil was flooded with 30 mL of water that had been purged with nitrogen for approximately 1 hour. This resulted in a water layer approximately 2.5 cm above the soil surface. The samples were placed in a sealed glass chamber connected to two traps in series for the collection of volatile components. Trap 1 contained ethylene glycol and Trap 2 contained 2-ethoxyethanol:ethanolamine (1:1) . A water trap was installed after the traps for volatile components to collect solvent vapors; the water from this trap was not analyzed. Nitrogen was introduced into the chamber at approximately 62 to 76 mL/minute through water in the chamber bottom to create a humid, anaerobic environment. The chamber was kept in a dark, temperature-controlled room at 24.4 ºC to 25.0 ºC.
- Sample Collection. The two samples collected and analyzed after 31 days of aerobic incubation were considered to be the Day 0 samples for the anaerobic phase of the study. Two samples were removed from test conditions after 36 and 71 days of anaerobic incubation (67 and 102 days of aerobic/anaerobic incubation, respectively). The remaining two samples were not analyzed. The order of sample collection was determined by a computer randomization program. The traps were sampled at Days 36 and 71 (anaerobic incubation time) . The total volume of each trapping medium was measured, and duplicate 1.0-mL aliquots were collected for analysis. The traps were emptied and refilled with fresh trapping media.
- Sample Extraction. Samples were centrifuged at approximately 2,200 rpm for 10 minutes. The supernatant (water layer) (approximately 25 mL) from each sample was decanted into a tared container. Methanol (100 mL) was added to the supernatant to a final ratio of 80:20 (MeOH:H2O). The residual soil was extracted with two 20-mL portions of the MeOH:H2O (80:20) solution. These extractions were done by stirring on a magnetic stir plate for 30 minutes followed by centrifugation. The extracts were combined with the original MeOH-diluted supernatant.
- Sample Analysis - MeOH:H2O (80:201 Extract of Soil. The radioactivity in duplicate aliquots of each MeOH:H2O (80:20) extract was quantitated by LSC. The distribution of radioactivity for the extracts was determined by TLC. A 20 µL aliquot of a nonradiolabeled test substance standard was applied to a silica gel TLC plate 2 cm from the plate bottom and overlaid with a 100 µL aliquot of ; the MeOH:H2O (80:20) extract. The TLC plates were predeveloped to approximately 0.5 cm above the origin in ACN for Day 36 (anaerobic) and Day 71 (anaerobic). The plates were then developed and analyzed as described in the Sample Analysis - MeOH:H2O (80:20) Extract section for the aerobic phase of the study.
- Sample Analysis - Extracted Soil: The level of radioactivity remaining in duplicate aliquots of the extracted soil was measured by LSC after oxidation.
- Sample Analysis - Traps for Volatile Components: The radioactivity in duplicate aliquots (1.00 mL) of the trapping media was quantitated by LSC. Validation of the Soil Oxidation Procedure The efficiency of the oxidation procedure was determined. Duplicate aliquots of the untreated soil were fortified with 9, 562, 20, 213, or 30,690 dpm (mean of duplicate aliquots of the fortification solution) of the radiolabelled test substance and oxidized by combustion. The resulting 14CO2 was trapped in Carbo-Sorb:Perma-Fluor V (1:1) and quantitated by LSC. The recovered radioactivity (dpm) was divided by the applied radioactivity (dpm) and multiplied by 100 to determine the percentage of recovery. Because the mean recovery values were higher than 95% (104.7%, 98.2%, and 103.3% for the low, middle, and high radioactivity levels, respectively), radioactivity values for extracted soil were not corrected for oxidation efficiency. - % Recovery:
- 100
- Parent/product:
- parent
- Key result
- Soil No.:
- #1
- % Degr.:
- 29.6
- Parameter:
- radiochem. meas.
- Sampling time:
- 102 d
- Key result
- Soil No.:
- #1
- DT50:
- 261 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 25 °C
- Remarks on result:
- other: under aerobic conditions
- Transformation products:
- not specified
- Details on transformation products:
- - HPLC Distribution of Radioactivity Expressed as the Percentage of Radioactivity Applied to Sample for the Reflux Extracts: No important differences in the distribution of radioactivity were observed between the aerobic and aerobic/anaerobic samples. The radioactivity found corresponding to the radiolabelled test substanceranged from 2.4% to 6.3% of that applied for the samples analyzed. The radioactivity found corresponding to M2 was less than 10% of that applied (except 10.3% for Sample No. 3 at Aerobic Day 102) for all samples. The radioactivity found corresponding to M13 and M4 was less than 2% of that applied for all samples.
- Stability of the radiolabelled test substance under Reflux Conditions: Whereas the major degradation product observed in the reflux extract of definitive study soil samples [previously extracted with MeOH:H2O (80:20)] was M2, the major degradation product observed in the soil sample used to determine the stability of the radiolabelled test substance under reflux conditions was M13. Several factors may have contributed to this difference. First, the radiolabelled test substanceapplied to the definitive study samples had been incubated under aerobic and aerobic/anaerobic conditions for 67 and 102 days before extraction. In contrast, radiolabeled test substance was applied to the soil sample used for stability analysis and extracted immediately by reflux. The degradation observed in the refluxed soil for the definitive study samples probably resulted at least in part because of the aerobic and aerobic/anaerobic study conditions. Second, the definitive study samples had been extracted with MeOH:H2O (80:20) and air dried before reflux extraction with MeOH:HCl (11:1). The stability sample was subjected to reflux extraction with MeOH:HCl (11:1) only. Finally, the definitive study samples were analyzed at different times from the stability samples. - Evaporation of parent compound:
- no
- Volatile metabolites:
- no
- Residues:
- no
- Details on results:
- PRELIMINARY STUDY
- The total mean radioactivity found (material balance) for the two samples used to determine extraction efficiency was 109.3%. Of the radioactivity applied to the samples, a mean of 97.7% and 11.6% was found in the extract and extracted soil, respectively. The TLC analysis of the extracts produced a single sharp peak that comigrated with the nonradiolabeled test substance standard. The extraction procedure' and analytical method were considered to be adequate for use in the definitive study.
DEFINITIVE STUDY - AEROBIC INCUBATION PHASE
- Distribution of Applied Radioactivity Found among the Sample Matrices: The individual sample values for the applied radioactivity found among the sample matrices are Table 1 in “Any other information on results incl tables”. The mean material balance ranged from 99.6% to 102.5% throughout the 102-day aerobic incubation period. The mean radioactivity found in the MeOH:H2O (80:20) extract decreased from 93.0% (Day 0) to 78.9% (Day 102) of that applied. Concurrently, the mean radioactivity found in the extracted soil increased from 9.2% (Day 0) to 23.6% (Day 102) of that applied. By the end of the 102-day study, the radioactivity found in each of the traps for volatile components was less than 0.1% of that applied.
