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Environmental fate & pathways

Biodegradation in soil

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Endpoint:
biodegradation in soil: simulation testing
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 2017-08-15 to 2018-10-18 with an actual exposure phase from 2017-11-27 to 2018-06-21
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)
Deviations:
no
GLP compliance:
yes
Remarks:
The14C-labelled test item was not retained due to its radioactivity and small sample amount.
Test type:
laboratory
Radiolabelling:
yes
Oxygen conditions:
aerobic
Soil classification:
DIN 19863 (Deutsche Industrie-Norm)
Year:
2018
Soil no.:
#1
Soil type:
loamy sand
% Clay:
8.6
% Silt:
15.7
% Sand:
75.7
% Org. C:
1.7
pH:
5.6
CEC:
9.8 meq/100 g soil d.w.
Bulk density (g/cm³):
1 201
Soil no.:
#2
Soil type:
other: silty sand
% Clay:
7.6
% Silt:
35.2
% Sand:
57.2
% Org. C:
0.55
pH:
5.8
CEC:
7.5 meq/100 g soil d.w.
Bulk density (g/cm³):
1 307
Soil no.:
#3
Soil type:
clay loam
% Clay:
26.6
% Silt:
46.1
% Sand:
27.3
% Org. C:
2.47
pH:
7.4
CEC:
26.5 meq/100 g soil d.w.
Bulk density (g/cm³):
1 251
Soil no.:
#4
Soil type:
clay loam
% Clay:
11.2
% Silt:
34.5
% Sand:
54.3
% Org. C:
0.88
pH:
7.3
CEC:
15.7 meq/100 g soil d.w.
Bulk density (g/cm³):
1 221
Details on soil characteristics:
Test system
Four different standard soils (LUFA 2.2, 2.3, 2.4 and 5M field fresh sampled) representing a range of relevant soils, were used. The soils varied in their organic carbon content, pH, clay content and microbial biomass. LUFA 2.2, 2.3, 2.4 and 5M were used for determination of the transformation rate. LUFA soil 2.2 was used for metabolite identification and evaluation of the transformation pathway.

Origin
Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer (LUFA), Obere Langgasse 40, 67346 Speyer, Germany

Reasons for the selection
The soils met the recommendations of the guideline.

Soil handling
The soils were manually cleared of large objects and then sieved to a particle size of 2 mm (carried out by LUFA Speyer). The WHCmax and the pH-value were determined (carried out by LUFA Speyer under GLP). The soil moisture content was determined at receipt of the soils at the test facility.

Soil storage
The soils were stored in the dark at 4 ± 2 °C: soil 2 .2 for 27 days, soil 2.3 and 5M for 25 days and soil 2.4 for 22 days.

Preincubation
The soil moisture content was adjusted to 40 % of its WHCmax (soil 2.2 and 2.4) with demineralized water. Soil 2.3 and 5M were adjusted to 35 % of its WHCmax because otherwise the soils were too wet after adjustment of soil moisture content at application. To allow germination and removal of seeds and to guarantee a temperature adaptation of the microorganisms, the soils were preincubated (prior to application) at room temperature (20 ± 2 °C) for 13 days (soil 2.2), 21 days (soil 2.3), 12 days (soil 2.4) and 7 days (soil 5M), respectively. The soil was checked for a detectable microbial biomass (result in terms of percentage of total organic carbon).
Soil No.:
#1
Duration:
122 d
Soil No.:
#2
Duration:
120 d
Soil No.:
#3
Duration:
128 d
Soil No.:
#4
Duration:
120 d
Soil No.:
#1
Initial conc.:
9.2 other: kBq/g soil DW
Based on:
test mat.
Soil No.:
#2
Initial conc.:
7.58 other: kBq/g soil DW
Based on:
test mat.
Soil No.:
#3
Initial conc.:
9.2 other: kBq/g soil DW
Based on:
test mat.
Soil No.:
#4
Initial conc.:
9.2 other: kBq/g soil DW
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
radiochem. meas.
Soil No.:
#1
Temp.:
20+/-2 °C
Humidity:
50 % (WHCmax)
Microbial biomass:
2.66 % of org. C (Day 0)
Soil No.:
#2
Temp.:
20+/-2 °C
Humidity:
45 % (WHCmax)
Microbial biomass:
3.94 % of org. C (Day 0)
Soil No.:
#3
Temp.:
20 +/- 2 °C
Humidity:
50 % (WHCmax)
Microbial biomass:
3.02 % of org. C (Day 0)
Soil No.:
#4
Temp.:
20 +/- 2 °C
Humidity:
45 % (WHCmax)
Microbial biomass:
3.09 % of org. C (Day 0)
Details on experimental conditions:
Test Item Concentration and Treatment Groups
Test item [ring-U-14C]Benzalkonium chloride
Working Solution 37 MBq/mL (provided by the sponsor)

Actual test item concentration
9.20 kBq/g soil DW (460.1 kBq/Replicate) (soil 2.3, 2.4, 5M) corresponding to 1500 µg test item/kg soil DW
C12 chain: 6.37 kBq/g soil DW
C14 chain: 2.83 kBq/g soil DW
7.58 kBq/g soil DW (378.8 kBq/Replicate) (soil 2.2) corresponding to 1235 µg test item/kg soil DW
C12 chain: 5.25 kBq/g soil DW
C14 chain: 2.33 kBq/g soil DW

Application solution of labelled test item
4.76 MBq/mL (soil 2.3, 2.4, 5M) (actual)
4.79 MBq/mL (soil 2.2) (actual)

Solvent for application Ultrapure water : ACN (90 : 10)
Carrier for application Quartz sand

Control
Control soil samples were not treated with the test item and were incubated under the same aerobic conditions as the treated soil samples. These samples were used for biomass measurements at test start, during and at the end of the study.

Reference Item
According to the guideline no reference item is recommended for this test.


Test Item Concentration for Identification of Metabolites and Evaluation of Transformation Pathway
Test Item Arquad MCB-50
Nominal test item concentration 2000 µg Benzalkonium chloride/kg soil DW (soil 2.2)

Solvent for application Ultrapure water : ACN (90:10)
Carrier for application Quartz sand


Test Procedures
Application
Quartz sand was used as a carrier for the application. Therefore, a deepening was made on the soil surface and filled with quartz sand (2 % of the total applied soil as dry weight, for soil 2.2 (treated with labelled test item) 1.65 % of the total applied soil as dry weight). Afterwards an appropriate volume of the working solution was applied on the quartz sand. After 15 minutes the respective volume of ultrapure water for adjusting the % of MWHC was applied on the surface of each soil. Immediately after that, the soils were mixed carefully (3 min.) to insure a homogeneous distribution of the test item in the soil. Subsequently, samples for combustion and LSC analysis were taken to check the homogeneous distribution of the test item. Afterwards the soils were distributed to the test replicates.

Frequency of application Application was carried out once at Day 0 for each soil type.

Test duration
Soil 2.2: 122 days
Soil 2.3: 120 days
Soil 2.4: 128 days
Soil 5M: 120 days

Incubation
All replicates were incubated at 20 ± 2 °C in the dark. Between 2018-02-26 and 2018-02-28 a deviation of 0.3 °C of the incubation temperature occurred. The incubation temperature was temporary lowest at 17.7 °C. Aerobic conditions (exchange of air) were maintained by diffusion from the headspace and ambient atmosphere.

Soil moisture content
At application the soils were adjusted to 45 – 50 % of the maximum water holding capacity. All replicates were checked at least in two weeks intervals for losses by evaporation

Type and Frequency of Measurements and Observations for Determination of the Transformation Rate
Sampling
To confirm the applied test item amount and the homogeneous distribution of the test item in the soil, directly after application, Five subsamples of the soils were combusted in a sample oxidizer and the evolved 14CO2 was analyzed by LSC.

Two test item replicates were sampled at each sampling time. At test start four samples of the total applied soil amount were analyzed.
Sampling was carried out directly after application and at 9 additional sampling points. The sampling points were chosen in such a way that the pattern of decline of the test item could be established.

Sampling Times
Soil Number of Samplings Sampling Times
2.2 10 0, 1, 2, 3, 4, 7, 10, 21, 74, 122 days
2.3 10 0, 1, 2, 3, 4, 8, 22, 46, 80, 120 days
2.4 10 0, 2, 3, 6, 8, 13, 20, 56, 87, 128 days
5M 10 0, 1, 3, 7, 11, 21, 36, 60, 94, 120 days

Test item andof transformation products
The amount of test item and transformation products (as % applied radioactivity) in the soil (after extraction) were determined by LC-FSA.

14CO2
The soda lime for trapping 14CO2 was acidified and the evolved 14CO2 was trapped in sodium hydroxide solution. The sodium hydroxide solution was analyzed for 14CO2 by LSC.

Volatile organic transformation products
Mass balances indicated that no volatile organic transformation products were formed. Therefore, polyurethane foam traps were only analyzed at test end.

NER
The non-extractable residues (NER) as % of the applied radioactivity were determined by LSC after combustion of the extracted soil.
Soil organic matter (humus) fractionation of the non-extractable residues was done at test end to characterize the radioactivity bound to the humic and fulvic acids as well as to the humin fraction of the soil.


Soil moisture
At least three representative replicates were checked at least in two week intervals for losses by evaporation. The replicates were weighed for this procedure. Compensation with demineralized and filtered water was done for all replicates if water loss was > 0.5 g.Care was taken to avoid any losses of volatile degradation products during moisture addition.

Biomass activity
To check the biomass activity glucose induced respiration rates of the controls were determined at test start, during the study (day 49 – 63) and test end.

Measurement of glucose induced respiration rates
The soil of each replicate was mixed with a sufficient amount of glucose (4000 mg/kg) to produce an immediate maximum respiratory response. 200 g soil dry weight were filled into 500 mL glass flasks and closed with Oxitop® sensors. CO2 was adsorbed by soda lime deposited in the headspace. Due to the adsorption of CO2 and the oxygen uptake by the soil the pressure in the glass flasks was reduced and measured. Based on the change of pressure the evolved CO2 and thus the consumed O2 was calculated. Incubation took place for 24 h in the dark at 20 ± 2°C. The pressure was measured 360 times in 24 hours after glucose supplement.

Sampling for Metabolite Identification and Evaluation of Transformation Pathway
Sampling for metabolite identification and evaluation of the transformation pathway (LC-HRMS and LC-MS/MS analysis) was done based on the metabolite information obtained from the extracts of soil 2.2. Two test item replicates as well as two controls were sampled, extracted and analysed on day 0, 3, 5 and 12. Two subsamples for analysis were prepared for each test item replicate.
Soil No.:
#1
% Recovery:
90.4
Soil No.:
#2
% Recovery:
91.5
Soil No.:
#3
% Recovery:
93.7
Soil No.:
#4
% Recovery:
94
Soil No.:
#1
% Degr.:
1.7
Parameter:
test mat. analysis
Remarks:
C12 BKC
Sampling time:
122 d
Soil No.:
#1
% Degr.:
0.9
Parameter:
test mat. analysis
Remarks:
C14 BKC
Sampling time:
122 d
Soil No.:
#1
% Degr.:
20.1
Parameter:
radiochem. meas.
Sampling time:
122 d
Soil No.:
#1
% Degr.:
21.5
Parameter:
other: Non Extractable Residues
Sampling time:
122 d
Soil No.:
#1
% Degr.:
48.8
Parameter:
CO2 evolution
Sampling time:
122 d
Soil No.:
#2
% Degr.:
2.2
Parameter:
test mat. analysis
Remarks:
C12 BKC
Sampling time:
120 d
Soil No.:
#2
% Degr.:
2.1
Parameter:
test mat. analysis
Remarks:
C14 BKC
Sampling time:
120 d
Soil No.:
#2
% Degr.:
10.5
Parameter:
radiochem. meas.
Sampling time:
120 d
Soil No.:
#2
% Degr.:
24.9
Parameter:
other: Non Extractable Residues
Sampling time:
120 d
Soil No.:
#2
% Degr.:
56.1
Parameter:
CO2 evolution
Sampling time:
120 d
Soil No.:
#3
% Degr.:
2.4
Parameter:
test mat. analysis
Remarks:
C12 BKC
Sampling time:
128 d
Soil No.:
#3
% Degr.:
2
Parameter:
test mat. analysis
Remarks:
C14 BKC
Sampling time:
128 d
Soil No.:
#3
% Degr.:
12
Parameter:
radiochem. meas.
Sampling time:
128 d
Soil No.:
#3
% Degr.:
31.2
Parameter:
other: Non Extractable Residues
Sampling time:
128 d
Soil No.:
#3
% Degr.:
50.6
Parameter:
CO2 evolution
Sampling time:
128 d
Soil No.:
#4
% Degr.:
3.9
Parameter:
test mat. analysis
Remarks:
C12 BKC
Sampling time:
120 d
Soil No.:
#4
% Degr.:
4
Parameter:
test mat. analysis
Remarks:
C 14 BKC
Sampling time:
120 d
Soil No.:
#4
% Degr.:
19.7
Parameter:
radiochem. meas.
Sampling time:
120 d
Soil No.:
#4
% Degr.:
29.4
Parameter:
other: Non Extractable Residues
Sampling time:
120 d
Soil No.:
#4
% Degr.:
44.9
Parameter:
CO2 evolution
Sampling time:
120 d
Soil No.:
#1
DT50:
2.2 d
Type:
other: Single First Order (SFO)
Temp.:
20 °C
Remarks on result:
other: DT90 = 7.2 d (Dissipation)
Remarks:
C12 BKC
Soil No.:
#1
DT50:
1.6 d
Type:
other: First Order Multi Compartment (FOMC)
Temp.:
20 °C
Remarks on result:
other: DT90 =15.0 d (Dissipation)
Remarks:
C12 BKC
Soil No.:
#1
DT50:
6.1 d
Type:
other: Single First Order (SFO)
Temp.:
20 °C
Remarks on result:
other: DT90 = 20.2 d (Dissipation)
Remarks:
C14 BKC
Soil No.:
#1
DT50:
5.5 d
Type:
other: First Order Multi Compartment (FOMC)
Temp.:
20 °C
Remarks on result:
other: DT90 = 35.8 d (Dissipation)
Remarks:
C14 BKC
Soil No.:
#2
DT50:
3.3 d
Type:
other: Single First Order (SFO)
Temp.:
20 °C
Remarks on result:
other: DT90 = 11.0 d (Dissipation)
Remarks:
C12 BKC
Soil No.:
#2
DT50:
3.2 d
Type:
other: First Order Multi Compartment (FOMC)
Temp.:
20 °C
Remarks on result:
other: DT90 = 16.5 d (Dissipation)
Remarks:
C12 BKC
Soil No.:
#2
DT50:
8.9 d
Type:
other: Single First Order (SFO)
Temp.:
20 °C
Remarks on result:
other: DT90 = 29.6 d (Dissipation)
Remarks:
C14 BKC
Soil No.:
#2
DT50:
8.3 d
Type:
other: First Order Multi Compartment (FOMC)
Temp.:
20 °C
Remarks on result:
other: DT90 = 54.4 d (Dissipation)
Remarks:
C14 BKC
Soil No.:
#3
DT50:
6.2 d
Type:
other: Single First Order (SFO)
Temp.:
20 °C
Remarks on result:
other: DT90 = 20.5 d (Dissipation)
Remarks:
C12 BKC
Soil No.:
#3
DT50:
5.2 d
Type:
other: First Order Multi Compartment (FOMC)
Temp.:
20 °C
Remarks on result:
other: DT90 = 34.0 d (Dissipation)
Remarks:
C12 BKC
Soil No.:
#3
DT50:
12.9 d
Type:
other: Single First Order (SFO
Temp.:
20 °C
Remarks on result:
other: DT90 = 42.7 d (Dissipation)
Remarks:
C14 BKC
Soil No.:
#3
DT50:
12.1 d
Type:
other: First Order Multi Compartment (FOMC)
Temp.:
20 °C
Remarks on result:
other: DT90 = 69.7 d (Dissipation)
Remarks:
C14 BKC
Soil No.:
#4
DT50:
8.7 d
Type:
other: Single First Order (SFO
Temp.:
20 °C
Remarks on result:
other: DT90 = 28.8 d (Dissipation)
Remarks:
C12 BKC
Soil No.:
#4
DT50:
7.2 d
Type:
other: First Order Multi Compartment (FOMC)
Temp.:
20 °C
Remarks on result:
other: Dt90 = 48.8 d (Dissipation)
Remarks:
C12 BKC
Soil No.:
#4
DT50:
28.7 d
Type:
other: Single First Order (SFO
Temp.:
20 °C
Remarks on result:
other: DT90 = 95.4 d (Dissipation)
Remarks:
C14 BKC
Soil No.:
#4
DT50:
23.3 d
Type:
other: First Order Multi Compartment (FOMC)
Temp.:
20 °C
Remarks on result:
other: DT90 = 164.3 d (Dissipation)
Remarks:
C14 BKC
DT50:
19.2 d
Type:
other: SFO
Temp.:
12 °C
Remarks on result:
other: weighted DT50 value for the major alkyl chains extrapolated at 12°C
DT50:
17.1 d
Type:
other: FOMC
Temp.:
12 °C
Remarks on result:
other: weighted DT50 value for the major alkyl chains extrapolated at 12°C
Transformation products:
yes
Details on transformation products:
Formation of Metabolites and Transformation Pathway
Metabolites determined during the Study by Radio-HPLC
Metabolites determined in soil extracts by radio-HPLC are given as % of AR in Table 21 to Table 24. Only metabolites detected at least once at > 1 % AR in both replicates were considered for further evaluation. Metabolites which were detected at ≥ 5 % AR at two consecutive sampling intervals or ≥ 10% AR at any sampling were identified by LC-HRMS. Extracts from soil 2.2 were used for metabolite identification and for evaluation of the transformation pathway. The maximum amount of all metabolites was determined until Day 21, thereafter all metabolites decreased steadily until the end of the study.

