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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

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Reference
Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 27 August 2018 to 08 April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 16 December 2015; Date of signature: 31 May 2016
Specific details on test material used for the study:
- Water solubility: 4.7 mg/L at 20°C (experimental study, OECD Guideline 105, flask method modified with slowstirring)
- Vapour pressure: Low volatility: 17.2 Pa at 20°C (experimental study, EU Method A.4, isoteniscope method)
- Log Kow: 4.68 (experimental study, EU Method A.8, flask method)
- Relative density (D 20/4): 0,938 (experimental study, Pycnometer method)
- Chemical stability: Stable in water (SDS)
- Storage conditions: Closed vessel, preferably full, container away from heat sources, and protected from extremes of temperature (SDS). Protected from light, in a refrigerator (temp 2-8 °C), store under Nitrogen in a closed container after first opening (CoA).
Analytical monitoring:
yes
Details on sampling:
Based on analytical results from the range finding test it was decided to verify test item concentration in the test matrix. For this, aliquots of the test matrix were sampled for analysis of the test substance at test start, day 1, 3, 7, 14, 21, 28, 42 and 56 (test end).
Vehicle:
no
Details on preparation and application of test substrate:
The quantity of liquid test item required to obtain the desired concentrations was mixed with an amount of quartz sand (10 g per kg test substrate). The mixture and the respective amount of water needed to adjust the test substrate to 40 – 60% water holding capacity was added to the test soil and mixed thoroughly. The test substrate was prorated to each test container.
Test organisms (species):
Eisenia andrei
Animal group:
annelids
Details on test organisms:
SOURCE
The test organisms were synchronized adult earthworms of the species Eisenia andrei, which within an age of two months and 1 year, with a clitellum, and a wet mass between 250 mg and 600 mg. Origin of the organisms is Regenwurmfarm Tacke, Klosterdiek 61, 46325 Borken. Specimens used in the test are bred in the laboratory of the Fraunhofer IME.

PRETREATMENT
The worms were conditioned for 4 days in the artificial soil before use. The same food which was also used in the test was given in a sufficient amount.

FOOD
Air-dried, finely ground horse manure was used as food. The manure originated from Elisabeth Hardebusch, 57392 Schmallenberg, Germany. Horses from which manure was obtained by self-collecting were not subject to medication or treatment with substances, such as growth promoters, nematicides or similar veterinary products that could adversely affect the worms during the test (not certified self-declaration by the owner).
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Test temperature:
The incubation temperature was measured continuously with a thermograph. With 18 – 22 °C the permitted range of 20 ± 2 °C was met.
pH:
The pH of the artificial soil in the presence of 1 mol/L KCl at test start was 6.3 in the control and decreasing from 6.2 - 5.6 with increasing test item concentration. At test end, pH of the artificial soil was 6.4 in the control and 6.0 - 6.2 in the test assays.
Moisture:
With 47.5 – 49.6% the permitted range of 40 – 60% Maximum Water Holding Capacity (WHC) for the artificial soil was maintained during incubation.
Since WHC is affected by food adding, no calculation for test end can be applied.
Details on test conditions:
TEST SYSTEM
- Test container (material, size): Plastic containers with an area of 17 x 12 cm and a height of 6 cm were used. The containers were covered with perforated transparent plastic caps to prevent worms from escaping but also permit gaseous exchange between the medium and the atmosphere and access of light.
- Amount of soil or substrate: The containers were filled with about 500 - 650 g dry mass of artificial soil to ensure a moist substrate depth of about 5-6 cm.
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8 (the control consists of test substrate adjusted to 60% of the maximum water holding capacity)
- No. of replicates per vehicle control: not applicable

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
Artificial soil components:
- Sphagnum peat, air-dried, finely ground = 10 %
- Kaolinite, air-dried = 20 %
- Industrial quartz sand, air-dried = 70 %
The test substrate was wetted with deionised water to a water content of between 40 % and 60 % by mass of the total water-holding capacity. The pH of the substrate was adjusted to 6.0 ± 0.5 (1 mol/L KCl) by adding pulverized calcium carbonate (CaCO3). The water content and the pH in the presence of 1 mol/L KCl of the test substrate are determined immediately before the start of the test and at the end of the test in one sample of the controls and one sample of each concentration tested.