- Distribution of Radioactivity in the MeOH:H2O (80:20) Extract Expressed as the Percentage of Radioactivity Applied to TLC Plate: The individual and mean percentage values for the TLC distribution of radioactivity in the MeOH:H2O (80:20) extract were determined. A single, sharp radioactive peak that comigrated with the nonradiolabeled test substance standard was observed throughout the 102-day study period, except at Day 102. At Day 102, a small amount of radioactivity designated as Peak 1 was observed at the TLC plate origin in addition to the peak corresponding to the radiolabelled test substance. [Some minor degradation products were also observed by autoradiography on Days 67 and 102; however, they were not of sufficient quantity to be detected by the linear analyzer].
- Distribution of Radioactivity in the MeOH:H2O (80:20) Extract Expressed as the of Radioactivity Applied to Sample: for the TLC distribution of radioactivity in the MeOH:H2O (80:20) extract expressed as the percentage of radioactivity applied to sample are in Table 2 in “Any other information on results incl tables”. The mean radioactivity found for the TLC peak corresponding to the radiolabelled test substance decreased from 94.5% (Day 0) to 70.4% (Day 102) of that applied. The mean radioactivity found corresponding to TLC Peak 1 (observed only at Day 102) was 5.1% of that applied.
- Degradation Half-Life of the radiolabelled test substance: The degradation of the radiolabelled test substance as measured in the MeOH:H2O (80:20) extract was assumed to follow first-order kinetics. The calculated half-life using linear regression analysis was 261 days.
Percentage The individual and mean values
DEFINITIVE STUDY - ANAEROBIC INCUBATION PHASE
- Distribution of Applied Radioactivity Found among the Sample Matrices: The individual sample values for the applied radioactivity found among the sample matrices are in Table 3 in “Any other information on results incl tables”. The mean material balance ranged from 99.1% to 100.6% throughout the 71-day anaerobic incubation period. The amount of radioactivity in the MeOH:H2O (80:20) extract remained relatively constant when compared with the anaerobic Day 0 value (aerobic Day 31); the mean radioactivity found ranged from 85.0% (anaerobic Day 0) to 88.4% (anaerobic Day 36) of that applied. Similarly, the amount of radioactivity in the MeOH:H2O (80:20) extracted soil did not increase from the anaerobic Day 0 value; the mean radioactivity found at anaerobic Day 71 was 11.9% of that applied compared with 15.6% at anaerobic Day 0. By the end of the 71-day anaerobic incubation period, the radioactivity found in each of the traps for volatile components was less than 0.1% of that applied.
- Distribution of Radioactivity in the MeOH:H2O (80:20) Extract Expressed as the Percentage of Radioactivity Applied to TLC Plate: A single, sharp radioactive peak that comigrated with the nonradiolabeled test substance standard was observed throughout the 71-day anaerobic incubation period.
- Distribution of Radioactivity in the MeOH:H2O (80:20) Extract Expressed as the Percentage of Radioactivity Applied to Sample: The individual and mean values for the TLC distribution of radioactivity in the MeOH:H2O (80:20) extract expressed as the percentage of radioactivity applied to sample are in Table 11. The amount of radioactivity found for the TLC peak corresponding to the radiolabelled test substance did not decline from the anaerobic Day 0 value; the mean radioactivity found at anaerobic Day 71 was 86.0% of that applied compared with 82.0% at anaerobic Day 0.
- Comparison of the radiolabelled test substance Degradation under Aerobic and Anaerobic Conditions: A slow degradation of the radiolabelled test substance over time was evident in the aerobic phase of the study from the change in the distribution of radioactivity among the MeOH:H2O (80:20) extract and extracted soil (Table 1) , and from the TLC distribution of radioactivity in the MeOH:H2O (80:20) extract (Table 2). A decrease over time in the radioactivity in the MeOH:H2O (80:20) extract was concurrent with an increase of radioactivity in the extracted soil ; the majority of the applied radioactivity (77.1% to 93.2%, Table 3), however, was present in the MeOH:H2O (80:20) extract. The total extractable radioactivity was increased to 87.5% to 104.8% of the applied radioactivity by refluxing the extracted soil of selected samples with MeOH:HCl (11:1).The radioactivity in the MeOH:H2O (80:20) extract corresponding to the radiolabelled test substance decreased over time and by Day 102, a small amount of radioactivity was observed at the TLC plate origin. However, when selected samples were removed from aerobic conditions at Day 31 and placed under anaerobic conditions, the degradation of the radiolabelled test substance was inhibited. The distribution of radioactivity among the MeOH:H20 (80:20) extract and extracted soil remained relatively unchanged when compared with anaerobic Day 0 over the 71-day period. The TLC analysis of the MeOH:H2O (80:20) extract did not show any degradation of the radiolabelled test substance (Table 14 in “Any other information on results incl tables”).
- Material Balance for the TLC Procedure: The recovery of radioactivity applied to selected TLC plates ranged from 96.8% to 105.2%. The recovery values indicate that no radioactivity was lost because of volatilization during the TLC procedure.
- Identification and Characterization of Radioactivity in Study Samples Identification of Radioactivity in the MeOH:H2O (80:20) Extract: Radiolabeled test substance in the MeOH:H2O (80:20) extract for Sample Nos. 16 (Aerobic Day 102) and 14 (Aerobic/Anaerobic Day 102; Anaerobic Day 71) comigrated by two-dimensional TLC with a nonradiolabeled test substance standard in Solvent System 1 and Solvent System 2. In addition to the radiolabelled test substance, minor degradation products in the MeOH:H2O (80:20) extract of both samples were detected by autoradiography. [Minor degradation products that were not of sufficient quantity to be detected by the linear analyzer but were detected by autoradiography were also observed in some of the MeOH:H2O (80:20) extracts analyzed by one-dimensional TLC.
- Characterization of Radioactivity in the MeOH:H2O (80:20) Extracted Soil: Almost all of the residual radioactivity in the MeOH:H2O (80:20) extracted soil was released by refluxing with MeOH:HCl (11:1). The radioactivity recovered from the MeOH:H2O (80:20) extracted soil by reflux extraction ranged from 87.5% to 104.8% for the aerobic samples and 97.9% to 118.4% for the anaerobic samples.
- HPLC Distribution of Radioactivity Expressed as the Percentage of Radioactivity Applied to HPLC Column for the Reflux Extracts: Comparison of the HPLC retention times of the radioactive peaks for the reflux extracts with the retention times of the nonradiolabeled standards, showed that four radioactive peaks had retention times comparable to the non-radiolabelled standards and the test substance. The four radioactive peaks were observed for all samples analyzed, except Sample Nos. 7 and 8 at anaerobic Day 36.
