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Registration Dossier
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EC number: 225-625-8 | CAS number: 4979-32-2
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:
- From 2016-02-18 until
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)
- Version / remarks:
- Version from 2002
- Deviations:
- no
- GLP compliance:
- yes
- Test type:
- laboratory
- Specific details on test material used for the study:
- - 14C-labelled Test item
Test item name: [benzene-ring-U-14C]-DCBS
Chemical name: 14C-N, N-Dicyclohexylbenzo-thiazole-2-sulphenamide
Purity (radiochemical): 99.6 %
Specific radioactivity: 6.92 MBq/mg (65 mCi/mmol)
Water solubility: 0.0019 mg/L (OECD SIDS)
Vapour pressure (100 °C): 7 x 10-7 hPa
Chemical stability (water/light): hydrolysis: DT50 (pH=7) = 53 h; photolysis: DT50 (air, indirect) = 3.38 h
Origin of the test item: Quotient Bioresearch, The Old Glassworks, Nettlefold Road, Cardiff, CF24 5JQ
- Unlabelled Test Item
Trade name: DCBS, Vulkacit DZ/EG-C
Chemical name: N, N-Dicyclohexylbenzothiazole-2-sulphenamide
Content: 93.9 % (according to substance characterisation report)
Water solubility: 0.0019 mg/L (OECD SIDS)
Vapour pressure (100 °C): 7 x 10-7 hPa
Chemical stability (water/light): hydrolysis: DT50 (pH=7) = 53 h; photolysis: DT50 (air, indirect) = 3.38 h
State of matter and appearance: light beige, solid
Origin of the reference item: LANXESS Deutschland GmbH - Radiolabelling:
- yes
- Remarks:
- 14C
- Oxygen conditions:
- anaerobic
- Soil classification:
- USDA (US Department of Agriculture)
- Year:
- 2 016
- Soil no.:
- #1
- Soil type:
- silt loam
- % Clay:
- 15.8
- % Silt:
- 80.1
- % Sand:
- 4.1
- % Org. C:
- 1.04
- pH:
- 6.63
- CEC:
- 59.1 other: mmol(c)/kg
- Details on soil characteristics:
- For the degradation study of the test item a freshly sampled soil was used (soil no. #1, refesol 02-A). The soil was taken by the testing facility. Characterization of the soil has been performed by Fraunhofer IME following GLP. The selected soil is representative for agriculturally used soils. Further information on the soil can be found under www.refesol.de.
Prior to incubation the soil was sieved (2 mm sieve). The moisture content of the soil was checked and adjusted to 50 % of their maximum water holding capacity (WHCmax). Additionally, pH-values and the amount of organic carbon were re-determined. The test soil was pre-incubated at 12 °C in the dark for 3 days. The microbial activity of the test soil was determined at the test start by system induced respiration (SIR) which is typically used as a measure for biological activity of soils under aerobic conditions. The requirement of the OECD test guideline 307 that the microbial biomass should be at least 1 % of the total organic carbon was met.
Further soil properties: maximum water holding capacity (WHC(max))
Soil no. #1 (refesol 02-A): 4 g/kg - Soil No.:
- #1
- Duration:
- 120 d
- Soil No.:
- #1
- Initial conc.:
- 1 mg/kg soil d.w.
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- radiochem. meas.
- Soil No.:
- #1
- Temp.:
- 12 °C +/-2°C
- Humidity:
- 50% WHC(max)
- Microbial biomass:
- > 1 % C(mic)/C(org)
- Details on experimental conditions:
- - Test system
The test apparatus consisted of a flow-through apparatus containing test vessels (two vessels in sequence per sampling point) of treated soil samples connected to traps for collection of volatile and mineralization products. Glassware was connected using a combination of glass connectors and plastic tubing. A constant stream of humidified air was passed over the sub-samples in order to achieve and maintain aerobic conditions for 28 days. The outgoing gas was bubbled through three absorption traps in sequence containing ethylene glycol (1 x), H2SO4 (1 x), and 1M NaOH (1 x) in order to quantify volatile reaction products and to determine the rate of mineralization (formation of 14CO2).
After 28 days, the samples were water-logged (ca. 1 – 3 cm water layer) and the incubation system was flushed with nitrogen in order to establish and maintain anaerobic conditions. First, the nitrogen was bubbled through water in order to humidify the gas. Thereafter, the humidified gas was passed over the soil subsamples. Once the gas had passed over the sub-samples it was bubbled through three absorption traps in sequence containing ethylene glycol, 0.5 N H2SO4 and 1 N NaOH (i.e., identical to the aerobic phase of incubation). Since the formation of 14CH4 was expected in such an anaerobic system the gas was furthermore passed through an oven at 850°C. 14CH4 was catalytically (CuO-catalyst + O2 feeding to the tube) converted to 14CO2 which again was trapped in a further NaOH trap.
Duplicate samples of the soil (ca. 50 g dry weight basis) were weighed into the vessels. For information on the abiotic transformation of the test substance additional sterilized samples (duplicates) were taken, processed and analyzed at 0 DAT, 28 DAT and 120 DAT.
During the whole test period the soil samples were incubated in the dark at a test temperature of 12 °C +/-2°C. The moisture content of the soil samples was checked periodically by weighting and, if necessary, re-adjusted to the initial moisture with distilled water in order to maintain constant moisture content of 50 %WHCmax.
- Treatment
The target concentration of the test item in soil was set to 1 mg/kg soil (dry mass). Therefore, each pre-incubated non-sterile soil sample was treated with 50 µg and 346 kBq per soil sample, taking into account the specific radioactivity of the test item of 6.92 MBq/mg.
