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Diss Factsheets

Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-10-27 to 2016-12-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Version / remarks:
2004
Deviations:
yes
Remarks:
Food was provided on day 0 instead of day 1 due to good experience with this procedure; Due to technical reasons, temperature increased temporarily to a maximum of 23 °C; Soil moisture deviated by more than 10 % from initial value
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2017-01-03

Test material

Constituent 1
Chemical structure
Reference substance name:
(R,S)-5-cyclohexyl-2-methyl-pentan-1-ol
EC Number:
700-146-1
Cas Number:
1141487-54-8
Molecular formula:
C12H24O
IUPAC Name:
(R,S)-5-cyclohexyl-2-methyl-pentan-1-ol

Sampling and analysis

Analytical monitoring:
no

Test substrate

Vehicle:
no
Details on preparation and application of test substrate:
- Method of mixing into soil: The respective test item amount was weighed out for each test item concentration and dissolved in demineralised water. The spiking solution was then added to the artificial soil and mixed thoroughly. Application was performed prior to the introduction of test organisms to the test vessels.
- Controls: Artificial soil moistened with demineralised water without test item, applied under the same conditions as the test item treatments.

Test organisms

Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Earthworm
- Source: Breeding stock culture maintained at the test facility.
- Method of breeding: Organisms are bred in covered plastic vessels containing potting compost.
- Age at test initiation (mean and range, SD): Adult earthworms (with clitellum) aged 2-12 months (difference in age did not deviate by more than one month).
- Weight at test initiation (mean and range, SD): 0.33-0.59 g

ACCLIMATION
- Acclimation period: 2 days
- Acclimation conditions (same as test or not): Medium same as test medium (artificial soil)
- Health during acclimation (any mortality observed): No mortality observed.

Study design

Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
8 wk

Test conditions

Test temperature:
18-23 °C
pH:
At test start (day 0): 5.80-6.05
At test end (day 56): 5.96-6.15
Moisture:
At test start (day 0): 17.7-18.9 % of dry weight
At test end (day 56): 22.7-24.9 % of dry weight
Details on test conditions:
TEST SYSTEM
- Test container (material, size): Round plastic boxes with transparent and perforated lids; inner diameter: 15 cm; bottom surface area: 177 cm2; height: 14 cm
- Amount of soil or substrate: 600 g soil dry weight
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate):
- 5 % peat (air-dried and finely ground);
- 20 % kaolin (kaolinite content > 30 %);
- 74 % air-dried quartz sand (sand with > 50 % particle size of 0.05-0.2 mm);
- 0.20 % calcium carbonate
- Maximum water holding capacity: 37.2 g/100 g dry weight
- Pretreatment of soil: Artificial soil was moistened to 27 % of the maximum water holding capacity two days prior to test initiation.
- Storage (condition, duration): Moistened soil was kept covered to prevent evaporative water losses, 2 days

OTHER TEST CONDITIONS
- Photoperiod: 16 h light:8 h dark
- Light intensity: 400-800 lx
- Feeding during test: Cattle manure (contamination status not specified), weekly
- Moistening: Replicates were weighed weekly. Evaporated water was replenished by adding demineralised water to maintain the soil moisture. Additional moistening was performed in case the soil appeared too dry.

EFFECT PARAMETERS MEASURED:
- Body weight (biomass) of adult earthworms, at 0 d (test start) and at 28 d
- Mortality of adult earthworms, at 28 d
- Morphological changes of adult earthworms, at 28 d
- Reproduction (number of offspring), at 56 d

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ~ 2

- Range finding study
- Test concentrations: 0 (control), 10, 100, 1000 mg test item/kg soil dry weight (SDW)
- Results used to determine the conditions for the definitive study:
- Mortality: 0 % at 0-100 mg test item/kg SDW; 25 % at 1000 mg test item/kg SDW
- Change of live weight: -12.5 % in control; -12.4 % at 10 mg test item/kg SDW; -10.6 % at 100 mg test item/kg SDW; -12.8 % at 1000 mg test item/kg SDW
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 15.625, 31.3, 62.5, 125, 250, 500 mg test item/kg soil dry weight
Reference substance (positive control):
yes
Remarks:
Carbendazim; test concentrations: 0.5, 1.0, 2.0, 4.0 mg active substance/kg soil dry weight

