Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

Biodegradation in soil

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14-Apr-2011 to 07-Dec-2011

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:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 95/36/EC of July 14, 1995; amending Council Directive 91/414/EEC, Annex I, 7.1.1.2 rate of degradation; 7.1.1.2.1 laboratory studies - aerobic degradation.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SETAC (Europe): Procedures for assessing the environmental fate and ecotoxicity of pesticides, March 1995, Part 1 - Aerobic degradation.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 22 to 26 March and 12 to 16 April 2010 Date of decision: 19 November 2010

Test type:

laboratory

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

other: Three representative field soils I (Fislis, France; silt loam); II (Speyer 6S, Germany; clay) and III (Mechtildshausen, Germany; loam) were used in the study.

Year:

2011

Soil no.:

#1

Soil type:

silt loam

% Clay:

25.83

% Silt:

65.38

% Sand:

8.79

% Org. C:

1.37

pH:

6.89

CEC:

20.4 other: (mmol/100 g soil)

Soil no.:

#2

Soil type:

clay

% Clay:

41.76

% Silt:

34.16

% Sand:

24.08

% Org. C:

1.66

pH:

7.16

CEC:

25.52 other: (mmol/100 g soil)

Soil no.:

#3

Soil type:

loam

% Clay:

17.57

% Silt:

44.47

% Sand:

37.96

% Org. C:

1.08

pH:

6.75

CEC:

12.82 other: (mmol/100 g soil)

Details on soil characteristics:

SOIL COLLECTION AND STORAGE

Three representative field soils I (Fislis, France; silt loam); II (Speyer 6S, Germany; clay) and III (Mechtildshausen, Germany; loam) were used in the study. The soils were collected from the field from the top 0-20 cm layer. Sampling and handling of the soils was performed in accordance with ISO 10381-6 [see References (1)]. For all soils, the top plant cover was removed during sampling and soil was put in containers with free access to air.

Soil Collection

Soil I (Fislis) was freshly sampled from a field in Fislis, France (47°30'N, 7°21'E) in March 2011 and transported to Harlan Laboratories Ltd. shortly after sampling.

Soil II (Speyer 6S) was freshly sampled from a field in Siebeldingen, Rheinland-Pfalz, Germany (49 °12’ N, 8 °03’ E) in March 2011 and transported to Harlan Laboratories Ltd. shortly after sampling.

Soil III (Mechtildshausen) was freshly sampled from a field in Mechtildshausen, Germany (50°02'N, 8°18'E) in March 2011 and transported to Harlan Laboratories Ltd. shortly after sampling.

All three soils had not been subjected to any pesticide, organic or inorganic fertiliser treatment for at least the last four years prior to sampling. There had also been no such treatment of the soils at Harlan Laboratories Ltd.

Soil Preparation

After transportation to Harlan Laboratories Ltd. the soils were stored at 4±2 °C for about one to two months until use. The soils were sieved through a 2-mm screen prior to testing, and characterized for pH, % organic carbon and matter, carbonate, cation exchange capacity, nitrogen content, particle size distribution, percent moisture at water holding capacity and microbial biomass.

Before use in the study, the soils were conditioned to room temperature for about three weeks. Then, the soil was finger-crumbled and turned over frequently to avoid excessive surface drying. The soil moisture content was determined for triplicate sub-samples by halogen drying and weighing. The moisture content was adjusted with purified water to approximately pF 2.5.

Soil No.:

#1

Duration:

124 d

Soil No.:

#2

Duration:

124 d

Soil No.:

#3

Duration:

124 d

Soil No.:

#1

Initial conc.:

0.2 other: mg a.i./kg dry soil

Based on:

test mat.

Soil No.:

#2

Initial conc.:

0.2 other: mg a.i./kg dry soil

Based on:

test mat.