Sterile samples were applied under sterile conditions. The soil samples had been sterilized previoulsy by γ-irradiation at the facility of Beta-Gamma-Service GmbH (51674 Wiehl, Germany). The treatment dose also was 1 mg/kg soil (dry mass). After application these samples were closed gas-tightly and no trapping of produced gases was performed. - Soil No.:
- #1
- % Recovery:
- 103.8
- St. dev.:
- 2.3
- Remarks on result:
- other: Applied Radioactivity after 0 days
- Remarks:
- Total mass balance
- Soil No.:
- #1
- % Recovery:
- 106.7
- St. dev.:
- 2.3
- Remarks on result:
- other: Applied Radioactivity after 28 days (measured before conversion to anaerobic conditions)
- Remarks:
- Total mass balance
- Soil No.:
- #1
- % Recovery:
- 102.1
- St. dev.:
- 2.3
- Remarks on result:
- other: Applied Radioactivity after 120 days
- Remarks:
- Total mass balance
- Key result
- Soil No.:
- #1
- DT50:
- 783 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 12 °C
- Remarks on result:
- other: The degradation rate (DegT50) of DCBS in refesol 02-A soil at 12°C was determined by means of KinGUI (version 1.1). The calculation was conducted using the fractions of the parent obtained from radio-HPLC analyses of the respective samples.
- Transformation products:
- no
- Remarks:
- No residues other than the parent DCBS could be identified by radio-HPLC
- Details on transformation products:
- For all samples, with few exceptions, which are caused by artifacts during the sample processing, no residues other than the parent DCBS could be identified by radio-HPLC. However, during the study the occurrence of transformation products was observed for some samples. In the parallel aerobic study it was observed that in repeatedly concentrated samples the distribution of radioactive residues showed a complete reversal in the respective radio-HPLC chromatograms. Thus, it was concluded that the appearance of transformation products was most likely induced by the sample concentration step. The graph for the distribution of radioactive residues in the anaerobic study shows a similar pattern for the anaerobic study with outliers of complete “transformation” of the parent followed. These observations are most likely artifacts as the analyses of extracts of the following sampling points showed again only the slight decrease of the parent fraction, due to the decreased extractability, and no transformation products.
To conclude, the appearance of transformation products only in a few samples, followed by samples at subsequent sample points with 100% parent in the residue distribution, and - in addition - the reversal of the residues distribution after repeated sample processing observed in the aerobic study is a strong indication that the apparent degradation of the parent is in fact an artifact induced by the sample concentration step. With other words: Disappearance of the parent is in fact not caused by microbial degradation in the soil but by so far unknown processes during the sample processing, most likely the concentration step. - Evaporation of parent compound:
- no
- Volatile metabolites:
- no
- Remarks:
- Only low amounts of radioactivity evolved as volatile degradation products. No radioactivity detected in H2SO4 adsorption traps and minor amounts measured in ethylene glycol containing traps. Only low amounts of 14CO2 to 1.5 % AR found at the end.
- Residues:
- yes
- Remarks:
- No residues other than the parent DCBS could be identified by radio-HPLC.
- Details on results:
- Microbial activity
Biomass results indicated viable microflora at study initiation with Cmic/Corg ratios > 1 %. The measurements during and at end of the study demonstrate that the test system remained viable throughout the study.
Mass balance
The total recoveries ranged from 102.0 to 108.4 % AR and are well within the range of 90-110 % AR required by the test guideline.
Extractable and bound residues
The total extractable radioactivity ranged from 91.4 to 101.0 % AR and decreased only slightly over time, remaining > 90% AR for all individual samples throughout the test. Only minor amounts of radioactivity could be found in the additional fourth aqueous extract, reaching a maximum level of 2.2 % AR at 21 DAT.
Non-extractable residues (NER), determined by combustion analysis of dried soil samples after extraction, increased throughout the study and reached 9.1 % AR at end of the test. - Conclusions:
- Half-life time of DCBS at 12°C under anaerobic conditions is greater than one year.
- Executive summary:
This study was designed to determine the rate of degradation of DCBS in soil under anaerobic conditions at 12 ± 2 ºC.
Freshly sampled refesol 02-A soil (silty loam) was used. The soil is representative for agriculturally used soils.
The moisture content of the soil was checked and adjusted to 50 % of their maximum water holding capacity (WHCmax). Additionally, pH-values and the amount of organic carbon was determined. The microbial activity of the test soil was determined at the test start by system induced respiration (SIR) which is typically used as a measure for biological activity of soils under aerobic conditions. The requirement of the OECD testguideline 307 that the microbial biomass should be at least 1 % of the total organic carbon was met. During the whole test period the soil samples were incubated in the dark at a test temperature of 12 °C +/-2°C. The target concentration of the test item in soil was set to 1 mg/kg soil (dry mass). Sterile samples were applied under sterile conditions.
The test apparatus consisted of a flow-through apparatus. Radio-HPLC was applied as primary method.
The study duration was 28 days (aerobic phase) plus 92 days (anaerobic phase) resulting in a total of 120 days. Samples were taken at 0 DAT, 1 DAT, 3 DAT, 7 DAT, 14 DAT, 21 DAT, 28 DAT (thereafter conversion to anaerobic conditions), 59 DAT, 91 DAT, and 120 DAT. Radioactivity in the ethyleneglycol, H2SO4 and NaOH containing traps was quantified at sampling date.
Mass balance: The total recoveries ranged from 102.0 to 108.4 % AR (applied radioactivity) and are well within the range of 90-110 % AR required by the test guideline.
Extractable and bound residues: The total extractable radioactivity ranged from 91.4 to 101.0 % AR and decreased only slightly over time, remaining > 90% AR for all individual samples throughout the test.
