Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.

REACH

Benzododecinium chloride

EC number: 205-351-5 | CAS number: 139-07-1

Life Cycle description
Uses advised against

Environmental fate & pathways

Biodegradation in soil

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference 1

Endpoint:

biodegradation in soil: simulation testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

November 27, 2017 to October 10, 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Deviations:

yes

Remarks:

The deviations were considered to have no impact on the integrity and quality of the study.

GLP compliance:

yes (incl. QA statement)

Test type:

laboratory

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

other: 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

Year:

2018

Soil no.:

Soil type:

loamy sand

% Clay:

ca. 8.6

% Silt:

ca. 14.9

% Sand:

ca. 75.7

% Org. C:

ca. 1.7

pH:

ca. 5.6

CEC:

ca. 9.8 meq/100 g soil d.w.

Bulk density (g/cm³):

ca. 1 201

Soil no.:

Soil type:

loamy sand

% Clay:

ca. 8.3

% Silt:

ca. 14.5

% Sand:

ca. 77.1

% Org. C:

ca. 0.939

pH:

ca. 5.6

CEC:

ca. 9.5 meq/100 g soil d.w.

Bulk density (g/cm³):

ca. 1 205

Soil no.:

Soil type:

other: silty sand

% Clay:

ca. 7.6

% Silt:

ca. 35.2

% Sand:

ca. 57.2

% Org. C:

ca. 0.55

pH:

ca. 5.8

CEC:

ca. 7.5 meq/100 g soil d.w.

Bulk density (g/cm³):

ca. 1 307

Soil no.:

Soil type:

other: clayey loam

% Clay:

ca. 26.6

% Silt:

ca. 46.1

% Sand:

ca. 27.3

% Org. C:

ca. 2.47

pH:

ca. 7.4

CEC:

ca. 26.5 meq/100 g soil d.w.

Bulk density (g/cm³):

ca. 1 251

Soil no.:

Soil type:

loamy sand

% Clay:

ca. 11.2

% Silt:

ca. 34.5

% Sand:

ca. 54.3

% Org. C:

ca. 0.88

pH:

ca. 7.3

CEC:

ca. 15.7 meq/100 g soil d.w.

Bulk density (g/cm³):

ca. 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.:

Duration:

ca. 122 d

Soil No.:

Duration:

ca. 120 d

Soil No.:

Duration:

ca. 128 d

Soil No.:

Duration:

ca. 120 d

Parameter followed for biodegradation estimation:

CO2 evolution

Details on experimental conditions:

(A) Test Substance Concentration and Treatment Groups
Test substance: [ring-U-14C]Benzalkonium chloride
Working Solution: 37 MBq/mL (provided by the sponsor)
Nominal test substance: 9.20 kBq/g soil DW (460.1 kBq/Replicate) (soil 2.3, 2.4, 5M) concentration corresponding to 1500 µg test substance/kg soil DW, C12 chain: 6.37 kBq/g soil DW (66.5%) C14 chain: 2.83 kBq/g soil DW (32.0%), 7.58 kBq/g soil DW (378.8 kBq/Replicate) (soil 2.2) corresponding to 1235 µg test substance/kg soil DW
C12 chain: 5.25 kBq/g soil DW (66.5%) C14 chain: 2.33 kBq/g soil DW (32.0%)
Application solution of labelled test substance: 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 substance 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 Substance: According to the guideline, no reference substance is recommended for this test.

(B) Test Substance Concentration for Identification of Metabolites and Evaluation of Transformation Pathway
Test Substance: Arquad MCB-50
Nominal test substance: 2000 µg test substance/kg soil DW, corresponding to 1000 µg concentration Benzalkonium chloride/kg soil DW (soil 2.2)
Solvent for application: Ultrapure water : ACN (90:10)
Carrier for application: Quartz sand

Key result

Soil No.:

DT50:

ca. 2.2 - ca. 8.7 d

Type:

other: Single-First-Order Model (SFO)

Temp.:

ca. 20 °C

Remarks on result:

other: C12 chain

Key result

Soil No.:

DT50:

ca. 1.6 - ca. 7.2 d

Type:

other: First-Order Multi-Compartment Model (FOMC)

