Methyl 2-naphthyl ether

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

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.10, 1.04 and 11.0 mg/kg dry soil
- Sampling method: GC-MS/MS
- Sample storage conditions before analysis:

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

- Method of mixing into soil (if used):
- Controls: For control, required volume of deionized water was mixed thoroughly in test substrate on dry weight basis.
- Vehicle conetrol acetone mixed industrial sand was used as the vechicle control.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone

Test solutoiin preperations:
To prepare test item concentration of 0.10, 0.18, 0.32, 0.58, 1.04, 1.88, 3.39, 6.11 and 11.00 mg test item/kg dry weight of artificial soil, 0.060, 0.108, 0.192, 0.348, 0.624, 1.128, 2.034, 3.666 and 6.600 mL of test item stock
(5 mg/mL test item stock - 250 mg test item made up to 50 mL with acetone using volumetric flask) was mixed with industrial sand (30 g). This mixture was allowed to evaporate followed by introduction into pre-moistened test substrate (equivalent to 3 kg dry test substrate excluding 30 g industrial sand) and this mixture was homogenously mixed by hand.
For control, required volume of deionized water (1620 mL) was mixed thoroughly in 6 kg of test substrate on dry weight basis. For vehicle control, 13.2 mL of acetone was mixed with industrial sand (60 g). This mixture was allowed to evaporate followed by introduction into pre-moistened test substrate (equivalent to 6 kg dry test substrate excluding 60 g industrial sand) and this mixture was homogenously mixed by hand.
The test medium so prepared was used for exposure of earthworms using 762 g of test medium (equivalent to 600 g dry test substrate). There were 4 replicates for each test concentration and 8 replicates for the control, vehicle control and reference substance test groups.

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) 490 and 496
Temperature (ºC) 20.4 and 20.6
Feeding with pasteurized cow manure Yes
Duration of acclimatization Above above 24 hours
Body weight range at start of test (mg) 301.5 to 500.2
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) 500 and 510
Temperature (ºC) 19.8 and 19.9
Feeding with pasteurized cow manure Yes
Duration of acclimatization Above above 24 hours
Body weight range at start of test (mg) 320.5 to 400.6
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:

19.8 to 20.1

pH:

Group Treatment(mg/kg dry soil)
pH
Day 0 Day 28
G1 Control 6.32 6.53
G2 Vehicle control 6.58 6.66
G3 0.1 6.71 6.78
G4 1 6.75 6.79
G5 10 6.79 6.78
G6 100 6.69 6.77
G7 250 6.73 6.79
G8 500 6.79 6.83
G9 1000 6.81 6.96

Definitive Test - pH of Test Medium
Group Treatment(mg/kg dry soil)
pH
Day 0 Day 56
(start of the test) (end of the test)

C Control 6.21 6.27
V Vehicle control 6.55 6.60
T1 0.10 6.69 6.71
T2 0.18 6.75 6.78
T3 0.32 6.57 6.65
T4 0.58 6.63 6.69
T5 1.04 6.62 6.70
T6 1.88 6.62 6.71
T7 3.39 6.69 6.73
T8 6.11 6.61 6.69
T9 11.0 6.55 6.63
R 3* 6.54 6.59
*: mg a.i./kg dry soil (reference substance)

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:

0.10, 0.18, 0.32, 0.58, 1.04, 1.88, 3.39, 6.11 and 11.00 mg/kg ( nominal concentrations)

Reference substance (positive control):

yes

Remarks:

Carbendazim

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

0.18 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

0.32 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

0.194 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Fucidal limits 0.07098 - 0.4143

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

1.811 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Fucidal limit (1.271 - 2.611)