Volatile degradation products: Only low amounts of radioactivity evolved from the incubation flasks as volatile degradation products.
Radioactive residues in soil extracts (non-sterile samples): For all samples, with few exceptions, which are caused by artifacts during the sample processing, no residues other than the parent DCBS could be identified by radio-HPLC.
The degradation rate (DegT50) of DCBS in refesol 02-A soil at 12°C was determined by means of KinGUI (version 1.1). The calculation was conducted using the fractions of the parent obtained from radio-HPLC analyses of the respective samples. DegT50- and DegT90-values for DCBS at 12°C under anaerobic conditions are:
DegT50: 783 days
DegT90: > 1000 days
Half-life time of DCBS at 12°C under anaerobic conditions is greater than one year.
- Endpoint:
- biodegradation in soil: simulation testing
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 2016-02-18 until 2016-07-12
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)
- Version / remarks:
- Version from 2002
- Deviations:
- no
- GLP compliance:
- yes
- Test type:
- laboratory
- Specific details on test material used for the study:
- - 14C-labelled Test item
Test item name: [benzene-ring-U-14C]-DCBS
Chemical name: 14C-N, N-Dicyclohexylbenzo-thiazole-2-sulphenamide
Purity (radiochemical): 99.6 %
Specific radioactivity: 6.92 MBq/mg (65 mCi/mmol)
Water solubility: 0.0019 mg/L (OECD SIDS)
Vapour pressure (100 °C): 7 x 10-7 hPa
Chemical stability (water/light): hydrolysis: DT50 (pH=7) = 53 h; photolysis: DT50 (air, indirect) = 3.38 h
Origin of the test item: Quotient Bioresearch, The Old Glassworks, Nettlefold Road, Cardiff, CF24 5JQ
- Unlabelled Test Item
Trade name: DCBS, Vulkacit DZ/EG-C
Chemical name: N, N-Dicyclohexylbenzothiazole-2-sulphenamide
Content: 93.9 % (according to substance characterisation report)
Water solubility: 0.0019 mg/L (OECD SIDS)
Vapour pressure (100 °C): 7 x 10-7 hPa
Chemical stability (water/light): hydrolysis: DT50 (pH=7) = 53 h; photolysis: DT50 (air, indirect) = 3.38 h
State of matter and appearance: light beige, solid
Origin of the reference item: LANXESS Deutschland GmbH - Radiolabelling:
- yes
- Remarks:
- 14C
- Oxygen conditions:
- aerobic
- Soil classification:
- USDA (US Department of Agriculture)
- Year:
- 2 016
- Soil no.:
- #1
- Soil type:
- clay loam
- % Clay:
- 25.8
- % Silt:
- 41.1
- % Sand:
- 33.2
- % Org. C:
- 1.92
- pH:
- 7.3
- CEC:
- 330 other: mmol(c)/kg
- Soil no.:
- #2
- Soil type:
- silt loam
- % Clay:
- 15.8
- % Silt:
- 80.1
- % Sand:
- 4.1
- % Org. C:
- 1.04
- pH:
- 6.63
- CEC:
- 59.1 other: mmol(c)/kg
- Soil no.:
- #3
- Soil type:
- silt loam
- % Clay:
- 22.6
- % Silt:
- 55.2
- % Sand:
- 22.3
- % Org. C:
- 3.19
- pH:
- 5.91
- CEC:
- 66.9 other: mmol(c)/kg
- Soil no.:
- #4
- Soil type:
- loamy sand
- % Clay:
- 4
- % Silt:
- 10.6
- % Sand:
- 85.4
- % Org. C:
- 2.65
- pH:
- 5.22
- CEC:
- 34 other: mmol(c)/kg
- Details on soil characteristics:
- For the degradation study of the test item freshly sampled soils were used. The refesol soils (Soil no. #2, #3 and #4) were taken by the testing facility and the additionally used LUFA 2.4 (Soil no. #1) soil freshly sampled by LUFA Speyer. The key soil physicochemical properties of the LUFA soil were determined in compliance with the GLP principles by LUFA Speyer. Characterization of the refesol soils has been performed by Fraunhofer IME following GLP. The selected soils are representative for agriculturally used soils.
Prior to incubation the soils were sieved (2 mm sieve). The moisture content of the soil was checked and adjusted to 50% of their maximum water holding capacity (WHC(max)). Additionally, pH-values and the amount of organic carbon were re-determined. The test soils were pre-incubated at 12 °C in the dark for 13 days. The microbial activity of the test soils was determined at the test start by system induced respiration (SIR) which is typically used as a measure for biological activity of soils under aerobic conditions. The requirement of the OECD testguideline 307 that the microbial biomass should be at least 1% of the total organic carbon was met.
Further soil properties: maximum water holding capacity (WHC(max))
Soil no. #2 (refesol 02-A): 4 g/kg
Soil no. #3 (refesol 03-G): 714 g/kg
Soil no. #4 (refesol 04-A): 375 g/kg - Soil No.:
- #1
- Duration:
- 120 d
- Soil No.:
- #2
- Duration:
- 120 d
- Soil No.:
- #3
- Duration:
- 120 d
- Soil No.:
- #4
- Duration:
- 120 d
- Soil No.:
- #1
- Initial conc.:
- 1 mg/kg soil d.w.
- Based on:
- test mat.
- Soil No.:
- #2
- Initial conc.:
- 1 mg/kg soil d.w.
- Based on:
- test mat.
- Soil No.:
- #3
- Initial conc.:
- 1 mg/kg soil d.w.
- Based on:
- test mat.
- Soil No.:
- #4
- Initial conc.:
- 1 mg/kg soil d.w.