Distribution of Metabolites as Percent of Applied Radioactivity
    % of Applied Radioactivity
RT   Sampling Day
(min) Repl. 0 1 2 3 4 7 10 21 74 122
Primary Metabolites
8.13 - 9.40 1 n.d. n.d. 14.1 32.2 42.1 52.4 63.6 65.5 60.9 24.9 12.9
2 n.d. n.d. 16.9 31.5 42.3 53.7 66.4 65.6 61.7 25.1 11.1
10.00 - 10.93 1 n.d. n.d. 1.4 1.7 1.3 1.3 n.d. 2.0 n.d. n.d. n.d.
2 n.d. n.d. 1.0 1.8 1.6 1.1 n.d. n.d. 0.4 n.d. n.d.
11.07 - 11.27 1 n.d. n.d. 8.4 8.2 6.5 5.3 3.4 n.d. 0.4 n.d. n.d.
2 n.d. n.d. 9.2 7.7 6.3 5.5 3.5 2.3 n.d. n.d. n.d.
RT = Retention time in minutes n.d. = not detected


Distribution of Metabolites as Percent of Applied Radioactivity
    % of Applied Radioactivity
RT   Sampling Day
(min) Repl. 0 1 2 3 4 8 22 46 80 120
    Primary Metabolites
8.13 - 9.80 1 n.d. n.d. 2.9 16.2 29.9 37.2 54.4 32.1 17.8 7.5 3.9
2 n.d. n.d. 5.5 17.4 30.6 36.1 52.6 37.3 18.0 6.9 4.2
10.00 - 10.93 1 n.d. n.d. 5.5 6.8 5.4 4.0 1.4 0.3 n.d. n.d. 0.2
2 n.d. n.d. 6.3 7.8 6.1 2.6 1.5 0.2 n.d. n.d. 0.1
18.47 - 19.00 1 n.d. n.d. 0.9 0.8 1.1 0.9 n.d. n.d. n.d. n.d. n.d.
2 n.d. n.d. 1.0 1.4 0.2 n.d. n.d. n.d. n.d. n.d. n.d.
RT = Retention time in minutes n.d. = not detected


Soil 2.4: Distribution of Metabolites as Percent of Applied Radioactivity
    % of Applied Radioactivity
RT   Sampling Day
(min) Repl. 0 2 3 6 8 13 20 56 87 128
    Primary Metabolites
7.47 - 9.40 1 n.d. n.d. 7.8 10.1 20.5 23.4 26.5 23.4 9.7 6.2 4.3
2 n.d. n.d. 6.3 10.6 20.5 29.1 30.1 25.5 9.2 5.5 4.0
10.00- 10.40 1 n.d. n.d. n.d. n.d. 1.4 1.4 1.6 1.1 0.8 0.2 n.d.
2 n.d. n.d. n.d. 0.9 1.4 2.4 1.4 1.2 n.d. n.d. n.d.
11.07 - 11.40 1 n.d. n.d. 7.2 6.5 6.5 5.0 3.0 1.4 0.2 n.d. n.d.
2 n.d. n.d. 7.0 6.9 5.8 6.4 3.3 1.4 0.2 n.d. n.d.
RT = Retention time in minutes n.d. = not detected


Soil 5M: Distribution of Metabolites as Percent of Applied Radioactivity
    % of Applied Radioactivity
RT   Sampling Day
(min) Repl. 0 1 3 7 11 21 36 60 94 120
    Primary Metabolites
7.60- 8.80 1 n.d. n.d. 1.3 6.8 17.6 22.9 27.1 22.6 14.5 9.7 9.1
2 n.d. n.d. 1.1 5.9 17.3 23.7 26.8 21.1 13.4 8.5 10.3
10.33 - 10.67 1 n.d. n.d. 2.3 3.7 3.2 n.d. n.d. n.d. 0.1 n.d. n.d.
2 n.d. n.d. 2.3 3.9 3.0 2.0 n.d. n.d. 0.1 n.d. n.d.
18.07 - 18.87 1 n.d. n.d. n.d. 1.0 0.7 n.d. n.d. n.d. 0.1 n.d. n.d.
2 n.d. n.d. n.d. 0.9 1.3 1.0 n.d. n.d. n.d. 0.1 n.d.
RT = Retention time in minutes n.d. = not detected

Due to the long study duration the retention capacity of the chromatographic column changed. As a result the retention times shifted slightly within the duration of the study. The correct assignment of the peaks was confirmed by re-analysis of selected samples after end of the study.



Metabolite Identification
Data Evaluation – LC-HRMS Analysis
Data obtained from the MS scan measurements were evaluated by the software tools Profinder and Mass Profiler Professional. The Profinder prepares the initial data set for the final evaluation with the Mass Profiler Professional. A batch recursive extraction was perfomed with the Profinder. This workflow consists of several steps allowing a refinement of the obtained large data set from the MS scan measurements. First, peaks (features) were extracted from the complex data set by an algorithm. This process is called molecular feature extraction (MFE) and the evaluation yields many individual peaks. The quality of the extracted peaks is described by a score which was used as a filter to exclude peaks with limited quality from the data set. A score of ≥ 70 is a suitable filter for this purpose. The obtained data set was subsequently refined by aligning these peaks and binning peaks with similar molecular weight and retention time. In a third step, the quality of the binning was assessed and improved by a process described as Find by Ion. This process optimizes the peak extraction and calculates a score for the binning by taking into the sameness of the detected mass, isotope abundance and isotope spacing. A threshold filter of 50 was applied for this score. Finally, a peak height filter excluded peaks with a height < 2000.
These processed data were then evaluated with the software Mass Profiler Professional. The Mass Profiler investigated in which of the analysed samples the binned peaks occurred and if their occurrence is increasing or decreasing. Furthermore, it is possible to form sample groups. This was done in this case and all control samples were summarized in one group whereas the samples of the test item replicates were grouped per day of sampling. The data were analysed then with the aim to find compounds which are unique for the samples of the test item replicates after day 0 or their intensity increase after day 0 and are not present in the control samples. Finally, molecular formulas were generated for the unique peaks. After that, further measurements in the targeted MS/MS mode were performed for the compounds which could be annotated with molecular formulas to obtain mass spectra for them followed by proposing chemical structures for them.

Parameters – Batch Recursive Extraction
Molecular Feature Extraction (MFE)
Spectral peak height ≥ 2000
Ion Species [M+ H, Na, NH4]+
Charge State z = 1 – 2, ions with undefined charge state were treated as singly charged, no halogen atoms in molecules
Compound Filters Two or more ions
Post Processing Filter Score ≥ 70

Compound Binning and Alignment
Retention time [min] ± 0.3
Mass error ± (20 ppm + 2 mDa)
Find by Ion
Expansion values extraction ± 35 ppm, symmetric, ± 0.5 min
Weighting for scoring [%] Mass score 100
Isotope abundance 60
Isotope spacing 50
Absolute height ≥ 2000
Score ≥ 50

Mass Profiler - Generator for Molecular Formulas
Ion electron state Allow both even and odd
Element limits: C: 3 – 30
H: 3 - 70
N: 0 - 1
O: 0 - 2
Double bond equivalents: 0 - 7
Mass error [ppm]: 7.5

Results and Data Assessment – LC-HRMS Analysis
The results of the final data assessment analysis with the Mass Profiler Professional are shown below in Table 25. The data show the occurrence of the unique peaks depending on the sampling day. All compounds were not present in the controls and either not present in samples of Day 0 or their intensity increased after Day 0. Plus and minus indicate increasing or decreasing peak intensity within two subsequent sampling days.

Results Data Assessment Mass Profiler Professional
Samples control group n = 8
Samples other groups n = 2

Metabolite Molecular
Number Formula Control Day 0 Day 3 Day 5 Day 12
1 C9 H13 N 0/8 0/2 2/2 2/2+ 2/2+
2 C12 H17 N O2 0/8 0/2 2/2 2/2+ 2/2+
3 C13 H18 N O2 0/8 0/2 2/2 2/2+ 2/2+
4 C13 H20 N O2 0/8 0/2 2/2+ 2/2- 2/2-
5 C15 H24 N O2 0/8 2/2 2/2+ 2/2- 2/2-
6 C16 H25 N O2 0/8 1/2 2/2+ 2/2- 2/2-
+/- minus indicate increasing or decreasing peak intensity within two subsequent sampling days

Targeted MS/MS experiments were conducted to record their mass spectra and to propose chemical structures for them. Diagnostic ions and the corresponding annotations are given. The diagnostic ions were used for the proposal of chemical structures for the metabolites.

Results Data Assessment Targeted MS/MS Experiments
Samples control group n = 8

Molecular Formula Diagnostic Ion 1 Annotation 1 Diagnostic Ion2 Annotation 2
m/z m/z
C9 H13 N 91.0542 [C7H7]+ -- --
C12 H17 N O2 91.0542 [C7H7]+ 116.0706 [C5H10NO2]+
C13 H18 N O2 91.0542 [C7H7]+ 128.0706 [C6H10NO2]+
C13 H20 N O2 91.0542 [C7H7]+ 130.0863 [C6H12NO2]+
C15 H24 N O2 91.0542 [C7H7]+ 158.1176 [C8H16NO2]+
C16 H25 N O2 91.0542 [C7H7]+ 172.1332 [C9H18NO2]+
Samples other groups n = 2


The diagnostic ion, [C7H7]+, is characteristic for molecules with a benzyl unit and is present in all metabolites. The other diagnostic ions in the metabolites 2 – 6 show alkyl chains with varying numer of carbon atoms which are oxidized to carboxylic acids. These metabolites have 5 – 6 (C13H18NO2) double bond equivalents (DBE). In contrast, the test item has only four DBE. The metabolites 2 – 6 indicate that the alkyl chain of the test item is stepwise degraded after an initial oxidation at the ω-end of the alkyl chain. It is assumed that the alkyl chain is degraded by a β-oxidation removing two carbon atoms form the alkyl chain in each step. The β-oxidation as catabolic pathway is ubiquitous. It is assumed that the metabolites 2 and 6 were demethylated. Otherwise, the alkyl chain must be odd numbered and that was assessed to be unlikely.
The metabolites 5 and 6 are already present in soil extracts of samples from Day 0. This fact shows that the β-oxidation takes place very fast. Dimethylbenzylamine was proposed as chemical structure for metabolite 1 after oxidative removal of the alkyl chain (see attachment)


Confirmatory – LC-MS/MS Analysis
Additional analysis with LC-MS/MS a was conducted to link the results of the LC-HRMS analysis with the study results of the radio-HPLC.
The radio-HPLC chromatograms peaks with highest % AR within the retention time window of 7.47 – 9.80 min. Dimethylbenzylamine, Methylbenzylamine and Benzylamine were regarded as possible metabolites eluting in this retention time window.
Methylbenzylamine was not detected as metabolite in the experiments of the previous section but the occurrence of the metabolites 2 and 6 indicate that this metabolite may be formed after oxidative degradation of the alkyl chain. Benzylamine was considered as possible metabolite prior to oxidative degradation of the benzyl unit. Based on these considerations, an MS/MS experiment was designed with expected mass transitions for the corresponding 14C-metabolites and 14C C12 BKC.
The results show the most distinct peaks within the retention time window of 6.0 -10.0 min of the LC-MS/MS analysis, matching the peaks with highest % AR of the radio-HPLC analysis. Dimethylbenzylamine and Methylbenzylamine are present as metabolites. Both compounds did not occur in soil extracts of a control and sample of day 0, but in all further analysed soil extracts. Dimethylbenzylamine is the predominant metabolite whereas Methylbenzylamine was present only in traces. The highest concentrations of Dimethylbenzylamine were determined until day 22, thereafter the concentrations deceased continuously until test end.

