Tetrabutylammonium bromide

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.91, 9.53 and 100.00 mg/kg dry soil
- Sampling method: Approximately 2 g of test sample of each concentrations, was transferred into 50 mL conical flask and 10 mL of diluent (Methanol) was added and the process was continued by shaking on a mechanical platform shaker at room temperature at an approximate speed of 180 rpm for 30 minutes. Then the sample were transferred to 15 mL Tarson tube and centrifuged (3000 rpm, at 25°C for 10 min). The supernatant was diluted with diluent (Methanol) as described below to obtain the final concentration of approximately 0.02 µg/mL of Tetra butyl ammonium bromide and injected to LC-MS/MS.
- Sample storage conditions before analysis: The test samples were analysed soon after sampling.

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): Test chemcial dissolved in water was mixed in soil homogenously
- Controls: For control, required volume of deionized water was mixed thoroughly in test substrate on dry weight basis.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not used/ Not applicable

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Red wiggler
- Source: The earthworm, Eisenia fetida bred at Eurofins Advinus Limited were procured from (initial source): Karthik Vermicompost Limited, Hebbal, Near GKVK, Bengaluru, India
- Age at test initiation (mean and range, SD):
- Weight at test initiation (mean and range, SD):

ACCLIMATION
Parameters Range Finding Test

Adults with well-developed clitellum of age
(difference in age of all worms is within 4 weeks) 4 to 4.5 months

Light intensity (Lux) 465 and 482
Temperature (ºC) 20.1 and 20.2
Feeding with pasteurized cow manure Yes
Duration of acclimatization Above 24 hours
Body weight range at start of test (mg) 305.0 to 520.1
Test containers weight
(to monitor moisture) Weekly once


Parameters Range Finding Test

Adults with well-developed clitellum of age
(difference in age of all worms is within 4 weeks) 4.5 to 5 months

Light intensity (Lux) 510 and 540
Temperature (ºC) 19.9
Feeding with pasteurized cow manure Yes
Duration of acclimatization Above 24 hours
Body weight range at start of test (mg) 322.4 to 400.3
Test containers weight
(to monitor moisture) Weekly once

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

19.6 to 20.4°C

pH:

range finding test::
Control: day 0: 6.21; day 56: 6.27
test concentrations: day 0: 6.50- 6.72 day 56: 6.55 - 6.76

Moisture:

Moisture content was in range of 40-60 % of WHCmax

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Crystallizing glass dishes (range finding test) and plastic containers (definitive test) having cross sectional area of approximately 200 cm2 of about one litre capacity were used as test vessels, so that a moist substrate depth of about 5-6 cm was achieved
- Amount of soil or substrate: 3kg of artificial soil
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- No. of replicates per vehicle control: not applicable


OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours dark
- Light intensity: 520 to 578

VEHICLE CONTROL PERFORMED: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study
- Test concentrations: 0.1, 1, 10, 100, 250, 500 and 1000 mg/kg dry soil
- Results used to determine the conditions for the definitive study:
Based on the observed mortality rate and number of coocon produtions after 28 days of exposure in rangefinding test was taken in consideration to decide the concentrations for definetive test.

Nominal and measured concentrations:

0.91, 1.63, 2.94, 5.29, 9.53, 17.15, 30.87, 55.56 and 100.00 mg/kg (nominal Concentrations)

Reference substance (positive control):

yes

Remarks:

Carbendazim

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

1.63 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

2.94 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

0.503 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Fucidal limits 0.2502-1.691

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

9.524 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Fucidal limit (3.128-36.93)

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

> 100 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Mortality at end of exposure period: No mortality was observed at the end test.
- Total mass of adults at beginning of test:
Control -3.6138 ± 0.0544 (gram)
0.91 mg/kg dry weight : 3.6797± 0.0351
1.63 mg/kg dry weight : 3.6851±0.0685
2.94 mg/kg dry weight : 3.5981±0.0811
5.29 mg/kg dry weight : 3.6073±0.0895
9.53 mg/kg dry weight : 3.6519±0.0549
17.15 mg/kg dry weight : 3.6615±0.0500
30.87mg/kg dry weight : 3.6673±0.0613
55.56 mg/kg dry weight : 3.6136±0.0318
100 mg/kg dry weight : 3.6892 ±0.0689