OTHER TEST CONDITIONS
The test was carried out at 20 °C ± 2 °C and a controlled light/dark cycle of 16 h : 8 h with a light intensity of 400 lx to 800 lx.
The light intensity was measured using an illuminance meter (MINOLTA) with photometric sensor in Lux. With 728 – 792 lx the permitted value of about 400 - 800 lx was kept.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
At the start of the test, batches of ten conditioned worms were weighed and placed into each container. One day after addition of the worms, an amount of 5 g air dried finely ground horse manure per test container was spread on the soil surface and moistened with water. Once a week during the first four weeks the adult earthworms were fed according to their food consumption. Feeding behaviour and the quantity of food applied over the test period for each test container was recorded. The water content of the soil substrate in the test containers was maintained during the test period by reweighing the test containers periodically and by replenishing lost water. The adult earthworms were kept in the substrate over a period of 4 weeks. At the end of this period, the adults were removed. For each container the total number and mass of living adult earthworms was recorded. To allow the offspring to develop, the test containers were kept for another period of 4 weeks in the test environment. After this period the number of offspring per test container hatched from the cocoons was counted by hand selection.

VEHICLE CONTROL PERFORMED: not applicable

TEST CONCENTRATIONS
The nominal concentration in the test containers with test item was 15.6, 31.3, 62.5, 125 and 250 mg/kg dry mass soil. The concentrations were chosen due to a non-GLP range finding test showing only a slight inhibition at 10 - 100 mg/kg dry mass soil, and indicating an EC10 and EC50 values in the range of 10 - 100 and 100 – 1000 mg/kg dry mass soil, respectively.
Nominal and measured concentrations:
The nominal concentration in the test containers with test item was 15.6, 31.3, 62.5, 125 and 250 mg/kg dry mass soil.
For the measured concentrations, refer to tables 6.3.1/1-2 in "Any other information on results incl. tables".
Reference substance (positive control):
yes
Remarks:
To assess health and sensitivity of the test organisms, the results of a previous non-GLP reference sub-acute toxicity test with carbendazim, not older than 12 months, were used.
Key result
Duration:
56 d
Dose descriptor:
EC10
Effect conc.:
61.8 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% CL: 42.5 - 78.3 mg/kg dry mass soil
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
170.1 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks on result:
other: 95% CL not determined
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
> 234.9 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL not determined
Details on results:
- Test concentrations: The measured values of the test substance concentrations deviated by more than 20% from nominal values (see Tables 6.3.1/1-2 in "Any other information on results incl. tables"). At test start, measured extractable test substance concentrations were between 68 and 112% of nominal concentrations. Time weighted average concentrations (TWA) relative to nominal concentrations were 5 to 8% after 56 d. Already after 28 d, extractable test substance concentrations in the artificial soil had decreased below LOQ (1.25 mg/kg dry mass soil). Time weighted average concentrations (TWA) relative to nominal concentrations were calculated as 9 to 12% after 28 d. Thus, not surprisingly, at 56 d, extractable test substance concentrations in the artificial soil were also found to be below LOQ. Effect concentrations were calculated on the basis of initially measured concentrations. The concentrations applied for statistical evaluation were 17.5, 26.4, 58.5, 85.3, and 234.9 mg test item/kg dry mass soil. The evaluation on the basis of TWA concentrations over the whole test duration or even over the first 28 days is assumed to overestimate the substance related effect due to a fast decrease of the test item concentration already within the first 7 – 14 days for the relevant treatment levels (15.6 – 62.5 mg test item/kg dry mass soil nominal concentrations). Therefore, the effects are assumed to be better related on the maximum concentrations at test start than on TWA concentrations considering recovery periods.
- Physical/Pathological symptoms and changes in behaviour: Neither any physical or pathological symptoms nor changes in behaviour were observed. All specimens gave the impression of healthy condition.
- Survival rate: See Table 6.3.1/3 in "Any other information on results incl. tables".
- Weight change: See Table 6.3.1/4 in "Any other information on results incl. tables".
- Reproduction: See Table 6.3.1/5 in "Any other information on results incl. tables".
- Effect concentration: NOECs and ECx values for weight change, survival rate and reproduction are reported in Table 6.3.1/6 in "Any other information on results incl. tables".
The survival of Eisenia andrei was obviously not affected by the test substance. The NOEC (no observed effect concentration) was found to be ≥ 234.9 mg test substance/kg dry mass soil initial
concentration. No significant effect on weight change due to the test substance was detected. The NOEC was also found to be ≥ 234.9 mg test substance/kg dry mass soil initial concentration. However, due to maximum effects of 9-13% reduction of weight change in the two highest treatments levels, an EC10 value was calculated at 170.1 (CL 95%: n.d.) mg test substance/kg dry mass soil initial concentration. The reproduction of Eisenia andrei was affected by the test substance. The NOEC was found to be 26.4 mg test substance/kg dry mass soil initial concentration. The EC10 and EC50 were
calculated to be 61.8 (95% CL: 42.5 – 78.3) and 221.0 (95% CL: 188.1 – 274.3) mg test substance/kg dry mass soil initial concentration, respectively.