Benzylamine was not detected during this experiment.
The peaks determined by radio-HPLC within the retention time window 10.0 – 10.93 min. could be assigned by the LC-MS/MS measurements as metabolites containing a partly degraded alkyl chain by β-oxidation. These metabolites were transient, the highest activity was determined on day 2 in soil 2.3. Within the course of the study, the activity decreased rapidly and was ≤ 0.2 % AR soil 2.3 and not detectable in all other soils.
Further peaks determined by radio-HPLC at retention times later than 11 min. are assumed to be metabolites containing also the partly degradated alkyl chain, but with a higher number of carbon atoms. These metabolites were transient as well and the determined activity was < 5 % AR at all samplings. Formation and decline of major metabolites (once occurring at > 5% AR) in comparison to the decline of C12 BKC and C14 BKC is graphically presented in Figure 4 to Figure 7 (see attachment)

Benzylamine was not detected during this experiment.
The peaks determined by radio-HPLC within the retention time window 10.0 – 10.93 min. could be assigned by the LC-MS/MS measurements as metabolites containing a partly degraded alkyl chain by β-oxidation. These metabolites were transient, the highest activity was determined on day 2 in soil 2.3. Within the course of the study, the activity decreased rapidly and was ≤ 0.2 % AR soil 2.3 and not detectable in all other soils.
Further peaks determined by radio-HPLC at retention times later than 11 min. are assumed to be metabolites containing also the partly degradated alkyl chain, but with a higher number of carbon atoms. These metabolites were transient as well and the determined activity was < 5 % AR at all samplings.

Residues:
yes
Remarks:
Non Extractable Residues
Details on results:
Transformation of [ring-U-14C]Benzalkonium chloride
The transformation of the C12 chain of the test item [ring-U-14C]Benzalkonium chloride was rapid in all four soils. The transformation of the C14 chain proceeded slightly slower. Within 7 – 21 days the concentration of the C12 chain decreased from initially 67.2 – 69.6 % to < 20 %. The concentration of the C14 chain decreased from initially 23.8 – 24.6 % to < 10 % within 10 – 36 days.

Distinct mineralization started between Day 8 and Day 21. Within this period the 14CO2 increased for all four soils to > 10 %. In the course of the study the 14CO2 formation increased steadily, and the evolved 14CO2 reached 44.9 – 56.1 % AR at the end of the respective incubation period.

The formation of NER started directly after application of the test item. Further on in the study formation of NER increased in parallel to the start of increased mineralisation, indicating that a major amount of NER is comprised by radioactivity incorporated in microbial biomass.
For soil 2.2 NER formation was lowest compared to the other test soils and was < 10 % AR until Day 10. Thereafter the NER increased in parallel to 14CO2 formation and reached a plateau after 74 days (20.8 – 21.5 % AR).
For soil 2.3 and soil 5M NER reached a maximum of 28.7 – 32.9 % AR within 36 – 46 days of incubation. Thereafter, the NER decreased relative to the maximum NER, simultaneously to further14CO2 formation, and was in a range of 24.9 and 29.4 % AR at test end. This indicated that a fraction of radioactivity bound in biomass was remobilised and mineralized by the soil microbacteria.
For soil 2.4 NER increased to 22.7 % until day 8, during start of 14CO2 formation in soil 2.4 (Day 13) the NER increased to 28.9 % AR and varied thereafter within this range (28.8 – 31.2 % AR) until the end of the incubation phase.

No radioactivity (all samples < LSC background) was determined in the polyurethane foam traps, indicating that no volatile organic transformation products were formed.


Degradation of [ring-U-14C]Benzalkonium chloride in Soil 2.2,expressed as Percentage of Total Applied Radioactivity [% AR]
Soil 2.2
Sampling Interval (Days)
Repl. 01) 1 2 3 4 7 10 21 74 122
C12 BKC
(LC-FSA) 1 71.2 38.1 32.5 24.9 20.4 13.2 8.3 5.1 2.1 1.7
2 68.7 42.8 34.7 23.8 21.2 12.0 9.1 4.3 2.4 1.7
3 70.2
4 68.1
mv 69.6 40.5 33.6 24.3 20.8 12.6 8.7 4.7 2.2 1.7
C14 BKC
(LC-FSA) 1 23.9 18.4 18.8 16.7 14.3 9.7 8.1 4.4 1.6 1.0
2 25.6 21.1 19.3 16.5 15.2 10.1 8.1 3.6 1.8 0.7
3 24.1
4 25.0
mv 24.6 19.8 19.1 16.6 14.8 9.9 8.1 4.0 1.7 0.9
Total Radio-activity Extract 1 90.1 93.5 95.6 87.6 91.2 87.8 88.9 71.7 32.4 22.2
2 98.6 88.4 94.9 93.6 93.8 92.3 87.4 73.4 34.2 18.1
3 98.1
4 98.5
mv 96.3 90.9 95.3 90.6 92.5 90.0 88.1 72.5 33.3 20.1
NER 1 4.1 9.3 6.0 6.7 6.5 8.4 8.5 12.8 20.3 22.5
2 4.7 5.2 6.1 7.2 6.6 7.7 9.7 12.1 21.3 20.5
3 5.5
4 4.0
mv 4.5 7.2 6.1 7.0 6.5 8.0 9.1 12.5 20.8 21.5
14CO2 1 n.d. 0.6 0.8 0.9 1.3 2.3 3.6 11.5 39.5 49.5
2 n.d. 0.7 0.6 0.7 1.3 2.3 3.2 11.0 39.2 48.0
mv n.d. 0.7 0.7 0.8 1.3 2.3 3.4 11.3 39.3 48.8
Volatiles 1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 2 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. mv n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
Mass Balance 1 94.1 103.5 102.5 95.2 99.0 98.5 101.0 96.0 92.3 94.2
2 103.2 94.2 101.7 101.5 101.7 102.3 100.3 96.5 94.6 86.5
3 103.5
4 102.5
mv 100.8 98.9 102.1 98.4 100.3 100.4 100.6 96.2 93.4 90.4
BKC = [ring-U-14C]Benzalkonium chloride
NER = Non-Extractable Residues
LOD = Limit of Detection
1) = at test start, four samples of the soil batch were taken for analysis
mv = mean value n.d. = not determined

Degradation of [ring-U-14C]Benzalkonium chloride in Soil 2.3, expressed as Percentage of Total Applied Radioactivity [% AR]
Soil 2.3
Sampling Interval (Days)
Repl. 01) 1 2 3 4 8 22 46 80 120
C12 BKC
(LC-FSA) 1 67.6 56.6 41.5 35.2 26.8 14.6 5.2 3.4 2.8 2.2
2 69.6 58.9 45.8 34.1 27.8 14.8 5.9 3.7 2.4 2.2
3 62.2
4 69.3
mv 67.2 57.7 43.6 34.7 27.3 14.7 5.5 3.5 2.6 2.2
C14 BKC
(LC-FSA) 1 24.0 22.5 19.9 20.0 17.2 11.6 4.5 3.1 2.6 2.2
2 24.8 23.8 21.5 18.3 17.4 11.7 5.1 3.2 2.4 2.0
3 22.1
4 25.2
mv 24.1 23.2 20.7 19.1 17.3 11.6 4.8 3.1 2.5 2.1
Total Radio-activity Extract 1 92.6 90.3 88.6 89.4 84.1 88.8 43.0 26.7 15.9 10.4
2 97.9 100.1 83.9 96.8 94.9 85.0 54.1 29.8 14.6 10.5
3 87.5
4 98.5
mv 94.1 95.2 86.3 93.1 89.5 86.9 48.5 28.2 15.2 10.5
NER
1 6.1 7.2 6.8 8.1 9.3 14.0 27.2 29.4 27.2 24.7
2 4.9 7.2 7.3 8.3 10.6 15.6 25.0 27.9 28.3 25.1
3 6.0
4 6.1
mv 5.8 7.2 7.1 8.2 9.9 14.8 26.1 28.7 27.7 24.9
14CO2 1 n.d. 0.3 0.4 0.7 0.7 2.4 19.3 36.9 49.4 56.2
2 n.d. 0.3 0.5 0.7 0.8 2.6 19.6 35.4 48.3 56.1
mv - 0.3 0.5 0.7 0.7 2.5 19.5 36.2 48.8 56.1
Volatiles 1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 2 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. mv n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n. d.
Mass Balance 1 98.6 97.9 95.9 98.2 94.1 105.2 89.5 93.0 92.4 91.3
2 102.8 107.6 91.8 105.8 106.3 103.3 98.7 93.1 91.2 91.8
3 93.5
4 104.6
mv 99.9 102.7 93.8 102.0 100.2 104.2 94.1 93.0 91.8 91.5
BKC = [ring-U-14C]Benzalkonium chloride
NER = Non-Extractable Residues
LOD = Limit of Detection
1) = at test start, four samples of the soil batch were taken for analysis
mv = mean value n.d. = not determined

Degradation of [ring-U-14C]Benzalkonium chloride in Soil 2.4, expressed as Percentage of Total Applied Radioactivity [% AR]
Soil 2.4
Sampling Interval (Days)
Repl. 01) 2 3 6 8 13 20 56 87 128
C12 BKC
(LC-FSA) 1 (41.5)* 54.9 39.1 33.9 24.4 18.4 9.9 4.5 2.6 2.3
2 66.7 54.1 43.0 29.2 32.0 17.3 11.9 4.1 2.7 2.5
3 66.5
4 73.9
mv 69.0 54.5 41.0 31.5 28.2 17.8 10.9 4.3 2.6 2.4
C14 BKC
(LC-FSA) 1 (14.7)* 24.3 18.7 18.2 13.9 12.0 7.1 3.5 2.4 1.8
2 23.0 24.4 20.6 16.3 18.9 11.5 7.9 3.2 2.2 2.3
3 23.4
4 26.3
mv 24.3 24.4 19.7 17.2 16.4 11.7 7.5 3.3 2.3 2.0
Total Radio-activity Extract 1 (51.3)* 93.3 77.6 76.5 68.3 59.8 41.2 20.7 13.6 12.1
2 92.3 93.8 82.5 72.6 91.7 67.3 50.6 20.2 13.5 11.8
3 90.3
4 100.3
mv 94.3 93.5 80.0 74.6 80.0 63.6 45.9 20.5 13.5 12.0
NER 1 5.4 10.7 14.4 18.6 21.3 25.2 30.6 29.5 28.9 30.9
2 6.1 12.4 15.3 19.0 24.1 32.6 26.9 31.0 29.6 31.5
3 4.7
4 6.9
mv 5.8 11.5 14.8 18.8 22.7 28.9 28.8 30.3 29.3 31.2
14CO2 1 n.d. 0.5 0.6 1.6 2.6 6.1 12.8 37.6 43.0 51.5
2 n.d. 0.5 0.6 1.5 2.3 6.7 13.5 35.6 43.1 49.6
mv n.d. 0.5 0.6 1.6 2.5 6.4 13.2 36.6 43.1 50.6
Volatiles 1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 2 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. mv n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
Mass Balance 1 (56.7) 104.4 92.5 96.8 92.2 91.1 84.6 87.9 85.5 94.6
2 98.4 106.8 98.4 93.2 118.1 106.5 91.0 86.8 86.1 92.9
3 95.0
4 107.2
mv 100.2 105.6 95.5 95.0 105.2 98.8 87.8 87.4 85.9 93.7
BKC = [ring-U-14C]Benzalkonium chloride
NER = Non-Extractable Residues
LOD = Limit of Detection
1) = at test start, four samples of the soil batch were taken for analysis
mv = mean value n.d. = not determined
()* = possibly failure during extraction, replicate not taken into account for mean value calculation
( ) replicate not taken into account for mass balance calculation

Degradation of [ring-U-14C]Benzalkonium chloride in Soil 5M, expressed as Percentage of Total Applied Radioactivity [% AR]
Soil 5M
Sampling Interval (Days)
Repl. 01) 1 3 7 11 21 36 60 94 120
C12 BKC
(LC-FSA) 1 68.6 60.4 49.6 34.5 25.4 16.5 8.4 5.8 3.8 3.8
2 64.6 63.9 48.9 34.6 27.8 15.4 8.2 5.1 3.9 4.0
3 68.1
4 72.0
mv 68.3 62.1 49.3 34.5 26.6 15.9 8.3 5.4 3.9 3.9
C14 BKC
(LC-FSA) 1 24.6 22.4 19.9 19.3 16.1 12.7 8.2 6.0 4.2 3.4
2 22.1 24.0 21.2 18.4 17.5 13.5 8.3 5.5 4.4 4.6
3 23.0
4 25.5
mv 23.8 23.2 20.6 18.9 16.8 13.1 8.3 5.8 4.3 4.0
Total Radio-activity Extract 1 90.6 85.1 79.0 72.1 70.9 59.9 39.1 28.1 21.2 17.7
2 89.6 95.6 84.6 77.9 75.9 59.3 40.0 27.6 18.5 21.6
3 97.7
4 97.4
mv 93.8 90.3 81.8 75.0 73.4 59.6 39.6 27.9 19.8 19.7
NER 1 9.7 11.3 13.5 19.0 23.4 26.3 33.8 31.1 33.0 29.4
2 12.5 8.9 15.0 19.7 23.5 28.6 31.9 32.6 30.1 29.5
3 4.4
4 9.8
mv 9.1 10.1 14.2 19.3 23.4 27.5 32.9 31.8 31.6 29.4
14CO2 1 n.d. 0.3 0.6 1.8 3.8 10.5 21.6 32.9 41.7 44.1
2 n.d. 0.3 0.6 1.8 3.9 10.7 21.4 32.1 40.7 45.7
mv n.d. 0.3 0.6 1.8 3.9 10.6 21.5 32.5 41.2 44.9
Volatiles 1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 2 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. mv n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
Mass Balance 1 100.3 96.7 93.1 92.9 98.1 96.8 94.5 92.2 95.8 91.3
2 102.1 104.7 100.2 99.4 103.3 98.6 93.3 92.3 89.3 96.8
3 102.1
4 107.3
mv 103.0 100.7 96.7 96.1 100.7 97.7 93.9 92.3 92.6 94.0
BKC = [ring-U-14C]Benzalkonium chloride
NER = Non-Extractable Residues
LOD = Limit of Detection
1) = at test start, four samples of the soil batch were taken for analysis
mv = mean value n.d. = not determined



Characterization of Non-Extractable Residues (NER)
The non-extractable residues from the last sampling of each soil were subjected to organic matter fractionation in order to measure the radioactivity bound to the humic and fulvic acids as well as to the humin fraction of the soil.