- Changes in body weight of live adults (% of initial weight) at end of exposure period:
% chnage in BW
Control 8.9
0.91 mg/kg dry weight : 10.4
1.63 mg/kg dry weight : 10.6
2.94 mg/kg dry weight : 7.2
5.29 mg/kg dry weight : 5.4
9.53 mg/kg dry weight : -2.9
17.15 mg/kg dry weight : -7.6
30.87mg/kg dry weight : -10.8
55.56 mg/kg dry weight : -13.8
100 mg/kg dry weight : -18.7
"-" indicates the decrease in the body weight from the initial body weight
- No. of offspring produced:
Number of offsprings produced% chnage in BW
Control 69.3
0.91 mg/kg dry weight : 70
1.63 mg/kg dry weight : 68.5
2.94 mg/kg dry weight : 58.3
5.29 mg/kg dry weight : 46
9.53 mg/kg dry weight : 34.5
17.15 mg/kg dry weight : 28.3
30.87mg/kg dry weight : 21.3
55.56 mg/kg dry weight : 17.5
100 mg/kg dry weight : 10
- Morphological abnormalities: No morphological and behavioural abnormalities were observed

Results with reference substance (positive control):

- Results with reference substance valid - yes valid
- Relevant effect levels: The percent mortality of earthworms at 3.0 mg a.i. of reference substance/kg dry soil was 17.5%, Reference substance (Carbendazim) group exhibited statistically significant reduction in juvenile production at 3 mg a.i./kg dry soil as compared with the control. Hence the test has met the validity acceptance criteria as per test guideline

Reported statistics and error estimates:

The statistical analysis of the average body weights and juvenile production was evaluated using licensed copies of SYSTAT Statistical package version 12.0. Data was tested for normality (Shapiro-Wilk test) and homogeneity of variance (Levene’s) before performing further analysis. When data was found as normal and homogeneous, ANOVA was performed for overall group comparison and Dunnett’s test for pairwise comparison. When data was found as non-normal or non-homogeneous, then Kruskal-Wallis was performed for overall group comparison and Mann-Whitney U test for pairwise comparison. Comparison of means between reference substance group and control group was done using two-sample t-test and Mann-Whitney U test. All analyses and comparisons were evaluated at the 5% (p≤0.05) level.

TABLE 1.            Definitive Test - pH of Test Medium

Group		Treatment (mg/kg dry soil)	pH
			Day 0 (start of the test)	Day 56 (start of the test)
C		Control	6.21	6.27
T1		0.91	6.50	6.55
T2		1.63	6.53	6.59
T3		2.94	6.50	6.56
T4		5.29	6.61	6.68
T5		9.53	6.70	6.73
T6		17.15	6.68	6.71
T7		30.87	6.63	6.69
T8		55.56	6.69	6.74
T9		100.00	6.72	6.76
R		3*	6.54	6.59

*: mg a.i./kg dry soil (reference substance)

TABLE 2.            Definitive Test - Moisture Content of Test Medium

Group	Treatment (mg/kg dry soil)	Moisture content (%)
		Day 0 (start of the test)	Day 56 (end of the test)
C	Control	28.0	27.1
T1	0.91	26.4	26.3
T2	1.63	26.9	26.7
T3	2.94	26.8	26.8
T4	5.29	26.7	26.4
T5	9.53	26.1	26.4
T6	17.15	26.7	26.8
T7	30.87	26.5	26.7
T8	55.56	26.2	26.1
T9	100.00	26.4	26.7
R	3*	26.2	26.9

*: mg a.i./kg dry soil (reference substance)

Note: Moisture content should be 40-60 % ofWHCmaxas per OECD 222 test guideline which corresponds to 21.6 to 32.4 % of moisture in the test medium based on WHCmax. The results of moisture in the test medium were found to be within this range.

