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Environmental fate & pathways

Biodegradation in soil

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Endpoint:
biodegradation in soil: simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)
GLP compliance:
yes
Test type:
laboratory
Radiolabelling:
yes
Oxygen conditions:
aerobic
Details on soil characteristics:
Soil 1 Soil 2 Soil 3 Soil 4
Sampling Depth 12-20 cm ~20 cm ~20 cm 0-30 cm
Texture Sand-loamy sand, sandy loam, clay, silt loam
Particle size (% w/w)
Clay (<2 µm) 6.83 % 10.37 % 40.38 % 15.61 %
Silt (50-2µm) 7.12 % 29.47 % 32.95 % 72.26 %
Sand (2000-50 µm) 80.05 % 60.16 % 26.67 % 12.13 %
pH (0.01 M CaCl2) 3.86 5.78 7.19 6.51
Organic carbon (%) 1.81 0.65 1.99 1.17
Organic matter (%)
= 1.74 x % organic carbon 3.12 1.12 3.43 2.02
CEC (meq/100 g Soil) 8.91 5.83 24.93 13.34
Carbonate as CaCO3 (%) 0.20 0.20 1.2 0.20
Nitrogen content 0.11 0.08 0.17 0.13
Moisture at pF 2.0 (w/w %) 13.94 22.78 37.21 32.04
Moisture at pF 2.5 (w/w %) 11.86 14.70 31.54 25.41
Biomass, start of study
(mg microbial carbon per 100 g soil) 17.5 19.4 35.4 34.5
Biomass, end of study
(mg microbial carbon per 100 g soil) 17.5 17.3 46.0 18.5
Soil No.:
#1
Temp.:
12°C
Humidity:
13.94 at pF 2.0 (w/w%)
Microbial biomass:
Biomass, start of study 17.5 mg microbial carbon per 100 g soil; Biomass, end of study 17.5 mg microbial carbon per 100 g soil
Soil No.:
#2
Temp.:
12°C
Humidity:
22.78 at pF 2.0 (w/w%)
Microbial biomass:
Biomass, start of study 19.4 mg microbial carbon per 100 g soil; Biomass, end of study 17.3 mg microbial carbon per 100 g soil
Soil No.:
#3
Temp.:
12°C
Humidity:
37.21 at pF 2.0 (w/w%)
Microbial biomass:
Biomass, start of study 35.4 mg microbial carbon per 100 g soil; Biomass, end of study 46.0 mg microbial carbon per 100 g soil
Soil No.:
#4
Temp.:
12°C
Humidity:
32.04 at pF 2.0 (w/w%)
Microbial biomass:
Biomass, start of study 34.5 mg microbial carbon per 100 g soil; Biomass, end of study 18.5 mg microbial carbon per 100 g soil
Details on experimental conditions:
Experimental Set-up
Fresh soil samples (100 g dry weight) in 1 liter metabolism glass flasks (≈ 11 cm Ø) were treated with the test item. Treated flasks were connected to a flow-through system for ventilation with moistened air and trapping of organic volatiles and CO2. The flow rate was sufficient for bubble formation in the trapping solutions. The radioactive content in the test system was analyzed in duplicate flasks at time points up to 56 days.

Application of the Test Item

Target Rate:
The target rate was 0.25 mg a.i. per kg dry soil (= 22.5 μg/flask), corresponding to an application rate of 250 g/ha, assuming an even distribution in the top 10 cm soil layer and a soil bulk density of 1.0 g/cm3.

Application Solutions and Applied Amounts:
14C-7PPD was dissolved in acetonitrile at a concentration adjusted to 21.8 × 106 dpm/300 μL. This concentration was derived from LSC measurements of 300 μL application aliquots taken before and after the application of the test item to the test vessels. Application aliquots were spread drop wise on the soil surface resulting in an applied amount of 21.8 × 106 dpm/flask or 25.39 μg parent equivalents/flask at the specific radioactivity of 14.32 MBq/mg.
Control flasks were treated with the same volume of solvent without test item. Treated flasks were swirled to mix the test item into the soil.

Radiochemical Purity and Stability of 14C-7PPD:
The radiochemical purity of 14C-7PPD dissolved in acetonitrile (application solution) was determined by HPLC before and after treatment of the soil.
The stability of 14C-7PPD in aqueous environments, notably in acetonitrile/water 8/2 and 1/1 (v/v), was determined in preliminary investigations in order to verify in advance the suitability of the acetonitrile/water mix used for HPLC analysis of study sampels. The stability of 14C-7PPD was also investigated in acetonitrile/0.1 N HCl 1/1 (v/v) after 4 h exposure to 100 °C to determined potential effects of the acidic reflux conditions.