Soil No.:

#3

Initial conc.:

0.2 other: mg a.i./kg dry soil

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

Soil No.:

#1

Temp.:

20

Humidity:

pF 2.0-2.5

Microbial biomass:

start of incubation 33.4 mg org. C/100 g dry soil end of incubation 27.3 mg org. C/100 g dry soil

Soil No.:

#2

Temp.:

20

Humidity:

pF 2.0-2.5

Microbial biomass:

start of incubation 33.4 mg org. C/100 g dry soil end of incubation 27.3 mg org. C/100 g dry soil

Soil No.:

#3

Temp.:

20

Humidity:

pF 2.0-2.5

Microbial biomass:

start of incubation 33.4 mg org. C/100 g dry soil end of incubation 27.3 mg org. C/100 g dry soil

Details on experimental conditions:

Test System
Rationale for the Selection of the Test System
Anthropogenic chemicals are frequently detected in the environment and wastewater and sewage sludge can be important emission pathways for them. Sludge from municipal wastewater treatment plant is sometimes used as an organic fertilizer, resulting in the transfer of any chemicals contained within it to the soil. Aerobic and anaerobic conditions occur in soils. The use of aerobic and anaerobic soil test systems enables the study of the fate of chemicals under laboratory conditions, while providing relevant information about natural systems.
Three fresh soil types were selected to evaluate the rate of degradation of the test item in the environment.

Anoxic Digested Sludge
The anoxic digested sludge (dewatered) was sampled from a municipal wastewater treatment plant (ARA Füllinsdorf, Switzerland). The sludge moisture content was determined for triplicate sub-samples by oven drying and weighing. This determination revealed a water content of 68.0% (312.4 g wet sludge corresponded to 100 g dry sludge).
The amount of sludge added to the soil reflected normal sludge loading to agricultural soils according to REACH Guidance (5000 kg/ha) and should correspond to about 0.17 g dry sludge/ 100 g dry soil. Therefore, an amount of 0.53 g sludge per sample was mixed with the soil.

Determination of Soil Microbial Biomass
The microbial biomass of each soil was determined for all soils prior to treatment and at the end of the incubation period. A modification of the respiratory method described by Anderson and Domsch [see References (2)] was used. For this purpose, sub-samples of the sieved soil were amended with increasing amounts of glucose and submitted to respiratory measurements to determine the microbial biomass of the soil. The determination of the microbial biomass was performed at 20 ± 2 °C in the dark.
The soil aliquots were packed into all-glass columns and measured semi-continuously for about 24 hours by means of an IR-gas-analyser (X STREAM R, Emerson Process Management) until CO2 decreased after the peak phase. The total volume of CO2 evolved during approximately one hour was calculated. For the calculations, the values which corresponded to a maximum initial and constant CO2-production, were used (i.e. the so-called plateau values). The value obtained was extrapolated to 100 g dry soil (for details see Section "Determination of the Microbial Biomass).

Experimental Conditions
Samples of 100 g soil based on the dry weight were incubated under aerobic conditions in all-glass metabolism flasks (inner diameter: about 10.6 cm, volume: ca. 1 litre, see Scheme 1) in the dark at 20 ± 2 °C. The flasks were equipped with an air inlet and outlet. The system was continuously ventilated with moistened air. For each flask, the exiting air was passed through a trapping system, equipped with two absorption traps containing about 50 mL of ethylene glycol and 50 mL 2N sodium hydroxide to trap organic volatiles and 14CO2, respectively.
For the time 0 samples, no absorption traps were set up.
Each test system was uniquely identified according to Harlan Laboratories identification system to assure unmistakable identification.
The soil moisture content was maintained at pF values of approximately 2.5. Soil water losses were kept to a minimum by circulating moistened air at a minimum volumetric flow rate sufficient enough to pass through the trapping system.
Samples were weighed periodically during the incubation period to determine the amount of water lost by evaporation. To compensate for this loss, an amount of water equal to that lost by evaporation was added.

Treatment and Sampling
Rationale for the Application Rate
The soil samples were treated with [14C]C22-ATQ at the rate of about 0.2 mg test item/per kg dry soil (= 0.02 mg a.i./100 g dry soil), sufficient to determine its degradation rate. The application rate was based on an exposure modeling using realistic use rates.