Amounts of 3.4 – 5.1 % AR of the non-extractable fraction were bound to the soluble fraction of humic acids in all four soils.
In soil 2.2 and 2.3 the majority of the non-extractable radioactivity was bound to the soluble fraction of fulvic acids (13.0 – 14.7 % of AR), followed by the insoluble fraction of humins (8.0 – 12.3 % AR). In soil 2.4 the majority of the non-extractable fraction was bound to the insoluble fraction of humins (15.9 and 20.0 % AR), followed by the soluble fraction of fulvic acids (14.2 and 15.3 % AR). In soil 5M comparable amounts were bound to fulvic acids (14.9 and 15.9 % AR) and the humin fraction (12.4 and 16.5 % AR).


Soil Organic Matter Fractionation
Soil
2.2 2.3 2.4 5M
% of AR
Repl. Repl. Repl. Repl.
1 2 1 2 1 2 1 2
Remaining NER in soil after ASE extraction 22.4 20.5 24.7 25.1 30.9 31.5 29.4 29.5
Fulvic acids
Soluble fraction at low pH 13.8 14.7 13.0 13.3 14.2 15.3 14.9 15.9
Humic acids
Soluble fraction at high pH 3.8 4.6 4.8 5.1 3.4 3.6 4.0 4.9
Humin
Insoluble fraction 8.0 8.7 8.5 12.3 20.0 15.9 16.5 12.4
Total NER 25.6 27.9 26.3 30.7 37.6 34.8 35.4 33.2
*Total NER = % AR from Fulvic Acids Fraction + % AR from Humic Acids Fraction + % AR from Humin Fraction


Microbial Biomass
The microbial biomass activity of the control replicates was determined for all soils by measurement of the glucose induced respiration rates at the respective application day, during and at the end of the definite exposure phase of each soil.
At test start the biomass concentration was in the range 2.66 – 3.94 % of the soil organic carbon content.
For soil 2.3 and soil 2.4 an initial decrease of the biomass concentration until day 57 and day 49, respectively, was detected. After that, the biomass concentration remained at that level until test end (day 120 and Day 128, respectively). For soil 2.2 and 5M the biomass concentration decreased evenly over the incubation time but was > 1 % of soil organic content at test end.

At test end, the biomass concentration was in the range 1.46 – 2.62 % of soil organic carbon content in all four soils, indicating that a viable microbial biomass was present throughout the incubation time.

Microbial Biomass Activity of the Controls
Soil Study SR Biomass
day mgO2/(kg DW) % of org. C
2.2 0 387.7 2.66
50 276.4 1.90
122 212.9 1.46
2.3 0 185.1 3.94
57 105.0 2.24
120 109.1 2.32
2.4 0 639.0 3.02
49 521.7 2.46
128 553.9 2.62
5M 0 232.0 3.09
63 188.5 2.51
120 118.8 1.58
SR = Soil Respiration Rate

Results

Table: Soil Parameters

 

Parameter

 

LUFA-soil 2.2

Batch-No. F2.2 0118

 

LUFA-soil 2.2

Batch-No. F2.2 1818

 

LUFA-soil 2.3

Batch-No. F2.3 4317

 

LUFA-soil 2.4

Batch-No. F2.4 0118

 

LUFA-soil 5M

Batch-No. F5M 4217

Sampling depth*

ca. 20 cm

ca. 20 cm

ca. 20 cm

ca. 20 cm

ca. 20 cm

pH-value*

5.6  0.4

5.6  0.4

5.8  0.6

7.4 ± 0.1

7.3 ± 0.1

Maximum water holding capacity* [g/100 g DW]

45.8 ± 1.9

44.8 ± 2.9

35.4 ± 1.0

44.6 ± 2.2

41.6 ± 2.6

Particle size distribution

 

 

 

 

 

(mm)#*

 

 

 

 

 

Sand:

 

 

 

 

 

0.63 - 2.0

0.7 ± 0.2

0.8 ± 0.2

2.8 ± 0.4

1.7 ± 0.2

1.0 ± 0.3

0.2 - 0.63

40.2 ± 1.7

41.0 ± 1.5

29.8 ± 1.0

5.9 ± 0.5

13.6 ± 1.2

0.063 - 0.2

34.8 ± 3.0

35.3 ± 2.0

24.6 ± 1.0

19.7 ± 1.0

39.7 ± 1.9

Silt:

 

 

 

 

 

0.02 - 0.063

7.5 ± 1.6

6.9 ± 0.7

18.5 ± 1.1

23.0 ± 1.1

21.3 ± 1.5

0.006 - 0.02

5.2 ± 0.8

4.8 ± 0.4

11.4 ± 0.7

15.2 ± 1.0

9.4 ± 0.7

0.002 - 0.006

3.0 ± 1.2

2.8 ± 0.9

5.3 ± 0.7

7.9 ± 0.4

3.8 ± 1.1

Clay:

 

 

 

 

 

< 0.002

8.6 ± 1.2

8.3 ± 0.9

7.6 ± 0.4

26.6 ± 0.6

11.2 ± 0.9

Organic carbon content

(%)1

 

1.70

 

0.939

 

0.55

 

2.47

 

0.88

Microbial biomass (%) of total organic carbon2)

2.66

2.65

3.94

3.02

3.09

Cation exchange capacity* [meq/100 g]*

 

9.8 ± 0.5

 

9.2 ± 1.4

 

7.5 ± 0.8

 

26.5 ± 15.5

 

15.7 ± 5.3

Weight per Volume (g/1000 mL)*

 

1201 ± 41

 

1205 ± 41

 

1307 ± 41

 

1251 ± 39

 

1221 ± 72

 

Soil texture*

loamy sand (lS)#

loamy sand (lS)#

silty sand (uS)#

clayey loam (tL)#

loamy sand (IS)#

Sampling date

2018-01-05

2018-05-02

2017-10-23

2018-01-05

2017-10-16

*) data provided by LUFA SPEYER       

1) data determined by AGROLAB AGRAR UND UMWELT GMBH (non GLP) #) acc. to German DIN classification           

2) determined at the respective application day of the respective soil

 

 

Soil site

2.2 Hanhofen, Großer Striet, Nr. 585, Rheinland-Pfalz, Germany

2.3: Offenbach, Rechts der Landauer Str., Nr. 826/7, Rheinland- Pfalz, Germany

2.4: Leimersheim, Hoher Weg, Nr. 3138, Rheinland-Pfalz, Germany

5M: Mechtersheim, In der Speyerer Hohl, Nr. 977, Rheinland- Pfalz, Germany

 

Table: Soil History (Crop Rotation and Fertilization

Soil

2.2

2.3

2.4

5M

Crop Rotation by year

 

2017

 

meadow

 

uncultivated

meadow with apple trees

 

meadow

 

2016

 

meadow

 

uncultivated

meadow with apple trees

 

meadow

 

2015

 

meadow

 

uncultivated

meadow with apple trees

 

meadow

2014

meadow

uncultivated

meadow with

apple trees

meadow

 

2013

 

meadow

 

uncultivated

meadow with apple trees

 

meadow

Fertilization by year

2017

none

none

none

none

2016

none

none

none

none

2015

none

none

none

none

 

 

 

 

2014

 

 

2000 kg/ha CaO

833 kg/ha MgO

(2014-12-15)

3500 kg/ha CaO

(2014-06-05)

3500 kg/ha CaO

(2014-09-24)

1463 kg/haMgO

3500 kg/haCaO

(2014-12-15)

 

 

 

 

none

 

 

 

 

none

2013

none

none

none

none

 

Pesticides        

No crop protection products applied during sampling year and four former years.

 

Microbial Biomass

The microbial biomass activity of the control replicates was determined for all soils by measurement of the glucose induced respiration rates at the respective application day, during and at the end of the definite exposure phase of each soil. At test start the biomass concentration was in the range 2.66 – 3.94 % of the soil organic carbon content. For soil 2.3 and soil 2.4 an initial decrease of the biomass concentration until Day 57 and Day 49, respectively, was detected. After that, the biomass concentration remained at that level until test end (Day 120 and Day 128, respectively). For soil 2.2 and 5M the biomass concentration decreased evenly over the incubation time but was > 1 % of soil organic content at test end. At test end, the biomass concentration was in the range 1.46 – 2.62 % of soil organic carbon content in all four soils, indicating that a viable microbial biomass was present throughout the incubation time.

 

Table: Microbial Biomass Activity of the Controls

Soil

Study day

SR

mgO2/(kg DW)

Biomass

% of org. C

 

0

387.7

2.66

2.2

50

276.4

1.90

 

122

212.9

1.46

 

0

185.1

3.94

2.3

57

105.0

2.24

 

120

109.1

2.32

 

0

639.0

3.02

2.4

49

521.7

2.46

 

128

553.9

2.62

 

0

232.0

3.09

5M

63

188.5

2.51

 

120

118.8

1.58

 

SR = Soil Respiration Rate

 

Stability (Radiochemical Purity) of the Test Substance (Application Solution)

LC-FSA analysis of the application solutions prior to experimental starting confirmed the stability of the test substance with a radiochemical purity of 99.1 % (74.4 % C12 and 24.7 % C14) and 96.3 % (70.3 % C12 and C14 25.9 % C14).

 

Homogeneity after Application

Five samples of 0.6 g (fresh weight) of the total applied soil batch were taken directly after application. The samples were combusted and analyzed by LSC. The results show a good homogeneity for soils 2.2 and 2.3 after mixing (CV < 10). The higher variation (CV > 10 %) of soil 2.4 and 5M was associated with the specific soil characteristics (carbon and clay/silt content). Due to the high clay content the soil formed larger agglomerations during mixing, making the sampling of representative 0.6 g samples for combustion challenging. Therefore, the higher variation was accepted for soils 2.4 and 5M. Furthermore, for ASE extraction 15 g soil (DW) was sampled, so that the agglomerating was not influencing the homogeneity of the samples during the study.

 

 

Table:Homogeneity of the Soils after Application

 

 

Sample No.

 

Soil 2.2

 

Soil 2.3

 

Soil 2.4

 

Soil 5M

 

% of AR

1

111.8

113.6

97.1

82.1

2

90.5

103.6

112.6

103.1

3

103.2

106.1

101.4

80.9

4

107.4

104.6

129.7

116.2

5

107.6

93.7

85.7

116.3

Mean

104.1

104.3

105.3

99.7

SD

8.2

7.1

16.7

17.5

CV

7.9

8.2

15.8

17.5

AR = Applied Radioactivity       

SD = Standard Deviation

CV = Coefficient of Variation

 

Mass Balance

At test start the mass balance was in the range 99.9 – 103.0 % for all four soils. Mass balances were > 90 % throughout the duration of the study for soil 2.2, 2.3 and 5M, only single replicates were < 90 % (86.5 %, 89.3%, 89.5 %). For soil 2.4 the mass balance was > 90 % until the start of mineralization. During the initial phase with high mineralization, the mass balance decreased to 85.9 – 87.8 %. Due to the high clay content of the soil slight amounts of 14CO2might have been entrapped in the soil and were not captured in the soda lime. At test end mass balance was in the range 90.4 – 94.0 % for all soils.

 

Transformation of [ring-U-14C]Benzalkonium chloride

Transformation of the C12 chain of the test substance [ring-U-14C]Benzalkonium chloride was rapid in all four soils. The transformation of the C14 chain started after short adaptation phase but was thereafter rapid as well. Within 7 – 21 days the concentration of the C12 chain decreased from initially 67.2 – 69.6 % to < 20 %. The concentration of the C14 chain decreased from initially 23.8 – 24.6 % to < 10 % within 10 – 36 days. Distinct mineralization started between Day 8 and Day 21. Within this period the 14CO2increased for all four soils to > 10 %. In the course of the study the 14CO2formation increased steadily, and the evolved 14CO2reached 44.9 – 56.1 % AR at the end of the respective incubation period. The formation of NER started directly after application of the test substance. Within the course of the study formation of NER increased in parallel to the start of increased mineralisation, indicating that a major amount of NER is comprised by radioactivity incorporated in microbial biomass. For soil 2.2 NER formation was lowest compared to the other test soils and was < 10 % AR until Day 10. Thereafter the NER increased in parallel to 14CO2formation and reached a plateau after 74 days (20.8 – 21.5 % AR). For soil 2.3 and soil 5M NER reached a maximum of 28.7 – 32.9 % AR within 36 – 46 days  of incubation. Thereafter, the NER decreased relative to the maximum NER, simultaneously to further 14CO2formation, and was in a range of 24.9 and 29.4 % AR at test end. This indicated that a fraction of radioactivity which was bound in biomass was remobilised and mineralized by the soil microbacteria. For soil 2.4 NER increased to 22.7 % until day 8, during start of 14CO2formation in soil 2.4 (Day 13) the NER increased to 28.9 % AR and varied thereafter within this range (28.8 – 31.2 % AR) until the end of the incubation phase. No radioactivity (all samples < LSC background) was determined in the polyurethane foam traps, indicating that no volatile organic transformation products were formed.