- Stability of the radiolabelled test substance under Reflux Conditions: The HPLC analysis showed that the radiolabelled test substance degraded when heated to reflux with MeOH:HCl (11:1). The degradation of the radiolabelled test substancewas more extensive in the presence of soil under reflux conditions than with just the reflux extract solvent. Also, differences in the retention time of the test substance and related compounds were observed between the two stability samples. The soil reflux extracts were concentrated by roto-evaporation and had a strong brown coloration suggesting the presence of soil organic matter. Because of the high molecular weight and chemical nature of soil organic matter, it would not be expected to be mobile under the HPLC conditions used. The presence of these impurities in the extracts may have affected the properties of the HPLC column to retain the solutes and thereby resulted in a decrease of retention times. The broad distribution of the radioactivity in the peaks was caused largely by the injection of large volumes of the extract (typically, 200 µL) , which was necessary to have detectable amounts of radioactivity for the HPLC/ LSC analysis. Differences in the degradation between the definitive study samples and stability samples were observed. - Conclusions:
- Radiolabeled test substance degraded slowly on soil under aerobic conditions. The calculated degradation half-life of the radiolabelled test substance under aerobic conditions was 261 days. The radioactivity was distributed among the unchanged the test substance, soil -bound residues, and a minor product that constituted a mean of 5.1% of the applied radioactivity. This minor product was observed only at Aerobic Day 102 of the study. The volatile radioactivity observed was less than 0.1% of that applied. The degradation of the radiolabelled test substance was inhibited under anaerobic conditions. The presence of the radiolabelled test substance in the MeOH:H2O (80:20) sample extracts at Aerobic Day 102 and Anaerobic Day 71 was confirmed by two-dimensional TLC in two solvent systems. The majority of the soil - bound radioactivity was released by reflux extraction. Three minor degradation products in addition to the parent compound were detected in the reflux extracts: M13, M4, and M2. Two separate experiments, however, showed that the radiolabelled test substance was not completely stable in the presence of soil under reflux conditions. Based on the results of this study, anaerobic soil metabolism would not be expected to be a major pathway of the test substance degradation on soil.
- Executive summary:
The soil metabolism of the 14C-substance was studied under aerobic/anaerobic conditions using a California sandy loam soil (Citrus Heights, California). The study was conducted in accordance with the Environmental Protection Agency Pesticide Assessment Guidelines, 40 CFR 158, Subdivision N, Section 162-2, Anaerobic Soil Metabolism. Soil samples were fortified with 14C-the test substance at 10.6 µg/g (test material/soil) and placed in a glass chamber maintained at 25ºC +1°C in a dark, temperature controlled room. The glass chamber was connected to traps containing ethylene glycol and 2-ethoxyethanol:ethanol amine (1:1) for the collection of organic volatiles and carbon dioxide, respectively. Air was passed through the system continuously to maintain aerobic conditions. Duplicate samples were collected for analysis on Days 0, 15, 31, 67, and 102 of aerobic incubation. On Day 31, four aerobic samples were flooded with water and placed under conditions similar to the aerobic samples, except that nitrogen was passed through the incubation chamber. Duplicate anaerobic samples were collected for analysis 36 and 71 days after flooding. Radiolabeled test substance degraded slowly in soil under aerobic conditions. The calculated degradation half-life of the radiolabelled test substance under aerobic conditions as measured in the methanol (MeOH):water (H2O) (80:20) extract was 261 days. A gradual decrease over time of the radioactivity in the MeOH:H,0 (80:20) extract was accompanied by a corresponding increase of radioactivity in the extracted soil; the majority of the applied radioactivity (77.1% to 93.2%), however, was present in the MeOH:H2O (80:20) extract. The total extractable radioactivity was increased to 87.5% to 104.8% of the applied radioactivity by refluxing the extracted soil of selected samples with MeOH:HCl (11:1). The volatile radioactivity observed was less than 0.1% of that applied. Analysis of the MeOH:H2O (80:20) extract by thin-layer chromatography (TLC) revealed only one minor peak at the TLC plate origin at Day 102 in addition to the parent compound. The mean material balance of the aerobic samples ranged from 99.6% to 102.5% throughout the study. The degradation of the radiolabelled test substance was inhibited when samples incubated 31 days under aerobic conditions were placed under anaerobic conditions for 36 and 71 days. When compared with anaerobic Day 0, the radioactivity distribution among the MeOH:H2O (80:20) extract and extracted soil remained relatively unchanged after /1 days of anaerobic incubation. Analysis of the MeOH:H2O (80:20) extract by TLC showed only parent compound. The mean material balance for the aerobic/anaerobic samples ranged from 99.1% to 100.6% throughout the study. The presence of the radiolabelled test substance in the MeOH:H2O (80:20) sample extracts at Aerobic Day 102 and Anaerobic Day 71 was confirmed by two-dimensional TLC in two solvent systems. Characterization of the MeOH:H2O (80:20) extracted soil of selected aerobic and aerobic/anaerobic samples by high-performance liquid chromatography after reflux extraction with MeOH:HCl (11:1) revealed thepresence of three minor degradation products in addition to the parent compound: M13, M4, and M2. Two separate experiments, however, showed that the radiolabelled test substance was not stable in the presenceof soil under reflux conditions. The degradation products observed in the MeOH:H2O (80:20) extracted soil were the result of several variables including the study incubation conditions and extraction procedures. Based on the results of this study, anaerobic soil metabolism would not be expected to be a major pathway of the test substance degradation on soil.
- Endpoint:
- biodegradation in soil: simulation testing
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2 May 1985 to 2 Dec 1985
- 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 was carried out in an all-glass metabolism apparatus with an open glass- flow system. Fresh, sieved samples of soil corresponding to 200 g dry soil were treated with 2.0 mg 14C-substance. For this purpose, the substance was dissolved in acetone. Thereafter , aliquots were added dropwise to the soil samples (200 g) by means of a Hamilton syringe and the soil mixed thoroughly. Finally, the soil moisture content was adjusted to 70% of the field capacity, the metabolism flask connected with the absorption bottles and the air flow adjusted to 60 mL/min.
- GLP compliance:
- not specified
- Test type:
- laboratory
- Radiolabelling:
- yes
- Oxygen conditions:
- aerobic
- Soil classification:
- USDA (US Department of Agriculture)
- Year:
- 1 986
- Soil no.:
- #1
- Soil type:
- sandy loam
- % Clay:
- 6.5
- % Silt:
- 46.7
- % Sand:
- 46.8
- % Org. C:
- 0.63
- pH:
- 7.5
- CEC:
- 6.2 meq/100 g soil d.w.
- Details on soil characteristics:
- - For the study a field soil ("Strassenacker") was used. It was classified as a sandy loam. The specifications and origin of the soil are presented in Table 1 in “Any other information on materials and methods incl tables”.
- Soil No.:
- #1
- Duration:
- 180 d
- Soil No.:
- #1
- Initial conc.:
- 24.75 other: microCi/mg
- Based on:
- other: based on specific radioactivity
- Parameter followed for biodegradation estimation:
- CO2 evolution
- radiochem. meas.
- Soil No.:
- #1
- Temp.:
- 23 ± 1 °C
- Humidity:
- 70% moisture of the field capacity
- Microbial biomass:
- 66.4 mg C/100 g soil dw
- Details on experimental conditions:
- TREATMENT OF SOIL
- Fresh, sieved (2 mm) samples of soil corresponding to 200 g dry soil were treated with 2.0 mg 14C-substance (spec. rad.: 24 75 microCi/mg). For this purpose, the substance (42.15 mg) was dissolved in 25.0 mL acetone. Thereafter , aliquots of 1.20 mL (= 2.02 mg substance) were added dropwise to the soil samples (200 g) by means of a Hamilton syringe and the soil mixed thoroughly. Finally, the soil moisture content was adjusted to 70% of the field capacity, the metabolism flask connected with the absorption bottles and the air flow adjusted to 60 mL/min.