Preparation of the Application Solution and Treatment of the Test System
The radiolabelled test item was solubilized in ethanol. Volumetric adjustments were made by adding or evaporating ethanol, in the latter case under a stream of nitrogen. Concentrations were determined by LSC in triplicate aliquots. The applied amount was determined by LSC in triplicate application aliquots before, during and after the application process. The average of all replicates was taken as the actual applied amount. Purity and stability of the test item in the stock and application solutions was determined by TLC (see Section "RESULTS AND DISCUSSION").

The quantitative details for solution preparation and the application of the test system are given below:

Application solution:
127'173'000 dpm/mL
Specific radioactivity: 5.29 MBq/mg
test item / ml: 0.401 mg/mL

Application aliquots
Purity: 95.9%
0.050 mL
6'490'871 dpm
0.108 MBq
0.020 mg test item

Application rate
0.205 mg/kg, test item / kg soil
102 % of Target

Treatment of the Test System
Samples containing 100 g soil were treated with the test item at the concentration of 0.20 mg test item/kg dry soil. In order to achieve a realistic exposure regime the test item was added to the soil via sewage sludge. The amount of sludge added to the soil reflected normal sludge loading to agricultural soils according to REACH Guidance (5000 kg/ha) and corresponds to about 0.17 g dry sludge / 100 g dry soil.

Due to the low amount of sludge which was used per sample, the soil was first transferred to the metabolism flask. Prior to application a small amount of the soil from the metabolism flask was placed on a weighing boat and the solid sludge was added on top. Thereafter, a defined aliquot of 50 µL of the application solution (or ethanol for the control samples) was applied to each solid sludge sample to reach the target concentration in the soil (see Section "Preparation of the Application Solution and Treatment of the Test System"). Care was taken to absorb the complete application volume into the sludge using a Hamilton syringe. After the application procedure, the soil and sludge were put back into the metabolism flask and then mixed with the soil using a glass rod. Afterwards, the soil moisture content was adjusted as described in Section "Preparation of the Application Solution and Treatment of the Test System". Each replicate was attached immediately to the trapping system after treatment and mixing. The glass rod used was rinsed with ethanol and the radioactivity in the rinsate was measured by LSC, to ensure that no significant amounts of radioactivity were removed from the metabolism flasks. The actual amount removed is given exemplarily for soil I were the radioactivity found on the glass rod amounted to 0.03% of the applied amount.


Sampling
Soil Samples
Duplicate samples from all three soils were taken for extraction and analysis immediately after treatment (day 0) and after 3, 7, 14, 29, 62 and 124 days of incubation.
The untreated samples were taken for the determination of the microbial biomass at the start and end of incubation.

Trapping Solutions
Radioactivity in the traps was periodically monitored by liquid scintillation counting (LSC). The traps were exchanged several times during the study. Prior to the determination of radioactivity, the volume of the liquid in each ethylene glycol and sodium hydroxide trap was recorded. In order to confirm the presence of 14CO2, the radioactivity contained in the sodium hydroxide traps was precipitated with barium hydroxide on pooled samples. For this purpose, 0.5 mL of the alkaline solutions was diluted with 3 mL of water and the precipitations were induced by addition of 3 mL of a saturated barium hydroxide solution. The suspensions were centrifuged and the supernatants were tested for quantitative precipitation by adding another one to two drops of the saturated Ba(OH)2 solution. If no turbidity developed upon the second addition of Ba(OH)2, the supernatants were counted by LSC measurement. If turbidity was observed, LSC measurements were performed after another precipitation step. The absence of radioactivity in the supernatants after precipitation was taken as proof that only 14CO2 was present in the sodium hydroxide solutions.