The chronic earthworm toxicity test fulfills the validity criteria of the guideline:
- With a mean value of 201 individuals, the rate of reproduction in the control(s) was ≥30 juveniles per test vessel at the end of the test.
- With 4% the CV of reproduction in the control does not exceed 30%.
- With 0% the percent mortality of the adults observed in the control(s) over the initial 4 weeks is ≤ 10%.
Results with reference substance (positive control):
In order to confirm the sensitivity of the test species Eisenia andrei, the influence of the reference item carbendazim on mortality and reproduction was investigated not longer than 12 months prior to this study. In the last reference test, survival was not affected up to and including 3 mg/kg. No concentration dependence occurred. Reproduction was significantly affected by carbendazim. The EC10 and EC50 values were 0.15 mg/kg and 3.92 mg/kg, respectively.
The results for reproduction are in agreement with the range of 1 – 5 mg carbendazim per kg required for the occurrence of significant effects in OECD 222. Weight change and mortality are in agreement with experience obtained in the test facility.

Table 6.3.1/1: Mean measured concentrations of the test substance (mg/kg DM)

 

Nominal concentration [mg/kg DM]

Date

Control

15.6

31.3

62.5

125

250

d0

<LOQ*

17.52

26.36

58.51

85.29

234.88

d1

<LOQ

11.16

21.68

34.72

80.47

170.27

d3

<LOQ

4.87

10.96

19.44

35.32

63.37

d7

<LOQ

<LOQ

2.51

4.77

7.26

19.43

d14

<LOQ

<LOQ

<LOQ

<LOQ

1.77

3.16

d21

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

1.73

d28

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

d42

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

d56

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

LOQ = 1.25 mg/kg DM

Table 6.3.2/2: Measured concentrations of the test substance and percentage from nominal values

Nominal concentrations (mg TI/kg DM)

Measured concentrations

(mg/kg)

Percentage from nominal

(%)

 

Start

56 d

TWA

Start

56 d

TWA

15.6

17.5

< LOQ

1.8

112

0

8

31.3

26.4

< LOQ

3.5

8

0

7

62.5

58.5

< LOQ

5.8

94

0

5

125

85.3

< LOQ

10.8

68

0

5

250

234.9

< LOQ

23.0

94

0

5

 

Start

28 d

TWA

Start

28 d

TWA

15.6

17.5

< LOQ

1.8

112

0

12

31.3

26.4

< LOQ

3.5

8

0

11

62.5

58.5

< LOQ

5.8

94

0

9

125

85.3

< LOQ

10.8

68

0

9

250

234.9

< LOQ

23.0

94

0

9

TI = Test item; DM = Dry mass; TWA = Time weighted average. TWA values were calculated over the whole test duration (56 days) and for the first 28 days. LOQ = 1.25 mg/kg dry mass soil.