- Based on:
- test mat.
- Parameter followed for biodegradation estimation:
- radiochem. meas.
- Soil No.:
- #1
- Temp.:
- 12 °C +/-2°C
- Humidity:
- 50% WHC(max)
- Microbial biomass:
- > 1 % C(mic)/C(org)
- Soil No.:
- #2
- Temp.:
- 12 °C +/-2°C
- Humidity:
- 50% WHC(max)
- Microbial biomass:
- > 1% C(mic)/C(org)
- Soil No.:
- #3
- Temp.:
- 12 °C +/-2°C
- Humidity:
- 50% WHC(max)
- Microbial biomass:
- > 1% C(mic)/C(org)
- Soil No.:
- #4
- Temp.:
- 12 °C +/-2°C
- Humidity:
- 50% WHC(max)
- Microbial biomass:
- > 0.9% C(mic)/C(org)
- Details on experimental conditions:
- - Test system
The test apparatus consisted of a flow-through apparatus containing test vessels (two vessels in sequence per sampling point) of treated soil samples connected to traps for collection of volatile and mineralization products. Glassware was connected using a combination of glass connectors and plastic tubing. A constant stream of humidified air was be passed over the sub-samples in order to achieve and maintain aerobic conditions. The outgoing gas was bubbled through three absorption traps in sequence containing ethylene glycol (1 x), H2SO4 (1 x), and 1M NaOH (2 x) in order to quantify volatile reaction products and to determine the rate of mineralization (formation of 14CO2).
Duplicate samples of the soil (ca. 50g dry weight basis) were weighed into the vessels. For information on the abiotic transformation of the test substance additional sterilized samples (duplicates) were taken, processed and analyzed at 0 DAT, 28 DAT and 120 DAT.
During the whole test period the soil samples were incubated in the dark at a test temperature of 12 °C +/-2°C. The moisture content of the soil samples was checked periodically by weighting and, if necessary, re-adjusted to the initial moisture with distilled water in order to maintain constant moisture content of 50% WHC(max).
- Treatment
The target concentration of the test item in soil was set to 1 mg/kg soil (dry mass). Therefore, each pre-incubated non-sterile soil sample was treated with 50 µg and 346 kBq per soil sample, taking into account the specific radioactivity of the test item of 6.92 MBq/mg.
Sterile samples were applied under sterile conditions. The soil samples had been sterilized previoulsy by γ-irradiation at the facility of Beta-Gamma-Service GmbH (51674 Wiehl, Germany). The treatment dose also was 1 mg/kg soil (dry mass). After application these samples were closed gas-tightly and no trapping of produced gases was performed. - Soil No.:
- #1
- % Recovery:
- 102.2
- St. dev.:
- 3.9
- Remarks on result:
- other: Applied Radioactivity after 0 days
- Remarks:
- Total mass balance
- Soil No.:
- #1
- % Recovery:
- 106
- St. dev.:
- 3.9
- Remarks on result:
- other: Applied Radioactivity after 28 days
- Remarks:
- Total mass balance
- Soil No.:
- #1
- % Recovery:
- 105.2
- St. dev.:
- 3.9
- Remarks on result:
- other: Applied Radioactivity after 120 days
- Remarks:
- Total mass balance
- Soil No.:
- #2
- % Recovery:
- 103.3
- St. dev.:
- 0.8
- Remarks on result:
- other: Applied Radioactivity after 0 days
- Remarks:
- Total mass balance
- Soil No.:
- #2
- % Recovery:
- 104.1
- St. dev.:
- 0.8
- Remarks on result:
- other: Applied Radioactivity after 28 days
- Remarks:
- Total mass balance
- Soil No.:
- #2
- % Recovery:
- 105.1
- St. dev.:
- 0.8
- Remarks on result:
- other: Applied Radioactivity after 120 days
- Remarks:
- Total mass balance
- Soil No.:
- #3
- % Recovery:
- 104.9
- St. dev.:
- 2.4
- Remarks on result:
- other: Applied Radioactivity after 0 days
- Remarks:
- Total mass balance
- Soil No.:
- #3
- % Recovery:
- 104.8
- St. dev.:
- 2.4
- Remarks on result:
- other: Applied Radioactivity after 28 days
- Remarks:
- Total mass balance
- Soil No.:
- #3
- % Recovery:
- 104.4
- St. dev.:
- 2.4
- Remarks on result:
- other: Applied Radioactivity after 120 days
- Remarks:
- Total mass balance
- Soil No.:
- #4
- % Recovery:
- 104.1
- St. dev.:
- 0.9
- Remarks on result:
- other: Applied Radioactivity after 0 days
- Remarks:
- Total mass balance
- Soil No.:
- #4
- % Recovery:
- 105.7
- St. dev.:
- 0.9
- Remarks on result:
- other: Applied Radioactivity after 28 days
- Remarks:
- Total mass balance
- Soil No.:
- #4
- % Recovery:
- 105.4
- St. dev.:
- 0.9
- Remarks on result:
- other: Applied Radioactivity after 120 days
- Remarks:
- Total mass balance
- Key result
- Soil No.:
- #1
- DT50:
- > 1 000 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 12 °C
- Remarks on result:
- other: The degradation rate (DegT50) of DCBS by means of KinGUI (version 1.1). First, kinetic analyses were performed using all available kinetic models SFO, FOMC, HS, DFOP. Calculation was conducted using fractions of parent obtained from radio-HPLC analyses.
- Key result
- Soil No.:
- #2
- DT50:
- 451.1 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 12 °C
- Remarks on result:
- other: The degradation rate (DegT50) of DCBS by means of KinGUI (version 1.1). First, kinetic analyses were performed using all available kinetic models SFO, FOMC, HS, DFOP. Calculation was conducted using fractions of parent obtained from radio-HPLC analyses.