 

Table:Degradation of [ring-U-14C]Benzalkonium chloride in Soil 2.2, expressed as Percentage of Total Applied Radioactivity [% AR]

 

 

 

 

Repl.

Soil 2.2 Sampling Interval (Days)

01)       1         2         3         4         7         10       21        74       122

 

1

71.2

38.1

32.5

24.9

20.4

13.2

8.3

5.1

2.1

1.7

 

C12 BKC (LC-FSA)

2

3

4

68.7

70.2

68.1

42.8

34.7

23.8

21.2

12.0

9.1

4.3

2.4

1.7

 

mv

69.6

40.5

33.6

24.3

20.8

12.6

8.7

4.7

2.2

1.7

 

1

23.9

18.4

18.8

16.7

14.3

9.7

8.1

4.4

1.6

1.0

 

C14 BKC (LC-FSA)

2

3

4

25.6

24.1

25.0

21.1

19.3

16.5

15.2

10.1

8.1

3.6

1.8

0.7

 

mv

24.6

19.8

19.1

16.6

14.8

9.9

8.1

4.0

1.7

0.9

 

1

90.1

93.5

95.6

87.6

91.2

87.8

88.9

71.7

32.4

22.2

Total

Radio-

activity Extract

2

3

4

98.6

98.1

98.5

88.4

94.9

93.6

93.8

92.3

87.4

73.4

34.2

18.1

 

mv

96.3

90.9

95.3

90.6

92.5

90.0

88.1

72.5

33.3

20.1

 

1

4.1

9.3

6.0

6.7

6.5

8.4

8.5

12.8

20.3

22.5

 

2

4.7

5.2

6.1

7.2

6.6

7.7

9.7

12.1

21.3

20.5

NER

3

5.5

 

 

 

 

 

 

 

 

 

 

4

4.0

 

 

 

 

 

 

 

 

 

 

mv

4.5

7.2

6.1

7.0

6.5

8.0

9.1

12.5

20.8

21.5

 

1

n.d.

0.6

0.8

0.9

1.3

2.3

3.6

11.5

39.5

49.5

14CO2

2

n.d.

0.7

0.6

0.7

1.3

2.3

3.2

11.0

39.2

48.0

 

mv

n.d.

0.7

0.7

0.8

1.3

2.3

3.4

11.3

39.3

48.8

 

1

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

<LOD

Volatiles

2

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

<LOD

 

mv

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

 

1

94.1

103.5

102.5

95.2

99.0

98.5

101.0

96.0

92.3

94.2

Mass Balance

2

3

4

103.2

103.5

102.5

94.2

101.7

101.5

101.7

102.3

100.3

96.5

94.6

86.5

 

mv

100.8

98.9

102.1

98.4

100.3

100.4

100.6

96.2

93.4

90.4

 

BKC    = [ring-U-14C]Benzalkonium chloride

NER    = Non-Extractable Residues

LOD    = Limit of Detection

= at test start, four samples of the soil batch were taken for analysis

mv       = mean value   

n.d.     = not determined

 

Table:Degradation of [ring-U-14C]Benzalkonium chloride in Soil 2.3, expressed as Percentage of Total Applied Radioactivity [% AR]

 

 

 

 

Repl.

Soil 2.3 Sampling Interval (Days)

01)        1          2         3          4         8         22        46        80       120

 

1

67.6

56.6

41.5

35.2

26.8

14.6

5.2

3.4

2.8

2.2

 

C12 BKC (LC-FSA)

2

3

4

69.6

62.2

69.3

58.9

45.8

34.1

27.8

14.8

5.9

3.7

2.4

2.2

 

mv

67.2

57.7

43.6

34.7

27.3

14.7

5.5

3.5

2.6

2.2

 

1

24.0

22.5

19.9

20.0

17.2

11.6

4.5

3.1

2.6

2.2

 

C14 BKC (LC-FSA)

2

3

4

24.8

22.1

25.2

23.8

21.5

18.3

17.4

11.7

5.1

3.2

2.4

2.0

 

mv

24.1

23.2

20.7

19.1

17.3

11.6

4.8

3.1

2.5

2.1

 

1

92.6

90.3

88.6

89.4

84.1

88.8

43.0

26.7

15.9

10.4

Total Radio- activity Extract

2

3

4

97.9

87.5

98.5

100.1

83.9

96.8

94.9

85.0

54.1

29.8

14.6

10.5

 

mv

94.1

95.2

86.3

93.1

89.5

86.9

48.5

28.2

15.2

10.5

 

1

6.1

7.2

6.8

8.1

9.3

14.0

27.2

29.4

27.2

24.7

 

NER

2

3

4.9

6.0

7.2

7.3

8.3

10.6

15.6

25.0

27.9

28.3

25.1

 

4

6.1

 

 

 

 

 

 

 

 

 

 

mv

5.8

7.2

7.1

8.2

9.9

14.8

26.1

28.7

27.7

24.9

 

1

n.d.

0.3

0.4

0.7

0.7

2.4

19.3

36.9

49.4

56.2

14CO2

2

n.d.

0.3

0.5

0.7

0.8

2.6

19.6

35.4

48.3

56.1

 

mv

-

0.3

0.5

0.7

0.7

2.5

19.5

36.2

48.8

56.1

 

1

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

<LOD

Volatiles

2

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

<LOD

 

mv

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n. d.

 

1

98.6

97.9

95.9

98.2

94.1

105.2

89.5

93.0

92.4

91.3

 

Mass Balance

2

3

4

102.8

93.5

104.6

107.6

91.8

105.8

106.3

103.3

98.7

93.1

91.2

91.8

 

mv

99.9

102.7

93.8

102.0

100.2

104.2

94.1

93.0

91.8

91.5

 

BKC    = [ring-U-14C]Benzalkonium chloride

NER    = Non-Extractable Residues

LOD    = Limit of Detection

= at test start, four samples of the soil batch were taken for analysis

mv       = mean value   

n.d.     = not determined

 

Table:Degradation of [ring-U-14C]Benzalkonium chloride in Soil 2.4, expressed as Percentage of Total Applied Radioactivity [% AR]

 

 

 

 

Repl.

Soil 2.4 Sampling Interval (Days)

01)       2        3         6         8        13       20       56       87      128

 

1

(41.5)*

54.9

39.1

33.9

24.4

18.4

9.9

4.5

2.6

2.3

 

C12 BKC (LC-FSA)

2

3

4

66.7

66.5

73.9

54.1

43.0

29.2

32.0

17.3

11.9

4.1

2.7

2.5

 

mv

69.0

54.5

41.0

31.5

28.2

17.8

10.9

4.3

2.6

2.4

 

1

(14.7)*

24.3

18.7

18.2

13.9

12.0

7.1

3.5

2.4

1.8

 

C14 BKC (LC-FSA)

2

3

4

23.0

23.4

26.3

24.4

20.6

16.3

18.9

11.5

7.9

3.2

2.2

2.3

 

mv

24.3

24.4

19.7

17.2

16.4

11.7

7.5

3.3

2.3

2.0

 

1

(51.3)*

93.3

77.6

76.5

68.3

59.8

41.2

20.7

13.6

12.1

Total Radio-

2

92.3

93.8

82.5

72.6

91.7

67.3

50.6

20.2

13.5

11.8

activity

3

90.3

 

 

 

 

 

 

 

 

 

Extract

4

100.3

 

 

 

 

 

 

 

 

 

 

mv

94.3

93.5

80.0

74.6

80.0

63.6

45.9

20.5

13.5

12.0

 

1

5.4

10.7

14.4

18.6

21.3

25.2

30.6

29.5

28.9

30.9

 

2

6.1

12.4

15.3

19.0

24.1

32.6

26.9

31.0

29.6

31.5

NER

3

4.7

 

 

 

 

 

 

 

 

 

 

4

6.9

 

 

 

 

 

 

 

 

 

 

mv

5.8

11.5

14.8

18.8

22.7

28.9

28.8

30.3

29.3

31.2

 

1

n.d.

0.5

0.6

1.6

2.6

6.1

12.8

37.6

43.0

51.5

14CO2

2

n.d.

0.5

0.6

1.5

2.3

6.7

13.5

35.7

43.1

49.6

 

mv

n.d.

0.5

0.6

1.6

2.5

6.4

13.2

36.6

43.1

50.6

 

1

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

<LOD

Volatiles

2

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

<LOD

 

mv

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

 

1

(56.7)

104.4

92.5

96.8

92.2

91.1

84.6

87.9

85.5

94.6

 

2

98.4

106.8

98.4

93.2

118.1

106.5

91.0

86.8

86.1

92.9

Mass Balance

3

95.0

 

 

 

 

 

 

 

 

 

 

4

107.2

 

 

 

 

 

 

 

 

 

 

mv

100.2

105.6

95.5

95.0

105.2

98.8

87.8

87.4

85.9

93.7

BKC = [ring-U-14C]Benzalkonium chloride

NER= Non-Extractable Residues

LOD = Limit of Detection

1) = at test start, four samples of the soil batch were taken for analysis

mv       = mean value   

n.d.     = not determined

()* = possibly failure during extraction, replicate not taken into account for mean value calculation 

( ) replicate not taken into account for mass balance calculation

 

 

Table:Degradation of [ring-U-14C]Benzalkonium chloride in Soil 5M, expressed as Percentage of Total Applied Radioactivity [% AR]

 

 

 

 

Repl.

Soil 5M Sampling Interval (Days)

01)       1         3         7         11        21       36        60       94       120

 

1

68.6

60.4

49.6

34.5

25.4

16.5

8.4

5.8

3.8

3.8

 

C12 BKC (LC-FSA)

2

3

4

64.6

68.1

72.0

63.9

48.9

34.6

27.8

15.4

8.2

5.1

3.9

4.0

 

mv

68.3

62.1

49.3

34.5

26.6

15.9

8.3

5.4

3.9

3.9

 

1

24.6

22.4

19.9

19.3

16.1

12.7

8.2

6.0

4.2

3.4

 

C14 BKC (LC-FSA)

2

3

4

22.1

23.0

25.5

24.0

21.2

18.4

17.5

13.5

8.3

5.5

4.4

4.6

 

mv

23.8

23.2

20.6

18.9

16.8

13.1

8.3

5.8

4.3

4.0

 

1

90.6

85.1

79.0

72.1

70.9

59.9

39.1

28.1

21.2

17.7

Total

Radio- activity Extract

2

3

4

89.6

97.7

97.4

95.6

84.6

77.9

75.9

59.3

40.0

27.6

18.5

21.6

 

mv

93.8

90.3

81.8

75.0

73.4

59.6

39.6

27.9

19.8

19.7

 

1

9.7

11.3

13.5

19.0

23.4

26.3

33.8

31.1

33.0

29.4

 

2

12.5

8.9

15.0

19.7

23.5

28.6

31.9

32.6

30.1

29.5

NER

3

4.4

 

 

 

 

 

 

 

 

 

 

4

9.8

 

 

 

 

 

 

 

 

 

 

mv

9.1

10.1

14.2

19.3

23.4

27.5

32.9

31.8

31.6

29.4

 

1

n.d.

0.3

0.6

1.8

3.8

10.5

21.7

32.9

41.7

44.1

14CO2

2

n.d.

0.3

0.6

1.8

4.0

10.7

21.4

32.1

40.7

45.7

 

mv

n.d.

0.3

0.6

1.8

3.9

10.6

21.5

32.5

41.2

44.9

 

1

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

<LOD

Volatiles

2

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

<LOD

 

mv

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

 

1

100.3

96.7

93.1

92.9

98.1

96.8

94.5

92.2

95.8

91.3

 

Mass Balance

2

3

4

102.1

102.1

107.3

104.7

100.2

99.4

103.3

98.6

93.3

92.3

89.3

96.8

 

mv

103.0

100.7

96.7

96.1

100.7

97.7

93.9

92.3

92.6

94.0

 

BKC = [ring-U-14C]Benzalkonium chloride

NER= Non-Extractable Residues

LOD = Limit of Detection

1) = at test start, four samples of the soil batch were taken for analysis

mv       = mean value   

n.d.     = not determined

 

Characterization of Non-Extractable Residues (NER)

The non-extractable residues from the last sampling of each soil were subjected to organic matter fractionation in order to measure the radioactivity bound to the humic and fulvic acids as well as to the humin fraction of the soil. Amounts of 3.4 – 5.1 % AR of the non-extractable fraction were bound to the soluble fraction of humic acids in all four soils. In soil 2.2 and 2.3 the majority of the non-extractable radioactivity was bound to the soluble fraction of fulvic acids (13.0 – 14.7 % of AR), followed by the insoluble fraction of humins (8.0 – 12.3 % AR). In soil 2.4 the majority of the non-extractable fraction was bound to the insoluble fraction of humins (15.9 and 20.0 % AR), followed by the soluble fraction of fulvic acids (14.2 and 15.3 % AR). In soil 5M comparable amounts were bound to fulvic acids (14.9 and 15.9 % AR) and the humin fraction (12.4 and 16.5 % AR).

 

Soil Organic Matter Fractionation

 

 

 

2.2

 

 

2.3

Soil

% of AR

 

 

2.4

 

 

5M

Repl.

Repl.

Repl.

Repl.