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
500 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
500 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass
Duration:
56 d
Dose descriptor:
other: EC20
Effect conc.:
343 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
250 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
56 d
Dose descriptor:
LOEC
Effect conc.:
500 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Mortality at end of exposure period: No significant mortality (< 10 %) was observed in the controls and all test concentrations
- Total mass of adults at beginning of test: 0.33-0.59 g per individual
- Changes in body weight of live adults (% of initial weight) at end of exposure period: 30.8 ± 7.22 % (control)
- No. of offspring produced: 79 ± 14 juveniles (control)
- Morphological abnormalities: No significant morphological symptoms were observed in the controls and all test concentrations.
- Behavioural abnormalities: No significant behavioural abnormalities were observed in the controls and all test concentrations.
Results with reference substance (positive control):
Tests with the reference substance are conducted once every 12 months. In the most recent test (2016-01-21 to 2016-03-18), no effects on the reproduction of Eisenia fetida after 56 days were observed at 0.5 and 1.0 mg a.s./kg soil dry weight. Significant effects (ANOVA, α = 0.05) occurred at 2.0 and 4.0 mg a.s./kg soil dry weight. These results are in accordance with OECD 222, which states that significant effects of Carbendazim should be observed between 1.0 and 5.0 mg a.s./ kg soil dry weight.
Reported statistics and error estimates:
For the effect on biomass, Williams Multiple Sequential t-test Procedure (α = 0.05) was carried out for the determination of significant differences in biomass compared to the control. A Normality Test and an Equal Variance Test were conducted prior to running this test. For the effect on reproduction, significant differences were determined in comparison with the control group by t-test (α = 0.05). A Shapiro-Wilk Normality Test and Equal Variance Test were run beforehand. For the determination of the EC20 value, a regression analysis was performed (coefficient of determination (R2) and equation of the linear regression are not reported).

Any other information on results incl. tables

Table 1: Range Finding Test (14 days): Mortality and Change of Live Weight

Concentration

[mg test item/kg soil dry weight]

Mortality of adult earthworms

[%]

Change of Live weight

[%]

Control

0

- 12.5

10

0

- 12.4

100

0

- 10.6

1000

25

- 12.8

Table 2: Mortality of Adult Earthworms in [%] after 28 Days of Exposure

Application rate

Replicate

[mg/kg SDW]

1

2

3

4

5

6

7

8

Mean

Control

0

0

10

0

0

0

0

0

1.25

15.625

10

0

0

0

---

2.5

31.3

0

0

0

0

0

62.5

0

0

0

0

0

125

0

0

0

0

0

250

0

0

0

0

0

500

0

0

0

0

0

Table 3: Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Application rate

Replicate

[mg/kg SDW]

Observation

parameter

1

2

3

4

5

6

7

8

Control

A

H

10/10

10/10

9/10

1/10

10/10

10/10

10/10

10/10

10/10

15.625

A

H

9/10

1/10

10/10

10/10

10/10

-

31.3

A

10/10

10/10

10/10

10/10

62.5

A

10/10

10/10

10/10

10/10

125

A

10/10

10/10

10/10

10/10

250

A

10/10

10/10

10/10

10/10

500

A

10/10

10/10

10/10

10/10

A = no obvious pathological symptoms; H = earthworm dead

Table 4: Body Weight Changes of the Adult Earthworms

Application rate
[mg/kg SDW]

Replicate

Mean body weights per replicate

Mean body weight change of earthworms

Test start

28 days

Per replicate

Mean ± SD

Sig.