Incubation:
Treated flasks were kept in a dark room at 12 ± 2 °C. Soil moisture was maintained at pF 2.0 - 2.5 by adding water to compensate for losses by evaporation.

Sampling and Work-up:
Duplicate flasks were sampled immediately after treatment (time 0) and after 1, 3, 7, 15, 28 and 56 days for analysis of the extractable, non-extractable and volatile radioactivity.
The study was not prolonged beyond day 56 since DT50 values for 7PPD were < 2 day, all metabolites were minor, individually < 2% AR by day 7, and mineralization reached a plateau beginning day 15. Storage of samples prior to analysis was avoided where possible. Where storage was required, samples were retained short time (overnight) at 4 °C or longer at -20 °C.

Extractable Radioactivity:
Entire soil samples were extracted repeatedly in acetone/dichloromethane 1/3 (v/v) for 30 min on a flatbed shaker (≈ 250 rpm) at ambient temperature until less than 5% of the applied radioactivity was released in a single extraction. About 150 mL solvent mix was used for the first extraction and 100 mL for subsequent extractions. Extract and soil were separated by centrifugation at 3000 rpm for 10 minutes. Since phase separations were noticed in some extracts, presumably due to residual water in the soil forming an aqueous phase above the organic solvent), 20 mL methanol was added to individual extracts (≈ 100 - 140 mL extract depending on the extraction, before adding methanol) to mix the phases. The radioactivity in the extracts was determined by LSC. Extracts exceeding 2% AR were pooled. Sub-samples of pooled extracts were dried under a stream of nitrogen and re-solubilised in acetonitrile/water 3/7 (v/v) for HPLC analysis.
Following ambient extractions, Soxhlet extraction (4 hr) using 250 mL acetone/dichloromethane 1/3 (v/v) was performed for samples of early and late intervals. Soxhlet extractions were not performed for intermediate intervals given the low levels extracted at the adjacent intervals. The total extractable radioactivity was calculated as the sum of the radioactivity released by ambient and Soxhlet extractions.

Non-Extractable Radioactivity:
Following ambient and Soxhlet extractions, the extracted soil was air-dried and homogenized using a disk swing mill. Sub-samples of the homogenized soil were combusted to determine the non-extractable radioactivity. Additionally, samples from day 7 onwards were submitted to acidic reflux extraction at 100 °C in acetonitrile/0.1 N HCl 50/50(v/v) for 4 hours. The effect of the reflux conditions on 7PPD was investigated by chromatographic analysis of 14C-7PPD solutions amended with 0.1 N HCl and exposed for 4 hr to 100 °C.
Reflux extracts of day 56 were analyzed chromatographically. For that purpose, the extracts were partitioned by salt-induced phase separation. The organic and aqueous phases were separated and measured by LSC. The organic phase was dried under a stream of nitrogen and solubilised in acetonitrile/water 3/7 (v/v) for HPLC analysis.
Following reflux extraction, samples of day 56 were submitted to organic matter fractionation according to a procedure published by Stevenson (1982)

Volatile Radioactivity:
The radioactivity in the adsorption traps was monitored by LSC at each sampling interval and between intervals depending on accumulation in the trapping solutions. Traps were exchanged several times during the study.
Soil No.:
#1
DT50:
0.03 d
Type:
other: First Order Multi Compartment kinetics
Remarks on result:
other: DT90 = 1.1 d
Soil No.:
#2
DT50:
1.4 d
Type:
other: First Order Multi Compartment kinetics
Remarks on result:
other: DT90 = 12.4 d
Soil No.:
#3
DT50:
1.4 d
Type:
other: First Order Multi Compartment kinetics
Remarks on result:
other: DT90 = 10.5 d
Soil No.:
#4
DT50:
1.9 d
Type:
other: First Order Multi Compartment kinetics
Remarks on result:
other: DT90 = 9.22 d
Transformation products:
yes
No.:
#1
Details on transformation products:
The metabolism of 14C-7PPD in aerobic soils is proposed to proceed via formation of minor transient metabolites and mineralization. The main portion of the residue is binding to the soil matrix and appears to become unavailable for further degradation/mineralization.
Conclusions:
The metabolism of 14C-7PPD in aerobic soils is proposed to proceed via formation of minor transient metabolites and mineralization. The main portion of the residue is binding to the soil matrix and appears to become unavailable for further degradation/mineralization. DT50 values of the parent, derived from FOMC kinetics, were 1.4 – 1.9 days for Soils II-IV and < 1 day for soil I.
Executive summary:

The metabolism and decline of 14C-7PPD was investigated in fresh soil samples (100 g dry weight) in metabolism flasks. The soil was treated with 14C-7PPD at a target rate of 0.25 mg per kg dry soil, corresponding to an application rate of 250 g/ha, assuming even distribution in the top 10 cm soil layer and a soil bulk density of 1.0 g/cm3. Treated flasks were placed in a dark room at 12 ± 2 °C and connected to a flow-through system for ventilation with moistened air and trapping of organic volatiles and CO2.

Duplicate flasks were sampled at time 0 and after 1, 3, 7, 15, 28 and 56 days to determine amounts and nature of the extractable, non-extractable and volatile radioactivity. This time frame was sufficient to establish the metabolism of the test item and transformation products.

Results are discussed as means of duplicates and in percent of the applied radioactivity [%AR].

The mass balance (sum of extractable, non-extractable and volatile radioactivity) was consistently > 90% for all intervals, soils and replicates.

The extractable radioactivity was defined as the sum of the ambient and Soxhlet extractions. The radioactivity released by acidic reflux extraction was considered as non-extractable due to the residue altering effect of the reflux conditions.

Since extraction procedures applied after ambient and Soxhlet extractions did not release significant amounts of radioactivity, except for the residue altering reflux extractions, it was concluded that the soils were exhaustively extracted and that the unextracted residue was bound and unavailable.

The extractable radioactivity decreased from 69.0%, 77.1%, 66.8% and 37.2% AR at time 0 for soils I-IV, respectively, to 4.5% - 9.1% AR by the end of the study. HPLC was used for analysis of soil extracts and quantification of parent and metabolites. The parent decreased from 31.6% - 68.4% AR at time 0 to below half-live values by day 2 in soils I-IV. All metabolic fractions were minor and transient with levels < 2 % AR by day 15.

The non-extractable radioactivity increased from 26.9 - 59.8% AR to 76.4 – 84.8% by the end of the study soils I-IV. Acidic reflux extraction following ambient and Soxhlet extraction released 8.9% – 13.0% AR. Given the residue-altering effect of the acidic reflux conditions, the extracted residue was considered part of the non-extractable residue. Nevertheless, reflux extracts of day 56 samples were analyzed by HPLC and found to consist of multiple fractions with retention times below the retention of the parent and for the most part also below the retention of the reference 4-hydroxy-diphenylamine (double peak near 12 min). The largest fraction seen in reflux extracts amounted to 5.2% AR.

The non-extractable residue (NER) released by reflux extraction was considered as part of the NER-type 1, defined as non extractable residue (NER) that is sorbed or entrapped within the soil organic matter. The residue remaining in the soil after refluxing was considered as bound residue, consisting of NER-type 2 (covalently bound) and NER-type 3 (biogenic NER).

Organic matter fractionation performed after reflux extraction of day 56 samples indicated that 7.8% – 16.4% AR was bound to fulvic acids, 9.8% – 30.5% AR to the humic acids and 23.3% – 57.3% AR to insoluble humins.

Of the volatile radioactivity, organic volatiles were consistently below 0.1% AR. Radioactive carbon dioxide reached 4.1% – 8.9% AR for soils I-IV by the end of the study with marginal change after day 15. The marginal mineralization towards the end of the study was indicative that the residue in the soil was no longer bioavailable.