- Key result
- Soil No.:
- #3
- DT50:
- 305.4 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 12 °C
- Remarks on result:
- other: The degradation rate (DegT50) of DCBS by means of KinGUI (version 1.1). First, kinetic analyses were performed using all available kinetic models SFO, FOMC, HS, DFOP. Calculation was conducted using fractions of parent obtained from radio-HPLC analyses.
- Key result
- Soil No.:
- #4
- DT50:
- > 1 000 d
- Type:
- (pseudo-)first order (= half-life)
- Temp.:
- 12 °C
- Remarks on result:
- other: The degradation rate (DegT50) of DCBS by means of KinGUI (version 1.1). First, kinetic analyses were performed using all available kinetic models SFO, FOMC, HS, DFOP. Calculation was conducted using fractions of parent obtained from radio-HPLC analyses.
- Transformation products:
- yes
- Remarks:
- No residues other than the parent DCBS could be identified by radio-HPLC chromatograms.
- No.:
- #1
- No.:
- #2
- No.:
- #3
- No.:
- #4
- No.:
- #5
- No.:
- #6
- No.:
- #7
- No.:
- #8
- Details on transformation products:
- Radioactive residues in soil extracts
Residues in organic extracts 1-3
For all samples, with few exceptions, which are caused by artifacts during the sample processing, no residues other than the parent DCBS could be identified by radio-HPLC. However, during the study the occurrence of transformation products was observed for some samples. Aliquots of these extracts were then subjected to repetition of sample concentration. For most samples the analysis of the repeatedly concentrated extract samples resulted in a complete reversal of the distribution observed for the first concentrate with no observed transformation products and 100% parent in the respective radio-HPLC chromatograms. Thus, it was concluded that the appearance of transformation products was most likely induced by the sample concentration step. However, it was not possible to avoid the observation of these artifacts in all cases by repetition of sample concentration. For example, the radio-HPLC analysis of the sample 02-A 28 DAT showed a decrease of the parent DCBS to 13.1 %AR and an increase of the transformation product BT (identified by retention time) up to 81.3 % AR.
To conclude, the appearance of transformation products only in a few samples, followed by samples at subsequent sample points with 100% parent in the residue distribution, and - in addition - the reversal of the residues distribution after repeated sample processing is a strong indication that the apparent degradation of the parent is in fact an artifact induced by the sample concentration step. With other words: Disappearance of the parent is in fact not caused by microbial degradation in the soil but by so far unknown processes during the sample processing, most likely the concentration step.
The residue found in most of the samples showing apparent degradation of the parent eluted at a retention time of 5.1-5.6 min, and was thus identified as transformation product Benzothiazole (BT). To confirm this allocation, selected samples (02-A 21 DAT repeated sample concentration and 02-A 28 DAT second sample concentration) were analyzed additionally by LC-HR-MS, equipped with additional 14C-detection to allow identification of peaks derived from the labelled test item and to confirm the peak pattern observed in the conventional radio-HPLC analysis.
Residues in aqueous extract 4
Only for samples 120 DAT for all soils and 91 DAT of soil 03-G amounts of radioactivity > 5 % AR were found in the aqueous extraction. Therefore only these extracts were subjected to chemical analysis by LC-HR-MS. The measured concentrations were converted into parent equivalents. In contrast to the results obtained for organic extracts the main residues the parent is not the dominating fraction. Instead, the formation of significant amounts of transformation products BTSO3H and DCHA could be identified by LC-HR-MS analysis. However, with exception of the 120 DAT sample of soil 03-G, all fractions are below 5 % AR. - Evaporation of parent compound:
- no
- Volatile metabolites:
- no
- Remarks:
- Only low amounts of radioactivity from incubation flasks as volatile degradation products. No radioactivity in ethylene glycol and H2SO4 adsorption traps. Only low amount of 14CO2 found to a range of 1.9 to 3.2 % AR at the end of the study.
- Residues:
- yes
- Remarks:
- No observed transformation products and 100% parent DCBS observed in the respective radio-HPLC chromatograms.
- Details on results:
- Microbial activity
Biomass results indicated viable microflora at study initiation with C(mic)/C(org) ratios > 1% for all soils with exception of the refesol 04-A, soil no. #4 (0.9%), which was slightly below the threshold set by the test guideline. The measurements during and at end of the study demonstrate that the test system remained viable throughout the study.
Mass balance
The total recoveries ranged from 92.5 to 106.0 % Applied Radioactivity (AR) and are well within the range of 90-110 % AR required by the test guideline.
Extractable and bound residues
The total extractable radioactivity ranged from 82.2 to 101.3 % AR and decreased only slightly over time, remaining < 90% AR for most individual samples throughout the test. Only minor amounts of radioactivity could be found in the additional fourth aqueous extract, reaching levels > 5% AR at 120 DAT for all four soils and additionally at 91 DAT for soil 03-G.
Non-extractable residues (NER), determined by combustion analysis of dried soil samples after extraction, increased throughout the study and reached a range of 11.7 (LUFA 2.4, soil no. #1) to 19.4 (03-G, soil no. #3) % AR at end of the test. - Conclusions:
- The half-life of DCBS in soils at 12°C is greater than one year.
- Executive summary:
This study was designed to determine the rate of degradation of DCBS in soil under aerobic conditions at 12°C.
4 samples were analyzed, representative for agriculturally used soils. For soil samples a constant moisture content of 50% WHC(max) was maintainend. Sterile samples were applied under sterile conditions. Each pre-incubated non-sterile soil sample was treated with 50 µg and 346 kBq per soil sample, taking into account the specific radioactivity of the test item of 6.92 MBq/mg. The requirement of the OECD testguideline 307 that the microbial biomass should be at least 1 % of the total organic carbon was met.
