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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

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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.