Extraction and Isolation of Radioactivity from Soil

Extractable and non-extractable radioactivity was determined by liquid scintillation counting the extract and combusting the extracted soil. The total amount of soil contained in each flask (about 100 g dry weight) was exhaustively extracted using the following work-up procedure:
- Acetone/0.1 M hydrochloric acid (4:1; v/v), up to four times.
- Acidic reflux extraction for four hours using Acetonitrile/0.1 M hydrochloric acid (1:1; v/v)

The amount of solvent used for each extraction step was about 0.8-1 mL per g soil. Each ambient extraction was performed in a shaker at about 250 vibrations per minute for about 30 minutes. The individual extracts were quantified by LSC and then combined. Acidic reflux extraction was conducted subsequent to the ambient extractions.
The residual radioactivity remaining in soil after the extraction procedure was quantified by LSC after combustion of aliquots of the air-dried and homogenized soil.


Characterization of the Non-Extractable Radioactivity
The residues from the reflux extractions from day 124 were submitted 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. A procedure based on Stevenson [see Reference (4)] was used.

Soil No.:

#1

% Recovery:

106.9

St. dev.:

3.7

Remarks on result:

other: The total mean recoveries in terms of percent of the applied radioactivity ± 3.7%

Soil No.:

#2

% Recovery:

105.4

St. dev.:

5.1

Remarks on result:

other: The total mean recoveries in terms of percent of the applied radioactivity ± 5.1%

Soil No.:

#3

% Recovery:

107.1

St. dev.:

3.9

Remarks on result:

other: The total mean recoveries in terms of percent of the applied radioactivity ± 3.9%

Soil No.:

#1

% Degr.:

4.5

Parameter:

radiochem. meas.

Sampling time:

124 d

Soil No.:

#2

% Degr.:

3.4

Parameter:

radiochem. meas.

Sampling time:

124 d

Soil No.:

#3

% Degr.:

13.5

Parameter:

radiochem. meas.

Sampling time:

124 d

Soil No.:

#1

DT50:

23.2

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: DT50 (d) at 20 °C

Soil No.:

#2

DT50:

24

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: DT50 (d) at 20 °C

Soil No.:

#3

DT50:

41.4

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: DT50 (d) at 20 °C

Transformation products:

no

Evaporation of parent compound:

not measured

Volatile metabolites:

yes

Residues:

yes

Details on results:

Radiochemical Purity and Stability of the Test Item.
The purity of [14C]C22-ATQ in the stock and application solutions was determined by TLC to be 95.9% , thereby confirming the purity and stability of the test item.


Material Balance (Overall Recovery of Radioactivity)
The results are summarized in Table 2 to Table 4 in terms of percent of the applied radioactivity and in Appendix II in mg test item equivalents/kg dry soil.

The total mean recoveries in terms of percent of the applied radioactivity were 106.9 ± 3.7% (soil I), 105.4 ± 5.1% (soil II), and 107.1 ± 3.9% (soil III).

On the whole, duplicate samples gave similar results; therefore the results in the following sections are expressed as the mean value.


Characterization of Radioactivity
Extractable Radioactivity
The extractable radioactivity decreased in soils I-III from 27.8 – 60.4% of the applied radioactivity at time 0 (immediately after application) to 3.4 – 13.5% by day 124 at the end of the study.


Non-Extractable Radioactivity
The non-extractable radioactivity decreased in soils I-III from 45.8 – 80.6% of the applied radioactivity at time 0 to 17.3 – 28.2% by day 124 .

Organic matter fractionation of the non-extractable radioactivity of soils I-III from day 124 indicated that 1.5 – 2.3% of applied was bound to fulvic acids, 2.5 – 2.9% to the immobile humic acids and 12.5 – 24.2% to humins (Table 5 and Table 6).


See tables 2-6 in section "Any other information on results incl. tables"

Volatiles
The levels of radioactive CO2 in soils I-III reached 63.1 – 72.6% of the applied radioactivity by the end of the study (Table 2 to Table 4 ).
Volatile radioactivity trapped in ethylene glycol was throughout the study below 0.1% the applied radioactivity.


Microbial Biomass
The microbial biomass in soils I-III was 23.6 – 35.2 mg microbial C/100 g dry soil at the start and 19.1 – 27.4 mg at the end of the study confirming thereby the viability of the soils during the study.