INCUBATION
The flasks were incubated in an air-conditioned room at a temperature of 23 ± 1°C. The moisture content of the soil samples was controlled and adjusted every two weeks.
SAMPLING INTERVALS
- Sodium Hydroxide Solutions: Sodium hydroxide solutions were exchanged weekly during the first month and thereafter every two weeks. Analyses for 14CO2 were performed accordingly.
- Soil Samples: Soil samples were taken for analysis 0, 13, 30, 60, 90, 120 and 180 days after the treatment.
EXTRACTION AND ISOLATION OF RADIOACTIVITY FROM SOIL
- Extraction of Radioactivity: Per sampling interval, subsamples of about 100 g soil (dry weight basis) were taken and extracted by shaking for one hour five times with acetone-water [8+2, v/v (amount of solvent used: 2mg/1 g soil)] followed by an exhaustive extraction with methanol in a Soxhlet apparatus. The respective extracts were combined and their radioactivity determined by liquid scintillation counting .
- Isolation of Radioactivity: Acetone-Water-Extracts: The combined extracts were concentrated by evaporation of the acetone and partitioned with dichloromethane. Metabolites in the dichloromethane-phase were isolated and analysed as described below. The aqueous phase was further purified by ultrafiltration. Radioactivity in the filtrate was also analysed as described below. Methanol-Soxhlet-Extract: The radioactivity in the methanol-extract was analysed by TLC . - Soil No.:
- #1
- % Recovery:
- 47.2
- Remarks on result:
- other: Extracted radioactivity after 180 days
- Soil No.:
- #1
- % Recovery:
- 45.9
- Remarks on result:
- other: Non-extracted radioactivity after 180 days
- Soil No.:
- #1
- % Recovery:
- 5.9
- Remarks on result:
- other: Volatiles (14CO2) after 180 days
- Soil No.:
- #1
- % Recovery:
- 99
- Remarks on result:
- other: Total radioactivity after 180 days
- Soil No.:
- #1
- % Degr.:
- 97.1
- Parameter:
- radiochem. meas.
- Sampling time:
- 180 d
- Key result
- Soil No.:
- #1
- DT50:
- 32.5 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 23 °C
- Transformation products:
- not specified
- Details on transformation products:
- - Pattern of Metabolites: Besides the parent molecule, 10 metabolites were isolated from the extractable soil radioactivity . The most prominent metabolites found were M18, M19, M17 and M26 (table 2 in “Any other information on results incl tables”). The other metabolites were of minor importance, i.e. ranging from 0.1%to 2.7% of the radioactivity applied. The most prominent metabolite was metabolite M19 mainly found in the aqueous phases of the acetone-water extracts after partitioning with dichloromethane. It reached its maximum after 90 days of incubation (30.0%). At the end of the study it still amounted to 24.1%. Metabolite M18 reached its maximum with 12.5% after 90 days and amounted, to 10.7% at the end of the study. It was also mainly found in the water phases. M17 represented a transient metabolite, reaching its maximum of 12.9% already after 30 days of incubation. It was mainly found in the dichloromethane extracts. After 180 days of incubation the concentration of M17 in the soil was 3.6% of the radioactivity initially applied. Finally, transient metabolite M26 was also mainly found in the dichloromethane phases. It reached its maximum with 7.3% after 30 days of incubation. At the end of the study , its amount was 1.0%. The minor metabolites observed were mainly found in the water phases and in the Soxhlet extracts.
- Characterization of Metabolites: Metabolite M20: M20 was a polar metabolite isolated first from the ultrafiltrate after 30 days of exposure and reached its highest concentration of 2.7 % in the soil 120 days after starting the experiment. On TLC, M20 behaved slightly less polar than the reference M28. Further characterizations were not done due to the low concentration of the metabolite in the soil. Metabolite M21: M21 mainly occurring in the ultrafiltrate, was shown to behave on TLC identical with the polar reference compound M5. Metabolite M18: M18 represented a metabolite present in the ultrafiltrate. In TLC the metabolite was behaving more polar than the reference M2. M18 was isolated from TLC plates and was further purified by preparative column chromatography on silica gel. With HVE, migration of M18 was followed by high-voltage electrophoresis at pH 1.94 and 10.0. From the electrophoretic mobility M18 showed an anionic character at pH 10.0. Direct mass-spectrometry was performed by using the solid probe inlet. The instrument was run in the El-mode. Metabolite M19: M19 represented a metabolite mainly present in the ultrafiltrate and in the dichloromethane phases. On TLC, M19 showed the same migration as reference M2. When M19 was submitted to reversed phase HPLC, it was simultaneously eluted with reference M2. Migration of M19 was followed by high-voltage electrophoresis at pH 1.94 and pH 10.0 . The migration observed was: pH 1.94 : 9.5 cm and pH 10.0 : + 2.0 cm. MS was performed by using the solid probe inlet of the instrument. The MS was run in the El-mode. The identity of M19 with the proposed structure was confirmed by comparative mass-spectrometry with reference M2 showing an identical fragmentation pattern. Metabolites M22 and M23: Due to their very low amounts no further analyses could be performed. Metabolite M17: M17 was mainly present in the dichloromethane phase of the acetone-water extract after partitioning. Metabolite M17 was isolated by two-dimensional TLC using solvent system S for the first dimension and solvent system V for the second dimension. By this procedure, it was separated from metabolites M22 and M23. MS was performed by using the solid probe inlet of the instrument. The MS was run in the El- and Cl-(methane) mode. Metabolites M24 and M25: M24 and M25 were minor metabolites only occurring in the methanol Soxhlet extracts. Due to their very low amounts, no characterization could be performed. Metabolite M26: M26 was found in the dichloromethane phase of the acetone-water extracts and in the methanol Soxhlet extracts. As mentioned, it represented a transient metabolite fraction which was of no further significance at the end of the study. It proved to be unpolar. By co-chromatography, it was tentatively characterized as M4. However , indication was given that M26 consisted of two metabolites, one representing M4 and the other representing an unknown metabolite. Metabolite M27: This radioactive fraction proved to be the parent molecule as shown by co-chromatography on TLC, HPLC on silica gel and on GLC. - Evaporation of parent compound:
- no
- Volatile metabolites:
- yes
- Residues:
- not specified
- Details on results:
- RECOVERY AND EXTRACTIBILITY OF RADIOACTIVITY
The total recoveries averaged 105.2 ± 4.4% (Table 1 in “Any other information on results incl tables”). Extraction of radioactivity from soil with the methods described under 2.5 was almost complete directly after the treatment (101.1%). With increasing exposure time, the amount of radioactivity which could be extracted continuously decreased so that after 180 days of exposure only 47.2% of the radioactivity applied could be extracted . Accordingly, the quantity of non-extracted radioactivity gradually increased up to 45.9% after 180 days of incubation. The amount of volatile radioactivity was low during the first 30 days of incubation representing 0.3% of the radioactivity applied. Thereafter, increasing amounts of 14CO2 were detected representing 3.5%and 5.9% after 90 and 180 days of incubation, respectively . The nature of the volatile radioactivity proved to be 14CO2. This was regularly shown by comparing the results of the direct 14C-measurement of the absorption solution with those obtained by liberation of CO2 from the 2N NaOH or by precipitation of the radioactivity with saturated barium hydroxide solution. The latter results demonstrate that at least a part of the triazine ring moiety was available for mineralization by soil microorganisms.