TABLE 3.            Definitive Test - Observations of Adult Earthworms on Day 28

Group	Treatment (mg/kg dry soil)	Adult Earthworm Observations on Day 28 (at the end of first 4-week)								Total worms in a group at start of test	Mortality of worms in a group
		R1	R2	R3	R4	R5	R6	R7	R8		CM	%
C	Control	N(10)	N(10)	N(10)	N(10)	N(10)	N(10)	N(10)	N(10)	80	0	0
T1	0.91	N(10)	N(10)	N(10)	N(10)	$	$	$	$	40	0	0
T2	1.63	N(10)	N(10)	N(10)	N(10)	$	$	$	$	40	0	0
T3	2.94	N(10)	N(10)	N(10)	N(10)	$	$	$	$	40	0	0
T4	5.29	N(10)	N(10)	N(10)	N(10)	$	$	$	$	40	0	0
T5	9.53	N(10)	N(10)	N(10)	N(10)	$	$	$	$	40	0	0
T6	17.15	N(10)	N(10)	N(10)	N(10)	$	$	$	$	40	0	0
T7	30.87	N(10)	N(10)	N(10)	N(10)	$	$	$	$	40	0	0
T8	55.56	N(10)	N(10)	N(10)	N(10)	$	$	$	$	40	0	0
T9	100.00	N(10)	N(10)	N(10)	N(10)	$	$	$	$	40	0	0
R	3*	N(8), M(2)	N(10)	N(8), M(2)	N(10)	N(8), M(2)	N(6), M(4)	N(10)	N(6), M(4)	80	14	17.5

*: mg a.i./kg dry soil (reference substance); Earthworms in each replicate at start of test: 10; CM: Cumulative group mortality of earthworms in a group; $: Four replicates for test item treated groups; Toxic signs: N: Normal, M: Mortality; Number in the parentheses denotes the total number of affected earthworms.

TABLE 1.            Definitive Test - Observations of Juveniles on Day 56[c1] [r2] 

Group	Treatment (mg/kg dry soil)	Observations of Juveniles on Day 56 (at the end of second 4-week)								No. of juveniles		% CV	Change in juvenile’s production (% of control)	Reduction in juveniles production (% of control)	Total number of cocoons in a group
		R1	R2	R3	R4	R5	R6	R7	R8	Average	SD
C	Control	N(69)	N(71)	N(69)	N(69)	N(72)	N(68)	N(69)	N(67)	69.3	1.6	2.3	-	-	0
T1	0.91	N(69)	N(68)	N(72)	N(71)	$	$	$	$	70.0	1.8	2.6	101.0	-1.0	0
T2	1.63	N(66)	N(68)	N(69)	N(71)	$	$	$	$	68.5	2.1	3.1	98.8	1.2	0
^#T3	2.94	N(58)	N(59)	N(61)	N(55)	$	$	$	$	58.3	2.5	4.3	84.1	15.9	0
^#T4	5.29	N(46)	N(47)	N(42)	N(49)	$	$	$	$	46.0	2.9	6.3	66.4	33.6	0
^#T5	9.53	N(33)	N(35)	N(32)	N(38)	$	$	$	$	34.5	2.6	7.5	49.8	50.2	0
^#T6	17.15	N(26)	N(27)	N(31)	N(29)	$	$	$	$	28.3	2.2	7.8	40.8	59.2	0
^#T7	30.87	N(23)	N(21)	N(19)	N(24)	$	$	$	$	21.8	2.2	10.1	31.5	68.5	0
^#T8	55.56	N(18)	N(19)	N(16)	N(17)	$	$	$	$	17.5	1.3	7.4	25.3	74.7	0
^#T9	100.00	N(8)	N(12)	N(9)	N(11)	$	$	$	$	10.0	1.8	18.0	14.4	85.6	0
^#R	3*	N(16)	N(11)	N(14)	N(15)	N(16)	N(9)	N(14)	N(10)	13.1	2.7	20.6	18.9	81.1	0

*: mg a.i./kg dry soil (reference substance); $: Four replicates for test item treated groups;SD: Standard Deviation; CV: Coefficient of variation; Toxic signs: N: Normal;^#: Statistically significant difference in juvenile production as compared with the control.

