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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

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Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2009-06-10 to 2009-08-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study compliant to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Version / remarks:
DIN ISO 11268-2: 1998 (E), BBA Guideline (1994)
Deviations:
yes
Remarks:
Room temperature increased once to about 22.5 °C for four hours. The soil moisture of all test vessels deviated by more than 10 % of the initial value at the end of the test.
GLP compliance:
yes (incl. certificate)
Remarks:
GLP according to German Chemikaliengesetz and Directive 88/320/EEC

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
no

Test substrate

Vehicle:
yes
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): The respective test item concentration was weighed out, dissolved in acetone (2.5 mL per replicate) and mixed thoroughly with quartz sand (10 g per replicate). After complete evaporation of the solvent, the treated quartz sand was given to the artificial soil. Demineralised water was added to adjust the humidity of the artificial soil to a moisture of 52 % of the maximum water holding capacity. Subsequently, the test medium will be mixed with a mixer for at least three minutes and 600 g soil dry weight were filled into each test vessel.
- Controls: Artificial soil with addition of quartz sand moistened with demineralised water without test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): organic solvent: acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 2.5 mL acetone per replicate, 10 g quartz sand per replicate
- Evaporation of vehicle before use: Yes

Test organisms

Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Eisenia fetida SAVIGNY 1826, subspecies Eisenia fetida fetida
- Source: Own breeding
- Age at test initiation (mean and range, SD): synchronized earthworm culture, not deviating more than 1 month
- Weight at test initiation (mean and range, SD): 0.44 +/- 0.05 g


ACCLIMATION
- Acclimation period: 2 days prior to test start
- Acclimation conditions (same as test or not): same as test
- Health during acclimation (any mortality observed): healthy

Study design

Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Remarks:
Removement of adults after 28 days

Test conditions

Test temperature:
18 - 22.5 deg C
pH:
Application rate
[mg test item/kg soil DW] pH-value
day 0 day 56
Control 5.42 6.13
Solv. Control 5.40 6.40
62.5 5.39 6.18
125 5.39 6.30
250 5.40 6.33
500 5.38 6.07
1000 5.42 6.14
Moisture:
Application rate
[mg test item/kg soil DW] Moisture [%] of dry weight

day 0 day 56
Control 35.6 42.5*
Solv. Control 35.1 45.8*
62.5 34.8 46.7*
125 35.5 46.8*
250 34.9 44.6*
500 35.2 45.2*
1000 34.6 44.6*

*Variation of the water content > 10 %, this deviation is considered to have no impact on the study
Details on test conditions:
TEST SYSTEM
- Test container (material, size): round plastic boxes, ID: 15 cm, bottom surface area: 177 cm3, height: 14 cm, transparent and perforated lids enabled sufficient gas exchange and light penetration and prevented the test substrate from drying
- Amount of soil or substrate: 600 g DW
- No. of organisms per container (treatment): 10 (40)
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control: 4


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 10 % peat, 20 % kaolin, 69 % air dried quartz sand, 0.4 % calcium carbonate
- Organic carbon (%): not determined
- Maximum water holding capacity (in % dry weigth): 68
- CEC: not determined
- Pretreatment of soil: none
- Storage (condition, duration): 8 days after preparation at test conditions in the dark



OTHER TEST CONDITIONS
- Photoperiod: 16 h light - 8 h dark
- Light intensity: 524 +/- 70 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Adult mortality, biomass, behaviour and pathological symptoms after 28 days
Reproduction (Number of juveniles) after 56 days

VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Nominal and measured concentrations:
Nominal application rates: 62.5 - 125 - 250 - 500 - 1000 mg test substance/kg soil dry weight (DW)
Reference substance (positive control):
yes
Remarks:
1.0 - 2.5 - 5.0 mg Carbendazim/ kg soil DW

Results and discussion

Effect concentrations
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
reduction of body weight, mortality of adult earthworms
Details on results:
- Mortality at end of exposure period: No mortality of adult earthworms was observed in the controls as well as at all application rates after 28 days of exposure to the test item
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: At test start the earthworms had an individual weight of 0.30 - 0.60 g. The body weights of the earthworms increased in the control and at the test item concentrations of 62.5, 125, 250, 500 and 1000 mg test item / kg soil dry weight throughout the first 28 days. The biomass did not differ statistically significant compared to the pooled control at all test item concentrations
- No. of offspring produced: The mean number of juveniles in the pooled control was 359. The average number of juveniles in the treated groups ranged between 288 and 365. Compared to the pooled control the reproduction of the earthworms did not statistically significantly differ at all test item application rates
- Behavioural abnormalities: No symptoms or changes in the behaviour of the earthworms were observed in the controls as well as at all application rates after 28 days of exposure to the test item
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: 2.5 mg Carbendazim/kg soil DW
Reported statistics and error estimates:
Differences between the control and the solvent control were statistically analysed using t-tests. No statistical differences occurred between the controls concerning biomass and reproduction. Thus, both controls were pooled for further statistical analyses.
One Way Analysis of Variance (ANOVA) was carried out for the determination of statistically significant differences compared to
the control. A Normality Test and an Equal Variance Test were conducted prior to running the ANOVA. P-values for both Normality and Equal Variance Test are 0.05. The a-value for ANOVA test (acceptable probability of incorrectly concluding that there is a difference) is a = 0.05.