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

> 11 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)
Vehicle control - 3.6372 ± 0.0471
0.10 mg/kg dry weight : 3.6405 ± 0.0610
0.18 mg/kg dry weight : 3.6370 ± 0.0463
0.32 mg/kg dry weight : 3.5655 ± 0.1089
0.58 mg/kg dry weight : 3.6021± 0.0897
1.04 mg/kg dry weight : 3.6364± 0.0712
1.88 mg/kg dry weight : 3.6093 ± 0.0563
3.39 mg/kg dry weight : 3.5610 ± 0.1197
6.11 mg/kg dry weight : 3.6216 ± 0.0273
11 mg/kg dry weight :3.5792 ± 0.0392

- Changes in body weight of live adults (% of initial weight) at end of exposure period:
% chnage in BW
Control 8.9
Vehicle conctrol 8.6
0.10 mg/kg dry weight : 8.5
0.18 mg/kg dry weight : 8.5
0.32 mg/kg dry weight : 5.1
0.58 mg/kg dry weight : 3.7
1.04 mg/kg dry weight : -2.7
1.88 mg/kg dry weight : -4.3
3.39 mg/kg dry weight : -8.9
6.11 mg/kg dry weight : -12.8
11 mg/kg dry weight :-15.4
"-" 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
Vehicle conctrol 69.8
0.10 mg/kg dry weight : 68.8
0.18 mg/kg dry weight : 68.3
0.32 mg/kg dry weight : 58.3
0.58 mg/kg dry weight : 53.8
1.04 mg/kg dry weight : 42.0
1.88 mg/kg dry weight : 35.5
3.39 mg/kg dry weight : 29.3
6.11 mg/kg dry weight : 22.5
11 mg/kg dry weight :13.3
- 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.            Range Finding Test - pHof Test Medium

Group	Treatment (mg/kg dry soil)	pH
		Day 0	Day 28
G1	Control	6.32	6.53
G2	Vehicle control	6.58	6.66
G3	0.1	6.71	6.78
G4	1	6.75	6.79
G5	10	6.79	6.78
G6	100	6.69	6.77
G7	250	6.73	6.79
G8	500	6.79	6.83
G9	1000	6.81	6.96

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TABLE 2.            Range Finding Test - Moisture Content of Test Medium

Group	Treatment (mg/kg dry soil)	Moisture content (%)
		Day 0 (start of the test)	Day 28 (end of the test)
G1	Control	26.9	28.0
G2	Vehicle control	27.0	27.5
G3	0.1	26.8	27.7
G4	1	27.7	27.0
G5	10	26.6	27.5
G6	100	28.2	26.8
G7	250	26.5	28.0
G8	500	27.7	27.5
G9	1000	27.0	26.5

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.            Range Finding Test - Summary Results

Group	Treatment (mg/kg dry soil)	Earthworms per group at start of test	Pathological and behavioral symptoms on Day 28	% Mortality on Day 28	Mean body weight of earthworm on Day 0 (mg)	Mean body weight of earthworm on Day 28 (mg)	% Body weight change from test start (%)	Unhatched cocoon on Day 28	Juvenile production on Day 28
G1	Control	10	N(10)	0	377.1	412.4	9.4	13	5
G2	Vehicle control	10	N(10)	0	389.3	426.6	9.6	14	3
G3	0.1	10	N(10)	0	394.4	430.8	9.2	13	4
G4	1	10	N(10)	0	373.5	365.5	-2.2	8	3
G5	10	10	N(10)	0	377.7	335.2	-11.3	5	2
G6	100	10	N(10)	0	386.5	298.3	-22.8	3	-
G7	250	10	-	100	393.3	-	-	-	-
G8	500	10	-	100	362.7	-	-	-	-
G9	1000	10	-	100	368.2	-	-	-	-

Toxic signs: N: Normal

Number in the parentheses denotes the total number of affected earthworms.