TABLE 4.            Definitive Test - Group Body Weight of Earthworms

Group	Treatment (mg/kg dry soil)	Body Weight on Day 0 (g)				Body Weight on Day 28 (g)			% Change (Day 0 to 28)
		Replicate		Mean	SD	Replicate	Mean	SD
C	Control	R1	3.6766	3.6138	0.0544	3.9851	3.9366	0.0485	8.9
		R2	3.6155			3.9401
		R3	3.5374			3.8714
		R4	3.6697			3.9962
		R5	3.5345			3.8645
		R6	3.6331			3.9615
		R7	3.6439			3.9524
		R8	3.5993			3.9214
T1	0.91	R1	3.6372	3.6797	0.0351	4.0204	4.0629	0.0410	10.4
		R2	3.7010			4.0958
		R3	3.6656			4.0351
		R4	3.7150			4.1001
T2	1.63	R1	3.6049	3.6851	0.0685	3.9948	4.0774	0.0682	10.6
		R2	3.6524			4.0482
		R3	3.7525			4.1381
		R4	3.7304			4.1284
T3	2.94	R1	3.5302	3.5981	0.0811	3.8096	3.8575	0.0561	7.2
		R2	3.5329			3.8095
		R3	3.6329			3.8944
		R4	3.6964			3.9165

Group	Treatment (mg/kg dry soil)	Body Weight on Day 0 (g)				Body Weight on Day 28 (g)			% Change (Day 0 to 28)
		Replicate		Mean	SD	Replicate	Mean	SD
T4	5.29	R1	3.4858	3.6073	0.0895	3.6852	3.8034	0.0872	5.4
		R2	3.7008			3.8954
		R3	3.6291			3.8218
		R4	3.6136			3.8110
T5	9.53	R1	3.7021	3.6519	0.0549	3.5952	3.5455	0.0564	-2.9^#
		R2	3.5827			3.4748
		R3	3.6894			3.5865
		R4	3.6334			3.5255
T6	17.15	R1	3.6713	3.6615	0.0500	3.4045	3.3844	0.0187	-7.6^#
		R2	3.6054			3.3601
		R3	3.7246			3.3915
		R4	3.6447			3.3815
T7	30.87	R1	3.7417	3.6673	0.0613	3.3895	3.2700	0.0864	-10.8^#
		R2	3.6935			3.2322
		R3	3.6179			3.1881
		R4	3.6162			3.2702
T8	55.56	R1	3.6202	3.6136	0.0318	3.1485	3.1156	0.0519	-13.8^#
		R2	3.6396			3.1658
		R3	3.6272			3.0514
		R4	3.5675			3.0968

Group	Treatment (mg/kg dry soil)	Body Weight on Day 0 (g)				Body Weight on Day 28 (g)			% Change (Day 0 to 28)
		Replicate		Mean	SD	Replicate	Mean	SD
T9	100.00	R1	3.7172	3.6892	0.0689	2.9574	2.9988	0.0759	-18.7^#
		R2	3.7031			3.0295
		R3	3.7470			3.0895
		R4	3.5895			2.9189
R	3* (reference substance)	R1	3.6784	3.6454	0.0531	2.2851	2.4029	0.564A2	-34.1^#
		R2	3.6591			3.0354
		R3	3.6502			2.2241
		R4	3.6228			2.9947
		R5	3.7019			2.5015
		R6	3.6395			1.5541
		R7	3.5297			2.8998
		R8	3.6815			1.7284

*: mg a.i./kg dry soil (reference substance); Negative value indicates reduction in body weight of earthworms; SD: Standard Deviation

^#: Statistically significant reduction in body weight as compared with the control.

