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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:
2011-09-28 to 2011-11-22
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:
Food was provided on day 0 (test start) instead of day 1. Additionally the earthworms were fed on day 35, 42 and 49. These deviations are considered to have no impact on quality and integrity of the study.
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 amount was weighed out for the limit test item concentration, 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. Additional demineralised water was added to the artificial soil 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 homogeneous 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): 0.39


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:
17 - 22 °C
During 10 nights the room temperature fell down to 17°C, this deviation is considered to have no impact on the quality and integrity of the study.
pH:
Application rate
[mg / kg soil dry weight] pH-value
day 0 day 54
Control 5.12 6.17
Solvent Control 5.18 6.12
1000 5.38 6.13
Moisture:
Application rate
[mg / kg soil dry weight] Moisture [%] of dry weight
day 0 day 55
Control 31.0 36.5*
Solvent Control 30.9 38.2*
1000 33.9 38.0*
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, 0.30 % calcium carbonate
- Organic carbon (%): not determined
- Maximum water holding capacity (in % dry weigth): 65
- 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: 615 +/- 100 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 mortality of adult earthworms was observed in the controls as well as at the limit test item concentration 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 adult earthworms increased throughout the first 28 days at the limit test item concentration with no statistically significant differences compared to the controls

- No. of offspring produced: The mean number of juveniles in the pooled control was 133. The average number of juveniles in the treated group was 156. Compared to the control the reproduction of the earthworms did not statistically significantly differ. The coefficient of variation calculated for the reproduction of the control was lower than 30 %.

- Behavioural abnormalities: No pathological symptoms nor changes in the behaviour of the earthworms were observed in the controls or at the limit test item concentration.
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.

Test Item

Application rate

[mg test item/kg soil dry weight]

NOECreproduction, mortality, body weight

1000

LOECreproduction, mortality, body weight

> 1000

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

0

0

0

0

0

0

0

0

0

Solvent Control

0

0

0

0

0

0

0

0

0

1000

0

0

0

0

0

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

5

6

7

8

Control

A)

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

Solvent Control

A)

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

1000

A)

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

Body Weight Changes of the Adult Earthworms      

Application rates

[mg /kg soil dry weight]

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

0.41

0.52

0.11

27.1

0.18 ± 0.04

45.9 ± 12.9

-

2

0.37

0.56

0.20

53.7

3

0.44

0.57

0.14

31.4

4

0.38

0.54

0.17

45.1

5

0.42

0.61

0.19

45.5

6

0.39

0.59

0.20

51.4

7

0.35

0.59

0.24

68.4

8

0.40

0.58

0.18

44.5

Solvent Control

1

0.40

0.60

0.20

50.6

0.18 ± 0.02

46.6 ± 8.49

No

2

0.41

0.57

0.16

38.1

3

0.37

0.57

0.20

55.1

4

0.44

0.59

0.15

33.3

5

0.38

0.57

0.19

50.8

6

0.42

0.61

0.19

44.1

7

0.39

0.55

0.17

42.7

8

0.35

0.55

0.20

57.9

1000

1

0.39

0.61

0.22

56.3

0.20 ± 0.03

50.3 ± 8.00

No

2

0.35

0.52

0.17

48.1

3

0.40

0.59

0.18

45.7

4

0.41

0.63

0.22

54.8

5

0.37

0.60

0.23

61.6

6

0.44

0.59

0.16

35.3

7

0.38

0.58

0.20

52.9

8

0.42

0.62

0.20

47.5

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

113

138 ± 29.9

21.6

2

173

3

182

4

127

5

137

6

100

7

- 1)

8

136

Solvent Control

1

109

128 ± 26.5

20.8

2

126

3

112

4

135

5

143

6

177

7

130

8

88

Pooled Control

133 ± 27.6

20.8

1000

1

182

155 ± 27.7

17.9

117

No

2

107

3

152

4

149

5

177

6

173

7

176

8

123

1) = Replicate was mistakenly excluded

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the test item did not induce mortality, pathological symptoms or changes in the behaviour of adult earthworms after 28 days of exposure at the limit test item concentration. As there were no statistically significant differences determined between the control and the solvent control for the body weight and the reproduction rate, the controls were pooled. The adult earthworm biomass increased in the control and at the limit test item concentration without any statistically significant differences. After eight weeks of exposure the earthworm reproduction at the limit test item concentration did not statistically significantly differ to the pooled control.

Thus, the No Observed Effect Concentration (NOEC) for the test item summarizing the endpoints mortality, body weight and reproduction of Eisenia fetida fetida after 8 weeks of exposure in artificial soil was determined to be 1000 mg /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, that 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 were inserted into 8 control, solvent control and test item replicates (10 earthworms per replicate). They had an individual body weight between 0.30 and 0.60 g at experimental starting.

After 28 days of exposure in soil, no evident earthworm mortalities as well as no pathological symptoms or changes in the behaviour of adult earthworms were observed in all treatments. After 28 days of exposure, no statistically significant differences in earthworm body weights at 1000 mg/kg soil dry weight were observed. After further four weeks the reproduction rate (average number of juveniles produced) was 133 in the pooled control and ranged between 107 and 182 in the test item treatment rate. Compared to the pooled control, reproduction rate was not statistically significantly reduced at 1000 mg/kg soil dry weight. As there were no statistically significant differences determined between the control and the solvent control for the body weight and the reproduction rate, the controls were pooled.

Overall, the NOEC of the test item concerning mortality, biomass and reproduction of earthworms was 1000 mg/kg soil dry weight.

All validity criteria recommended by the test guideline were fulfilled.