1

2

1

2

1

2

1

2

Remaining NER in soil after ASE extraction

 

22.5

 

20.5

 

24.7

 

25.1

 

30.9

 

31.5

 

29.4

 

29.5

Fulvic Acids

Soluble fraction at low pH

 

13.8

 

14.7

 

13.0

 

13.3

 

14.2

 

15.3

 

14.9

 

15.9

Humic Acids

Soluble fraction at high pH

 

3.8

 

4.6

 

4.8

 

5.1

 

3.4

 

3.6

 

4.0

 

4.9

Humin

Insoluble fraction

 

8.0

 

8.7

 

8.5

 

12.3

 

20.0

 

15.9

 

16.5

 

12.4

 

Total NER

 

25.6

 

27.9

 

26.2

 

30.7

 

37.6

 

34.8

 

35.4

 

33.2

* Total NER = % AR from Fulvic Acids Fraction + % AR from Humic Acids Fraction + % AR from Humin Fraction

 

 

Formation of Metabolites and Transformation Pathway

 

Metabolites determined during the Study by Radio-HPLC

Metabolites determined in soil extracts by radio-HPLC are given as % of AR. Only metabolites detected at least once at > 1 % AR in both replicates were considered for further evaluation. Metabolites which were detected at ≥ 5 % AR at two consecutive sampling intervals or ≥ 10% AR at any sampling were identified by LC-HRMS. Extracts from soil 2.2 were used for metabolite identification and for evaluation of the transformation pathway. The maximum amount of all metabolites was determined until Day 21, thereafter all metabolites decreased steadily until the end of the study.

 

Table: Soil 2.2: Distribution of Metabolites as Percent of Applied Radioactivity

 

 

RT

 

(min)

 

 

 

 

Repl.

% of Applied Radioactivity

 

Sampling Day

0

1

2

3

4

7

10

21

74

122

 

 

8.13 - 9.40

 

 

1

2

Primary Metabolites

n.d.  n.d.

n.d.  n.d.

14.1

16.9

32.2

31.5

42.1

42.3

52.4

53.7

63.6

66.4

65.5

65.6

60.9

61.7

24.9

25.1

12.9

11.1

10.00 - 10.93

1

2

n.d.  n.d.

n.d.  n.d.

1.4

1.0

1.7

1.8

1.3

1.6

1.3

1.1

n.d.

n.d.

2.0

n.d.

n.d.

0.4

n.d.

n.d.

n.d.

n.d.

11.07 - 11.27

1

2

n.d.  n.d.

n.d.  n.d.

8.4

9.2

8.2

7.7

6.5

6.3

5.3

5.5

3.4

3.5

n.d.

2.3

0.4

n.d.

n.d.

n.d.

n.d.

n.d.

RT = Retention time in minutes 

n.d. = not detected

 

Table: Soil 2.3: Distribution of Metabolites as Percent of Applied Radioactivity

 

RT

(min)

 

 

Repl.

% of Applied Radioactivity

Sampling Day

0

1

2

3

4

8

22

46

80

120

 

 

8.13 - 9.80

 

1

2

Primary Metabolites

n.d.   n.d.

n.d.   n.d.

2.9

5.5

16.2

17.4

29.9

30.6

37.2

36.1

54.4

52.6

32.1

37.3

17.8

18.0

7.5

6.9

3.9

4.2

10.00 - 10.93

1

2

n.d.   n.d.

n.d.   n.d.

5.5

6.3

6.8

7.8

5.4

6.1

4.0

2.6

1.4

1.5

0.3

0.2

n.d.

n.d.

n.d.

n.d.

0.2

0.1

18.47 - 19.00

1

2

n.d.   n.d.

n.d.   n.d.

0.9

1.0

0.8

1.4

1.1

0.2

0.9

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

RT = Retention time in minutes 

n.d. = not detected

 

Table: Soil 2.4: Distribution of Metabolites as Percent of Applied Radioactivity

 

RT

(min)

 

 

Repl.

% of Applied Radioactivity

Sampling Day

0

2

3

6

8

13

20

56

87

128

 

 

7.47 - 9.40

 

1

2

Primary Metabolites

n.d.  n.d.

n.d.  n.d.

7.8

6.3

10.1

10.6

20.5

20.5

23.4

29.1

26.5

30.1

23.4

25.5

9.7

9.2

6.2

5.5

4.3

4.0

10.00- 10.40

1

2

n.d.  n.d.

n.d.  n.d.

n.d.

n.d.

n.d.

0.9

1.4

1.4

1.4

2.4

1.6

1.4

1.1

1.2

0.8

n.d.

0.2

n.d.

n.d.

n.d.

11.07 - 11.40

1

2

n.d.  n.d.

n.d.  n.d.

7.2

7.0

6.5

6.9

6.5

5.8

5.0

6.4

3.0

3.3

1.4

1.4

0.2

0.2

n.d.

n.d.

n.d.

n.d.

 

RT = Retention time in minutes 

n.d. = not detected

 

Table: Soil 5M: Distribution of Metabolites as Percent of Applied Radioactivity

 

RT

 

(min)

 

 

 

Repl.

% of Applied Radioactivity

Sampling Day

0

1

3

7

11

21

36

60

94

120

 

 

7.60- 8.80

 

1

2

Primary Metabolites

n.d.   n.d.

n.d.   n.d.

1.3

1.1

6.8

5.9

17.6

17.3

22.9

23.7

27.1

26.8

22.6

21.1

14.5

13.4

9.7

8.5

9.1

10.3

10.33 - 10.67

1

2

n.d.   n.d.

n.d.   n.d.

2.3

2.3

3.7

3.9

3.2

3.0

n.d.

2.0

n.d.

n.d.

n.d.

n.d.

0.1

0.1

n.d.

n.d.

n.d.

n.d.

18.07 - 18.87

1

2

n.d.   n.d.

n.d.   n.d.

n.d.

n.d.

1.0

0.9

0.7

1.3

n.d. 1.0

n.d.

n.d.

n.d.

n.d.

0.1

n.d.

n.d. 0.1

n.d.

n.d.

 

RT = Retention time in minutes 

n.d. = not detected

 

Due to the long study duration the retention capacity of the chromatographic column changed. As a result, the retention times shifted slightly within the duration of the study. The correct assignment of the peaks was confirmed by re-analysis of selected samples after end of the study.

 

Metabolite Identification

 

Data Evaluation – LC-HRMS Analysis

Data obtained from the MS scan measurements were evaluated by the software tools Profinder and Mass Profiler Professional. The Profinder prepares the initial data set for the final evaluation with the Mass Profiler Professional. A batch recursive extraction was perfomed with the Profinder. This workflow consists of several steps allowing a refinement of the obtained large data set from the MS scan measurements. First, peaks (features) were extracted from the complex data set by an algorithm. This process is called molecular feature extraction (MFE) and the evaluation yields many individual peaks. The quality of the extracted peaks is described by a score which was used as a filter to exclude peaks with limited quality from the data set. A score of ≥ 70 is a suitable filter for this purpose. The obtained data set was subsequently refined by aligning these peaks and binning peaks with similar molecular weight and retention time. In a third step, the quality of the binning was assessed and improved by a process described as Find by Ion. This process optimizes the peak extraction and calculates a score for the binning by taking into the sameness of the detected mass, isotope abundance and isotope spacing. A threshold filter of 50 was applied for this score. Finally, a peak height filter excluded peaks with a height < 2000. These processed data were then evaluated with the software Mass Profiler Professional. The Mass Profiler investigated in which of the analysed samples the binned peaks occurred and if their occurrence is increasing or decreasing. Furthermore, it is possible to form sample groups. This was done in this case and all control samples were summarized in one group whereas the samples of the test substance replicates were grouped per day of sampling. The data were analysed then with the aim to find compounds which are unique for the samples of the test substance replicates after day 0 or their intensity increase after day 0 and are not present in the control samples. Finally, molecular formulas were generated for the unique peaks. After that, further measurements in the targeted MS/MS mode were performed for the compounds which could be annotated with molecular formulas to obtain mass spectra for them followed by proposing chemical structures for them.

 

Results and Data Assessment – LC-HRMS Analysis

The results of the final data assessment analysis with the Mass Profiler Professional are shown. The data show the occurrence of the unique peaks depending on the sampling day. All compounds were not present in the controls and either not present in samples of Day 0 or their intensity increased after Day 0. Plus, and minus indicate increasing or decreasing peak intensity within two subsequent sampling days.

 

Results Data Assessment Mass Profiler Professional

Samples control group 

n = 8 Samples

 

other groups    

n = 2

 

Metabolite Number

Molecular Formula

Control

Test Substance Day 0

Test Substance Day 3

Test Substance Day 5

Test Substance Day 12

1

C9H13N

0/8

0/2

2/2

2/2+

2/2+

2

C12H17N O2

0/8

0/2

2/2

2/2+

2/2+

3

C13H18N O2

0/8

0/2

2/2

2/2+

2/2+

4

C13H20N O2

0/8

0/2

2/2+

2/2-

2/2-

5

C15H24N O2

0/8

2/2

2/2+

2/2-

2/2-

6

C16H25N O2

0/8

1/2

2/2+

2/2-

2/2-

+/- minus indicate increasing or decreasing peak intensity within two subsequent sampling days

 

Targeted MS/MS experiments were conducted to record their mass spectra and to propose chemical structures for them. Diagnostic ions and the corresponding annotations are given. The diagnostic ions were used for the proposal of chemical structures for the metabolites.

 

Results Data Assessment Targeted MS/MS Experiments

Samples control group 

n = 8

 

Samples other groups  

n = 2

 

Metabolite Number

Molecular Formula

Diagnostic Ion 1

m/z

Annotation 1

Diagnostic Ion 2

m/z

Annotation 2

1

C9H13N

 

 

 

 

91.0542

 

 

 

 

[C7H7]+

--

--

2

C12H17N O2

116.0706

[C5H10NO2]+

3

C13H18N O2

128.0706

[C6H10NO2]+

4

C13H20N O2

130.0863

[C6H12NO2]+

5

C15H24N O2

158.1176

[C8H16NO2]+

6

C16H25N O2

172.1332

[C9H18NO2]+

 

The diagnostic ion, [C7H7]+, is characteristic for molecules with a benzyl unit and is present in all metabolites. The other diagnostic ions in the metabolites26show alkyl chains with varying number of carbon atoms which are oxidized to carboxylic acids. These metabolites have 5 – 6 (C13H18NO2) double bond equivalents (DBE). In contrast, the test substance has only four DBE. The metabolites26indicate that the alkyl chain of the test substance is stepwise degraded after an initial oxidation at the ω-end of the alkyl chain. It is assumed that the alkyl chain is degraded by a β-oxidation removing two carbon atoms form the alkyl chain in each step. The β-oxidation as catabolic pathway is ubiquitous. It is assumed that the metabolites2and6were demethylated. Otherwise, the alkyl chain must be odd numbered and that was assessed to be unlikely. The metabolites5and6are already present in soil extracts of samples from Day 0. This fact shows that the β-oxidation takes place very fast. Dimethylbenzylamine was proposed as chemical structure for metabolite1after oxidative removal of the alkyl chain.

 

 

Confirmatory – LC-MS/MS Analysis

Additional analysis with LC-MS/MS according to the experimental parameters was conducted to link the results of the LC-HRMS analysis with the study results of the radio-HPLC. Dimethylbenzylamine, Methylbenzylamine and Benzylamine were regarded as possible metabolites eluting in the radio-HPLC chromatogram peaks with highest % AR within the retention time window of 7.47 – 9.80 min. Methylbenzylamine was not detected by LC-HRMS as metabolite in the experiments of the previous section but the occurrence of the metabolites2and6indicate that this metabolite may be formed after oxidative degradation of the alkyl chain. Benzylamine was considered as possible metabolite prior to oxidative degradation of the benzyl unit. Based on these considerations, an MS/MS experiment was designed with expected mass transitions for the corresponding 14C-metabolites and 14C-C12 BKC. The results show the most distinct peaks within the retention time window of 6.0 -10.0 min of the LC-MS/MS analysis, matching the peaks with highest % AR of the radio-HPLC analysis. Dimethylbenzylamine and Methylbenzylamine are present as metabolites. Both compounds did not occur in soil extracts of a control and sample of Day 0, but in all further analysed soil extracts. Dimethylbenzylamine is the predominant metabolite whereas Met hylbenzylamine was present only in traces. The highest concentrations of Dimethylbenzylamine were determined between Day 10 and Day 22, thereafter the concentrations deceased continuously until test end. Benzylamine was not detected during this experiment. The peaks determined by radio-HPLC within the retention time window 10.0 – 10.93 min. could be assigned by the LC-MS/MS measurements as metabolites containing a partly degraded alkyl chain by β-oxidation. These metabolites were transient, the highest activity was determined on Day 2 in soil 2.3. Within the course of the study, the activity decreased rapidly and was ≤ 0.2 % AR in soil 2.3 and not detectable in all other soils. Further peaks determined in soil 2.2 and 2.4 by radio-HPLC at retention times later than 11 min. are assumed to be metabolites containing also the partly degradated alkyl chain, but with a higher number of carbon atoms. These metabolites were transient as well, the highest activity was determined up to two day after application. Thereafter the activity decreased continuously and was not determined at samplings beyond Day 74.

 

Kinetic Analysis

The kinetic evaluations were done based on the FOCUS guidance document on estimating persistence and degradation kinetics. The kinetic models were chosen based on the following criteria:

·        Visual assessment of the fitted and observed data versus time

·        Visual assessment of the residuals

·        Estimation of the error percentage at which the x2-test was passed

 

The transformation of [ring-U-14C]Benzalkonium chloride started directly after application with a fast decrease of the C12 chain, the transformation of the C14 chain started after an adaptation phase of approx. 2 days and showed thereafter a fast decrease as well. The transformation showed a slight bi-phasic pattern, therefore the Single First Order Model (SFO) and the First-Order Multi-Compartment Model (FOMC) were compared. Based on the visual fit and x2 error, the transformation of [ring-U-14C]Benzalkonium chloride met the requirements for both models well for all four soils.

 

Table:Data for Kinetic Evaluations: Dissipation of [ring-U-14C]Benzalkonium chloride

 

Soil 2.2

% of Applied Radioactivity Day

 

Repl.

0#

1

2

3

4

7

10

21

74

122

 

C12 BKC

1

2

71.2   68.7

70.2   68.1

38.1

42.8

32.5

34.7

24.9

23.8

20.4

21.2

13.2

12.0

8.3

9.1

5.1

4.3

2.1

2.4

1.7

1.7

 

C14 BKC

1

2

23.9   25.6

24.1   25.0

18.4

21.1

18.8

19.3

16.7

16.5

14.3

15.2

9.7

10.1

8.1

8.1

4.4

3.6

1.6

1.8

1.0

0.7

 

Soil 2.3

% of Applied Radioactivity Day

 

Repl.

