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Toxicity to soil macroorganisms except arthropods

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Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-07-20 to 2011-09-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to relevant guidelines and compliant to GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Deviations:
yes
Remarks:
see: principles of method if other than guideline
Principles of method if other than guideline:
Erroneously, 11 instead of 10 earthworms were inserted in replicate 3 of the limit test item concentration. The additional
earthworm was not included into the calculations.
The soil moisture of all test vessels deviated by more than 10 % of the initial moisture at the end of the test since it was decided to add additional water during the study due to a dry soil surface.
GLP compliance:
yes (incl. QA statement)
Remarks:
according to German Chemikaliengesetzt and directive 88/320/EEC
Analytical monitoring:
no
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. 790 mL demineralised water were added to adjust the humidity of the artificial soil to a moisture of 52 % of the maximum water holding capacity. Subsequently, the test medium was thoroughly mixed to
ensure a homogenous distribution and about 600 g soil dry weight were filled into each test vessel.
- Controls: Artificial soil moistened with demineralised water without test item was used as control medium.
- Solvent control: Artificial soil with addition of quartz sand treated with acetone and moistened with demineralised water without test item was used as solvent control.
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): 0.42


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 type:
laboratory study
Substrate type:
artificial soil
Limit test:
yes
Total exposure duration:
56 d
Remarks:
Removement of adults after 28 days
Test temperature:
19 - 22 °C
pH:
Application rate
[mg test item/kg soil dry weight] pH-value
day 0 day 56
Control 5.90 6.63
Solvent Control 5.82 6.59
1000 5.79 6.40
Moisture:
Application rate
[mg test item/kg soil dry weight] Moisture [%] of dry weight

day 0 day 56
Control 36.7 44.8*
Solvent Control 37.8 45.0*
1000 38.6 40.5*

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 (80)
- No. of replicates per treatment group: 8
- No. of replicates per control: 8



SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 10 % peat, 20 % kaolin, 69 % air dried quartz sand, 0.35 % calcium carbonate
- Organic carbon (%): not determined
- Maximum water holding capacity (in % dry weigth): 85
- 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: 621 +/- 43 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


Nominal and measured concentrations:
Nominal application rate: 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
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:
mortality, body weight
Details on results:
- Mortality at end of exposure period: No noteworthy mortality of adult earthworms was observed in the controls as well as at the test item concentration of 1000 mg test item/kg soil dry weight after 28 days of exposure

- 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 controls and at the limit test item concentration of 1000 mg test item/kg soil dry weight throughout the first 28 days. As no statistically significant difference was found between control and solvent control, both controls were pooled for further statistics. Compared to the pooled control, no statistically significant difference in the increase of biomass was found

- No. of offspring produced: The mean reproduction rate was 200 in the pooled control and 185 at the limit test item concentration of 1000 mg test item/kg soil dry weight (Table 7). As no statistically significant difference was found between control and solvent control, both controls were pooled for further statistics. Compared to the pooled control, the reproduction rate was not statistically significantly reduced at 1000 mg test item/kg soil dry weight.

- Behavioural abnormalities: No symptoms or changes in the behaviour of the earthworms were observed in the controls as well as at the limit test item concentration of 1000 mg test item/kg soil dry weight after 28 days of exposure
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: 4.02 mg Carbendazim/kg soil DW
Reported statistics and error estimates:
t-tests were 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 t-tests. P-values for both Normality and Equal Variance Test were 0.05. The a-value for ANOVA test (acceptable probability of incorrectly concluding that there is a difference) was a = 0.05.

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

Application rate

[mg test item/kg soil dry weight]

Mortality

[%]

Replicate

1

2

3

4

5

6

7

8

Mean

Control

10

10

0

0

0

0

0

0

2.5

Solvent Control

0

0

0

10

0

0

0

0

1.25

1000

0

0

0

0

0

10

0

0

1.25

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

5

6

7

8

Control

A)

9/10

9/10

10/10

10/10

10/10

10/10

10/10

10/10

H)

1/10

1/10

Solvent Control

A)

10/10

10/10

10/10

9/10

10/10

10/10

10/10

10/10

H)

1/10

1000

A)

10/10

10/10

10/10

10/10

10/10

9/10

10/10

10/10

H)

1/10

A) no observation of symptoms or behavioural changes; H) dead earthworm

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 [%]

Significance compared to pooled control

[g]

[g]

[g]

[%]