Rate of Degradation of [14C]C22-ATQ in Soil
The rate of disappearance of [14C]C22-ATQ was based on the total extractable radioactivity measured at each interval, thereby assuming the entire extractable radioactivity to consist of [14C]C22-ATQ. Fitted curves were calculated using simple first order kinetics (Section "Calculation of DT50 and DT90 values).

The following DT50 and DT90 values were calculated:
Soil I DT50 (days): 23.2 DT90 (days): 77.1
Soil II DT50 (days): 24.0 DT90 (days): 79.9
Soil III DT50 (days): 41.4 DT90 (days): 137.4

Results with reference substance:

Reference Item
Only the unlabeled test item was used as reference item. The latter was used to confirm the identity and radiochemical purity of the test item by 1D-TLC.

Tables 2 to 6.

Table2      Balance of the Applied Radioactivity in Soil I Treated with [¹⁴C]C22-ATQ. Values in Percent of Applied Radioactivity

^14C C22-ATQ	Sample	INCUBATION TIME IN DAYS
Fislis
(% of applied)		0	3	7	14	29	62	124
Ambient Extractions	A	43.1	40.2	37.6	23.0	10.8	6.1	2.4
	B	42.5	40.6	39.5	24.1	12.4	6.0	2.6
Reflux Extraction	A	3.1	2.0	5.1	4.3	5.2	3.5	2.0
	B	2.5	1.9	5.1	4.3	5.1	3.9	2.0
Total Extractables	A	46.2	42.2	42.7	27.3	16.0	9.6	4.4
	B	45.0	42.5	44.7	28.4	17.5	10.0	4.5
	Mean	45.6	42.4	43.7	27.9	16.7	9.8	4.5
Non-extractables	A	62.6	61.2	57.8	57.6	47.6	41.3	23.5
	B	63.5	63.9	55.1	56.3	45.6	38.7	20.5
	Mean	63.1	62.5	56.5	57.0	46.6	40.0	22.0
^14CO2	A	n.p.	2.4	7.9	23.7	42.3	59.3	71.4
	B	n.p.	2.4	9.4	22.6	44.8	60.5	73.8
	Mean	n.p.	2.4	8.6	23.2	43.6	59.9	72.6
Organic Volatiles	A	n.p.	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
	B	n.p.	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
	Mean	n.p.	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
T O T A L	A	108.8	105.8	108.4	108.7	105.8	110.2	99.3
	B	108.5	108.8	109.1	107.4	108.0	109.1	98.9
TOTAL MEAN ± SD			106.9	±	3.7

 n.p.    not performed

 

 

Table3      Balance of the Applied Radioactivity in Soil II Treated with [¹⁴C]C22-ATQ.Values in Percent of Applied Radioactivity

^14C C22-ATQ	Sample	INCUBATION TIME IN DAYS
Speyer 6S
(% of applied)		0	3	7	14	29	62	124
Ambient Extractions	A	26.6	23.6	27.9	13.2	7.6	5.3	2.5
	B	27.4	23.3	25.2	16.0	8.4	4.5	2.3
Reflux Extraction	A	0.8	0.6	1.5	2.4	1.8	1.3	1.0
	B	0.8	0.6	1.8	2.3	1.8	1.5	0.9
Total Extractables	A	27.4	24.2	29.5	15.6	9.4	6.6	3.5
	B	28.2	23.9	27.0	18.3	10.2	6.0	3.2
	Mean	27.8	24.1	28.2	17.0	9.8	6.3	3.4
Non-extractables	A	81.2	77.9	76.6	74.8	59.9	54.2	28.0
	B	80.1	80.2	74.6	72.0	62.1	51.3	28.4
	Mean	80.6	79.0	75.6	73.4	61.0	52.7	28.2
^14CO2	A	n.p.	0.6	4.2	14.9	38.3	48.7	63.4
	B	n.p.	1.5	6.6	15.9	34.6	50.4	62.8
	Mean	n.p.	1.0	5.4	15.4	36.5	49.5	63.1
Organic Volatiles	A	n.p.	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
	B	n.p.	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
	Mean	n.p.	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
T O T A L	A	108.6	102.7	110.2	105.4	107.6	109.5	94.9
	B	108.3	105.6	108.2	106.2	106.9	107.7	94.4
TOTAL MEAN ± SD			105.4	±	5.1

 n.p.     not performed

 