0#

1

2

3

4

8

22

46

80

120

 

C12 BKC

1

2

67.6   69.6

62.2   69.3

56.6

58.9

41.5

45.8

35.2

34.1

26.8

27.8

14.6

14.8

5.2

5.9

3.4

3.7

2.8

2.4

2.2

2.2

 

C14 BKC

1

2

24.0   24.8

22.1   25.2

22.5

23.8

19.9

21.5

20.0

18.3

17.2

17.4

11.6

11.7

4.5

5.1

3.1

3.2

2.6

2.4

2.2

2.0

 

Soil 2.4

% of Applied Radioactivity Day

 

Repl.

0#

2

3

6

8

13

20

56

87

128

 

C12 BKC

1

2

-      66.7

66.5   73.9

54.9

54.1

39.1

43.0

33.9

29.2

24.4

32.0

18.4

17.3

9.9

11.9

4.5

4.1

2.6

2.7

2.3

2.5

 

C14 BKC

1

2

-      23.0

23.4   26.3

24.3

24.4

18.7

20.6

18.2

16.3

13.9

18.9

12.0

11.5

7.1

7.9

3.5

3.2

2.4

2.2

1.8

2.3

 

Soil 5M

% of Applied Radioactivity Day

 

Repl.

0#

1

3

7

11

21

36

60

97

120

 

C12 BKC

1

2

68.6   64.6

68.1   72.0

60.4

63.9

49.6

48.9

34.5

34.6

25.4

27.8

16.5

15.4

8.4

8.2

5.8

5.1

3.8

3.9

3.8

4.0

 

C14 BKC

1

2

24.6   22.1

23.0   25.5

22.4

24.0

19.9

21.2

19.3

18.4

16.1

17.5

12.7

13.5

8.2

8.3

6.0

5.5

4.2

4.4

3.4

4.6

 

# at test start, four samples of the soil batch were taken for analysis

 

Table: Kinetic Data for [ring-U-14C]Benzalkonium chloride (C12 chain)

Endpoint / Statistic

Soil 2.2

Soil 2.3

Soil 2.4

Soil 5M

 

Model

 

Single First Order (SFO)

C0(% AR)

66.66

± 2.065

67.55

± 1.366

66.71

± 1.979

66.30

± 1.488

Initial value for fitting

69.55

67.18

69.03

68.33

kp

0.3185

0.2095

0.1125

0.0799

Initial value for fitting

0.1

0.1

0.1

0.1

ffM1

(as a fraction)

 

1

 

1

 

1

 

1

t-test

passed

passed

passed

passed

x2error

 

15.4

 

8.0

 

10.0

 

9.7

Model

 

First-Order Multi-Compartment (FOMC)

C0

69.38

± 0.8287

68.35

± 1.283

69.42

± 1.613

68.77

± 0.7344

Initial value for fitting

69.55

67.18

69.03

68.33

p

 

Initial value for fitting

0.9702

 

 

1

2.012

 

 

1

1.389

 

 

1

1.330

 

 

1

p

 

Initial value for fitting

1.546

 

 

10

7.691

 

 

10

7.996

 

 

10

10.490

 

 

10

t-test

passed

passed

passed

passed

x2error

 

4.8

 

6.7

 

5.7

 

2.3

 

Table:Kinetic Data for [ring-U-14C]Benzalkonium chloride (C14 chain)

Endpoint / Statistic

Soil 2.2

Soil 2.3

Soil 2.4

Soil 5M

Model

 

Single First Order (SFO)

C0(% AR)

23.80

± 0.4955

24.09

± 0.5917

24.57

± 0.8084

22.97

± 0.5151

Initial value for fitting

24.65

24.03

24.23

23.80

kp

0.1142

0.0779

0.05391

0.02413

Initial value for fitting

0.1

0.1

0.1

0.1

ffM1

(as a fraction)

 

1

 

1

 

1

 

1

t-test

passed

passed

Passed

passed

2error

 

7.1

 

8.7

 

9.1

 

7.5

Model

 

First-Order Multi-Compartment (FOMC)

C0

24.53

± 0.4014

24.71

± 0.5057

25.14

± 0.8134

23.83

± 0.3833

Initial value for fitting

24.65

24.03

24.23

23.80

p

 

Initial value for fitting

1.391

 

 

1

1.378

 

 

1

1.682

 

 

1

1.272

 

 

1

p

 

Initial value for fitting

8.447

 

 

10

12.620

 

 

10

23.770

 

 

10

32.160

 

 

10

t-test

passed

passed

passed

passed

2error

 

4.4

 

5.5

 

7.1

 

3.2

 

 

Table:DTx Values for [ring-U-14C]Benzalkonium chloride (C12 chain)

 

 

Endpoint / Statistic

DTxValues in Days

Soil

2.2

2.3

2.4

5M

 

Single First Order (SFO)

DT50

2.2

3.3

6.2

8.7

DT90

7.2

11.0

20.5

28.8

 

First-Order Multi-Compartment (FOMC)

DT50

1.6

3.2

5.2

7.2

DT90

15.0

16.5

34.0

48.8

 

Table: DTx Values for [ring-U-14C]Benzalkonium chloride (C14 chain)

 

 

Endpoint / Statistic

DTxValues in Days

Soil

2.2

2.3

2.4

5M

 

Single First Order (SFO)

DT50

6.1

8.9

12.9

28.7

DT90

20.2

29.6

42.7

95.4

 

First-Order Multi-Compartment (FOMC)

DT50

5.5

8.3

12.1

23.3

DT90

35.8

54.5

69.6

164.3

 

 Conclusions

• Transformation of the C12 chain of the test substance [ring-U-14C]Benzalkonium chloride was rapid in all four soils. The transformation of the C14 chain started after a short adaptation phase but was thereafter rapid as well. Within 7 - 21 days the concentration of the C12 chain decreased from initially 67.2 – 69.6 % of AR to < 20 % of AR. The concentration of the C14 chain decreased from initially 23.8 – 24.6 % of AR to < 10 % of AR within 10 – 36 days.

• Formation of NER started directly after application of the test substance. Further formation of NER increased in parallel to the start of increased mineralisation, indicating that a major amount of NER is comprised by radioactivity incorporated in microbial biomass.

• The mass balance was in the range 99.9 – 103.0 % at test start and 90.4 – 94.0 % at test end.

• The predominant initial degradation step was the oxidative removal of the alkyl chain. Dimethylbenzylamine was determined as the major metabolite, the highest concentrations of Dimethylbenzylamine were determined until Day 22, thereafter the concentrations deceased continuously until test end. Methylbenzylamine was transient and only present in traces. Benzylamine, a suspected metabolite, was not detected. Further metabolites containing partly degraded alkyl chain were all transient and were not detected or only < 0.2 % of AR (soil 2.3) at test end.

• Based on the visual fit and x2 error, the transformation of [ring-U-14C]Benzalkonium chloride met the requirements for both models well for all four soils.

• The calculated DT50 values with the Single-First-Order Model (SFO) for the dissipation of [ring-U-14C]Benzalkonium chloride were 2.2 – 8.7 days (C12 chain) and 6.1 – 28.7 days (C14 chain), the DT90 values were 7.2 – 28.8 (C12 chain) days and 20.2 – 95.4 days (C14 chain).

• The calculated DT50 values with the First-Order Multi-Compartment Model (FOMC) for the dissipation of [ring-U-14C]Benzalkonium chloride were 1.6 – 7.2 days (C12 chain) and 5.5 – 23.3 days (C14 chain) , the DT90 values were 15.0 – 48.8 days (C12 chain) and 35.8 – 164.3 days (C14 chain).

Conclusions:
Under the study conditions, transformations of both C12 and C14 carbon chains of the read across substance were determined to be rapid in all four soils and the DT50 values were determined to be 2.2 – 8.7 days [C12 chain] and 6.1 – 28.7 days [C14 chain] at 20°C with the SFO model and 1.6 – 7.2 days [C12 chain] and 5.5 – 23.3 days [C14 chain] at 20°C with the FOMC model. Further, in the biocides dossier, a weighted estimate of the DT50 value at 12°C was extrapolated for C12-16 ADBAC by assuming the highest allowable concentrations for the major chains. These calculations resulted in the estimated FOMC DT50 of 17.1 days at 12°C and SOF DT50 of 19.2 days at 12°C. The DT50 of 17.1 days at 12°C based on the biphasic model (FOMC) showing better visual fit and lower error (2) was used further for risk assessment.
Executive summary:

A study was conducted to determine the aerobic transformation/dissipation in the soil of the read across substance, C12 -16 ADBAC (radiochemical purity: 98.5%), according to the OECD Guideline 307, in compliance with GLP. Four different standard soils (LUFA 2.2, 2.3, 2.4 and 5M, field fresh sampled), varying in their organic carbon content, pH, clay content, cation exchange capacity and microbial biomass, were treated with [ring-U-14C] Benzalkonium chloride. Soil samples were incubated in the dark under aerobic conditions for up to 128 days under controlled laboratory conditions. After appropriate time intervals, soil samples were extracted, and the extracts were analysed for read across substance and transformation products to calculate DT50 and DT90 values. The mineralization was determined by trapping and analysis of the evolved 14CO2. Non-extractable residues (NER) were determined after combustion of the extracted soil samples. The total radioactivity of the soil extracts, the extracted soil (NER) and evolved 14CO2 was determined by LSC. Read across substance and transformation products in the soil extracts were analysed by LC-FSA (radio-HPLC). Evaluation of the transformation pathway was done by LC-HRMS. Transformation of the C12 chain of the read across substance [ring-U-14C]Benzalkonium chloride was rapid in all four soils. The transformation of the C14 chain started after a short adaptation phase but was thereafter rapid as well. Within 7 - 21 days the concentration of the C12 chain decreased from initially 67.2 – 69.6% of applied radioactivity (AR) to < 20 % of AR. The concentration of the C14 chain decreased from initially 23.8 – 24.6 % of AR to < 10 % of AR within 10 – 36 days. Formation of NER started directly after application of the read across substance. Further formation of NER increased in parallel to the start of increased mineralisation, indicating that a major amount of NER is comprised by radioactivity incorporated in microbial biomass. At the test end, the biomass concentration was in the range of 1.46 – 2.62 % of soil organic carbon content in all four soils, indicating that viable microbial biomass was present throughout the incubation time. The mass balance was in the range 99.9 – 103.0 % at test start and 90.4 – 94.0 % at test end.The predominant initial degradation step was the oxidative removal of the alkyl chain. Dimethylbenzylamine was determined as the major metabolite, the highest concentrations of dimethylbenzylamine were determined until Day 22, thereafter the concentrations deceased continuously until test end. Methylbenzylamine was transient and only present in traces. Benzylamine, a suspected metabolite, was not detected. Further metabolites containing partly degraded alkyl chains were all transient and were not detected or only <0.2 % of AR (soil 2.3) at the test end. With regard to the kinetics, the transformation showed a slight bi-phasic pattern, therefore the ‘Single First Order Model’ (SFO) and the ‘First-Order Multi-Compartment Model’ (FOMC) were compared. Based on the visual fit and x2 error, the transformation of [ring-U-14C]Benzalkonium chloride met the requirements for both models well for all four soils. The calculated DT50 values with the Single-First-Order Model (SFO) for the dissipation of [ring-U-14C]Benzalkonium chloride were 2.2 – 8.7 days (C12 chain) and 6.1 – 28.7 days (C14 chain), the DT90 values were 7.2 – 28.8 (C12 chain) days and 20.2 – 95.4 days (C14 chain). The calculated DT50 values with the FOMC model for the dissipation of [ring-U-14C]Benzalkonium chloride were 1.6 – 7.2 days (C12 chain) and 5.5 – 23.3 days (C14 chain), the DT90 values were 15.0 – 48.8 days (C12 chain) and 35.8 – 164.3 days (C14 chain).

The read across substance is predominantly C12-ADBAC and C14-ADBAC, with low to negligible amounts of C16-ADBAC. The chain length distribution is defined as follows:C12 (35-80%), C14 (20-55%), C16 (0-15%). C16-ADBAC was not included in this study because it is present in very low amounts; there are technical difficulties with having sufficient radioactivity for substances present in small amounts relative to other constituents. C16-ADBAC would be expected to degrade by the same route but at a slower rate than its C12 and C14 counterparts, as degradation rate tends to decrease with increasing chain lengths.Under the study conditions, transformations of both C12 and C14 carbon chains of the read across substance were determined to be rapid in all four soils and the DT50 values were determined to be 2.2 – 8.7 days [C12 chain] and 6.1 – 28.7 days [C14 chain] with the SFO model and 1.6 – 7.2 days [C12 chain] and 5.5 – 23.3 days [C14 chain] with the FOMC modelat 20°C (Fiebig, 2019).

 

Further, in the biocides dossier, to account for the potential contribution of C16 ADBAC to the overall DT50 of ADBAC, a geometric mean of SFO and FOMC DT50s for C12 and C14 ADBAC in the four soils (as recommended in BPR Vol IV Part B and C) was calculated and converted to 12° using the following equation (DT50 (12°) = DT50 (20°) * e(0.08*(20-12)). This was followed by linear extrapolation of the geometric mean DT50s for C12 and C14 ADBAC, to estimate the DT50 for C16 ADBAC. See tables below:

 

Soil 2.2

Soil 2.3

Soil 2.4

Soil 5M

Geo. Mean

Adj. to 12° C

SFO DT50s

C12 ADBAC

2.2

3.3

6.2

8.7

4.4

8.4

C14 ADBAC

6.1

8.9

12.9

28.7

11.9

22.6

C16 ADBAC

--

--

--

--

--

36.7

FOMC DT50s

C12 ADBAC

1.6

3.2

5.8

7.2

3.8

7.3

C14 ADBAC

5.5

8.3

12.1

23.3

10.7

20.2

C16 ADBAC

--

--

--

--

--

33.1

 

A weighted estimate of the DT50 of ADBAC (C12-C16) at 12°C was calculated by assuming the highest allowable concentrations of C14- and C16- ADBAC and the balance of C12-ADBAC (i.e., 12% C16, 52% C14 and 36% C12), which resulted in the following estimated DT50s:

SFO DT50 = 19.2d at 12°C; FOMC DT50 = 17.1d at 12°C

However, due to the relatively low levels of C16-ADBAC, the overall estimated DT50s were considered rather insensitive to the assumed DT50 for C16-ADBAC.The DT50 of 17.1 days at 12°C based on the biphasic model (FOMC) showing better visual fit and lower error was used further for risk assessment. Based on the results of the read across study, similar degradation potential and half-life is considered for the test substance.