Endpoint:
biodegradation in soil: simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)
GLP compliance:
yes
Test type:
laboratory
Radiolabelling:
yes
Oxygen conditions:
anaerobic
Details on soil characteristics:
Sampling Depth ~20 cm
Texture sandy loam
Clay (<2 µm) 10.37 % (w/w)
Silt (50-2µm) 29.47 % (w/w)
Sand (2000-50 µm) 60.16 % (w/w)
pH (0.01 M CaCl2) 5.78
Organic carbon (%) 0.65
Organic matter (%)
= 1.74 x % organic carbon 1.12
CEC (meq/100 g Soil) 10.9
Carbonate as CaCO3 (%) 0.20
Nitrogen content 0.08
Moisture at pF 2.0 (w/w %) 22.78
Moisture at pF 2.5 (w/w %) 14.70
Biomass, start of study
(mg microbial carbon per 100 g soil) 20.4
Biomass, end of study
(mg microbial carbon per 100 g soil) 162.9
Soil No.:
#1
Duration:
120 d
Soil No.:
#1
Initial conc.:
0.225 mg/kg soil d.w.
Based on:
test mat.
Soil No.:
#1
Temp.:
12°C
Humidity:
Moisture at pF 2.0 (w/w %)
Microbial biomass:
Biomass, start of study 20.4 mg microbial carbon per 100 g soil; Biomass, end of study 162.9 mg microbial carbon per 100 g soil
Details on experimental conditions:
Experimental Set-up:
Fresh soil samples (100 g dry weight) were placed into 500 mL metabolism glass flasks (≈ 5.5 cm Ø) and treated with the test item. Treated flasks were placed at 12 ± 2 °C in the dark and connected to a flow-through system for trapping organic volatiles and CO2. The flow consisted of moistened air during the aerobic phase and nitrogen during the anaerobic phase. The flow rate was sufficient for bubble formation in the trapping solutions. Anaerobic conditions were induced after 4 h aerobic incubation by flooding the soil with 150 mL deoxygenated water (submersing soil by ≈ 3.0 cm water). The radioactive content in the test system was analyzed in duplicate flasks at time points up to 120 days.

Application of the Test Item
Target Rate:
The target rate was 225 μg test item per kg dry soil (= 22.5 μg/flask), corresponding to an application rate of 225 g/ha, assuming an even distribution in the top 10 cm soil layer and a soil bulk density of 1.0 g/cm3.

Application Solution and Applied Amount:
14C-7PPD was dissolved in acetonitrile. The concentration was adjusted to 23.2 × 106 dpm/300 μL (determined in application aliquots before and after the application), resulting in an applied amount of 23.2 × 106 dpm/flask or 27.0 μg parent equivalents/flask at the specific radioactivity of 14.32 MBq/mg.
The application aliquots were spread drop wise on the soil surface. Control flasks were treated with the same volume of solvent without test item. Treated flasks were swirled to mix the test item into the soil.

Radiochemical Purity and Stability:
The radiochemical purity of the test item in the application solution (acetonitrile) was determined by HPLC before and after treatment of the soil.
The stability of the test item in acetonitrile/water mixtures (8/2 and 1/1; v/v) was verified in Envigo study (D93897) in order to confirm stability in the HPLC eluent.

Physicochemical Parameters:
Oxygen concentration, pH and redox potential were measured at each sampling interval prior to removing the water for sample work up. The redox potential was measured using a platinum electrode (SenTix® ORP) and measured values were converted to the standard hydrogen electrode by adding 211 mV.

Sampling and Work-up:
Duplicate flasks were sampled immediately after treatment (time 0) and after 4 h aerobic incubation as well as after 1, 3, 14, 28, 56 and 120 days anaerobic incubation. Treated flasks were placed at 12 ± 2 °C in the dark and connected to a flow-through system for trapping organic volatiles and CO2.
Sample storage prior to analysis was avoided where possible. Where storage was required, samples were retained short time (overnight) at 4 °C or longer at -20 °C.

Water-soluble Radioactivity (Flood Water):
At each sampling interval of the anaerobic phase, the flood water was removed using a glass pipette and the radioactive content was determined by LSC. Residual water left in the soil was considered as part of the soil for subsequent processing.
Due to the low levels of reactivity in the flood water, the water phase was analyzed only for the sample with the highest level of radioactivity, namely day 3 replicate B (4.1% AR) (AR = applied radioactivity). For that sample, the flood water was partitioned twice with dichloromethane. The two partition steps were sufficient to recover the expected radioactivity in the pool of both organic phases. The pool was dried under a stream of nitrogen and reconstituted in the appropriate solvent for HPLC analysis.