TABLE 4.            Definitive Test - pH of Test Medium

Group		Treatment (mg/kg dry soil)		pH
				Day 0 (start of the test)	Day 56 (end of the test)
C		Control		6.21		6.27
V		Vehicle control		6.55		6.60
T1		0.10		6.69		6.71
T2		0.18		6.75		6.78
T3		0.32		6.57		6.65
T4		0.58		6.63		6.69
T5		1.04		6.62		6.70
T6		1.88		6.62		6.71
T7		3.39		6.69		6.73
T8		6.11		6.61		6.69
T9		11.0		6.55		6.63
R		3*		6.54		6.59

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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TABLE 5.            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
V	Vehicle control	26.0	26.8
T1	0.10	26.0	27.6
T2	0.18	26.6	28.3
T3	0.32	26.7	27.5
T4	0.58	26.5	27.9
T5	1.04	26.4	28.0
T6	1.88	26.5	27.6
T7	3.39	26.5	26.7
T8	6.11	26.2	26.9
T9	11.0	26.1	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 6.            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
V	Vehicle control	N(10)	N(10)	N(10)	N(10)	N(10)	N(10)	N(10)	N(10)	80	0	0
T1	0.10	N(10)	N(10)	N(10)	N(10)	$	$	$	$	40	0	0
T2	0.18	N(10)	N(10)	N(10)	N(10)	$	$	$	$	40	0	0
T3	0.32	N(10)	N(10)	N(10)	N(10)	$	$	$	$	40	0	0
T4	0.58	N(10)	N(10)	N(10)	N(10)	$	$	$	$	40	0	0
T5	1.04	N(10)	N(10)	N(10)	N(10)	$	$	$	$	40	0	0
T6	1.88	N(10)	N(10)	N(10)	N(10)	$	$	$	$	40	0	0
T7	3.39	N(10)	N(10)	N(10)	N(10)	$	$	$	$	40	0	0
T8	6.11	N(10)	N(10)	N(10)	N(10)	$	$	$	$	40	0	0
T9	11.0	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 7.            Definitive Test - Observations of Juveniles on Day 56

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
V	Vehicle control	N(69)	N(65)	N(68)	N(72)	N(75)	N(71)	N(68)	N(70)	69.8	3.0	4.3	-	-	0
T1	0.10	N(67)	N(69)	N(68)	N(71)	$	$	$	$	68.8	1.7	2.5	98.6	1.4	0
T2	0.18	N(69)	N(70)	N(66)	N(68)	$	$	$	$	68.3	1.7	2.5	97.9	2.1	0
^#T3	0.32	N(61)	N(58)	N(55)	N(59)	$	$	$	$	58.3	2.5	4.3	83.5	16.5	0
^#T4	0.58	N(53)	N(54)	N(58)	N(50)	$	$	$	$	53.8	3.3	6.1	77.1	22.9	0
^#T5	1.04	N(45)	N(41)	N(39)	N(43)	$	$	$	$	42.0	2.6	6.2	60.2	39.8	0
^#T6	1.88	N(39)	N(37)	N(32)	N(34)	$	$	$	$	35.5	3.1	8.7	50.9	49.1	0
^#T7	3.39	N(28)	N(32)	N(31)	N(26)	$	$	$	$	29.3	2.8	9.6	42.0	58.0	0
^#T8	6.11	N(23)	N(26)	N(21)	N(20)	$	$	$	$	22.5	2.6	11.6	32.2	67.8	0
^#T9	11.0	N(17)	N(16)	N(9)	N(11)	$	$	$	$	13.3	3.9	29.3	19.1	80.9	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	18.8	81.2	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 or vehicle control.