 

Table4      Balance of the Applied Radioactivity in Soil III Treated with [¹⁴C]C22-ATQ.Values in Percent of Applied Radioactivity

^14C C22-ATQ	Sample	INCUBATION TIME IN DAYS
Mechthildshausen
(% of applied)		0	3	7	14	29	62	124
Ambient Extractions	A	54.6	57.8	56.3	38.9	28.0	17.3	11.0
	B	59.0	57.2	57.4	40.0	27.0	16.6	10.0
Reflux Extraction	A	3.6	2.3	4.9	7.8	5.8	4.2	3.1
	B	3.6	2.3	4.8	7.6	5.9	4.5	2.8
Total Extractables	A	58.3	60.1	61.2	46.7	33.8	21.4	14.1
	B	62.5	59.5	62.2	47.5	32.9	21.1	12.8
	Mean	60.4	59.8	61.7	47.1	33.4	21.3	13.5
Non-extractables	A	45.7	45.5	40.8	42.4	40.9	31.5	17.5
	B	45.9	49.3	41.3	41.6	41.5	31.4	17.1
	Mean	45.8	47.4	41.1	42.0	41.2	31.5	17.3
^14CO2	A	n.p.	<0.1	6.6	19.8	34.9	57.4	67.8
	B	n.p.	0.1	5.4	20.0	35.4	56.7	68.9
	Mean	n.p.	0.1	6.0	19.9	35.2	57.0	68.3
Organic Volatiles	A	n.p.	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
	B	n.p.	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
	Mean	n.p.	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
T O T A L	A	104.0	105.7	108.7	108.9	109.6	110.3	99.3
	B	108.4	108.8	108.9	109.2	109.8	109.3	98.8
TOTAL MEAN ± SD			107.1	±	3.9

 n.p.   not performed

 

Table5      Organic Matter Fractionation of the Day 124 Soil Samples after Ambient and Reflux Extraction in Soil I (Top) and Soil II (Bottom)

Soil Organic Matter Fractionation	Soil I, day 124
	Duplicate A			Duplicate B			Mean
	%*		%**	%*		%**	%*		%**
Non-extractables (after ambient and relfux extraction)	--		23.5	--		20.5	--		22.0
Remaining non-extractable	100.0		23.5	100.0		20.5	100.0		22.0
Soluble fraction at low pH (fulvic acids)	10.3		2.4	10.7		2.2	10.5		2.3
Soluble fraction at high pH (humic acids)	13.0		3.1	12.9		2.6	12.9		2.9
Insoluble fraction (humin)	76.7		18.0	76.4		15.7	76.6		16.9
Total	100.0		23.5	100.0		20.5	100.0		22.0
Total (humic acids and humin)	89.7		21.1	89.3		18.3	89.5		19.7

Soil Organic Matter Fractionation	Soil II, day 124
	Duplicate A			Duplicate B			Mean
	%*		%**	%*		%**	%*		%**
Non-extractables (after ambient and relfux extraction)	--		28.0	--		28.4	--		28.2
Remaining non-extractable	100.0		28.0	100.0		28.4	100.0		28.2
Soluble fraction at low pH (fulvic acids)	5.3		1.5	5.3		1.5	5.3		1.5
Soluble fraction at high pH (humic acids)	8.6		2.4	9.2		2.6	8.9		2.5
Insoluble fraction (humin)	86.2		24.1	85.4		24.3	85.8		24.2
Total	100.0		28.0	100.0		28.4	100.0		28.2
Total (humic acids and humin)	94.7		26.5	94.7		26.9	94.7		26.7