[g]

[g]

[g]

[%]

[g]

[%]

Control

1

0.43

0.54

0.11

25.6

0.13 ± 0.03

30.8 ± 7.22

No

2

0.44

0.55

0.11

25.0

3

0.43

0.57

0.14

32.6

4

0.44

0.57

0.13

29.5

5

0.43

0.56

0.13

30.2

6

0.44

0.54

0.10

22.7

7

0.43

0.58

0.15

34.9

8

0.44

0.64

0.20

45.5

15.625

1

0.43

0.57

0.14

32.6

0.15 ± 0.02

35.1 ± 3.96

No

2

0.44

0.59

0.15

34.1

3

0.43

0.57

0.14

32.6

4

0.44

0.62

0.18

40.9

31.3

1

0.43

0.61

0.18

41.9

0.15 ± 0.04

35.1 ± 8.82

No

2

0.44

0.57

0.13

29.5

3

0.43

0.54

0.11

25.6

4

0.44

0.63

0.19

43.2

62.5

1

0.43

0.61

0.18

41.9

0.16 ± 0.04

37.4 ± 9.77

No

2

0.44

0.65

0.21

47.7

3

0.43

0.58

0.15

34.9

4

0.44

0.55

0.11

25.0

125

1

0.43

0.54

0.11

25.6

0.16 ± 0.04

36.7 ± 9.15

No

2

0.44

0.65

0.21

47.7

3

0.43

0.58

0.15

34.9

4

0.44

0.61

0.17

38.6

250

1

0.43

0.60

0.17

39.5

0.15 ± 0.02

35.1 ± 4.33

No

2

0.44

0.57

0.13

29.5

3

0.43

0.59

0.16

37.2

4

0.44

0.59

0.15

34.1

500

1

0.43

0.61

0.18

41.9

0.18 ± 0.03

42.0 ± 6.52

No

2

0.44

0.61

0.17

38.6

3

0.43

0.65

0.22

51.2

4

0.44

0.60

0.16

36.4

SD = Standard deviation

Sig. = Statistical Significance (Williams Multiple Sequential t-test Procedure, a = 0.05) compared to control

Table 5: Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate [mg/kg dry soil weight]

Replicate

Number of juveniles

Mean ± SD

CV

[%]

[%]

of control

Sig.

Control

1

61

79 ± 14.0

17.7

-

-

2

98

3

64

4

74

5

92

6

83

7

89

8

67

15.625

1

60

68 ± 13.7

20.1

86.1

No

2

59

3

88

4

64

31.3

1

65

64 ± 11.6

18.2

81.0

No

2

58

3

79

4

52

62.5

1

56

66 ± 11.2

16.9

83.5

No

2

63

3

63

4

82

125

1

44

58 ± 9.88

17.0

73.4

Yes*

2

62

3

57

4

67

250

1

70

67 ± 10.6

15.8

84.8

No

2

70

3

75

4

51

500

1

39

55 ± 12.3

22.4

69.6

Yes

2

63

3

51

4

66

SD = Standard deviation CV = Coefficient of variation

*) = No dose-response relationship determinable, therefore not affecting the determination of NOEC/LOEC

Sig. = Statistical Significance (t - Test, a = 0.05) compared to the control

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Adult mortality in control < 10 %; Average number of juveniles in control > 30 per replicate; Coefficient of variation for reproduction of control < 30 %
Conclusions:
The test item showed no effect on the mortality and biomass of adult Eisenia fetida after 28 days exposure in this earthworm toxicity test conducted in artificial soil according to OECD test guideline 222 (28 d NOEC (mortality/biomass): 500 mg test item/kg dry soil weight (nominal)). An effect on the reproduction rate of Eisenia fetida was observed after 56 days exposure and an EC20 of 343 mg test item/kg dry soil weight (nominal) was determined, as well as a NOEC of 250 mg test item/kg dry soil weight (nominal) and a LOEC of 500 mg test item/kg dry soil weight (nominal).