Table 6.3.1/3: Survival rate after 28 days

 Test item concentration  replicate control   17.5 mg/kg  26.4 mg/kg  58.5 mg/kg  85.3 mg/kg  234.9 mg/kg
 Living earthworms [ind.]

1

2

3

4

5

6

7

8

10

10

10

10

10

10

10

10 

10

10

10

10

 

10

10

10

10

 

10

10

10

10

 

10

10

10

10

 

10

10

10

10

 

 Mortality (%)    0%  0% 0%   0%  0%  0%

Table 6.3.1/4: Weight change after 28 days

Test item concentration

Control

17.5 mg/kg

26.4 mg/kg

58.5 mg/kg

85.3 mg/kg

234.9 mg/kg

compared to start value [%]

65

59

63

62

54

55

compared to control [%]

 

6

-2

5

13

9

Table 6.3.1/5: Reproduction after 56 days

Test item concentration

replicate

Control

17.5 mg TI

per kg DM

26.4 mg TI

per kg DM

58.5 mg TI

per kg DM

85.3 mg TI

per kg DM

234.9 mg TI

per kg DM

 

Juveniles
[Ind.]

1

204

197

204

182

161

112

 

2

208

201

189

193

169

98

 

3

193

192

194

194

155

89

 

4

205

199

209

186

163

87

 

5

189

 

 

 

 

 

 

6

201

 

 

 

 

 

 

7

212

 

 

 

 

 

 

8

197

 

 

 

 

 

 
 Mean Juveniles [Ind.] Mean (CV) 201.1 (3.8)  196.6 (1.9) 196.0 (5.3) 189.0 (2.6) 161.2 (3.3)  93.0 (13.5)
 Reproduction inhibition (%)      2% 3%  6%  20%*  54%*

* significant when compared with control (Williams test, one-sided).

Table 6.3.1/6: Effect concentrations; NOECs and ECx values for weight change, survival rate and reproduction (mg test item/kg dry mass)

Parameter

Weight change

Survival rate

Reproduction

mg test item/kg dry mass soil [initial]

NOEC

≥ 234.9

≥ 234.9

26.4

LOEC

> 234.9

> 234.9

58.5

EC10

170.1

≥ 234.9

61.8

(95% CL)

(n.d.)

> 234.9

(42.5 – 78.3)

EC20

> 234.9

≥ 234.9

95.8

(95% CL)

(n.d.)

> 234.9

(75.0 – 113.8)

EC50

> 234.9

≥ 234.9

221.0

(95% CL)

(n.d.)

> 234.9

(188.1 – 274.3)

n.d.: not determined

Validity criteria fulfilled:
yes
Conclusions:
The test substance has chronic effects on Eisenia andrei under the chosen test conditions. The relevant EC10 values, based on initial concentrations, are 170.1 (CL 95%: n.d.) and 61.8 (CL 95%: 42.5 – 78.3) mg test substance/kg dry mass soil for growth and reproduction, respectively.
The supplementary analytical results on the transformation product of the test substance confirm the total and rapid loss of the test substance in the soil and rapid increase followed by the dissipation of the relevant transformation product at the end of the earthworm test (56 days). These results show that well before the end of the test days there was nor parent substance nor degradation product remaining at any concentration by day 30. Ultimately, the ecotox earthworm results should be based on the inital measured concentration of the parent substance as the effects found at the end of the study would be more a result of this concentration than a TWA of concentrations over the study period.
Executive summary:

A GLP study (reported in FIR-001/4 -80/V) was performed at the Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) to determine the effect of the test substance on survival rate, biomass, and reproduction of adult earthworms of the species Eisenia andrei according to the OECD guideline 222. For this, the worms were placed in a defined artificial soil substrate containing the test item in different concentrations. A single application of test item was made at the beginning of the

test. Effects on survival rate and biomass were determined after 28 days, the effects on reproduction were determined after 56 days. The test substance was applied in concentrations of 15.6, 31.3, 62.5, 125 and 250 mg/kg dry mass soil. Eight replicates were prepared for a control and four for each concentration. At test start, the recovery rate was found to be 112, 84, 94, 68 and 94% of nominal concentrations for the treatment levels 15.6, 31.3, 62.5, 125 and 250 mg/kg test

item/kg dry mass soil, respectively. After 7 – 14 days, recovery rate was < LOQ (1.25 mg/kg dry mass soil) for the three lower treatment levels. Recovery rate for the two highest treatment levels were 1%. On day 21, recovery rate was also < LOQ for the two highest treatment levels. Due to recovery rates of the test item in the artificial soil matrix outside the range of 80 – 120 % from nominal, effect concentrations were calculated on the basis of initially measured concentrations. The initially measured concentrations applied for statistical evaluation were 17.5, 26.4, 58.5, 85.3, and 234.9 mg test item/kg dry mass soil.

No decreased survival rate due to initial concentration of 234.9 mg test item/kg dry mass soil (highest concentration in the test) was detected when compared to the control worms. The NOEC for the tested species was assumed to be ≥ 234.9 mg/kg dry mass soil (initial concentration).

No significant effect on weight change was found up to and including an initial concentration of 234.9 mg test item/kg dry mass soil (highest concentration in the test). The NOEC for the tested species was assumed to be ≥ 234.9 mg/kg dry mass soil (initial concentration). However, due to maximum effects of 9 -13% reduction of weight change in the two highest treatment levels, a slight concentration-effect relationship was observed. The EC10 was 170.1 (CL 95%: n.d.) mg/kg soil (initial concentration). The EC50 was > 234.9 mg/kg dry mass soil (initial concentration), the highest concentration in the test.

A significant decrease in reproduction was found starting at an initial concentration of 58.5 mg test item/kg dry mass soil. The NOEC for the tested species was 26.4 mg/kg dry mass soil (initial concentration) while the LOEC was 58 mg/kg dry mass soil (initial concentration). The EC10 was 61.8 (CL 95%: 42.5 – 78.3) mg/kg dry mass soil (initial concentration). The EC50 was 221.0 (CL 95%: 188.1 – 274.3) mg/kg dry mass soil (initial concentration).

The test substance has chronic effects on Eisenia andrei under the chosen test conditions. The relevant EC10 values based on initial concentrations are 170.1 (CL 95%: n.d.) and 61.8 (CL 95%: 42.5 – 78.3) mg test substance/kg dry mass soil for growth and reproduction, respectively.

The earthworm reproduction test on the test substance was conducted under the request of the European authorities, because of its suspected soil hazard category. However, the results from the biodegradation test OECD 301C (Yokohama Lab, 2009) show that the test substance degrades into a transformation product (at a rate of 78 -80%) and Propionic acid (at ca. 20%) but this latter finally mineralizing. Therefore, at the request of the Sponsor, it was decided to include analytical measurements of the transformation product (non-GLP) , along with the measurements of the parent substance (GLP and reported in FIR-001/4-80/V) in the soil samples at the same sampling points. It was expected that the test substance would degrade to the transformation product in soil similarly to the biodegradation test OECD 301C, and that it was important to have analytical measurements of the major degradation product of the registered substance. This latest compound will be the product that will be in the sludge and amended o soil, thus in contact with soil organisms (not the registered substance). Also, the results are likely going to clarify how to express the ecotoxicological earthworms values.

According to this supplementary analytical results, the test substance, as expected, degraded very rapidly. The concentrations of the transformation product increased at a rate inversely proportional to the loss of the parent substance and then itself diminished until both substances were no longer analytically measurable. The peak of concentrations of the transformation product was measured at day 3 -7. The reproductive toxicity of the test substance to earthworms was nevertheless based on initial concentrations of the test substance as this was the parent substance under review, and as the parent substance represented an acute dose at the beginning of the experiment which could account for the effects found. Use of a time weighted average for either the parent and transformation product was considered inappropriate as a means to measure chronic toxicity due to the prolonged absence of the parent and transformation product over a large portion of the study period.