CHARACTERIZATION OF RADIOACTIVITY
- Half-Life of the substance: The degradation of the substance is shown in Table 2 in “Any other information on results incl tables”. The concentration of the substance in the soil rapidly decreased . Based on the radioactivity applied, substance amounted to 101.1%, 91.7%, 48.3%, 19.9%, 3.6%, 4.5% and 2.9% after 0, 13, 30, 60, 90, 120 and 180 days, respectively. The degradation during 180 days of exposure excluding the value of 90 days of incubation could be satisfactorily described by first order reaction kinetics. The slope of this linear equation (0.0213) represents the reaction rate constant, k1. - Conclusions:
- The substance rapidly degraded in the soil with a half- life of about one month (32.5 days). During degradation of the substance, numerous metabolites were formed. Prominent metabolites found, mere the transient metabolite M17, representing a possible precursor for the formation of M18 which might represent one possible precursor of the corresponding M5.
- Executive summary:
In the present study , the aerobic degradation of the substance in a field soil under laboratory conditions was investigated for 180 days. The concentration of the test substance in soil rapidly decreased. Based on the radioactivity applied, the test substance amounted to 101.1, 91.7, 48.3, 19.9, 3.6, 4.5 and 2.9% after 0, 13, 30, 60, 90, 120 and 180 days, respectively. The half- life of the compound was found to be about one month (32.5 days). Degradation of the substance proceeded via the hydroxylation of the 2-methylthio-group farming the transient metabolite M17. This metabolite reached its highest concentration with 12.9% in the soil after 30 days and is thought to be a precursor of M2, the most prominent metabolite found in the soil with 30.0 / and 24.1 / after 90 and 180 days, respectively. A further transient metabolite, M18. It reached its highest concentration (12.5 %) in the soil after 90 days. M5, accounted at highest for 3.3% after 180 days . Finally, M4 was found in significant amounts (about 7.0%) after 30 days of incubation. However, after 180 days the amount of M4 in the soil was of no further significance. Besides these major metabolites further 5 metabolites of lower significance were isolated from the soil. Significant amounts of 14CO2 were liberated from the soil by mineralization of the 14C-triazine ring. After 180 days 14CO2 accounted for 5.9% of the dose applied. Non-extractable radioactivity was low during the first two weeks of the study. Thereafter, it continuously increased with time reaching with 44.1%. an apparent plateau after 90 days of incubation. Total recoveries of radioactivity averaged 105.2% ± 4.4%.
Referenceopen allclose all
Table 1. Radioactivity Measurements: Extractable Amounts, Not-extractable Residues and 14CO2 [All Values Given as % of Applied Radioactivity]
Time after application (days) |
Sample No. |
Temperature = 10°C |
Temperature = 20°C |
||||||
Extract-ables |
NER (3) |
14CO2 |
Sum (Mass balance) |
Extract-ables |
NER (3) |
14CO2 |
Sum (Mass balance) |
||
0.01 |
1 |
94.0 |
2.8 |
n.d. (2) |
96.8 |
93.1 |
2.5 |
n.d. (2) |
95.6 |
2 |
92.9 |
4.3 |
n.d. (2) |
97.2 |
92.7 |
3.0 |
n.d. (2) |
95.7 |
|
Av. |
93.5 |
3.6 |
- |
97.0 |
92.9 |
2.8 |
- |
95.7 |
|
7 |
1 |
92.1 |
2.9 |
n.d. (2) |
95.0 |
92.7 |
3.7 |
n.d. (2) |
96.4 |
2 |
92.6 |
3.5 |
n.d. (2) |
96.1 |
92.3 |
3.8 |
n.d. (2) |
96.1 |
|
Av. |
92.4 |
3.2 |
- |
95.6 |
92.5 |
3.8 |
- |
96.3 |
|
14 (1) |
1 |
93.7 |
0.77 |
0.00 |
94.5 |
91.7 |
3.2 |
0.02 |
94.9 |
2 |
93.6 |
0.76 |
0.00 |
94.4 |
91.7 |
2.5 |
0.02 |
94.2 |
|
Av. |
93.7 |
0.77 |
0.0 |
94.5 |
91.7 |
2.9 |
0.02 |
94.6 |
|
21 (1) |
1 |
93.6 |
1.2 |
0.001 |
94.8 |
91.7 |
6.5 |
0.07 |
98.3 |
2 |
93.4 |
1.3 |
0.001 |
94.7 |
92.6 |
6.6 |
0.08 |
99.3 |
|
Av. |
93.5 |
1.3 |
0.001 |
94.8 |
92.2 |
6.6 |
0.08 |
98.8 |
|
28 (1) |
1 |
94.5 |
1.7 |
0.001 |
96.2 |
84.6 |
10.2 |
0.15 |
95.0 |
2 |
94.6 |
2.0 |
0.001 |
96.6 |
85.1 |
9.6 |
0.15 |
94.9 |
|
Av. |
94.6 |
1.9 |
0.001 |
96.4 |
84.9 |
9.9 |
0.15 |
95.0 |
|
56 (1) |
1 |
88.7 |
6.2 |
0.07 |
95.0 |
74.5 |
17.4 |
0.61 |
92.5 |
2 |
89.6 |
5.4 |
0.02 |
95.0 |
75.6 |
19.5 |
0.56 |
95.7 |
|
Av. |
89.2 |
5.8 |
0.05 |
95.0 |
75.1 |
18.5 |
0.59 |
94.1 |
|
90 (1) |
1 |
84.2 |
11.9 |
0.18 |
96.3 |
61.8 |
28.6 |
2.0 |
92.4 |
2 |
83.2 |
11.4 |
0.14 |
94.7 |
66.2 |
24.7 |
1.2 |
92.1 |
|
Av. |
83.7 |
11.7 |
0.16 |
95.5 |
64.0 |
26.7 |
1.60 |
92.3 |
|
120 (1)extraction steps 4+5 |
1 |
77.3 |
16.9 |
0.26 |
94.5 |
61.2 |
33.0 |
2.2 |
96.4 |
0.69 |
|
|
|
1.0 |
|
|
|
||
Extraction steps4+5 |
2 |
78.0 |
17.6 |
0.39 |
96.0 |
56.6 |
32.1 |
2.5 |
91.2 |
0.64 |
|
|
|
1.1 |
|
|
|
||
|
Av. |
78.3 |
17.3 |
0.33 |
95.3 |
60.0 |
32.6 |
2.4 |
93.8 |
179 (1) |
1 |
76.2 |
21.1 |
0.51 |
97.8 |
n.d. |
n.d. |
n.d. |
n.d. |
|
2 |
69.8 |
20.8 |
0.86 |
91.5 |
n.d. |
n.d. |
n.d. |
n.d. |
|
Av. |
73.0 |
21.0 |
0.7 |
94.7 |
- |
- |
- |
- |
Mean |
|
95.9 |
|
95.0 |
|||||
Table 2. Results of Thin-Layer Chromatography; All Results Refer to the Parent Compound Found in Extracts from Soil Gartenacker at 10°C.