Active Ingredient	Range of mean Analyzed content of active compound (% of nominal)
	Day 0	Day 28	Day 56
Tetra butyl ammonium bromide	97.548 - 99.365	47.392 - 79.084	47.075 - 78.535

Validity criteria fulfilled:

yes

Remarks:

• Each replicate (containing 10 adults) produced minimum of 67 juveniles by the end of the test. • The coefficient of variation of reproduction is 2.3%. • There was no mortality of the earthworms in the control during the experimental period of 28 days.

Conclusions:

The effect of test chemical was studied to understand the reprodutive outcome after 56 days of study. After exposure tenure, NOEC, LOEC, EC10 and EC50 was observed to be 1.63, 2.94, 0.5026, and 9.524 mg/kg dry soil, respectively.

Executive summary:

The toxic effect of Test substance was studied on the earthworm,Eisenia fetida by artificial soil test. By following OECD 222 guidelies in the artificial soil. Artificial soil was prepared by mixing 5% of sphagnum peat, 20% of kaolin clay (kaolinite content > 30%) and 75% industrial sand (predominantly fine sand with more than 50% of the particles between 50 and 200 microns) on dry weight basis (mass/mass). The pH of the test substrate was 6.21 during the definitive test. A range finding test was carried out to determine test concentrations for the definitive test at 0.1, 1, 10, 100, 250, 500 and 1000 mg/kg dry soil along with control. There were mortalities of 0, 0, 0, 0, 0, 0, 50 and 100 % at the tested concentrations of 0 (control), 0.1, 1, 10, 100, 250, 500 and 1000 mg/kg dry soil on Day 28. No pathological and behavioral symptoms were observed during the test period in the control and treated groups (except decrease in size in 500 mg/kg dry soil test group). Based on the results of range finding test, the definitive test was carried with the test concentrations of0.91, 1.63, 2.94, 5.29, 9.53, 17.15, 30.87, 55.56 and 100.00mg/kg dry soil along with a control (deionized water) and reference substance (Carbendazim).

Before initiating the study the test system was acclimitised under the conditions mentioned below:

Parameters	Range Finding Test	Definitive Test
Adults with well-developed clitellum of age (difference in age of all worms is within 4 weeks)	4 to 4.5 months	4.5 to 5 months
Light intensity (Lux)	465 and 482	510 and 540
Temperature (ºC)	20.1 and 20.2	19.9
Feeding with pasteurized cow manure	Yes	Yes
Duration of acclimatization	Above 24 hours	Above 24 hours
Body weight range at start of test (mg)	305.0 to 520.1	322.4 to 400.3
Test containers weight (to monitor moisture)	Weekly once	Weekly once

There was no mortality of earthworms in control and at the tested concentrations of 0.91, 1.63, 2.94, 5.29, 9.53, 17.15, 30.87, 55.56 and 100.00 mg/kg on Day 28. The percent mortality of earthworms at 3.0 mg a.i. of reference substance/kg dry soil was 17.5%. No pathological and behavioral symptoms were observed in adult worms during the test period in the control and treated groups (including reference substance). No statistically significant reduction in body weight was observed in treated groups (except 5.29, 9.53, 17.15, 30.87, 55.56 and 100.00 mg/kg dry soil and reference substance test group) as compared with the control. No pathological and behavioral symptoms were observed in juveniles on Day 56 in the control and treated groups. No statistically significant reduction in juvenile production was observed in treated groups (except 2.94, 5.29, 9.53, 17.15, 30.87, 55.56 and 100.00 mg/kg dry soil and reference substance test group) as compared with the control. Cocoons were not present in any of tested groups and reference substance group.