TABLE 8.            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
V	Vehicle control	R1	3.5919	3.6372	0.0471	3.9095	3.9487	0.0464	8.6
		R2	3.6531			3.9684
		R3	3.5572			3.8673
		R4	3.6836			3.9862
		R5	3.6858			3.9991
		R6	3.6345			3.9440
		R7	3.6130			3.9229
		R8	3.6785			3.9918
T1	0.10	R1	3.5572	3.6405	0.0610	3.8755	3.9499	0.0542	8.5
		R2	3.6860			3.9871
		R3	3.6863			3.9931
		R4	3.6325			3.9437

Note: Body weight range of earthworms at the start of test is from 320.5 to 400.6 mg; SD: Standard Deviation

TABLE 8 contd. 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
T2	0.18	R1	3.5967	3.6370	0.0463	3.8981	3.9462	0.0451	8.5
		R2	3.6015			3.9204
		R3	3.6567			3.9693
		R4	3.6932			3.9971
T3	0.32	R1	3.4775	3.5655	0.1089	3.6832	3.7470	0.1411	5.1^#
		R2	3.5671			3.7682
		R3	3.7184			3.9331
		R4	3.4988			3.6035
T4	0.58	R1	3.5998	3.6021	0.0897	3.7049	3.7351	0.1096	3.7^#
		R2	3.4887			3.5929
		R3	3.7079			3.8188
		R4	3.6121			3.8238
T5	1.04	R1	3.6981	3.6364	0.0712	3.5956	3.5382	0.0435	-2.7^#
		R2	3.5395			3.5041
		R3	3.6276			3.5047
		R4	3.6802			3.5485
T6	1.88	R1	3.6127	3.6093	0.0563	3.4914	3.4528	0.0302	-4.3^#
		R2	3.6590			3.4599
		R3	3.5297			3.4215
		R4	3.6358			3.4384

SD: Standard Deviation;^#: Statistically significant difference in body weight as compared with the vehicle control.

TABLE 8 contd. 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
T7	3.39	R1	3.6575	3.5610	0.1197	3.3954	3.2454	0.1258	-8.9^#
		R2	3.4295			3.1705
		R3	3.4898			3.1168
		R4	3.6673			3.2987
T8	6.11	R1	3.6543	3.6216	0.0273	3.1581	3.1594	0.0347	-12.8^#
		R2	3.5935			3.1351
		R3	3.6326			3.1355
		R4	3.6058			3.2089
T9	11.0	R1	3.5460	3.5792	0.0392	3.0357	3.0278	0.0361	-15.4^#
		R2	3.5553			2.9842
		R3	3.6333			3.0199
		R4	3.5823			3.0712
R	3* (reference substance)	R1	3.6784	3.6454	0.0531	2.2851	2.4029	0.5642	-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 difference in body weight as compared with the control or vehicle control.

Date	Temperature (ºC)	Light intensity (Lux)	Date	Temperature (ºC)	Light intensity (Lux)
22 July 20	19.8	540	20 August 20	19.9	536
23 July 20	19.7	550	21 August 20	20.1	534
24 July 20	20.0	535	22 August 20	20.1	539
25 July 20	19.9	520	23 August 20	20.0	540
26 July 20	19.7	532	24 August 20	20.0	541
27 July 20	19.8	540	25 August 20	20.1	538
28 July 20	19.7	530	26 August 20	20.1	540
29 July 20	19.9	543	27 August 20	19.9	535
30 July 20	20.0	556	28 August 20	20.0	542
31 July 20	19.8	550	29 August 20	19.8	533
01 August 20	20.0	548	30 August 20	19.9	539
02 August 20	19.7	542	31 August 20	20.0	540
03 August 20	19.9	560	01 September 20	20.1	546
04 August 20	20.0	533	02 September 20	20.0	530
05 August 20	20.3	536	03 September 20	19.9	538
06 August 20	20.1	539	04 September 20	20.0	540
07 August 20	20.0	543	05 September 20	19.9	538
08 August 20	19.9	539	06 September 20	19.9	540
09 August 20	20.0	537	07 September 20	20.0	545
10 August 20	20.1	540	08 September 20	20.1	535
11 August 20	20.0	532	09 September 20	19.9	540
12 August 20	20.3	539	10 September 20	20.1	538
13 August 20	19.9	540	11 September 20	19.9	540
14 August 20	20.0	541	12 September 20	20.0	530
15 August 20	19.9	539	13 September 20	20.2	539
16 August 20	20.0	544	14 September 20	20.2	540
17 August 20	20.1	536	15 September 20	20.1	535
18 August 20	20.0	541	16 September 20	19.9	520
19 August 20	19.9	530