Time /Sample (days) |
Total amount extracted [% of applied] |
Solvent System 1 Amount of a.i. in extract |
Solvent system 2 Amount of a.i. in extract |
Average of solvent system 1+2 |
||||
ROI [%] |
Extract [% of applied] |
Mean [% of applied] |
ROI [%] |
Extract [% of applied] |
Mean [% of applied] |
[% of applied] |
||
0.01/1 |
94.0 |
91.5 |
86.0 |
85.8 |
100.0 |
94.0 |
93.5 |
89.7 |
0.01/2 |
92.9 |
92.2 |
85.7 |
100.0 |
92.9 |
|||
7/1 |
92.1 |
93.2 |
85.8 |
86.1 |
97.8 |
90.0 |
90.4 |
88.3 |
7/2 |
92.6 |
93.3 |
86.4 |
98.1 |
90.9 |
|||
14/1 |
90.6 |
86.4 |
78.2 |
78.9 |
96.9 |
90.8 |
89.9 |
84.4 |
14/2 |
91.5 |
87.0 |
79.7 |
97.3 |
89.0 |
|||
21/1 |
90.1 |
86.6 |
78.0 |
78.3 |
96.4 |
86.8 |
86.7 |
82.5 |
21/2 |
89.8 |
87.6 |
78.7 |
96.4 |
86.6 |
|||
28/1 |
91.4 |
83.2 |
76.0 |
77.0 |
93.6 |
85.6 |
86.3 |
81.6 |
28/2 |
91.5 |
85.2 |
77.9 |
95.1 |
87.0 |
|||
56/1 |
84.2 |
66.4 |
55.9 |
57.4 |
77.6 |
65.4 |
66.5 |
61.9 |
56/2 |
85.2 |
69.0 |
58.842.6 |
79.4 |
67.7 |
|||
90/1 |
84.2 |
53. |
44.1 |
43.3 |
60.8 |
48.2 |
51.7 |
47.5 |
90/2 |
83.2 |
56.4 |
28.0 |
70.5 |
55.1 |
|||
120/1 |
71.7 |
39.0 |
29.1 |
28.5 |
53.6 |
38.4 |
39.2 |
33.8 |
120/2 |
72.6 |
40.0 |
21.2 |
55.1 |
40.0 |
|||
179/1 |
68.5 |
30.9 |
17.0 |
19.1 |
44.6 |
30.5 |
26.0 |
22.5 |
179/2 |
61.9 |
27.4 |
|
34.6 |
21.4 |
|||
Table 3. Results of Thin-Layer Chromatography; All Results Refer to the Parent Compound Found in Extracts from Soil Gartenacker at 20°C.
Time /Sample (days) |
Total amount extracted [% of applied] |
Solvent System 1 Amount of a.i. in extract |
Solvent system 2 Amount of a.i. in extract |
Average of solvent system 1+2 |
||||
ROI [%] |
Extract [% of applied] |
Mean [% of applied] |
ROI [%] |
Extract [% of applied] |
Mean [% of applied] |
[% of applied] |
||
0.01/1 |
93.1 |
92.6 |
86.2 |
86.4 |
100.0 |
93.1 |
92.9 |
89.7 |
0.01/2 |
92.7 |
93.4 |
86.6 |
100.0 |
92.7 |
|||
7/1 |
92.7 |
91.4 |
84.7 |
84.3 |
97.0 |
89.9 |
90.0 |
87.1 |
7/2 |
92.3 |
90.9 |
83.8 |
97.6 |
90.0 |
|||
14/1 |
88.5 |
75.2 |
66.6 |
67.7 |
94.3 |
86.4 |
85.3 |
76.5 |
14/2 |
89.6 |
76.9 |
68.9 |
93.8 |
84.1 |
|||
21/1 |
88.1 |
69.9 |
61.6 |
61.6 |
89.5 |
78.9 |
79.5 |
70.5 |
21/2 |
89.1 |
69.2 |
61.7 |
89.9 |
80.1 |
|||
28/1 |
81.6 |
65.7 |
53.6 |
51.7 |
85.1 |
69.4 |
68.8 |
60.2 |
28/2 |
82.0 |
60.6 |
49.7 |
83.1 |
68.1 |
|||
56/1 |
68.5 |
40.7 |
27.9 |
27.7 |
51.7 |
35.4 |
36.6 |
32.2 |
56/2 |
69.7 |
39.5 |
27.5 |
54.3 |
37.8 |
|||
90/1 |
56.3 |
16.7 |
9.4 |
13.7 |
19.8 |
11.2 |
17.1 |
15.4 |
90/2 |
59.8 |
30.1 |
18..0 |
38.4 |
22.9 |
|||
120/1 |
54.5 |
12.6 |
6.9 |
4.3 |
16.6 |
9.1 |
12.2 |
8.2 |
120/2 |
50.1 |
3.3 |
1.6 |
30.6 |
15.3 |
|||
Table 4. Degradation of 14C-substance in Soil at 10°C and 20°C: Half-Lives and DT-90 Values.
Temperature |
Correlation coefficient r2 |
Intercept [%] |
Rate constant [days-1] |
Half-life [days] |
DT90 [days] |
10°C |
0.992 |
95.4 |
0.0081 |
85.6 |
284 |
20°C |
0.996 |
101.1 |
0.0207 |
33.5 |
111 |
PURITY AND STABILITY OF THE TEST SUBSTANCE
- Purity: HPLC-analysis of the test substance in the application solution before treatment showed a purity of 98.92 %. Besides the parent molecule one impurity fraction was found to amount to 1.08 %.
- Stability: HPLC-analysis of the application solution after treatment procedure proved the stability of the radiolabelled test compound (purity 98.92 %). Besides the parent molecule one impurity fraction was found to amount to 1.08 %.
MICROBIAL BIOMASS
- The initial microbial biomass of Gartenacker, Pappelacker, Speyer 2.2 and Weide soil amounted to 37.5, 39.4, 32.1 and 37.9 mg microbial carbon / 100 g soil, respectively. After 120 days the microbial biomass of the corresponding soils dropped to 23.5, 20.6, 19.8 and 18.6 mg microbial carbon / 100 g soil (Table 1 in “Any other information on materials and methods incl tables”).
PHYSICOCHEMICAL CHARACTERISTICS
- During the equilibration and incubation period, the water layer of the anaerobic system was monitored for redox potential, oxygen content and pH. Due to a technical error positive redox potentials were recorded (402 ± 13 mV), although the oxygen concentration was monitored with a mean value of 0.10 ± 0.03 mg / L, thus, clearly demonstrating anaerobic conditions. However, partial repetition of the experiment with new samples over 120 days showed negative redox potentials, thus, confirming the low oxygen concentration (0.2 ± 0.1 mg / L) and anaerobic conditions. The pH remained constant with a mean value of 8.12 ± 0.24.