Any other information on results incl. tables

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

Application rate

[mg test item / kg soil dry weight]

Mortality

[%]

Replicate 1

Replicate 2

Replicate 3

Replicate 4

Control

0

0

0

0

Solvent Control

0

0

0

0

62.5

0

0

0

0

125

0

0

0

0

250

0

0

0

0

500

0

0

0

0

1000

0

0

0

0

Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Application rate

[mg test item / kg soil dry weight]

Effect

Replicate

1

2

3

4

Control

a)

10/10

10/10

10/10

10/10

Solvent Control

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

1000

a)

10/10

10/10

10/10

10/10

Body Weight Changes of the Adult Earthworms      

Application rate
[mg test item/kg soil dry weight]

Rep.

Mean body weights per replicate

Mean body weight change of earthworms

Test start

28 days

Per replicate

Mean ± SD

Sign.*

[g]

[g]

[g]

[%]

[g]

[%]

Control

1

0.481

0.541

0.06

12.5

0.10 ± 0.03  

 22.5 ± 6.42

-

2

0.448

0.541

0.09

20.8

3

0.439

0.565

0.13

28.7

4

0.453

0.552

0.10

21.9

Solvent Control

1

0.441

0.570

0.13

29.3

2

0.479

0.563

0.08

17.5

3

0.447

0.583

0.14

30.4

4

0.449

0.534

0.09

18.9

62.5

1

0.479

0.593

0.11

23.8

 0.13 ± 0.03

28.2 ± 7.74

No

2

0.444

0.600

0.16

35.1

3

0.451

0.605

0.15

34.2

4

0.452

0.540

0.09

19.5

125

1

0.451

0.580

0.13

28.6

0.13 ± 0.01

28.2 ± 3.63

No

2

0.451

0.565

0.11

25.3

3

0.478

0.601

0.12

25.7

4

0.437

0.582

0.15

33.2

250

1

0.477

0.575

0.10

20.5

0.12 ± 0.03

27.1 ± 6.57

No

2

0.448

0.594

0.15

32.6

3

0.453

0.554

0.10

22.3

4

0.444

0.590

0.15

32.9

500

1

0.444

0.581

0.14

30.9

0.12 ± 0.03

27.3 ± 7.81

No

2

0.442

0.581

0.14

31.4

3

0.454

0.596

0.14

31.3

4

0.475

0.549

0.07

15.6

1000

1

0.454

0.599

0.15

31.9

0.14 ± 0.02

29.9 ± 5.07

No

2

0.445

0.606

0.16

36.2

3

0.480

0.606

0.13

26.3

4

0.441

0.553

0.11

25.4

Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate
[mg test item/kg soil dry weight]

Rep.

Number of juveniles

Mean ± SD

CV               [%]

[%]

of pooled control

Significance compared to pooled control

Control

1

359

359 ± 49.6

13.8

-

-

2

338

3

456

4

323

Solvent Control

1

354

2

388

3

287

4

365

62.5

1

338

365 ± 28.5

7.81

102

No

2

356

3

360

4

405

125

1

284

355 ± 60.2

17.0

98.9

No

2

327

3

393

4

415

250

1

378

347 ± 88.3

25.5

96.6

No

2

442

3

334

4

232

500

1

303

288 ± 49.1

17.1

80.3

No

2

250

3

351

4

248

1000

1

344

346 ± 83.8

24.2

96.4

No

2

385

3

424

4

230

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this earthworm reproduction test according to OECD 222 the submission substance did not induce mortality or pathological symptoms of adult earthworms after an exposure to the test item concentrations of 62.5, 125, 250, 500 and 1000 mg/kg soil dry weight for 28 days. Also, the biomass increase of the adult earthworms after 28 days did not statistically significantly differ compared to the pooled control at all test item concentrations. In addition, after eight weeks of exposure the earthworm reproduction was not statistically significantly reduced at all applied test item concentrations.

Thus the No Observed Effect Concentration (NOEC) for the submission substance with respect to mortality, body weight and reproduction of Eisenia fetida fetida after 8 weeks of exposure in artificial soil was determined to be 1000 mg test item/kg soil dry weight.
Executive summary:

Effects of the submission substance on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Savigny) were determined compliant to GLP according to the guidelines DIN ISO 11268-2 (1998), OECD 222 (2004) and BBA (1994) (reliability category 1). The study was conducted under static conditions over 8 weeks with five different application rates (62.5 - 125 - 250 - 500 - 1000 mg test item/kg soil dry weight), which were mixed into artificial soil containing 10 % peat. 40 test organisms, with an individual weight of 0.30 to 0.60 g, were divided into 4 replicate groups with 10 test organisms per replicate.

After 28 days no mortality as well as no pathological symptoms or changes in the behaviour of adult earthworms were seen in the control, in the solvent control and at all tested application rates. The earthworm biomass increased in the control and at all application rates over 28 days without any statistically significant differences compared to the control. After further four weeks the reproduction rate (average number of juveniles) was 359 in the control (control and solvent control) and ranged between 288 and 365 at the test item application rates. Compared to the control, the reproduction rate did not statistically significantly differ at all application rates.

Overall, the NOEC of the submission substance concerning mortality, biomass and reproduction of earthworms was determined to be >= 1000 mg test item/kg soil dry weight (Table 2).

All validity criteria recommended by the test guidelines were fulfilled.

Summary of All Observed Effects in the Tested Doses

Effects

Test item

mg test item/kg soil dry weight

62.5

125

250

500

1000

Mortality of adult earthworms

No

No

No

No

No

Reduction of body weight

No

No

No

No

No

Reduced reproduction rate

  (Number of juveniles)

No

No

No

No

No

Summary of all effects

No

No

No

No

No

NOEC and LOEC for Reproduction, Mortality and Body Weight

Test item

Application rate

mg test item/kg soil dry weight

NOECreproduction, mortality, body weight

  1000

LOECreproduction, mortality, body weight

> 1000