Temp.:

ca. 20 °C

Remarks on result:

other: C12 chain

Key result

Soil No.:

DT50:

ca. 6.1 - ca. 28.7 d

Type:

other: Single-First-Order Model (SFO)

Temp.:

ca. 20 °C

Remarks on result:

other: C14 chain

Key result

Soil No.:

DT50:

ca. 5.5 - ca. 23.3 d

Type:

other: First-Order Multi-Compartment Model (FOMC)

Temp.:

ca. 20 °C

Remarks on result:

other: C14 chain

Transformation products:

Evaporation of parent compound:

yes

Volatile metabolites:

Residues:

yes

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 carbon 2)	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
Sample No.	% 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

Repl.

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

(min)

Repl.

% of Applied Radioactivity

Sampling Day

122

8.13 - 9.40

Primary Metabolites

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

n.d. n.d.

1.4

1.0

1.7

1.8

1.3

1.6

1.3

1.1

n.d.

2.0

n.d.

0.4

n.d.

11.07 - 11.27

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.

RT = Retention time in minutes

n.d. = not detected

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

(min)

Repl.

% of Applied Radioactivity

Sampling Day

120

8.13 - 9.80

Primary Metabolites

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

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.

0.2

0.1

18.47 - 19.00

n.d. n.d.

0.9

1.0

0.8

1.4

1.1

0.2

0.9

n.d.

RT = Retention time in minutes

n.d. = not detected

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

(min)

Repl.

% of Applied Radioactivity

Sampling Day

128

7.47 - 9.40

Primary Metabolites

n.d. n.d.

7.8

6.3

10.1

10.6

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

n.d. n.d.

n.d.

0.9

1.4

2.4

1.6

1.4

1.1

1.2

0.8

n.d.

0.2

n.d.

11.07 - 11.40

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

0.2

n.d.

RT = Retention time in minutes

n.d. = not detected

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

(min)

Repl.

% of Applied Radioactivity

Sampling Day

120

7.60- 8.80

Primary Metabolites

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

n.d. n.d.

2.3

3.7

3.9

3.2

3.0

n.d.

2.0

n.d.

0.1

n.d.

18.07 - 18.87

n.d. n.d.

n.d.

1.0

0.9

0.7

1.3

n.d. 1.0

n.d.

0.1

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 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 metabolites 2–6 show 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 metabolites 2– 6 indicate 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 metabolites 2 and 6 were 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 metabolite 1 after 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.

122

C12 BKC

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

C14 BKC

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

4.4

3.6

1.6

1.8

1.0

0.7

Soil 2.3

% of Applied Radioactivity Day

Repl.

120

C12 BKC

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

C14 BKC

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.

128

C12 BKC

- 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

- 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.

120

C12 BKC

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

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
k_p	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
k_p	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	DT_xValues in Days Soil
Endpoint / Statistic	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	DT_xValues in Days Soil
Endpoint / Statistic	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:

Based on the results of the read across study, the transformation of the C12 carbon chain of the test substance can be considered to be rapid with DT50 values ranging from 2.2-8.7 days with the SFO model and 1.6 – 7.2 days with the FOMC model.

Executive summary:

A study was conducted to determine the aerobic transformation/dissipation in 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 were 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. 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. 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 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). 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. Under the study conditions, transformations of both C12 and C14 carbon chains of the read across substance were determined to rapid in all four soils and the DT50 values were determined to 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 model (Fiebig, 2019).

Reference 2

Endpoint:: biodegradation in soil: simulation testing
Type of information:: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:: supporting 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 section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements.
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 using a closed bottle test for 28 days according to the US FDA Environmental Assessment Handbook, Technical Assistance Document 3.12 (1987).
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:: 70 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.
Conclusions:: Based on the results of the read across study, the test substance can also be expected to undergo 64% biodegradation in loam at Day 70, with DT50 of 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, using a closed bottle test for 28 days 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 (van Ginkel, 1994).

Reference 3

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 27, 2017 to October 10, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Deviations:

yes

Remarks:

The deviations were considered to have no impact on the integrity and quality of the study.