Description of key information

OECD Guideline 222, GLP, key study, validity 1:

56d-EC10 reproduction (Eisenia andrei) = 61.8 (CL 95%: 42.5 – 78.3) mg/kg dry mass soil, based on initial measured concentrations.

Key value for chemical safety assessment

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

Additional information

An earthworm reproduction test performed on the registered substance was conducted under the request of the European authorities. This GLP study was performed to determine the effect of the registered substance on survival rate, biomass, and reproduction of adult earthworms of the species Eisenia andrei according to the OECD guideline 222. For this, the worms were placed in a defined artificial soil substrate containing the registered substance in different nominal concentrations: 15.6, 31.3, 62.5, 125 and 250 mg/kg dry mass soil. A single application of test substance was made at the beginning of the test. Effects on survival rate and biomass were determined after 28 days, the effects on reproduction were determined after 56 days.

As the registered substance degrades into a transformation product at a rate of 78 -80% (and Propionic acid at ca. 20%, but this latter finally mineralizing) in a ready biodegradation test (OECD 301C, Yokohama Lab, 2009), it was decided to include supplementary analytical measurements of the relevant transformation product (non-GLP) , along with the measurements of the parent substance (GLP) in the soil samples at the same sampling points. It was expected that the registeredt substance would degrade to the transformation product in soil similarly to the biodegradation test OECD 301C, and that it was important to have analytical measurements of the major degradation product of the registered substance. This latest compound will be the product that will be in the sludge and amended o soil, thus in contact with soil organisms.

According to the analytical results, the registered substance, as expected, degraded very rapidly. Indeed, after 7 -14 days, recovery rates of the test substance were <LOQ and 1% for the three lower treatment levels and the two highest treatment levels, respectively. On day 21, recovery rate was also <LOQ for the two highest treatment levels. This rapid loss of parent substance is completely in line with the expected degradation profile of an ester. Esters are rapidly metabolised to an alcohol and an acid, but the acid moiety is extremely small and not expected to contribute to the observed toxicity (here, a complete mineralization of the Propionic acid moiety was observed). The concentrations of the relevant transformation product (the residual alcohol) increased at a rate inversely proportional to the loss of the parent substance and then itself diminished until both substances were no longer analytically measurable. The peak of concentrations of the transformation product was measured at day 3 -7. The reproductive toxicity of the registered substance to earthworms was based on initial concentrations of the test substance (measured at 17.5, 26.4, 58.5, 85.3 and 234.9 mg test item/kg dry mass soil) as this was the parent substance under review, and as the parent substance represented an acute dose at the beginning of the experiment which could account for the effects found. Use of a time weighted average for either the parent and transformation product was considered inappropriate as a means to measure chronic toxicity due to the prolonged absence of the parent and transformation product over a large portion of the study period and as the relevant transformation product, which is the residual alcohol of the registered substance, is a non-polar narcotic (MechoA 1.1) and is expected to be less toxic than the ester (the registered substance).

No decreased survival rate when compared to the control worms and no significant effect on weight change were found up to and including the highest tested concentration. However, due to maximum effects of 9 -13% reduction of weight change in the two highest treatment levels, a slight concentration-effect relationship was observed and an EC10 value was determined at 170.1 mg/kg soil (initial concentration). Regarding reproduction effects, a significant decrease in reproduction was found starting at an initial concentration of 58.5 mg test item/kg dry mass soil. The EC10 value was determined at 61.8 (CL 95%: 42.5 – 78.3) mg/kg dry mass soil (initial concentration).

In conclusion, the registered substance has chronic effects on Eisenia andrei under the chosen test conditions. The relevant EC10 values based on initial concentrations are 170.1 (CL 95%: n.d.) and 61.8 (CL 95%: 42.5 – 78.3) mg test substance/kg dry mass soil for growth and reproduction, respectively.