The results of active ingredientanalysisof test concentrations of 0.91, 9.53 and 100.00 mg/kg dry are detailed in addtional information. Reference substance (Carbendazim) group exhibited statistically significant reduction in juvenile production at 3 mg a.i./kg dry soil as compared with the control. Hence the test has met the validity acceptance criteria as per test guideline OECD 222, that significant effects should be observed between 1 and 5 mg a.i./kg dry soil in a test. This result infers that the obtained results during this test are valid andhencetest is acceptable. The Day 28 LC₅₀of Tetra butyl ammonium bromide on adult earthworms is more than 100 mg test item/kg dry soil. The day 56 juvenile production NOEC, LOEC and Effective Concentrations value based on the nominal concentration of the test item 1.63, 2.94, 0.5026, and 9.524 mg/kg dry soil, respectively.

Description of key information

The toxic effect of Test substance was studied on the earthworm,Eisenia fetidaby artificial soil test. By following OECD 222 guidelies in the artificial soil. Artificial soil was prepared by mixing 5% of sphagnum peat, 20% of kaolin clay (kaolinite content > 30%) and 75% industrial sand (predominantly fine sand with more than 50% of the particles between 50 and 200 microns) on dry weight basis (mass/mass). The pH of the test substrate was 6.21 during the definitive test. A range finding test was carried out to determine test concentrations for the definitive test at 0.1, 1, 10, 100, 250, 500 and 1000 mg/kg dry soil along with control. There were mortalities of 0, 0, 0, 0, 0, 0, 50 and 100 % at the tested concentrations of 0 (control), 0.1, 1, 10, 100, 250, 500 and 1000 mg/kg dry soil on Day 28. No pathological and behavioral symptoms were observed during the test period in the control and treated groups (except decrease in size in 500 mg/kg dry soil test group). Based on the results of range finding test, the definitive test was carried with the test concentrations of0.91, 1.63, 2.94, 5.29, 9.53, 17.15, 30.87, 55.56 and 100.00mg/kg dry soil along with a control (deionized water) and reference substance (Carbendazim).

Before initiating the study the test system was acclimitised under the conditions mentioned below:

Parameters	Range Finding Test	Definitive Test
Adults with well-developed clitellum of age (difference in age of all worms is within 4 weeks)	4 to 4.5 months	4.5 to 5 months
Light intensity (Lux)	465 and 482	510 and 540
Temperature (ºC)	20.1 and 20.2	19.9
Feeding with pasteurized cow manure	Yes	Yes
Duration of acclimatization	Above 24 hours	Above 24 hours
Body weight range at start of test (mg)	305.0 to 520.1	322.4 to 400.3
Test containers weight (to monitor moisture)	Weekly once	Weekly once

There was no mortality of earthworms in control and at the tested concentrations of 0.91, 1.63, 2.94, 5.29, 9.53, 17.15, 30.87, 55.56 and 100.00 mg/kg on Day 28. The percent mortality of earthworms at 3.0 mg a.i. of reference substance/kg dry soil was 17.5%. No pathological and behavioral symptoms were observed in adult worms during the test period in the control and treated groups (including reference substance). No statistically significant reduction in body weight was observed in treated groups (except 5.29, 9.53, 17.15, 30.87, 55.56 and 100.00 mg/kg dry soil and reference substance test group) as compared with the control. No pathological and behavioral symptoms were observed in juveniles on Day 56 in the control and treated groups. No statistically significant reduction in juvenile production was observed in treated groups (except 2.94, 5.29, 9.53, 17.15, 30.87, 55.56 and 100.00 mg/kg dry soil and reference substance test group) as compared with the control. Cocoons were not present in any of tested groups and reference substance group.

The results of active ingredientanalysisof test concentrations of 0.91, 9.53 and 100.00 mg/kg dry are detailed in addtional information. Reference substance (Carbendazim) group exhibited statistically significant reduction in juvenile production at 3 mg a.i./kg dry soil as compared with the control. Hence the test has met the validity acceptance criteria as per test guideline OECD 222, that significant effects should be observed between 1 and 5 mg a.i./kg dry soil in a test. This result infers that the obtained results during this test are valid andhencetest is acceptable. The Day 28 LC₅₀of Tetra butyl ammonium bromide on adult earthworms is more than 100 mg test item/kg dry soil. The day 56 juvenile production NOEC, LOEC and Effective Concentrations value based on the nominal concentration of the test item. After exposure tenure, NOEC, LOEC, EC10 and EC50 was observed to be 1.63, 2.94, 0.5026, and 9.524 mg/kg dry soil, respectively.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:

