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Toxicity to soil macroorganisms except arthropods

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Reference
Endpoint:
toxicity to soil macroorganisms except arthropods
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
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))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
None.
Analytical monitoring:
not required
Details on sampling:
Analytical analysis not required by OECD 222.
Vehicle:
no
Details on preparation and application of test substrate:
ARTIFICIAL SOIL COMPOSITION
- Sphagnum Peat: 10 % of dry matter of artificial soil. The peat was air dried before use.
- Kaolin Clay: 20 % of dry matter of artificial soil. Bolus alba (white clay), pharmaceutical quality.
- Quartz Sand: 70 % of dry matter of artificial soil. Fine quartz sand (round grains) was used.
- Calcium Carbonate CaCO3: Analytical grade, used for pH adjustment

Preparation of Artificial Soil: the dry constituents of the soil were mixed thoroughly in a well-ventilated area.

The pH of the final mixture was measured as 5.9. A small part of this mixture was used for the 24 hours acclimatisation of the animals used in this test. For this it was adjusted with demineralised water to a calculated water content of 50 % of the maximum water holding capacity calculated in relation to dry matter.

For the test the artificial soil described above was divided into 10 portions of an appropriate amount (4400 g for the blank control resp. 2200 g for each treatment dry weight). The required amount of test item was given to each portion using demineralised water which was used for adjustment of the artificial soil to a calculated water content of 50% of the maximum water holding capacity calculated in relation to dry matter. Afterwards, it was mixed and 659.8 g wet weight of each portion was introduced into a 1 L glass preserving jar.
Test organisms (species):
Eisenia andrei
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: earthworm (Eisenia andrei)
- Source: Bred at LAUS GmBH facilities
- Age at test initiation (mean and range, SD): at least 2 months
- Weight at test initiation (mean and range, SD): weight was in the range 400 - 600 mg (min-max)

ACCLIMATION
- Acclimation period: 24 hours
- Acclimation conditions (same as test or not): Yes
- Health during acclimation (any mortality observed): Not reported.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Post exposure observation period:
None.
Test temperature:
18 - 22.5°C (min-max), monitored continuously in the room.
pH:
5.5 - 6.1 (min-max)
Moisture:
33.3 - 37.4% (min-max)
Details on test conditions:
TEST SYSTEM
- Test container (material, size): 1 L glass jar
- Amount of soil or substrate: 500 g of soil dry weight per jar
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

OTHER TEST CONDITIONS
- Photoperiod: 16L:8D
- Light intensity: 800 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

- Adult survival after 28 days
- Adult growth after 28 days
- Juvenile production after 56 days (28 days after adult removal)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Test concentrations: 0.1 ; 0.32 ; 1 ; 3.2 ; 10 ; 32 ; 100 ; 320 ; 1000
Nominal and measured concentrations:
0.1 / 0.32 / 1.0 / 3.2 / 10 / 32 / 100 / 320 / 1000 mg/kg soil (dry matter). Concentrations not measured (not required by OECD 222 guideline).
Reference substance (positive control):
yes
Remarks:
Benomyl
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Key result
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Details on results:
- Mortality of parents at end of 28-d exposure period: Not more 2.5% of the introduced earthworm in any treatement.
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: -2 to 35% growth across treatements (min - max).
- No. of offspring produced: 52 - 100 juveniles across treatments (min - max).
Results with reference substance (positive control):
The 56d-EC50 of Benomyl (CAS-No. 17804-35-2) was tested in a current reference test. The value was determined as 1.9 mg/kg dry soil, lying within the required range of OECD Guideline 222 of 1 – 5 mg/kg.
Reported statistics and error estimates:
Calculation of results was performed with the help of validated software (Microsoft Ex-cel®). The estimation of the biological data was accomplished using the software Tox-Rat® Professional, version 3.2.1

Table 1: Number of juveniles produced after 56 days

Concentration in mg/kg

Vessel No.