*:   % of bound radioactivity after ambient and reflux extraction

**: % of applied radioactivity

Table6      Organic Matter Fractionation of the Day 124 Soil Samples after Ambient and Reflux Extraction in Soil III

Soil Organic Matter Fractionation		Soil III, day 124
		Duplicate A			Duplicate B			Mean
		%*		%**		%*		%**		%*		%**
Non-extractables (after ambient and relfux extraction)		--		17.5		--		17.1		--		17.3
Remaining non-extractable		100.0		17.5		100.0		17.1		100.0		17.3
Soluble fraction at low pH (fulvic acids)		12.3		2.1		13.0		2.2		12.6		2.2
Soluble fraction at high pH (humic acids)		15.3		2.7		15.3		2.6		15.3		2.6
Insoluble fraction (humin)		72.5		12.7		71.7		12.3		72.1		12.5
Total		100.0		17.5		100.0		17.1		100.0		17.3
Total (humic acids and humin)		87.7		15.3		87.0		14.9		87.4		15.1

 *:   % of bound radioactivity after ambient and reflux extraction

**: % of applied radioactivity

Soil Organic Matter Fractionation	Soil III, day 124
	Duplicate A			Duplicate B			Mean
	%*		%**	%*		%**	%*		%**
Non-extractables (after ambient and relfux extraction)	--		17.5	--		17.1	--		17.3
Remaining non-extractable	100.0		17.5	100.0		17.1	100.0		17.3
Soluble fraction at low pH (fulvic acids)	12.3		2.1	13.0		2.2	12.6		2.2
Soluble fraction at high pH (humic acids)	15.3		2.7	15.3		2.6	15.3		2.6
Insoluble fraction (humin)	72.5		12.7	71.7		12.3	72.1		12.5
Total	100.0		17.5	100.0		17.1	100.0		17.3
Total (humic acids and humin)	87.7		15.3	87.0		14.9	87.4		15.1

Conclusions:

[14C]C22-ATQ dissipated rapidly in all three soils with DT50 values of 23, 24 and 41 days in soils I, II and III, respectively. The rate of dissipation was based on the total extractable radioactivity measured at each interval, thereby assuming the entire extractable radioactivity to consist of [14C]C22-ATQ.

The levels of radioactive CO2 in soils I-III reached 63.1 – 72.6% of the applied radioactivity by the end of the study. The non-extractable radioactivity decreased in soils I-III from 45.8 – 80.6% of the applied radioactivity at time 0 to 17.3 – 28.2% by day 124.

Organic matter fractionation of the non-extractable radioactivity of soils I-III from day 124 indicated that 1.5 – 2.3% of applied was bound to fulvic acids, 2.5 – 2.9% to the immobile humic acids and 12.5 – 24.2% to humins.

Degradation of [14C]C22-ATQ in soil incubated under aerobic conditions at 20 °C proceeds via the formation significant amounts of radioactive carbon dioxide and bound residues.

Executive summary:

SUMMARY

The rate of degradation of[¹⁴C]C22-ATQ(C22-Trimethylammonium chloride)was investigated in three soils incubated under aerobic conditions for 124 days.The following soils were used for the study: soil I (,; silt loam), soil II (6S,; clay) and soil III (,; loam).

 

The freshly collected soils were passed through a 2 mm sieve.Samples containing 100 g dry soil were treated with[¹⁴C]C22-ATQat the concentration of 0.205 mg a.i./kg dry soil(= 0.020 mg a.i./100 g dry soil), sufficient to determine its degradation rate. The application rate was based on an exposure modeling using realistic use rates.In order to achieve a realistic exposure regime the test item was added to the soil via sewage sludge.The amount of sludge added to the soil reflected normal sludge loading to agricultural soils according to REACH Guidance (5000 kg/ha) and corresponds to about 0.17 g dry sludge / 100 g dry soil.