The study duration was 120 days. The test consisted of a flow-through apparatus.
The total recoveries ranged from 92.5 to 106.0 % AR and are well within the range of 90-110 % AR required by the test guideline. The total extractable radioactivity ranged from 82.2 to 101.3 % AR and decreased only slightly over time, remaining < 90% AR for most individual samples throughout the test. Non-extractable residues (NER), determined by combustion analysis of dried soil samples after extraction, increased throughout the study and reached a range of 11.7 ( Lufa 2.4, soil no. #1) to 19.4 ( Refesol 03-G, soil no. #3) % AR at end of the test.
Only low amounts of radioactivity evolved from the incubation flasks as volatile degradation products. Only a low amount of 14CO2 was found, increasing slightly throughout the study to a range of 1.9 to 3.2 % AR at the end of the study.
For the analysis of the combined organic soil extracts (extracts 1-3) the radio-HPLC method was applied as primary method. For all samples, with few exceptions, which are caused by artifacts during the sample processing, no residues other than the parent DCBS could be identified by radio-HPLC. However, during the study the occurrence of transformation products was observed for some samples. Aliquots of these extracts were then subjected to repetition of sample concentration. For most samples the analysis of the repeatedly concentrated extract samples resulted in a complete reversal of the distribution observed for the first concentrate with no observed transformation products and 100% parent in the respective radio-HPLC chromatograms. Thus, it was concluded that the appearance of transformation products was most likely induced by the sample concentration step.
The degradation rate (DegT50) of DCBS in four different soils at 12°C was determined by means of KinGUI (version 1.1). First, kinetic analyses were performed using all available kinetic models, namely single first order (SFO), first order multi compartment (FOMC), hockey stick (HS), and double first order in parallel (DFOP). The calculation was conducted using the fractions of the parent obtained from radio-HPLC analyses of the respective samples.
DegT50- and DegT90-values for DCBS in four soils at 12°C are:
Soil DegT50 [days] DegT90 [days]
Soil no.#2 (Refesol 02 -A) 462.2 >1000
Soil no.#3 (Refesol 03 -G) 314.8 >1000
Soil no.#4 (Refesol 04 -A) 528.2 >1000
Soil no.#1 (Lufa 2.4) 614.5 >1000
The half-life of DCBS in soils at 12°C is greater than one year.
Referenceopen allclose all
Microbial biomass determined by means of substrate induced respiration (SIR) at different stages of the test
| start of test (0 DAT) | During test (28 DAT before conversion to anaerobic conditions) | End of test (120 DAT) |
| Substrate induced respiration (SIR) method | Fumigation method (microbial biomass) | Substrate induced respiration (SIR) method | Fumigation method (microbial biomass) | Fumigation method (microbial biomass) |
| mg Cmic/kg dry mass | Cmic/Corg (%) | mg C /g | mg Cmic/kg dry mass | Cmic/Corg (%) | mg C /g | mg C /g | |
| untreated | 172.7 | 1.7 | 0.9 | 167.1 | 1.6 | 1.0 | 1.8 |
| treated with solvent | NS | NS | NS | 386.3 | 3.7 | 0.9 | 1.6 |
NS: not sampled
Radioactive residues in soil extracts
| Identification of Radioactive Residues in organic extracts 1-3: Soil refesol 02-A |
| 14C-Residues in soil 02-A | Rep | HPLC retention time | % AR per DAT |
| 0 | 1 | 3 | 7 | 14 | 21 | 28 | 59 | 91 | 120 | |||
| dead time | Mean | 0.0 - 0.2 |
ND |
ND |
32.4 |
ND |
ND |
ND |
5.0 |
ND |
ND |
ND |
Parent |
Mean |
23.2 |
100.0 |
101.0 |
20.7 |
98.9 |
94.6 |
36.4 |
ND |
92.9 |
92.1 |
89.5 |
BT |
Mean |
5.1 - 5.4 |
ND |
ND |
18.1 |
ND |
ND |
59.8 |
88.4 |
ND |
ND |
ND |
MBT |
Mean |
6.1 - 6.5 |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
Unidentified metabolite 3 |
Mean |
11.7 |
ND |
ND |
15.7 |
ND |
3.1 |
ND |
ND |
ND |
ND |
ND |
MBTS |
Mean |
19.5 |
ND |
ND |
4.6 |
ND |
1.3 |
ND |
2.3 |
ND |
ND |
ND |
Unidentified metabolite 2 |
Mean |
3.6 |
ND |
ND |
9.3 |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
Unidentified metabolite 1 |
Mean |
19.1 |
ND |
ND |
ND |
ND |
0.7 |
ND |
1.4 |
ND |
ND |
ND |
ND = not detected,
LOD = 2.64 %
AR = 26.36 µg/kg
Residues in aqueous extract 4
| Identification of Radioactive Residues in Extracts refesol 02-A |
| Soil 02-A | Retention time / min | replicate | % AR per DAT, 120 DAT |
| Parent | Mean | 0.9 | |
| DCHA | Mean | 0.9 | |
| MBT | Mean | 0.3 | |
| MeSBT | Mean | 0.2 | |
| BTSO3H | Mean | 3.6 |
Microbial biomass of soil samples
Microbial biomass determined by means of substrate induced respiration (SIR) at different stages of the test
| Soil | Soil sample | start of test (0 DAT) | During test (28 DAT) | End of test (142/143 DAT) |
| mg C(mic)/kg dry mass | C(mic)/C(org) (%) | mg C(mic)/kg dry mass | C(mic)/C(org) (%) | mg C(mic)/kg dry mass | C(mic)/C(org) (%) |
| 02-A, soil no. #2 | untreated | 233.3 | 2.2 | 143.0 | 1.4 | 207.6* | 2.0 |
| 02-A, soil no. #2 | treated with solvent | NS | NS | 434.9 | 4.2 | 100.3** | 1.0 |
| 03-G, soil no. #3 | Untreated | 569.0 | 1.8 | 483.3 | 1.5 | 426.0* | 1.3 |
| 03-G, soil no. #3 | Treated with solvent | NS | NS | 960.7 | 3.0 | 252.2** | 0.8 |
| 04-A, soil no. #4 | Untreated | 247.9 | 0.9 | 170.2 | 0.6 | 173.3* | 0.7 |
| 04-A, soil no. #4 | Treated with solvent | NS | NS | 479.5 | 1.8 | 166. ** | 0.6 |
| LUFA 2.4, soil no. #1 | Untreated | 432.1 | 2.4 | 276.8 | 1.5 | 412.4* | 2.2 |
| LUFA 2.4, soil no. #1 | Treated with solvent | NS | NS | 674.2 | 3.5 | 467.5** | 2.5 |
* sampled at 142 DAT
** sampled at 143 DAT
NS: not sampled
Remark: untreated and solvent control samples were analyzed on two following days. The time delay between the last sampling at 120 DAT and analysis of the biomass control samples at stage “end of test (142/143 DAT) was caused by the decision of the study monitor against further sampling of spare samples at 150 DAT and to end the incubation at this time point.