GLP compliance:

yes (incl. QA statement)

Test type:

laboratory

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

other: 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

Year:

2018

Soil no.:

Soil type:

loamy sand

% Clay:

ca. 8.6

% Silt:

ca. 14.9

% Sand:

ca. 75.7

% Org. C:

ca. 1.7

pH:

ca. 5.6

CEC:

ca. 9.8 meq/100 g soil d.w.

Bulk density (g/cm³):

ca. 1 201

Soil no.:

Soil type:

loamy sand

% Clay:

ca. 8.3

% Silt:

ca. 14.5

% Sand:

ca. 77.1

% Org. C:

ca. 0.939

pH:

ca. 5.6

CEC:

ca. 9.5 meq/100 g soil d.w.

Bulk density (g/cm³):

ca. 1 205

Soil no.:

Soil type:

other: silty sand

% Clay:

ca. 7.6

% Silt:

ca. 35.2

% Sand:

ca. 57.2

% Org. C:

ca. 0.55

pH:

ca. 5.8

CEC:

ca. 7.5 meq/100 g soil d.w.

Bulk density (g/cm³):

ca. 1 307

Soil no.:

Soil type:

other: clayey loam

% Clay:

ca. 26.6

% Silt:

ca. 46.1

% Sand:

ca. 27.3

% Org. C:

ca. 2.47

pH:

ca. 7.4

CEC:

ca. 26.5 meq/100 g soil d.w.

Bulk density (g/cm³):

ca. 1 251

Soil no.:

Soil type:

loamy sand

% Clay:

ca. 11.2

% Silt:

ca. 34.5

% Sand:

ca. 54.3

% Org. C:

ca. 0.88

pH:

ca. 7.3

CEC:

ca. 15.7 meq/100 g soil d.w.

Bulk density (g/cm³):

ca. 1 221

Details on soil characteristics:

Soil No.:

Duration:

ca. 122 d

Soil No.:

Duration:

ca. 120 d

Soil No.:

Duration:

ca. 128 d

Soil No.:

Duration:

ca. 120 d

Parameter followed for biodegradation estimation:

CO2 evolution

Details on experimental conditions:

Key result

Soil No.:

DT50:

ca. 2.2 - ca. 8.7 d

Type:

other: Single-First-Order Model (SFO)

Temp.:

ca. 20 °C

Remarks on result:

other: C12 chain

Key result

Soil No.:

DT50:

ca. 1.6 - ca. 7.2 d

Type:

other: First-Order Multi-Compartment Model (FOMC)

Temp.:

ca. 20 °C

Remarks on result:

other: C12 chain

Key result

Soil No.:

DT50:

ca. 6.1 - ca. 28.7 d

Type:

other: Single-First-Order Model (SFO)

Temp.:

ca. 20 °C

Remarks on result:

other: C14 chain

Key result

Soil No.:

DT50:

ca. 5.5 - ca. 23.3 d

Type:

other: First-Order Multi-Compartment Model (FOMC)

Temp.:

ca. 20 °C

Remarks on result:

other: C14 chain

Transformation products:

Evaporation of parent compound:

yes

Volatile metabolites:

Residues:

yes

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 carbon 2)	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

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)

Homogeneity after Application

Table:Homogeneity of the Soils after Application

Sample No.	Soil 2.2	Soil 2.3	Soil 2.4	Soil 5M
Sample No.	% 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

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

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)

Soil Organic Matter Fractionation

2.2

2.3

Soil

% of AR

2.4

Repl.

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

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

(min)

Repl.

% of Applied Radioactivity

Sampling Day

122

8.13 - 9.40

Primary Metabolites

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

n.d. n.d.

1.4

1.0

1.7

1.8

1.3

1.6

1.3

1.1

n.d.

2.0

n.d.

0.4

n.d.

11.07 - 11.27

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.

RT = Retention time in minutes

n.d. = not detected

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

(min)

Repl.

% of Applied Radioactivity

Sampling Day

120

8.13 - 9.80

Primary Metabolites

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

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.

0.2

0.1

18.47 - 19.00

n.d. n.d.

0.9

1.0

0.8

1.4

1.1

0.2

0.9

n.d.