 

Adequate moisture contents were adjusted with purified water. A pF value of 2.5 was adjusted, since the soils agglomerated at water contents corresponding to pF values below 2.5. Nevertheless, the soil moisture was sufficient to support microbial degradation. In addition, the water content had to be kept low enough to allow for thorough mixing of the soils with the sewage sludge.The treated soil samples were incubated at 20 ± 2 °C in the dark under continuous ventilation with moistened air. The exiting air was passed through a trapping system consisting of flasks of ethylene glycol and sodium hydroxide in series. Prior to treatment and at the end of the incubation period, the microbial biomass was determined for each soil. The results showed that the soils were viable during the study.

 

Duplicate samples treated with the test item were taken immediately from each soil after treatment (day 0) and after 3, 7, 14 29, 62 and 124 days. Each sample was submitted to the following extraction procedure: ambient extraction using acetone/0.1 M HCl (4:1; v/v) for up to four times, followed by acidic reflux extraction using acetonitrile/0.1 M HCl (1:1; v/v) for 4 hours. The individual extracts were measured by LSC and combined.

 

The total mean recoveries in terms of percent of the applied radioactivity were 106.9 ± 3.7% (soil I), 105.4 ± 5.1% (soil II), and 107.1 ± 3.9% (soil III). On the whole, duplicate samples gave similar results; therefore the results in the following sections are expressed as the mean value.

 

The extractable radioactivity decreased in soils I-III from 27.8 – 60.4% of the applied radioactivity at time 0 (immediately after application) to 3.4 – 13.5% by day 124at the end of the study.

 

The non-extractable radioactivity decreasedin soils I-III from 45.8 – 80.6% of the applied radioactivity at time 0 to 17.3 – 28.2% by day 124.

 

Organic matter fractionation of the non-extractable radioactivity of soils I-III from day 124 indicated that 1.5 – 2.3% of applied was bound to fulvic acids, 2.5 – 2.9% to the immobile humic acids and 12.5 – 24.2% to humins.

 

The levels of radioactive CO₂in soils I-III reached 63.1 – 72.6%of the applied radioactivity by the end of the study.

 

Volatile radioactivity trapped in ethylene glycol was throughout the study below 0.1% the applied radioactivity.

 

The microbial biomass in soils I-III was 23.6 – 35.2 mg microbial C/100 g dry soil at the start and 19.1 – 27.4 mg at the end of the study confirming thereby the viability of the soils during the study.

 

The rate of disappearance of[¹⁴C]C22-ATQ was based on the total extractable radioactivity measured at each interval, thereby assuming the entire extractable radioactivity to consist of [¹⁴C]C22-ATQ.Fitted curves were calculated using simple first order kinetics.The following DT₅₀and DT₉₀values were determined:

 

Soil	[^14C]C22-ATQ
	DT50(days)	DT90(days)
Soil I	23.2	77.1
Soil II	24.0	79.9
Soil	41.4	137.4

 

Degradation of[¹⁴C]C22-ATQin soil incubated under aerobic conditions at 20 °C proceeds via the formation radioactive carbon dioxide and bound residues.

Description of key information

OECD 307 Aerobic Transformation in Soil, Key study

According Annex IX, Section 9.2.1.3 column 2 of the REACH Regulation 1907/2006/EC a Soil simulation test need not to be carried out if the substance is readily biodegradable. Nevertheless available information is provided using read across from the supporting substance C22-ATQ (CAS 17301-53-0). The degradation rate of 14C-C22 -ATQ in three aerobic soils was investigated during 124 days. The 14C-labelled substance was applied at a rate of 0.2 mg a.i./kg soil dw. using sewage sludge as carrier. The application rate was determined from an exposure modelling using realistic use rates. Soil sampling was done after 3, 7, 14, 29, 62 and 124 days. Significant amounts of radioactive carbon dioxide and bound residues were formed. The total mean recoveries of radioactivity were in the range of 105 to 107% for the three soils. From the measurements the following DT50 for biotransformation were calculated:

DT50 soil 1: 23.2 d;

DT50 soil 2: 24.9 d;

DT50 soil 3: 41.4 d,

Key value for chemical safety assessment

Half-life in soil:

28.5 d

at the temperature of:

20 °C