N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-2-[(4-nitrophenyl)azo]-3-oxobutyramide

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

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

Reference 2

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

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

Justification for type of information:

See read across justification document in chapter 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

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

Description of key information

Toxicity to soil organisms

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.