RT = Retention time in minutes

n.d. = not detected

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

(min)

Repl.

% of Applied Radioactivity

Sampling Day

128

7.47 - 9.40

Primary Metabolites

n.d. n.d.

7.8

6.3

10.1

10.6

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

n.d. n.d.

n.d.

0.9

1.4

2.4

1.6

1.4

1.1

1.2

0.8

n.d.

0.2

n.d.

11.07 - 11.40

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

0.2

n.d.

RT = Retention time in minutes

n.d. = not detected

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

(min)

Repl.

% of Applied Radioactivity

Sampling Day

120

7.60- 8.80

Primary Metabolites

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

n.d. n.d.

2.3

3.7

3.9

3.2

3.0

n.d.

2.0

n.d.

0.1

n.d.

18.07 - 18.87

n.d. n.d.

n.d.

1.0

0.9

0.7

1.3

n.d. 1.0

n.d.

0.1

n.d.

n.d. 0.1

n.d.

RT = Retention time in minutes

n.d. = not detected

Metabolite Identification

Data Evaluation – LC-HRMS Analysis

Results and Data Assessment – LC-HRMS Analysis

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

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]+

Confirmatory – LC-MS/MS Analysis

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

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

Soil 2.2

% of Applied Radioactivity Day

Repl.

122

C12 BKC

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

C14 BKC

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

4.4

3.6

1.6

1.8

1.0

0.7

Soil 2.3

% of Applied Radioactivity Day

Repl.

120

C12 BKC

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

C14 BKC

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.

128

C12 BKC

- 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

- 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.

120

C12 BKC

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

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
k_p	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
k_p	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	DT_xValues in Days Soil
Endpoint / Statistic	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	DT_xValues in Days Soil
Endpoint / Statistic	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

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

Conclusions:

Under the study conditions, transformations of both C12 and C14 carbon chains of the test substance were determined to rapid in all four soils and the DT50 values were determined to 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 model

Executive summary:

A study was conducted to determine the aerobic transformation/dissipation in soil of the test 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 test 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 were determined by LSC. Test 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 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 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 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. 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 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). 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. Under the study conditions, transformations of both C12 and C14 carbon chains of the test substance were determined to rapid in all four soils and the DT50 values were determined to 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 model (Fiebig, 2019).

Reference 4

Endpoint:: biodegradation in soil: simulation testing
Type of information:: experimental study
Adequacy of study:: supporting study
Study period:: 1994
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: guideline study without detailed documentation
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 using a closed bottle test for 28 days according to the US FDA Environmental Assessment Handbook, Technical Assistance Document 3.12 (1987).
GLP compliance:: no
Test type:: laboratory
Specific details on test material used for the study:: - Purity: 50% (in water)
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:: 70 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.
Conclusions:: Under the study conditions, there was 64% degradation of the test 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 test substance, C12-16 ADBAC (50% active in water) in loamy soil, using a closed bottle test for 28 days 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 test 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 test 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 test 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 test substance after 70 days. This percentage of the theoretical carbon dioxide production presumes complete mineralization. The DT50 was estimated to be 40 days (van Ginkel, 1994).

Description of key information

Key value for chemical safety assessment

Half-life in soil:: 8.7 d
at the temperature of:: 20 °C

Additional information

Study 1: A study was conducted to determine the aerobic transformation/dissipation in 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 were 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. 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. 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 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). 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. Under the study conditions, transformations of both C12 and C14 carbon chains of the read across substance were determined to rapid in all four soils and the DT50 values were determined to 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 model (Fiebig, 2019).

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, using a closed bottle test for 28 days 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 (van Ginkel, 1994).

Based on the most recent and radiolabelled read across study in soil, the transformation of the C12 carbon chain of the test substance can be considered to be rapid with DT50 values ranging from 2.2-8.7 days with the SFO model and 1.6 – 7.2 days with the FOMC model.

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Registration Dossier

Registration Dossier

Data platform availability banner - registered substances factsheets

Diss Factsheets

Benzododecinium chloride

Biodegradation in soil

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Reference 1

Reference 2

Reference 3

Reference 4

Description of key information

Key value for chemical safety assessment

Additional information

Referenceopen all close all