Radioactive residues in soil extracts
| Identification of Radioactive Residues in Extracts: Soil LUFA 2.4, soil no. #1 |
| 14C-Residues | Rep | HPLC retention time | % AR per DAT |
| 0 | 1 | 3 | 7 | 14 | 21 | 28 | 59 | 91 | 120 |
| dead time | Mean | 0 -2 | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND |
| Parent | Mean | 22.8 | 97.4 | 97.8 | 98.5 | 66.1* | 95.3 | 94.9 | 75.5* | 89.9 | 88.1 | 85.1 |
| MBT | Mean | 6.1 - 6.5 | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND |
| MBTS | Mean | 19.5 | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND |
| BT | Mean | 5.1-5.4 | ND | ND | ND | 19.3 | ND | ND | 19.7 | ND | ND | ND |
| Unidentified metabolite 1 | Mean | 19.1 | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND |
* data points not used for KinGUI rate calculation
| Identification of Radioactive Residues in Extracts: Soil refesol 02-A, soil no. #2 |
| 14C-Residues | Rep | HPLC retention time | % AR per DAT |
| 0 | 1 | 3 | 7 | 14 | 21 | 28 | 59 | 91 | 120 | |||
| dead time | Mean | 0 - 2 | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND |
| Parent | Mean | 22.8 | 99.6 | 98.0 | 98.1 | 96.3 | 94.9 | 93.7 | 13.1* | 89.9 | 86.5 | 80.8 |
| MBT | Mean | 6.1 - 6.5 | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND |
| MBTS | Mean | 19.5 | ND | ND | ND | ND | 3.3 | ND | ND | ND | ND | ND |
| BT | Mean | 5.1-5.4 | ND | ND | ND | ND | ND | ND | 81.3 | ND | ND | ND |
| Unidentified metabolite 1 | Mean | 19.1 | ND | ND | ND | ND | 2.4 | ND | ND | ND | ND | ND |
* data point not used for KinGUI rate calculation
| Identification of Radioactive Residues in Extracts: Soil refesol 03-G, soil no. #3 |
| 14C-Residues | Rep | HPLC retention time | % AR per DAT |
| 0 | 1 | 3 | 7 | 14 | 21 | 28 | 59 | 91 | 120 | |||
| dead time | Mean | 0 - 2 | ND | ND | ND | ND | ND | 1.6 | ND | ND | ND | ND |
| Parent | Mean | 22.8 | 100.4 | 99.0 | 99.5 | 91.7 | 97.5 | 94.8 | 95.9 | 83.3 | 83.3 | 74.4 |
| MBT | Mean | 6.1 - 6.5 | ND | ND | ND | ND | ND | 3.9 | ND | ND | ND | ND |
| MBTS | Mean | 19.5 | ND | ND | ND | ND | ND | ND | ND | 3.9 | ND | ND |
| BT | Mean | 5.1-5.4 | ND | ND | ND | ND | ND | 3.7 | ND | ND | ND | ND |
| Unidentified metabolite 1 | Mean | 19.1 | ND | ND | ND | ND | ND | 4.7 | ND | ND | ND | ND |
| Identification of Radioactive Residues in Extracts: Soil 04-A, soil no. #4 |
| 14C-Residues | Rep | HPLC retention time | % AR per DAT |
| 0 | 1 | 3 | 7 | 14 | 21 | 28 | 59 | 91 | 120 | |||
| dead time | Mean | 0 - 2 | ND | ND | ND | ND | ND | ND | 3.7 | ND | ND | ND |
| Parent | Mean | 22.8 | 101.3 | 97.5 | 99.7 | 66.0* | 51.7* | 61.4* | 63.6* | 92.7 | 88.2 | 85.1 |
| MBT | Mean | 6.1 - 6.5 | ND | ND | ND | ND | ND | ND | 7.0 | ND | ND | ND |
| MBTS | Mean | 19.5 | ND | ND | ND | ND | ND | ND | 3.0 | ND | ND | ND |
| BT | Mean | 5.1-5.4 | ND | ND | ND | 23.9 | 34.0 | 17.5 | 14.7 | ND | ND | ND |
| Unidentified metabolite 1 | Mean | 19.1 | ND | 3.3 | ND | 1.4 | ND | ND | 6.0 | ND | ND | ND |
ND = not detected, LOD = 2.64 % AR = 26.36 µg/kg
* data points not used for KinGUI rate calculation
Residues in aqueous extract 4
Only for samples 120 DAT for all soils and 91 DAT of soil 03-G amounts of radioactivity > 5 % AR were found in the aqueous extraction. Therefore only these extracts were subjected to chemical analysis by LC-HR-MS. The measured concentrations were converted into parent equivalents. In contrast to the results obtained for organic extracts the main residues the parent is not the dominating fraction. Instead, the formation of significant amounts of transformation products BTSO3H and DCHA could be identified by LC-HR-MS analysis. However, with exception of the 120 DAT sample of soil 03-G, all fractions are below 5 % AR.