Analytical monitoring:

Active Ingredient	Range of mean Analyzed content of active compound (% of nominal)
	Day 0	Day 28	Day 56
Methyl-2-Naphthyl Ether	89.199 - 102.603	4.451 - 59.000	0.588 - 8.227

APPENDIX 1.          Definitive Test - Maximum Water Holding Capacity (WHCmax) of the Artificial Soil

Constituents		Weight
Mass of water-saturated substrate plus the dry mass of filter paper (g)	(^mS)	167.3113
Mass of Keens Cup + Filter Paper (g)	(^mT)	154.6635
Dry Mass of Substrate (g)	(^mD)	8.2116
WHCmax (Maximum Water Holding Capacity)		54.0%

 

Validity criteria fulfilled:

yes

Remarks:

• Each replicate (containing 10 adults) produced minimum of 65 juveniles by the end of the test. • The coefficient of variation of reproduction is 4.3%. • 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 0.18, 0.32, 0.1942, and 1.811 mg/kg dry soil, respectively.

Executive summary:

The toxic effect of test chemical was studied on the earthworm, Eisenia fetida by artificial soil test. the study was conducted in two phases ie., range finding test and definitive test. The artificial soil was used as the substrate which was prepared as per the OECD 222 guidelines. As the test chemical was insoluble, acetone was used as carrier vehicle, simultaneously along with test concentrations both control (deionized water) and vehicle control (acetone) were also studied. Before initiation of the definitive study a range finding test was conducted at concentrations of 0.1, 1, 10, 100, 250, 500 and 1000 mg/kg dry. Based on number of cocoon and juvenile production, the concentrations for the main study was decided as 0.10, 0.18, 0.32, 0.58, 1.04, 1.88, 3.39, 6.11 and 11.00 mg/kg. Adult earthwormswith well-developed clitellum of age ranging from 4.5 to 5 months were exposed for definitive test concentrations with minimum number of 10 test organisms per replicate. The test was conducted for the period of 56 days with 8 replicates in control groups and 4 replicates in exposure groups. After mixing of test chemical with artificial soil at a wide range of test item concentrations in which the adult earthworms were placed for 28 days exposure. The mortality and growth effects of the test item on the adult earthworms were examined after 4 weeks of exposure. The adults are then removed from the soil and effects on reproduction was assessed after a further 4 weeks by counting the number of offspring present in the soil. The reproductive output of the worms exposed to the test item is compared to that of the control(s) to determine the no observed effect concentration (NOEC) / effective concentrations.

There was no mortality of earthworms in control, vehicle control and at the tested concentrations of0.10, 0.18, 0.32, 0.58, 1.04, 1.88, 3.39, 6.11 and 11.00mg/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 behavioralsymptomswere observed in adult worms during the test period in the control, vehicle control and treated groups (including reference substance). No statistically significant reduction in body weight was observed in treated groups (except 0.32, 0.58, 1.04, 1.88, 3.39, 6.11 and 11.00 mg/kg dry soil and reference substance test group) as compared with the control or vehicle control.

No pathological and behavioral symptoms were observed in juveniles on Day 56 in the control, vehicle control and treated groups. No statistically significant reduction in juvenile production was observed in treated groups (except 0.32, 0.58, 1.04, 1.88, 3.39, 6.11 and 11.00 mg/kg dry soil and reference substance test group) as compared with the control or vehicle control. Cocoons were not present in any of tested groups and reference substance group. After obtaining all the required necessary outcomes, the generated data was treated for statistical analysis. 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.