Number of Juvenile Worms after 56 Days

Mean Number of Juvenile Worms after 56 Days

Inhibition of Reproduction Compared to Control

after 56 Days

in %

Blank control

1

67

78

-

2

77

3

58

4

88

5

105

6

79

7

80

8

72

0.1

9

38

51

34.82%

10

63

11

69

12

34

0.32

13

93

70

10.86%

14

69

15

64

16

53

1.0

17

139

100

-28.12%

18

115

19

91

20

56

3.2

21

96

77

2.24%

22

83

23

57

24

70

10

25

71

71

8.95%

26

106

27

54

28

54

32

29

76

51

34.50%

30

63

31

25

32

41

100

33

102

75

4.15%

34

61

35

66

36

71

320

37

80

69

11.82%

38

73

39

62

40

61

1000

41

52

53

32.27%

42

48

43

64

44

48

 

Table 2: Weight change adult worms in 28 days

Concentration in mg/kg

Vessel No.

Total Weight Worms Day 0 in g

Total Weight Worms Day 28 in g

Weight Change Worms after 28 Days in %

Mean Weight Change Worms after 28 Days in %

Mean Weight Worms Day 28 in g

Blank

control

1

4.484

5.325

19%

14%

5.122

2

4.531

5.037

11%

3

4.367

5.372

23%

4

4.473

4.887

9%

5

4.436

5.066

14%

6

4.655

5.393

16%

7

4.322

4.598

6%

8

4.517

5.299

17%

0.1

9

4.374

5.260

20%

19%

5.322

10

4.498

5.615

25%

11

4.677

5.104

9%

12

4.424

5.310

20%

0.32

13

4.609

5.450

18%

17%

5.541

14

4.710

5.910

25%

15

5.026

5.431

8%

16

4.640

5.374

16%

1.0

17

4.981

5.780

16%

16%

5.361

18

4.626

5.334

15%

19

4.674

5.094

9%

20

4.296

5.237

22%

3.2

21

4.377

5.124

17%

8%

4.888

22

4.370

4.953

13%

23

4.774

5.262

10%

24

4.596

4.216

-8%

10

25

4.358

4.729

9%

3%

4.595

26

4.710

4.830

3%

27

4.561

4.553

0%

28

4.275

4.269

0%

32

29

4.690

4.975

6%

-2%

4.340

30

4.335

4.547

5%

31

4.379

3.907

-11%

32

4.337

3.931

-9%

100

33

4.313

4.173

-3%

3%

4.455

34

4.375

4.398

1%

35

4.195

4.644

11%

36

4.450

4.604

3%

320

37

4.423

4.767

8%

15%

5.153

38

4.368

4.839

11%

39

4.405

5.115

16%

40

4.634

5.893

27%

1000

41

4.337

4.910

13%

19%

5.210

42

4.449

5.402

21%

43

4.346

5.318

22%

44

4.331

5.211

20%

 


Validity criteria fulfilled:
yes
Conclusions:
Eisenia andrei adults were exposed to 9 graded concentrations of dipentamethylenethiuram hexasulfide for 28 days and cocoon/juveniles for another 28 day period. Adult survival and growth and number of juvenile produced per treatment were examined. No significant effect on parental survival and juvenile production was seen up to 1000 mg/kg soil dw. NOEC for the whole study is therefore considered superior or equal to 1000 mg/kg soil dw.
Executive summary:

The study was performed using 9 concentrations ranging from 0.1 to 1000 mg/kg dry soil (4 replicates) and a control (8 replicates), following OECD 222 guideline protocol. In each replicate, 10 adult animals were exposed to the test item (except control group) for 28 days in an artificial soil. After 28 days, the living adult animals were counted and weighted, and 28 days later, the number of juveniles produced was determined.

No significant effect on parental survival and juvenile production was seen up to 1000 mg/kg soil dw. NOEC for the whole study is therefore considered superior or equal to 1000 mg/kg soil dw.

Description of key information

Key value for chemical safety assessment

Additional information

One reliable Klimisch 1 study is available for long-term toxicity to soil macroorganisms.The study was performed using 9 concentrations ranging from 0.1 to 1000 mg/kg dry soil (4 replicates) and a control (8 replicates), following OECD 222 guideline protocol. In each replicate, 10 adult animals were exposed to the test item (except control group) for 28 days in an artificial soil. After 28 days, the living adult animals were counted and weighted, and 28 days later, the number of juveniles produced was determined.No significant effect on parental survival and juvenile production was seen up to 1000 mg/kg soil dw. NOEC for the whole study is therefore considered superior or equal to 1000 mg/kg soil dw.