Identification of Radioactive Residues in Extracts refesol 02-A, soil no. #2
| Soil 02-A | Retention time / min | replicate | % AR per DAT, 91 DAT | % AR per DAT, 120 DAT |
| Parent | Mean | NS | 0.7 | |
| DCHA | Mean | NS | 2.6 | |
| MBT | Mean | NS | 0.3 | |
| BTon | Mean | NS | 0.0 | |
| MeSBT | Mean | NS | 0.2 | |
| BTSO3H | Mean | NS | 3.6 |
NS: not analysed, radioactivity in extract 4 below 5%AR
Identification of Radioactive Residues in Extracts refesol 03-G, soil no. #3
| Soil 03-G | Retention time / min | replicate | % AR per DAT, 91 DAT | % AR per DAT, 120 DAT |
| Parent | Mean | 0.7 | 0.6 | |
| DCHA | Mean | 1.5 | 0.7 | |
| MBT | Mean | 0.3 | 0.3 | |
| BTon | Mean | 0.0 | 1.2 | |
| MeSBT | Mean | 0.2 | 0.2 | |
| BTSO3H | Mean | 3.2 | 5.0 |
Identification of Radioactive Residues in Extracts refesol 04-A, soil no. #4
| Soil 04-A | Retention time / min | replicate | % AR per DAT, 91 DAT | % AR per DAT, 120 DAT |
| Parent | Mean | NS | 0.2 | |
| DCHA | Mean | NS | 0.2 | |
| MBT | Mean | NS | 0.2 | |
| BTon | Mean | NS | 0.0 | |
| MeSBT | Mean | NS | 0.2 | |
| BTSO3H | Mean | NS | 3.7 |
NS: not analysed, radioactivity in extract 4 below 5%AR
Identification of Radioactive Residues in Extracts soil LUFA 2.4, soil no. #1
| LUFA 2.4 | Retention time / min | replicate | % AR per DAT, 91 DAT | % AR per DAT, 120 DAT |
| Parent | Mean | NS | 1.5 | |
| DCHA | Mean | NS | 0.5 | |
| MBT | Mean | NS | 0.4 | |
| BTon | Mean | NS | 0.9 | |
| MeSBT | Mean | NS | 0.2 | |
| BTSO3H | Mean | NS | 2.3 |
NS: not analysed, radioactivity in extract 4 below 5%AR
Parent fractions in soil extracts 1-4
The combined fractions of the parent from extracts 1-4 are summarized. Finally, the data with artifact-affected results were omitted from further consideration and not used for the determination of the half-life. These data points that were not taken into account for the rate-calculations are highlighted.
Amount of parent DCBS in organic and aqueous soil extracts
Refesol 02-A, soil no. #2 |
Replicate |
% AR per DAT |
|
|
0 |
1 |
3 |
7 |
14 |
21 |
28 |
59 |
91 |
120 |
Parent |
Mean |
99.6 |
98.0 |
98.1 |
96.3 |
94.9 |
93.7 |
13.1* |
89.9 |
86.5 |
81.5 |
| Refesol 03-G, soil no. #3 |
Replicate |
% AR per DAT |
|
0 |
1 |
3 |
7 |
14 |
21 |
28 |
59 |
91 |
120 |
|
Parent |
Mean |
100.4 |
99.0 |
99.5 |
91.7 |
97.5 |
94.8 |
95.9 |
83.3 |
83.9 |
75.0 |
| Refesol 04-A, soil no. #4 |
Replicate |
% AR per DAT |
|
0 |
1 |
3 |
7 |
14 |
21 |
28 |
59 |
91 |
120 |
|
Parent |
Mean |
101.3 |
97.5 |
99.7 |
66.0* |
51.7* |
61.4* |
63.6* |
92.7 |
88.2 |
85.3 |
| LUFA 2.4, soil no. #1 |
Replicate |
% AR per DAT |
|
0 |
1 |
3 |
7 |
14 |
21 |
28 |
59 |
91 |
120 |
|
Parent |
Mean |
97.4 |
97.8 |
98.5 |
66.1* |
95.3 |
94.9 |
75.5* |
89.9 |
88.1 |
86.7 |
* not used for KinGUI-calculations
Description of key information
In a study according to OECD 307, 14C-labelled DCBS was used and investigated at 12°C. The aerobic test performed with 4 different test soils showed only limited degradation within 120 days of study period. Final values showed DCBS to be in the range of 75 to 87% of its original concentration. Most of the degradation was caused by NER formation. Mineralisation was found to be not relevant. The half-lives were in the range of 315 to 615 days. The anaerobic study showed similar findings with a half-life of 783 days at 12°C.
Key value for chemical safety assessment
- Half-life in soil:
- 615 d
- at the temperature of:
- 12 °C
Additional information
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