To ensure the bio-availability of test chemical, the active ingredientof test concentrations of low, mid and high test concentrations ie., 0.10, 1.04 and 11.0 mg/kg dry soil were analysed using GC-MS-MS. All the validity criteria, ie., 1.Each replicate (containing 10 adults) produced minimum of 65 juveniles by the end of the test. 2. The coefficient of variation of reproduction is 4.3%. 3. There was no mortality of the earthworms in the control during the experimental period of 28 days, were met, andReference substance (Carbendazim) group exhibited statistically significant reduction in juvenile production at 3 mg a.i./kg dry soil as compared with the control making study valid.

Upon 28 days exposure there was no mortality in the adult earthworms, resulting LC50 >11mg/kg dry soil. After 56 days of exposure NOEC, LOEC, EC10 and EC50 was determined to be 0.18, 0.32, 0.1942 (0.07098-0.4143) and 1.811 (1.271-2.611). respectively.

Description of key information

The toxic effect of test chemical was studied on the earthworm, Eisenia fetida by artificial soil test. the study was conducted in two phases ie., range finding test and definitive test. The artificial soil was used as the substrate which was prepared as per the OECD 222 guidelines. As the test chemical was insoluble, acetone was used as carrier vehicle, simultaneously along with test concentrations both control (deionized water) and vehicle control (acetone) were also studied. Before initiation of the definitive study a range finding test was conducted at concentrations of 0.1, 1, 10, 100, 250, 500 and 1000 mg/kg dry. Based on number of cocoon and juvenile production, the concentrations for the main study was decided as 0.10, 0.18, 0.32, 0.58, 1.04, 1.88, 3.39, 6.11 and 11.00 mg/kg. Adult earthwormswith well-developed clitellum of age ranging from 4.5 to 5 months were exposed for definitive test concentrations with minimum number of 10 test organisms per replicate. The test was conducted for the period of 56 days with 8 replicates in control groups and 4 replicates in exposure groups. After mixing of test chemical with artificial soil at a wide range of test item concentrations in which the adult earthworms were placed for 28 days exposure. The mortality and growth effects of the test item on the adult earthworms were examined after 4 weeks of exposure. The adults are then removed from the soil and effects on reproduction was assessed after a further 4 weeks by counting the number of offspring present in the soil. The reproductive output of the worms exposed to the test item is compared to that of the control(s) to determine the no observed effect concentration (NOEC) / effective concentrations.

There was no mortality of earthworms in control, vehicle control and at the tested concentrations of0.10, 0.18, 0.32, 0.58, 1.04, 1.88, 3.39, 6.11 and 11.00mg/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 behavioralsymptomswere observed in adult worms during the test period in the control, vehicle control and treated groups (including reference substance). No statistically significant reduction in body weight was observed in treated groups (except 0.32, 0.58, 1.04, 1.88, 3.39, 6.11 and 11.00 mg/kg dry soil and reference substance test group) as compared with the control or vehicle control.

No pathological and behavioral symptoms were observed in juveniles on Day 56 in the control, vehicle control and treated groups. No statistically significant reduction in juvenile production was observed in treated groups (except 0.32, 0.58, 1.04, 1.88, 3.39, 6.11 and 11.00 mg/kg dry soil and reference substance test group) as compared with the control or vehicle control. Cocoons were not present in any of tested groups and reference substance group. After obtaining all the required necessary outcomes, the generated data was treated for statistical analysis. 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.

To ensure the bio-availability of test chemical, the active ingredientof test concentrations of low, mid and high test concentrations ie., 0.10, 1.04 and 11.0 mg/kg dry soil were analysed using GC-MS-MS. All the validity criteria, ie., 1.Each replicate (containing 10 adults) produced minimum of 65 juveniles by the end of the test. 2. The coefficient of variation of reproduction is 4.3%. 3. There was no mortality of the earthworms in the control during the experimental period of 28 days, were met, andReference substance (Carbendazim) group exhibited statistically significant reduction in juvenile production at 3 mg a.i./kg dry soil as compared with the control making study valid.