Extractable Radioactivity:
Entire soil samples were extracted repeatedly in acetone and/or acetone/dichloromethane 1/3 (v/v) for 30 min on a flatbed shaker (≈ 250 rpm) at ambient temperature until less than 5% of the applied radioactivity was released in a single extraction.
About 150 mL organic solvent was typically used for the first extraction and 100 mL for subsequent extractions. Extract and soil were separated by centrifugation at 3000 rpm for 10 minutes.
Up to three consecutive extractions were performed in acetone/dichloromethane 1/3 for samples time 0, 4 h and day 1. Due to phase separation noticed in the first extract (presumably due to residual water in the soil forming an aqueous phase above the organic solvent), 20 mL (time 0 and 4 h) or 40 mL (day 1) methanol was added to the first extract (e.g. 120 mL extract plus 20 mL methanol) to achieve phase miscibility. The methanol amendment was not required for the second and subsequent extractions (presumably due to the removal of residual water in the first extraction).
The extractions in acetone/dichloromethane 1/3 were followed by an extraction in pure acetone for early samples (4 h and 1 day) in order to verify the exhaustively of preceding extractions in the less polar solvent mix acetone/dichloromethane. Since the final extraction in acetone released only small amounts of radioactivity (0.7% AR for 4 h and 2.6% AR for 4 h), it was discontinued.
To avoid the phase separation in the initial extraction, samples of intervals ≥ 1 day were first extracted in acetone, followed by acetone/dichloromethane 1/3.
The radioactivity in individual extracts was determined by LSC. Room temperature extracts exceeding 2% AR were pooled for further work-up. Sub-samples of pooled extracts were dried under a stream of nitrogen or by rotary evaporation at 30 - 35 °C and re-solubilized in acetonitrile/water 3/7 (v/v) for HPLC analysis. Recoveries of > 90% were expected for submitting concentrated samples to HPLC.
Following the ambient extractions, Soxhlet extraction using about 250 mL acetone/dichloromethane 1/3 (v/v) per soil sample was performed for time 0 and day 28. Soxhlet extraction was not performed for other intervals due to the small amount of radioactivity released by this extraction type.
Acidic reflux extractions (performed on day 28 and day 56 samples) are described under Nonextractable Radioactivity due to their residue altering effect observed in Envigo Study D93897).

Non-Extractable Radioactivity:
The extracted soil was air-dried and homogenized using a soil grinder. Sub-samples of the homogenized soil were combusted to determine the non-extractable radioactivity. Additionally, selected samples (day 28 and day 56) were submitted to Reflux extraction for 4 h at 100 °C in 0.1 N HCl/acetonitrile (1/1; v/v). Following reflux extraction, day 56 samples were subjected to organic matter fractionations according to a procedure published by Stevenson (1982).

Volatile Radioactivity:
The radioactivity in the trapping solutions was determined by LSC at the time of sampling and between sampling intervals depending on accumulation in the trapping solutions. Traps were exchanged during the study.
Soil No.:
#1
% Degr.:
68.4
Parameter:
test mat. analysis
Sampling time:
0 h
Soil No.:
#1
% Degr.:
36.9
Parameter:
test mat. analysis
Sampling time:
4 h
Soil No.:
#1
% Degr.:
23.2
Parameter:
test mat. analysis
Sampling time:
1 d
Soil No.:
#1
% Degr.:
13.1
Parameter:
test mat. analysis
Sampling time:
3 d
Soil No.:
#1
% Degr.:
13.1
Parameter:
test mat. analysis
Sampling time:
7 d
Soil No.:
#1
% Degr.:
17.7
Parameter:
test mat. analysis
Sampling time:
14 d
Soil No.:
#1
% Degr.:
19.4
Parameter:
test mat. analysis
Sampling time:
28 d
Soil No.:
#1
% Degr.:
13.5
Parameter:
test mat. analysis
Sampling time:
56 d
Soil No.:
#1
% Degr.:
17
Parameter:
test mat. analysis
Sampling time:
120 d
Soil No.:
#1
DT50:
1.5 d
Type:
other: HS kinetic model
Remarks on result:
other: DT90 >1000 d. The short DT50 of the parent is attributed to aerobic degradation.
Transformation products:
yes
No.:
#1
Details on transformation products:
The metabolism of 7PPD in anaerobic soils is proposed to proceed via formation of 1-N-(5-methyl-hexan-2-yl)-4-N-phenylcyclohexa-2,5-dione-4,4-diimine (7QD, mixture of I cis-/trans isomers), several minor metabolites and modest mineralization. 7PPD declines also by residue binding to the soil matrix.
Stated degradation products were:
Hydroquinone, p-Benzoquinone, 4-nitroaniline, Aniline, 4-Hydroxy-diphenylamine and 1-N-(5-methyl-hexan-2-yl)-4-Nphenylcyclohexa-2,5-dione-1,4-diimine also known as 7QDI.
Evaporation of parent compound:
no
Volatile metabolites:
no
Residues:
yes
Details on results:
The decline of 7PPD was best described by the HS kinetic model.
The rapid decline of 7PPD in the initial aerobic phase was likely to extend into the anaerobic phase of the test system, that the transition to anaerobic conditions by soil flooding is progressive. Therefore, the short DT50 of the parent (1.5 days) in the flooded test system was attributed to aerobic degradation. Following that rapid initial decline, the parent dissipated slowly, indicating a persistent nature under steady anaerobic conditions.