Table 1. Disappearance Rates of the substance from Different Soil Systems (aerobic and anaerobic, 20 °C)
Parameter* |
|
Soil |
||||
Gartenacker (aerobic) |
Pappelacker |
Speyer 2.2 |
Weide |
Gartenacker / (anaerobic system) |
Gartenacker / (anaerobic system) repeition |
|
Group 1 |
Group 2 |
Group 3 |
Group 4 |
Group 5 |
Group 5 |
|
C01 |
95.7 |
101.3 |
100.9 |
108.2 |
103.8 |
97.3 |
K1 (1/days) |
0.02627 |
0.02619 |
0.00573 |
0.03362 |
0.00065 |
0.00067 |
DT50 (days) |
26.4 |
26.5 |
121.0 |
20.6 |
1066 |
1035 |
DT90 (days) |
87.7 |
87.9 |
401.8 |
68.5 |
3542 |
3437 |
Chi square |
13.3877 |
32.1569 |
13.7618 |
35.1510 |
7.7659 |
4.8249 |
|
||||||
Comparison of the Half-lives evaluated by different Models: |
||||||
Soil |
Origin* |
Model maker** |
||||
Parent |
Parent |
M3 |
M2 |
M29 |
||
Gartenacker |
26.4 |
25.9 |
14.1 |
74.8 |
140.9 |
|
Papperlacker |
26.5 |
26.2 |
19.3 |
70.7 |
113.9 |
|
Speyer 2.2 |
121.0 |
109.6 |
11.9 |
- |
- |
|
Weide |
20.6 |
20.9 |
13.8 |
142.3 |
100.3 |
|
* MicroCal Origin Vers. 3.0 was used for this evaluation. The degradation rates of the substance were fitted by ct = c01 – eE(k1-t). The disappearance times for 50% (DT50) and 90% (DT90) were calculated by: DT50 =ln(2)/k1 and DT90 = ln(10)/K1 ** ModelMaker, Vers. 3.03 |
||||||
In addition, the data of decline of the parent molecule and formation and decline of the metabolites were fitted. For simplification, the amounts of metabolite M1, the non-extractable fraction and the evolved CO2 were combined in compartment Rest_NE_C02 for each sampling date. |
||||||
|
||||||
For modelling normalised data were used. The fit-results are shown below |
||||||
Gartenacker |
||||||
|
Parameter |
k |
DT50 |
DT90 |
||
Substance |
P1 |
0.015246 |
|
|
|
|
|
P2 |
0.011500 |
0.02675 |
25.9 |
86.1 |
|
M3 |
P4 |
0.029327 |
|
|
|
|
|
P8 |
0.019892 |
0.04922 |
14.1 |
46.8 |
|
M2 |
P7 |
0.009271 |
0.00927 |
74.8 |
248.4 |
|
M29 |
P5 |
0.001342 |
|
|
|
|
|
P6 |
0.003578 |
0.00492 |
140.9 |
468.0 |
|
Pappelacker |
||||||
|
Parameter |
k |
DT50 |
DT90 |
||
Substance |
P1 |
0.015242 |
|
|
|
|
|
P2 |
0.011208 |
0.02645 |
26.2 |
87.1 |
|
M3 |
P4 |
0.019987 |
|
|
|
|
|
P8 |
0.015972 |
0.03596 |
19.3 |
64.0 |
|
M2 |
P7 |
0.009800 |
0.00980 |
70.7 |
235.0 |
|
M29 |
P5 |
0.003155 |
|
|
|
|
|
P6 |
0.002932 |
0.00609 |
11.9 |
378.3 |
|
Speyer 2.2. |
||||||
|
Parameter |
k |
DT50 |
DT90 |
||
Substance |
P1 |
0.003152 |
|
|
|
|
|
P2 |
0.003172 |
0.00632 |
109.6 |
364.1 |
|
M3 |
P4 |
0.054937 |
|
|
|
|
|
P8 |
0.003490 |
0.05843 |
11.9 |
39.4 |
|
M2 |
P7 |
2.34E-9 |
2.34E-9 |
- |
- |
|
M29 |
P5 |
0.003 |
|
|
|
|
|
P6 |
0.003 |
0.006 |
- |
- |
|
Weide |
||||||
|
Parameter |
k |
DT50 |
DT90 |
||
Substance |
P1 |
0.018987 |
|
|
|
|
|
P2 |
0.014229 |
0.03322 |
20.9 |
69.3 |
|
M3 |
P4 |
0.021465 |
|
|
|
|
|
P8 |
0.028908 |
0.05037 |
13.8 |
45.7 |
|
M2 |
P7 |
0.004870 |
0.00487 |
142.3 |
472.8 |
|
M29 |
P5 |
0.001192 |
|
|
|
|
|
P6 |
0.005718 |
0.00691 |
100.3 |
333.2 |
|
Table 1. Mean Distribution of Radioactivity Found among the Matrices of the Samples Incubated under Aerobic Conditions.
Sample interval (day) |
Percent of radioactivity applied to sample |
||||
MeOH:H2O (80:20) Extract |
Extracted Soil |
Traps for Volatile Components |
|||
Ethylene glycol |
2-Ethoxyethanol: Ethanolamine (1:1) |
Total (a) |
|||
0 |
93.0 |
9.2 |
NA |
NA |
102.2 |
15 |
87.3 |
13.2 |
<0.1 |
ND |
100.5 |
31 |
85.0 |
15.6 |
<0.1 |
ND |
100.6 |
67 |
82.8 |
16.8 |
<0.1 |
<0.1 |
99.6 |
102 |
78.9 |
23.6 |
<0.1 |
<0.1 |
102.5 |
NA: Not applicable.
ND: Not detected.
(a)Sum of mean values in this table.
Table 2. Individual and Mean Distribution of Radioactivity Expressed as the Percentage of Radioactivity Applied to Sample for the MeOH:H2O (80:20) Extract of the Samples Incubated under Aerobic Conditions.
Sample interval (day) |
Sample Number |
Percent radioactivity applied to sample |
|||
14C-substance |
Peak 1 |
||||
Individual |
Mean |
Individual |
Mean |
||
0 |
C |
94.6 |
94.5 |
ND |
ND |
D |
94.4 |
ND |
|||
15 |
10 |
88.3 |
87.9 |
ND |
ND |
15 |
87.4 |
ND |
|||
31 |
1 |
80.8 |
82.0 |
ND |
ND |
9 |
83.2 |
ND |
|||
67 |
4 |
79.8 |
78.7 |
ND |
ND |
5 |
77.6 |
ND |
|||
102 |
3 |
67.0 |
70.4 |
5.9 |
5.1 |
16 |
73.7 |
4.2 |
|||
ND: Not detected.
(a) Percentage of radioactivity recovered from TLC plate multiplied by the applied radioactivity recovered from the MeOH:H2O (80:20) extract of the sample divided by 100.
Table 3.Mean Distribution of Radioactivity among the Matrices of Samples Incubated under Aerobic/Anaerobic Conditions.
Sample interval (day) (a) |
Percent of radioactivity applied to sample |
||||
MeOH:H2O (80:20) Extract |
Extracted Soil |
Traps for Volatile Components |
|||
Ethylene glycol |
2-Ethoxyethanol: Ethanolamine (1:1) |
Total (b) |
|||
31 [0] |
85.0 |
15.6 |
<0.1 |
ND |
100.6 |
67 [36] |
88.4 |
11.7 |
<0.1 |
<0.1 |
100.1 |
102 [71] |
87.2 |
11.9 |
<0.1 |
<0.1 |
99.1 |
ND: Not detected.