Endpoint:
biodegradation in soil: simulation testing
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
The aerobic biodegradation of the substance in loamy soil (at 10 mg carbon per 50 g soil) was determined by measuring carbon dioxide evolution in biometers for 90 days according to the US FDA Environmental Assessment Handbook, Technical Assistance Document 3.12 (1987). The amount of carbon dioxide (CO2) produced by the test substance was calculated by subtracting the mean CO2 production in the test systems containing the test substance and the mean CO2production level in the control blank. The biodegradation was calculated as the ratio of the experimental CO2 production to theoretical CO2 (ThCO2P which was based on the carbon content of the test substance).
GLP compliance:
no
Test type:
laboratory
Oxygen conditions:
aerobic
Soil classification:
other: loam
Details on soil characteristics:
COMPOSITION OF SOIL:
- %OM: 2.4
- % sand: 35.4
- % silt: 49.3
- % clay: 15.3
- CEC: 13.1 meq/100 g
- Additional substrate: No
- Test temperature: 22  3 degree C
- pH: 7.6
- Aeration of dilution water: No
Duration:
90 d
Initial conc.:
10 other: mg C per 50 g soil
Based on:
other: carbon
Parameter followed for biodegradation estimation:
CO2 evolution
Details on experimental conditions:
TEST SYSTEM:
- Culturing apparatus: Slightly modified biometers described by Bartha and Pramer. Both the Erlenmeyer and the side tube were closed with Mininert valves to enable direct sampling from the side tube and to provide pure oxygen in the Erlenmeyer. The Erlenmeyer and the side tube were fused together with an air- tight coupling so that the side tube and the Erlenmeyer could be weighed separately
- Number of culture flasks/concentration: 3
- Measuring equipment: Dohrmann DC-190 NPOC apparatus
- Test performed in closed vessels due to significant volatility of TS: No

INOCULUM:
- Nature: Loam
- Source: Heino, The Netherlands
- Sampling site: The soil was collected from below the litter layer
- Laboratory culture: No
- Preparation of inoculum for exposure: Stones and plant fragments were removed by hand. After collection the soil was air dried for approximately 2 days and sieved through a 2 mm sieve
- Pretreatment: No pretreatment, the soil was stored at room temperature in polyethylene bags until use in the experiment

SAMPLING: Once a week

STATISTICS:
The theoretical CO2 production of the test substance was calculated from the carbon content of the test substance. The amount of carbon dioxide produced by the test substance was calculated by subtracting the mean carbon dioxide production level in the control blank. The biodegradation was calculated as the ratio of experimental dioxide production to the theoretical carbon dioxide production (ThCO2P).


Key result
% Degr.:
64
Parameter:
CO2 evolution
Sampling time:
70 d
Key result
DT50:
40 d
Remarks on result:
other: DT50
Transformation products:
no
Details on results:
The test substance was biodegraded in loam. The percentage reached at Day 70 was 64. This percentage of the theoretical carbon dioxide production presumes complete mineralisation. The DT50 was estimated to be 40d.

No validity criteria are available for this test, but the test was performed according to a standard guideline and therefore considered to be valid without restrictions.

Conclusions:
Under the study conditions, there was 64% degradation of the read across substance after 70 days. This percentage of the theoretical carbon dioxide production presumes complete mineralization. The DT50 was estimated to be 40 days
Executive summary:

A study was conducted to determine the aerobic biodegradation of the read across substance, C12-16 ADBAC (50% active in water) in loamy soil, according to the US FDA Environmental Assessment Handbook, Technical Assistance Document 3.12 (1987). The study comprised two treatments: test and chemical blank control group, each with three replicates. The read across substance was added into biometers at a concentration of 10 mg carbon per 50 g soil using appropriate amount of deionised water required for bringing the soils to 50-70% of the moisture capacity. Loam was added to the biometers after the test solutions to facilitate uniform moistening of the soils by capillary action. The test was then incubated at 22 ± 3°C and run for approximately 90 d. The side tube of the biometer contained 20 mL 0.2 M KOH for absorbing carbon dioxide produced by the microorganisms. The theoretical CO2 production of the read across substance was calculated from its carbon content. The amounts of carbon dioxide were calculated by subtracting the mean carbon dioxide production in the test systems containing the read across substance and the mean carbon dioxide production level in the control blank. Biodegradation was calculated as the ratio of experimental carbon dioxide production to theoretical carbon dioxide production [ThCO2P]. Under the study conditions, there was 64% degradation of the read across substance after 70 days. This percentage of the theoretical carbon dioxide production presumes complete mineralization. The DT50 was estimated to be 40 days (Ginkel, 1994). Based on the results of the read across study, similar degradation potential and half-life is considered for the test substance.  ​

Description of key information

Based on the most recent and radiolabelled aerobic biodegradation study in soil with the read across substance, C12-16 ADBAC, the transformation of the substance was considered to be rapid with DT50 values ranging from 2.2-28.7 days with the SFO model and 1.6 – 23.3 days with the FOMC model at 20°C.​ Further, in the biocides dossier, a weighted estimate of the DT50 value at 12°C was extrapolated for C12-16 ADBAC by assuming the highest allowable concentrations for the major chains. These calculations resulted in the estimated FOMC DT50 of 17.1 days at 12°C and SOF DT50 of 19.2 days at 12°C. The DT50 of 17.1 days at 12°C based on the biphasic model (FOMC) showing better visual fit and lower error (x2)compared to the SFO model was used further for risk assessment. 

Key value for chemical safety assessment

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

Additional information

Study 1: A study was conducted to determine the aerobic transformation/dissipation in the soil of the read across substance, C12 -16 ADBAC (radiochemical purity: 98.5%), according to the OECD Guideline 307, in compliance with GLP. Four different standard soils (LUFA 2.2, 2.3, 2.4 and 5M, field fresh sampled), varying in their organic carbon content, pH, clay content, cation exchange capacity and microbial biomass, were treated with [ring-U-14C] Benzalkonium chloride. Soil samples were incubated in the dark under aerobic conditions for up to 128 days under controlled laboratory conditions. After appropriate time intervals, soil samples were extracted, and the extracts were analysed for read across substance and transformation products to calculate DT50 and DT90 values. The mineralization was determined by trapping and analysis of the evolved 14CO2. Non-extractable residues (NER) were determined after combustion of the extracted soil samples. The total radioactivity of the soil extracts, the extracted soil (NER) and evolved 14CO2 was determined by LSC. Read across substance and transformation products in the soil extracts were analysed by LC-FSA (radio-HPLC). Evaluation of the transformation pathway was done by LC-HRMS. Transformation of the C12 chain of the read across substance [ring-U-14C]Benzalkonium chloride was rapid in all four soils. The transformation of the C14 chain started after a short adaptation phase but was thereafter rapid as well. Within 7 - 21 days the concentration of the C12 chain decreased from initially 67.2 – 69.6% of applied radioactivity (AR) to < 20 % of AR. The concentration of the C14 chain decreased from initially 23.8 – 24.6 % of AR to < 10 % of AR within 10 – 36 days. Formation of NER started directly after application of the read across substance. Further formation of NER increased in parallel to the start of increased mineralisation, indicating that a major amount of NER is comprised by radioactivity incorporated in microbial biomass. At the test end, the biomass concentration was in the range of 1.46 – 2.62 % of soil organic carbon content in all four soils, indicating that viable microbial biomass was present throughout the incubation time. The mass balance was in the range 99.9 – 103.0 % at test start and 90.4 – 94.0 % at test end.The predominant initial degradation step was the oxidative removal of the alkyl chain. Dimethylbenzylamine was determined as the major metabolite, the highest concentrations of dimethylbenzylamine were determined until Day 22, thereafter the concentrations deceased continuously until test end. Methylbenzylamine was transient and only present in traces. Benzylamine, a suspected metabolite, was not detected. Further metabolites containing partly degraded alkyl chains were all transient and were not detected or only <0.2 % of AR (soil 2.3) at the test end. With regard to the kinetics, the transformation showed a slight bi-phasic pattern, therefore the ‘Single First Order Model’ (SFO) and the ‘First-Order Multi-Compartment Model’ (FOMC) were compared. Based on the visual fit and x2 error, the transformation of [ring-U-14C]Benzalkonium chloride met the requirements for both models well for all four soils. The calculated DT50 values with the Single-First-Order Model (SFO) for the dissipation of [ring-U-14C]Benzalkonium chloride were 2.2 – 8.7 days (C12 chain) and 6.1 – 28.7 days (C14 chain), the DT90 values were 7.2 – 28.8 (C12 chain) days and 20.2 – 95.4 days (C14 chain). The calculated DT50 values with the FOMC model for the dissipation of [ring-U-14C]Benzalkonium chloride were 1.6 – 7.2 days (C12 chain) and 5.5 – 23.3 days (C14 chain), the DT90 values were 15.0 – 48.8 days (C12 chain) and 35.8 – 164.3 days (C14 chain).

The read across substance is predominantly C12-ADBAC and C14-ADBAC, with low to negligible amounts of C16-ADBAC. The chain length distribution is defined as follows:C12 (35-80%), C14 (20-55%), C16 (0-15%). C16-ADBAC was not included in this study because it is present in very low amounts; there are technical difficulties with having sufficient radioactivity for substances present in small amounts relative to other constituents. C16-ADBAC would be expected to degrade by the same route but at a slower rate than its C12 and C14 counterparts, as degradation rate tends to decrease with increasing chain lengths.Under the study conditions, transformations of both C12 and C14 carbon chains of the read across substance were determined to be rapid in all four soils and the DT50 values were determined to be 2.2 – 8.7 days [C12 chain] and 6.1 – 28.7 days [C14 chain] with the SFO model and 1.6 – 7.2 days [C12 chain] and 5.5 – 23.3 days [C14 chain] with the FOMC modelat 20°C (Fiebig, 2019).

 

Further, in the biocides dossier, to account for the potential contribution of C16 ADBAC to the overall DT50 of ADBAC, a geometric mean of SFO and FOMC DT50s for C12 and C14 ADBAC in the four soils (as recommended in BPR Vol IV Part B and C) was calculated and converted to 12° using the following equation (DT50 (12°) = DT50 (20°) * e(0.08*(20-12)). This was followed by linear extrapolation of the geometric mean DT50s for C12 and C14 ADBAC, to estimate the DT50 for C16 ADBAC. See table below:

 

Soil 2.2

Soil 2.3

Soil 2.4

Soil 5M

Geo. Mean

Adj. to 12° C

SFO DT50s

C12 ADBAC

2.2

3.3

6.2

8.7

4.4

8.4

C14 ADBAC

6.1

8.9

12.9

28.7

11.9

22.6

C16 ADBAC

--

--

--

--

--

36.7

FOMC DT50s

C12 ADBAC

1.6

3.2

5.8

7.2

3.8

7.3

C14 ADBAC

5.5

8.3

12.1

23.3

10.7

20.2

C16 ADBAC

--

--

--

--

--

33.1

A weighted estimate of the DT50 of ADBAC (C12-C16) at 12°C was calculated by assuming the highest allowable concentrations of C14- and C16- ADBAC and the balance of C12-ADBAC (i.e., 12% C16, 52% C14 and 36% C12), which resulted in the following estimated DT50s:

SFO DT50 = 19.2d at 12°C; FOMC DT50 = 17.1d at 12°C

However, due to the relatively low levels of C16-ADBAC, the overall estimated DT50s were considered rather insensitive to the assumed DT50 for C16-ADBAC.The DT50 of 17.1 days at 12°C based on the biphasic model (FOMC) showing better visual fit and lower errorwas used further for risk assessment.

Based on the results of the read across study, similar degradation potential and half-life is considered for the test substance.

 

Study 2:A study was conducted to determine the aerobic biodegradation of the read across substance, C12-16 ADBAC (50% active in water) in loamy soil, according to the US FDA Environmental Assessment Handbook, Technical Assistance Document 3.12 (1987). The study comprised two treatments: test and chemical blank control group, each with three replicates. The read across substance was added into biometers at a concentration of 10 mg carbon per 50 g soil using appropriate amount of deionised water required for bringing the soils to 50-70% of the moisture capacity. Loam was added to the biometers after the test solutions to facilitate uniform moistening of the soils by capillary action. The test was then incubated at 22 ± 3°C and run for approximately 90 d. The side tube of the biometer contained 20 mL 0.2 M KOH for absorbing carbon dioxide produced by the microorganisms. The theoretical CO2 production of the read across substance was calculated from its carbon content. The amounts of carbon dioxide were calculated by subtracting the mean carbon dioxide production in the test systems containing the read across substance and the mean carbon dioxide production level in the control blank. Biodegradation was calculated as the ratio of experimental carbon dioxide production to theoretical carbon dioxide production [ThCO2P]. Under the study conditions, there was 64% degradation of the read across substance after 70 days. This percentage of the theoretical carbon dioxide production presumes complete mineralization. The DT50 was estimated to be 40 days (Ginkel, 1994). Based on the results of the read across study, similar degradation potential and half-life is considered for the test substance.  ​

Based on the most recent and radiolabelled aerobic biodegradation study in soil with the read across substance, C12-16 ADBAC, the transformation of the C12 and C14 carbon chains of the substance was considered to be rapid with DT50 values ranging from 2.2-28.7 days with the SFO model and 1.6 – 23.3 days with the FOMC model at 20°C.​ Further, in the biocides dossier, a weighted estimate of the DT50 value at 12°C was extrapolated for C12-16 ADBAC by assuming the highest allowable concentrations for the major chains. These calculations resulted in the estimated FOMC DT50 of 17.1 days at 12°C and SOF DT50 of 19.2 days at 12°C. The DT50 of 17.1 days at 12°C based on the biphasic model (FOMC) showing better visual fit and lower error (x2)compared to the SFO model was used further for risk assessment. Therefore, in line with the biocides dossier, the DT50 of 17.1 days at 12°C derived for the read across substance based on the biphasic model (FOMC) also has been considered further for hazard/risk assessment of the test substance.