Metabolite 1-N-(5-methyl-hexan-2-yl)-4-N-phenylcyclohexa-2,5-dione-4,4-diimine also known as 7QDI. This metabolite consists of cis and a trans isomer.

7QDI Isomer 1: DT50 = 57.9 d; DT90 = 192 d

7QDI Isomer 2: DT50 = 79.7 d; DT90 = 265 d

7QDI Isomer 1+2: DT50 = 66.9 d; DT90 = 222 d

Decline was described by SFO (Single First Order) model.

Conclusions:
The rapid decline of 7PPD in the initial aerobic phase was likely to extend into the anaerobic phase of the test system given, that the transition to anaerobic conditions by soil flooding is progressive. Therefore, the short DT50 of 7PPD (1.5 days) in the flooded test system was attributed to aerobic degradation. Following that rapid initial decline, the parent dissipated slowly, indicating persistent nature under steady anaerobic conditions.
Executive summary:

The metabolism and decline of 14C-7PPD was investigated under anaerobic conditions in fresh soil samples (100 g dry weight) contained in metabolism flasks. The soil was treated at a target rate of 0.225 mg per kg dry soil (= 22.5 μg/flask), corresponding to an application rate of 225 g/ha, assuming an even distribution in the top 10 cm soil layer and a soil bulk density of 1.0 g/cm3. Treated flasks were placed at 12 ± 2 °C in the dark and connected to a flow-through system for trapping organic volatiles and CO2. The flow consisted of moistened air during the aerobic phase and nitrogen during the anaerobic phase. The flow rate was sufficient for bubble formation in the trapping solutions. Anaerobic conditions were induced after 4 h aerobic incubation by flooding the soil with 150 mL deoxygenated water (submersing soil by ≈ 3.0 cm water).

The radioactive content in the test system was analyzed in duplicate flasks at multiple time points during the aerobic and anaerobic phase. Results are discussed in percent of the applied radioactivity [%AR].

The mass balance was consistently > 90% AR for all sampling intervals.

The radioactivity in the flood water increased from 2.8% AR on day 1 of the anaerobic phase to 3.5% AR on day 3 and decreased thereafter to 1.9% AR by day 120.

The extractable radioactivity decreased from 77.0% AR at time 0 to 49.5% AR by the end of the aerobic phase (4 h) and amounted to 50.0 – 59.4% AR during the anaerobic phase.

The non-extractable radioactivity increased from 26.9% AR at time 0 to 45.6% AR by the end of the aerobic phase (4 h) and amounted to 33.3 – 43.6% AR during the anaerobic phase.

Radioactive carbon dioxide formation was modest, reaching up to 0.7% AR by the end of the study while organic volatiles were consistently < 0.1 %.

Acidic reflux extractions at 100 °C, following ambient extractions, released 4.6% AR on day 28 and 5.9% AR on day 56. The acidic reflux conditions were found to be residue altering (Envigo Study D93897). The residue released by reflux extraction was considered as part of the NERtype 1, defined as non extractable residue (NER) that is sorbed or entrapped within the soil organic matter. The residue remaining in the soil after refluxing was considered as bound residue, consisting of NER-type 2 (covalently bound) and NER-type 3 (biogenic NER).

Organic matter fractionation performed after reflux extraction of day 56 samples indicated that 7.1% AR was bound to fulvic acids, 12.9% AR to humic acids and 17.7% AR to insoluble humins.

Due to the small amount of radioactivity in the flood water (maximal 4.1% AR on day 3) the metabolism and decline of 14C-7PPD was derived from the chromatographic analysis of the soil extracts.

The parent decreased from 68.4% AR at time 0 to 36.9% AR by the end of the aerobic phase. Thereafter, the parent decreased from 23.2% AR on day 1 of the anaerobic phase to levels fluctuating between 13.1% and 19.4% AR until day 120.