Upon 28 days exposure there was no mortality in the adult earthworms, resulting LC50 >11mg/kg dry soil. After 56 days of exposure NOEC, LOEC, EC10 and EC50 was determined to be 0.18, 0.32, 0.1942 (0.07098-0.4143) and 1.811 (1.271-2.611). respectively.

Key value for chemical safety assessment

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

0.18 mg/kg soil dw

Additional information

The toxic effect of test chemical was studied on the earthworm, Eisenia fetida by artificial soil test. the study was conducted in two phases ie., range finding test and definitive test. The artificial soil was used as the substrate which was prepared as per the OECD 222 guidelines. As the test chemical was insoluble, acetone was used as carrier vehicle, simultaneously along with test concentrations both control (deionized water) and vehicle control (acetone) were also studied. Before initiation of the definitive study a range finding test was conducted at concentrations of 0.1, 1, 10, 100, 250, 500 and 1000 mg/kg dry. Based on number of cocoon and juvenile production, the concentrations for the main study was decided as 0.10, 0.18, 0.32, 0.58, 1.04, 1.88, 3.39, 6.11 and 11.00 mg/kg. Adult earthwormswith well-developed clitellum of age ranging from 4.5 to 5 months were exposed for definitive test concentrations with minimum number of 10 test organisms per replicate. The test was conducted for the period of 56 days with 8 replicates in control groups and 4 replicates in exposure groups. After mixing of test chemical with artificial soil at a wide range of test item concentrations in which the adult earthworms were placed for 28 days exposure. The mortality and growth effects of the test item on the adult earthworms were examined after 4 weeks of exposure. The adults are then removed from the soil and effects on reproduction was assessed after a further 4 weeks by counting the number of offspring present in the soil. The reproductive output of the worms exposed to the test item is compared to that of the control(s) to determine the no observed effect concentration (NOEC) / effective concentrations.

There was no mortality of earthworms in control, vehicle control and at the tested concentrations of0.10, 0.18, 0.32, 0.58, 1.04, 1.88, 3.39, 6.11 and 11.00mg/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 behavioralsymptomswere observed in adult worms during the test period in the control, vehicle control and treated groups (including reference substance). No statistically significant reduction in body weight was observed in treated groups (except 0.32, 0.58, 1.04, 1.88, 3.39, 6.11 and 11.00 mg/kg dry soil and reference substance test group) as compared with the control or vehicle control.

No pathological and behavioral symptoms were observed in juveniles on Day 56 in the control, vehicle control and treated groups. No statistically significant reduction in juvenile production was observed in treated groups (except 0.32, 0.58, 1.04, 1.88, 3.39, 6.11 and 11.00 mg/kg dry soil and reference substance test group) as compared with the control or vehicle control. Cocoons were not present in any of tested groups and reference substance group. After obtaining all the required necessary outcomes, the generated data was treated for statistical analysis. 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.

To ensure the bio-availability of test chemical, the active ingredientof test concentrations of low, mid and high test concentrations ie., 0.10, 1.04 and 11.0 mg/kg dry soil were analysed using GC-MS-MS. All the validity criteria, ie., 1.Each replicate (containing 10 adults) produced minimum of 65 juveniles by the end of the test. 2. The coefficient of variation of reproduction is 4.3%. 3. There was no mortality of the earthworms in the control during the experimental period of 28 days, were met, andReference substance (Carbendazim) group exhibited statistically significant reduction in juvenile production at 3 mg a.i./kg dry soil as compared with the control making study valid.

Upon 28 days exposure there was no mortality in the adult earthworms, resulting LC50 >11mg/kg dry soil. After 56 days of exposure NOEC, LOEC, EC10 and EC50 was determined to be 0.18, 0.32, 0.1942 (0.07098-0.4143) and 1.811 (1.271-2.611). respectively.