Control

1

0.451

0.608

0.16

34.8

42.6
±
7.12

2

0.431

0.644

0.21

49.5

3

0.458

0.649

0.19

41.7

4

0.399

0.612

0.21

53.4

5

0.423

0.569

0.15

34.5

6

0.440

0.647

0.21

47.0

7

0.381

0.548

0.17

43.8

8

0.407

0.554

0.15

36.1

Solvent Control

1

0.407

0.620

0.21

52.3

47.4
 ±
8.74

2

0.450

0.649

0.20

44.2

3

0.429

0.610

0.18

42.2

4

0.456

0.620

0.16

36.0

5

0.397

0.645

0.25

62.5

6

0.424

0.639

0.22

50.7

7

0.436

0.605

0.17

38.8

8

0.384

0.587

0.20

52.9

1000

1

0.383

0.630

0.25

64.5

43.6
±
 14.2

No

2

0.410

0.631

0.22

53.9

3

0.454

0.534

0.08

17.6

4

0.430

0.582

0.15

35.3

5

0.455

0.644

0.19

41.5

6

0.395

0.607

0.21

53.6

7

0.419

0.591

0.17

41.1

8

0.443

0.625

0.18

41.1

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

213

190 ± 47.1

24.7

2

251

3

164

4

175

5

192

6

230

7

97

8

201

Solvent Control

1

162

209 ± 38.4

18.4

2

253

3

208

4

268

5

166

6

190

7

226

8

       196

1000

1

168

185 ± 37.3

20.1

92.9

No

2

220

3

145

4

201

5

220

6

214

7

120

8

194

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the test item did not induce mortality nor pathological symptoms to adult earthworms after an exposure to the test item concentration of 1000 mg test item/kg soil dry weight for 28 days. The biomass increase of the adult earthworms after 28 days was not statistically significantly reduced compared to the pooled control at the limit test item concentration of 1000 mg test item/kg soil dry weight. After eight weeks of exposure the earthworm reproduction was not statistically significantly reduced at the limit test item concentration of 1000 mg test item/kg soil dry weight.

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

Effects of the test item on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Savigny) were determined according to the guideline OECD 222 (2004). The study was conducted under static conditions over 8 weeks with a limit test item concentration of 1000 mg test item/kg soil dry weight, which was mixed into artificial soil containing 10 % peat. A control using untreated artificial soil and a solvent control with the solvent mixed into the artificial soil were set up. 80 test organisms, with an individual weight of 0.30 to 0.60 g, were divided into 8 replicate groups with 10 test organisms per replicate.

After 28 days no evident mortality as well as no pathological symptoms or changes in the behaviour of adult earthworms were seen in the controls as well as at the limit test item concentration of 1000 mg test item/kg soil dry weight. The biomass of the adult earthworms increased in the pooled control and at the limit test item concentration without a statistically significant difference (Table 1). After further four weeks the reproduction rate (average number of juveniles) was 200 in the pooled control and 185 at the limit test item concentration. Compared to the pooled control, the reproduction rate was not statistically significantly reduced at 1000 mg test item/kg soil dry weight.

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

All validity criteria recommended by the test guidelines were fulfilled.

Table 1:                       Summary of All Observed Effects in the Tested Dose 

Effects

 

Test Item

[mg test item/kg soil dry weight]

Mortality of adult earthworms

> 1000

Reduction of body weight

 > 1000

Reduction of reproduction rate

  (Number of juveniles)

> 1000

Summary of all effects

> 1000

Table 2:                 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

Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Acetolone Pigments – Nanoforms and Bulk Forms (Chapter 13)
Reason / purpose for cross-reference:
read-across source
Key result
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:
mortality; weight
Remarks on result:
other:
Remarks:
Read across from PY154
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the test item did not induce mortality nor pathological symptoms to adult earthworms after an exposure to the test item concentration of 1000 mg test item/kg soil dry weight for 28 days. The biomass increase of the adult earthworms after 28 days was not statistically significantly reduced compared to the pooled control at the limit test item concentration of 1000 mg test item/kg soil dry weight. After eight weeks of exposure the earthworm reproduction was not statistically significantly reduced at the limit test item concentration of 1000 mg test item/kg soil dry weight.
Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Acetolone Pigments – Nanoforms and Bulk Forms (Chapter 13)
Reason / purpose for cross-reference:
read-across source
Key result
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:
mortality; weight
Remarks on result:
other:
Remarks:
Read across from PY154
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the test item did not induce mortality nor pathological symptoms to adult earthworms after an exposure to the test item concentration of 1000 mg test item/kg soil dry weight for 28 days. The biomass increase of the adult earthworms after 28 days was not statistically significantly reduced compared to the pooled control at the limit test item concentration of 1000 mg test item/kg soil dry weight. After eight weeks of exposure the earthworm reproduction was not statistically significantly reduced at the limit test item concentration of 1000 mg test item/kg soil dry weight.
Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term study does not need to be conducted because an appropriate long-term toxicity study on terrestrial organisms is available

Description of key information

222

Under the conditions of this study, the test item did not induce mortality nor pathological symptoms to adult earthworms after an exposure to the test item concentration of 1000 mg test item/kg soil dry weight for 28 days. The biomass increase of the adult earthworms after 28 days was not statistically significantly reduced compared to the pooled control at the limit test item concentration of 1000 mg test item/kg soil dry weight. After eight weeks of exposure the earthworm reproduction was not statistically significantly reduced at the limit test item concentration of 1000 mg test item/kg soil dry weight.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:
1 000 mg/kg soil dw

Additional information