Summary of All Observed Effects in the Tested Application Rates

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

NOEC and LOEC values

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
Justification for type of information:
see Read-across justification in section 13 for details
Reason / purpose for cross-reference:
read-across source
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 mortality of adult earthworms was observed in the controls as well as at the limit test item concentration 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 adult earthworms increased throughout the first 28 days at the limit test item concentration with no statistically significant differences compared to the controls

- No. of offspring produced: The mean number of juveniles in the pooled control was 133. The average number of juveniles in the treated group was 156. Compared to the control the reproduction of the earthworms did not statistically significantly differ. The coefficient of variation calculated for the reproduction of the control was lower than 30 %.

- Behavioural abnormalities: No pathological symptoms nor changes in the behaviour of the earthworms were observed in the controls or at the limit test item concentration.
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.

Test Item

Application rate

[mg test item/kg soil dry weight]

NOECreproduction, mortality, body weight

1000

LOECreproduction, mortality, body weight

> 1000

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

0

0

0

0

0

0

0

0

0

Solvent Control

0

0

0

0

0

0

0

0

0

1000

0

0

0

0

0

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

5

6

7

8

Control

A)

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

Solvent Control

A)

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

1000

A)

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

Body Weight Changes of the Adult Earthworms      

Application rates

[mg /kg soil dry weight]

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

0.41

0.52

0.11

27.1

0.18 ± 0.04

45.9 ± 12.9

-

2

0.37

0.56

0.20

53.7

3

0.44

0.57

0.14

31.4

4

0.38

0.54

0.17

45.1

5

0.42

0.61

0.19

45.5

6

0.39

0.59

0.20

51.4

7

0.35

0.59

0.24

68.4

8

0.40

0.58

0.18

44.5

Solvent Control

1

0.40

0.60

0.20

50.6

0.18 ± 0.02

46.6 ± 8.49

No

2

0.41

0.57

0.16

38.1

3

0.37

0.57

0.20

55.1

4

0.44

0.59

0.15

33.3

5

0.38

0.57

0.19

50.8

6

0.42

0.61

0.19

44.1

7

0.39

0.55

0.17

42.7

8

0.35

0.55

0.20

57.9

1000

1

0.39

0.61

0.22

56.3

0.20 ± 0.03

50.3 ± 8.00

No

2

0.35

0.52

0.17

48.1

3

0.40

0.59

0.18

45.7

4

0.41

0.63

0.22

54.8

5

0.37

0.60

0.23

61.6

6

0.44

0.59

0.16

35.3

7

0.38

0.58

0.20

52.9

8

0.42

0.62

0.20

47.5

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

113

138 ± 29.9

21.6

2

173

3

182

4

127

5

137

6

100

7

- 1)

8

136

Solvent Control

1

109

128 ± 26.5

20.8

2

126

3

112

4

135

5

143

6

177

7

130

8

88

Pooled Control

133 ± 27.6

20.8

1000

1

182

155 ± 27.7

17.9

117

No

2

107

3

152

4

149

5

177

6

173

7

176

8

123

1) = Replicate was mistakenly excluded

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the test item did not induce mortality, pathological symptoms or changes in the behaviour of adult earthworms after 28 days of exposure at the limit test item concentration. As there were no statistically significant differences determined between the control and the solvent control for the body weight and the reproduction rate, the controls were pooled. The adult earthworm biomass increased in the control and at the limit test item concentration without any statistically significant differences. After eight weeks of exposure the earthworm reproduction at the limit test item concentration did not statistically significantly differ to the pooled control.

Thus, the No Observed Effect Concentration (NOEC) for the test item summarizing the endpoints mortality, body weight and reproduction of Eisenia fetida fetida after 8 weeks of exposure in artificial soil was determined to be 1000 mg /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, that 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 were inserted into 8 control, solvent control and test item replicates (10 earthworms per replicate). They had an individual body weight between 0.30 and 0.60 g at experimental starting.

After 28 days of exposure in soil, no evident earthworm mortalities as well as no pathological symptoms or changes in the behaviour of adult earthworms were observed in all treatments. After 28 days of exposure, no statistically significant differences in earthworm body weights at 1000 mg/kg soil dry weight were observed. After further four weeks the reproduction rate (average number of juveniles produced) was 133 in the pooled control and ranged between 107 and 182 in the test item treatment rate. Compared to the pooled control, reproduction rate was not statistically significantly reduced at 1000 mg/kg soil dry weight. As there were no statistically significant differences determined between the control and the solvent control for the body weight and the reproduction rate, the controls were pooled.

Overall, the NOEC of the test item concerning mortality, biomass and reproduction of earthworms was 1000 mg/kg soil dry weight.

All validity criteria recommended by the test guideline were fulfilled.

Summary of All Observed Effects in the Tested Application Rates

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

NOEC and LOEC values

Test Item

Application rate

[mg test item/kg soil dry weight]

NOECreproduction, mortality, body weight

1000

LOECreproduction, mortality, body weight

> 1000

Description of key information

RA from PBr25

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, that was mixed into artificial soil containing 10 % peat. After 28 days of exposure in soil, no evident earthworm mortalities as well as no pathological symptoms or changes in the behaviour of adult earthworms were observed in all treatments. After 28 days of exposure, no statistically significant differences in earthworm body weights at 1000 mg/kg soil dry weight were observed. After further four weeks,reproduction rate was not statistically significantly reduced at 1000 mg/kg soil dry weight.Overall, the NOEC of the test item concerning mortality, biomass and reproduction of earthworms was 1000 mg/kg soil dry weight.

Key value for chemical safety assessment

Additional information