1.63 mg/kg soil dw

Additional information

The toxic effect of Test substance was studied on the earthworm,Eisenia fetidaby artificial soil test. By following OECD 222 guidelies in the artificial soil. Artificial soil was prepared by mixing 5% of sphagnum peat, 20% of kaolin clay (kaolinite content > 30%) and 75% industrial sand (predominantly fine sand with more than 50% of the particles between 50 and 200 microns) on dry weight basis (mass/mass). The pH of the test substrate was 6.21 during the definitive test. A range finding test was carried out to determine test concentrations for the definitive test at 0.1, 1, 10, 100, 250, 500 and 1000 mg/kg dry soil along with control. There were mortalities of 0, 0, 0, 0, 0, 0, 50 and 100 % at the tested concentrations of 0 (control), 0.1, 1, 10, 100, 250, 500 and 1000 mg/kg dry soil on Day 28. No pathological and behavioral symptoms were observed during the test period in the control and treated groups (except decrease in size in 500 mg/kg dry soil test group). Based on the results of range finding test, the definitive test was carried with the test concentrations of0.91, 1.63, 2.94, 5.29, 9.53, 17.15, 30.87, 55.56 and 100.00mg/kg dry soil along with a control (deionized water) and reference substance (Carbendazim).

Before initiating the study the test system was acclimitised under the conditions mentioned below:

Parameters	Range Finding Test	Definitive Test
Adults with well-developed clitellum of age (difference in age of all worms is within 4 weeks)	4 to 4.5 months	4.5 to 5 months
Light intensity (Lux)	465 and 482	510 and 540
Temperature (ºC)	20.1 and 20.2	19.9
Feeding with pasteurized cow manure	Yes	Yes
Duration of acclimatization	Above 24 hours	Above 24 hours
Body weight range at start of test (mg)	305.0 to 520.1	322.4 to 400.3
Test containers weight (to monitor moisture)	Weekly once	Weekly once

There was no mortality of earthworms in control and at the tested concentrations of 0.91, 1.63, 2.94, 5.29, 9.53, 17.15, 30.87, 55.56 and 100.00 mg/kg on Day 28. The percent mortality of earthworms at 3.0 mg a.i. of reference substance/kg dry soil was 17.5%. No pathological and behavioral symptoms were observed in adult worms during the test period in the control and treated groups (including reference substance). No statistically significant reduction in body weight was observed in treated groups (except 5.29, 9.53, 17.15, 30.87, 55.56 and 100.00 mg/kg dry soil and reference substance test group) as compared with the control. No pathological and behavioral symptoms were observed in juveniles on Day 56 in the control and treated groups. No statistically significant reduction in juvenile production was observed in treated groups (except 2.94, 5.29, 9.53, 17.15, 30.87, 55.56 and 100.00 mg/kg dry soil and reference substance test group) as compared with the control. Cocoons were not present in any of tested groups and reference substance group.

The results of active ingredientanalysisof test concentrations of 0.91, 9.53 and 100.00 mg/kg dry are detailed in addtional information. Reference substance (Carbendazim) group exhibited statistically significant reduction in juvenile production at 3 mg a.i./kg dry soil as compared with the control. Hence the test has met the validity acceptance criteria as per test guideline OECD 222, that significant effects should be observed between 1 and 5 mg a.i./kg dry soil in a test. This result infers that the obtained results during this test are valid andhencetest is acceptable. The Day 28 LC₅₀of Tetra butyl ammonium bromide on adult earthworms is more than 100 mg test item/kg dry soil. The day 56 juvenile production NOEC, LOEC and Effective Concentrations value based on the nominal concentration of the test item. After exposure tenure, NOEC, LOEC, EC10 and EC50 was observed to be 1.63, 2.94, 0.5026, and 9.524 mg/kg dry soil, respectively.