(a)Sample interval expressed as the length of aerobic/anaerobic and anaerobic incubation time; values in brackets [ ] represent anaerobic incubation time.
(b)Sum of mean values in this table.
Table 4. Individual and Mean Distribution of Radioactivity Expressed as the Percentage of Radioactivity Applied to Sample for the MeOH:H2O (80:20) Extract of the Soil from the Samples Incubated under Aerobic/Anaerobic Conditions.
Sample interval (day) |
Sample Number |
Percent radioactivity applied to sample |
|||
14C-substance |
Peak 1 |
||||
Individual |
Mean |
Individual |
Mean |
||
31 [0] |
1 |
80.8 |
82.0 |
ND |
ND |
9 |
83.2 |
ND |
|||
67 [36] |
7 |
90.0 |
88.5 |
ND |
ND |
8 |
87.0 |
ND |
|||
102 [71] |
14 |
84.9 |
86.0 |
ND |
ND |
13 |
87.0 |
ND |
|||
ND: Not detected.
a Percentage of radioactivity recovered from TLC plate multiplied by the applied radioactivity recovered from the MeOH:H2O (80:20) extract of the soil divided by 100.
b Sample interval expressed as the length of aerobic/anaerobic and anaerobic incubation time; values in brackets [ ] represent anaerobic incubation time.
Table 5. HPLC Distribution of Radioactivity Expressed as the Percentage of Radioactivity Applied to Sample for the MeOH:HCl (11:1) Reflux Extracts.
Sample interval (b) |
Sample number |
Percent of Radioactivity Applied to Sample |
|||
M13 |
M4 |
M2 |
14C-substance |
||
Aerobic Samples |
|||||
67 |
4 |
0.5 |
1.0 |
7.6 |
5.8 |
|
5 |
0.5 |
0.9 |
4.9 |
6.3 |
102 |
3 |
0.8 |
1.8 |
10.3 |
6.4 |
|
16 |
0.6 |
1.5 |
9.5 |
4.9 |
Anaerobic samples |
|||||
67 [36] |
7 |
ND |
0.2 |
2.9 |
6.2 |
|
8 |
ND |
0.2 |
3.2 |
6.6 |
102 [71] |
14 |
0.3 |
0.3 |
8.5 |
2.4 |
|
13 |
0.2 |
0.3 |
4.2 |
5.4 |
ND: Not detected.
(a)Percentage of applied radioactivity found in the reflux extract multiplied by the percentage of radioactivity recovered from the HPLC column divided by 100.
(b)Sample interval for the aerobic/anaerobic samples expressed as the length of aerobic/anaerobic and anaerobic incubation time; values in brackets [ ] represent anaerobic incubation time.
Table 1. Extractability of radioactivity from soil treated with 14C-substance (values in % of the radioactivity applied).
|
Incubation time in days |
||||||
|
0 |
13 |
30 |
60 |
90 |
120 |
180 |
Volatiles (14CO2) |
0 |
0.2 |
0.3 |
1.6 |
3.5 |
4.0 |
5.9 |
Extracted radioactivity |
101.3 |
105.3 |
85.9 |
70.1 |
56.5 |
53.1 |
47.2 |
Non extracted radioactivity |
0.2 |
7.0 |
18.9 |
36.4 |
44.1 |
49.0 |
45.9 |
Total |
101.3 |
112.5 |
105.1 |
108.1 |
104.1 |
106.1 |
99.0 |
Table 2. Distribution of substance and its metabolites in the extracts of soil treated with 14C-substance (values in % of the radioactivity applied).
Identity |
Incubation time |
||||||
0 |
13 |
30 |
60 |
90 |
120 |
180 |
|
M20 |
n.d. |
n.d. |
0.6 |
0.9 |
n.d. |
2.7 |
n.d. |
M5 |
n.d. |
n.d. |
0.5 |
09 |
2.7 |
0.7 |
3.3 |
M18 |
n.d. |
n.d. |
5.2 |
10.4 |
12.5 |
11.5 |
10.7 |
M2 |
n.d. |
6.5 |
9.6 |
22.3 |
30.0 |
24.7 |
24.124.1 |
M22 |
n.d. |
n.d. |
1.5 |
1.0 |
0.8 |
0.7 |
|
M23 |
n.d. |
n.d. |
n.d. |
0.6 |
0.7 |
0.4 |
|
M17 |
n.d. |
7.0 |
12.9 |
9.5 |
3.1 |
4.1 |
3.6 |
M24 |
n.d. |
n.d. |
<0.1 |
0.1 |
0.2 |
0.2 |
n.d. |
M25 |
n.d. |
n.d. |
n.d. |
0.6 |
n.d. |
n.d. |
1.6 |
M26 |
n.d. |
0.1 |
7.3 |
3.9 |
3.5 |
3.6 |
1.0 |
Substance |
101.1 |
91.7 |
48.3 |
19.9 |
3.6 |
4.5 |
2.9 |
Total |
101.1 |
105.3 |
85.9 |
70.1 |
56.5 |
53.1 |
47.2 |
Description of key information
All available data were assessed and the studies representing the worst-case effects are included here in a weight of evidence approach.
The geometric mean of the DT50 values for the biodegradation of the substance in seven different soils was determined to be 46.9 days, BBVA Part IV, 4 -1 (or similar), Jonas 2002; Saxena 1989; Reischmann 1998; Ellgehausen 1986.
Key value for chemical safety assessment
- Half-life in soil:
- 46.9 d
- at the temperature of:
- 20 °C
Additional information
DT50 values for the substance in soil under aerobic standard(a) test conditions
USDA / Name / Origin |
OC [%] / |
T. [°C] / |
DT50[d] - |
Author / Year |
Loam, Gartenacker, CH |
2.35 / 7.23 |
20 / 40% MWHC |
33.5 (SFO) |
Jonas, 2002 |
Silt loam, Gartenacker, CH |
2.2 / 7.25 |
20 / 40% MWHC |
26.4 (SFO) |
Reischmann, 1998 |
Loamy sand, Pappelacker, CH |
1.1 / 7.6 |
20 / 40% MWHC |
26.5 (SFO) |
|
Loamy sand, Speyer 2.2, D |
2.2 / 5.65 |
20 / 40% MWHC |
121.0 (SFO) |
|
Sandy loam, Weide, CH |
1.3 / 7.5 |
20 / 40% MWHC |
20.6 (SFO) |
|
Sandy Loam, Strassenacker, CH |
0.63 / 7.5 |
23 / 70% FC |
32.5 (linear SFO) |
Ellgehausen, 1986 |
Sandy Loam, California, USA |
0.4 / 4.6 |
25 / 75%FC |
261 (linear SFO) |
Saxena, 1989 |
Geomean |
|
|
46.9 |
- |
Median |
|
|
32.5 |
- |
(a)Criteria for standard test conditions: Meet OECD, SETAC, OPPTS and JMAFF guideline requirements
Note: In case of SFO kinetics, the DT90 can be calculated by multiplying the DT50 by 3.32. This is not valid for other kinetic models.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