Metabolite M1 "1-N-(5-methyl-hexan-2-yl)-4-N-phenylcyclohexa-2,5-dione-4,4-diimine" also known as 7QDI appeared as a single fraction in the aerobic phase, increasing from 2.8% AR at time 0 to 5.7% AR by 4 h. In the subsequent anaerobic phase, M1 could be integrated as a double peak M1a/M1b (7QDI cis-/trans isomers).

The LC-MS investigation of M1a/M1b (Envigo Study KAH0012, not finalized yet) revealed two components with the mass of 7QDI, suggesting that M1a/M1b are the cis-/trans isomers of 7QDI. Based on this finding, 7QDI was synthesized and submitted as cis-/trans mixture for co-chromatography. In the primary HPLC method, 7QDI was found to co-elute with M1a/M1b. In the confirmatory HPLC method, 7QDI coeluted with a peak of similar size (27.75%) as M1a/M1b in primary HPLC method (27.99%). M1a/M1b was therefore considered identified as 7QDI cis-/trans isomers, based on the matching mass and coelution with 7QDI in two dissimilar chromatographic methods.

Rates of decline were determined for the parent (7PPD) and the transformation products M1a, M1b (7QDI isomers) and the sum of M1a and M1b (total 7QDI). The decline of the parent was best described by the HS kinetic model. FOMC and DFOP models gave acceptable fits while the SFO model was inadequate. The decline of M1a, M1b and M1 (M1a + M1b) was adequately described by SFO. Goodness of fit and DT50 and DT90 values are given below for the preferred models applied to the present study performed at 12 ± 2 °C.

The rapid decline of 7PPD in the initial aerobic phase was likely to extend into the anaerobic phase of the test system given that the transition to anaerobic conditions by soil flooding is progressive. Therefore, the short DT50 of the parent (1.5 days) in the flooded test system was attributed to aerobic degradation. Following that rapid initial decline, the parent dissipated slowly, indicating a persistent nature under steady anaerobic conditions.

The metabolism of 7PPD in anaerobic soils is proposed to proceed via formation of 7QDI cis-/trans isomers, several minor metabolites and modest mineralization. 7PPD declines also by residue binding to the soil matrix.

Description of key information

Aerobic conditions: The metabolism of 14C-7PPD in aerobic soils is proposed to proceed via formation of minor transient metabolites and mineralization. The main portion of the residue is binding to the soil matrix and appears to become unavailable for further degradation/mineralization. DT50 values of the parent, derived from FOMC (First-Order Multi Compartment kinetics), were 1.4 – 1.9 days for Soils II-IV and < 1 day for soil I.
Anaerobic conditions: The rapid decline of 7PPD in the initial aerobic phase was likely to extend into the anaerobic phase of the test system given, that the transition to anaerobic conditions by soil flooding is progressive. Therefore, the short DT50 of 7PPD (1.5 days) in the flooded test system was attributed to aerobic degradation. Following that rapid initial decline, the parent dissipated slowly, from 23.2% AR on day 1 to 17.0 AR on day 120.

Key value for chemical safety assessment

Half-life in soil:
1.9 d
at the temperature of:
12 °C

Additional information

Aerobic conditions:

DT50 values derived from First Order Multi Compartment kinetics (FOMC) kinetics were 1.4 – 1.9 days for Soils II-IV and < 1 day for soil I. The faster decline in acid soil I (pH 3.8) was explained by the instability of 7PPD under acidic conditions.

The metabolism of 14C-7PPD in aerobic soils is proposed to proceed via formation of minor transient metabolites and mineralization.The main portion of the residue is binding to the soil matrix and appears to become unavailable for further degradation/mineralization.

Anaerobic conditions:

The short DT50 of the parent (1.5 days) in the flooded test system was attributed to aerobic degradation. Following that rapid initial decline, 7PPD dissipated slowly. A DT90 of >1000 days was calculated. The metabolite 1-N-(5-methyl-hexan-2-yl)-4-N-phenylcyclohexa-2,5-dione-4,4-diimine also known as 7QDI, (cis/trans isomers) was observed.

7QDI Isomer 1: DT50 = 57.9 d; DT90 = 192 d

7QDI Isomer 2: DT50 = 79.7 d; DT90 = 265 d

7QDI Isomer 1+2: DT50 = 66.9 d; DT90 = 222 d

Degradation was described by SFO (Single First Order) model.