1-Butanone, 3-(dodecylthio)-1-[(1R,2S)-2,6,6-trimethyl-3-cyclohexen-1-yl]-, rel-

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 24 May 2011 to 26 May 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study was performed according to OECD guideline no. 207 but was not conducted under GLP. Only the filter paper test was performed. All validity criteria were fulfilled but results with a reference substance was not specified.

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Remarks:

Only a screening test was performed.

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of application to filter paper (if used): Dropping of first 1 mL of application solution onto the paper of the test unit. Mixing for 10 minutes on a roller mixer. Dropping of second 1 mL of application solution onto the paper of the test unit. Mixing for 10 minutes on a roller mixer and evaporation for additional 60 minutes. After evaporating the solvent, moisten the filter paper with 1 mL deionised water.
- Controls: one solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution): no data
- Evaporation of vehicle before use: no data

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: earthworm
- Source: Breeding stock maintained at Harlan Laboratories Ltd.
- Age at test initiation (mean and range, SD): Adults at least two months old
- Weight at test initiation (mean and range, SD): individual weight of approx. 300 to 600 mg.

ACCLIMATION
- Acclimation period: 3 hours on moist filter paper for voiding gut content.
- Acclimation conditions (same as test or not): no data
- Health during acclimation (any mortality observed): no data

Study type:

laboratory study

Substrate type:

filter paper

Limit test:

no

Total exposure duration:

48 h

Remarks:

none

Post exposure observation period:

None

Test temperature:

18.9-20.0°C

pH:

No data

Moisture:

No data

Details on test conditions:

TEST SYSTEM
- Test container (material, size): flat-bottomed glass vials (6cm in height and 3.5 cm in diameter) with filter paper inside (area of the filter paper: 40 cm²).
- Amount of soil or substrate: not applicable
- No. of organisms per container (treatment): 1
- No. of replicates per treatment group: 10
- No. of replicates per control: not applicable
- No. of replicates per vehicle control: 10

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
not applicable

OTHER TEST CONDITIONS
In the dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The mortality and symptoms of toxicity of the exposed test organisms were assessed after 48 hours of exposure.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: none
- Test concentrations: 0.25, 0.5, 1.0, 2.0 and 4.0 mg test item/ cm² (corresponding to 5, 10, 20, 40 and 80 g/L acetone).
- Results used to determine the conditions for the definitive study: none

Nominal and measured concentrations:

Nominal concentrations: 0.25, 0.5, 1.0, 2.0 and 4.0 mg test item/ cm² corresponding to 5, 10, 20, 40 and 80 g/L acetone.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 4 mg/cm²

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 4 mg/cm²

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

See table 6.3.1/1 in "Any other information on results incl. tables".

Results with reference substance (positive control):

None

Reported statistics and error estimates:

None

Table 6.3.1/1: Mortality of Eisenia fetida exposed to the test item for 48 hours in the paper test.

Nominal test item concentration		Number of earthworms		% mortality
		exposed	Dead after 48 hours	(average per group)
g/L	mg/cm²
Solvent control	-	10	0	0
5	0.25	10	0	0
10	0.5	10	0	0
20	1.0	10	0	0
40	2.0	10	0	0
80	4.0	10	0	0

Validity criteria fulfilled:

yes

Conclusions:

No mortality was observed in the solvent control at the end of the test and for all test concentrations used. The 48h-EC50 was > 4.0 mg/cm² and the 48h-NOEC was >= 4.0 mg/cm².

Executive summary:

This study was performed according to OECD guideline no. 207 but was not conducted under GLP. The purpose of this 2 -day toxicity study was to assess the acute toxic effect of the test item to earthworms Eisenia fetida.

For this purpose, the filter paper was treated with the test item. Adult worms (at least two months old) were exposed to different test item concentrations under defined conditions. The test item was applied in acetone to filter paper. The nominal test concentrations were 0.25, 0.5, 1.0, 2.0 and 4.0 mg/cm², corresponding to 5, 10, 20, 40 and 80 g/L acetone (based on a paper area of 40cm² and an application volume of 2 mL per replicate). Additionally, a solvent control was tested in parallel. In each treatment and the solvent control, 10 worms were tested with one worm per test unit. The temperature during the test was comprised between 18.9 and 20°C and the test was performed in the dark.

The mortality and symptoms of toxicity of the exposed test organisms were assessed after 48 hours of exposure. All results are related to nominal concentrations of the test item.

No mortality was observed in the solvent control at the end of the test and for all test concentrations used. The 48h-EC50 was > 4.0 mg/cm² and the 48h-NOEC was >= 4.0 mg/cm².

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 24 February 2015 to 10 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to OECD Guideline 222 with GLP statement. All validity criteria were fulfilled and no deviations were observed. Therefore, this study is considered reliable without restriction.

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection 14.09.2010 / Data of signature 07.02.2011

Analytical monitoring:

yes

Details on sampling:

According to the guideline, no verification of the test item concentration is strictly required. However, to verify test item concentration in the test matrix, start and end concentrations in the artificial soil were measured in all treatment levels. Additionally, samples of the 250 mg/kg treatment were measured on a weekly basis. For this, individual “analytic” test containers were prepared (incl. earthworms and feeding).

EXTRACTION PROCEDURE AND SAMPLE PREPARATION:
Subsamples were transferred into centrifuge tubes. After the addition of 50 mL methanol (HPLC grade) the subsamples were shaken for 30 minutes at 180 shakes per minute, treated with ultrasound for 10 minutes and centrifuged at 2000 rpm for 10 minutes. The supernatant layer was separated and the process was repeated threefold. After combining the extracts the volume was determined. About 1.5 mL of each combined extract was separated, transferred into an Eppendorf vial and centrifuged at 14000 rpm for 10 minutes. This extraction procedure was optimized in pretests to recover at least 70 % to 110 % of the test compound. After that, dilutions with methanol (HPLC grade) of the combined extracts were accomplished. Two dilutions step were performed. The solutions were transferred in HPLC vials during the dilution step 2. The samples were quantified by LC-MS/MS.
At incubation start (0 DAT sample) and incubation end (56 DAT sample) the dilution and quantification were accomplished without storing. The extracts of all other samples (7, 14, 21, 28, 35, 42, 49 DAT) were stored at -18 °C in the freezer. After 1 week of the last sampling (56 DAT sample) the frozen extracts were taken from the freezer and diluted and quantified.

Vehicle:

yes

Details on preparation and application of test substrate:

The quantity of test item required to obtain the desired concentrations was dissolved in 5 mL acetone (solvent) and applied onto 70 g quartz sand. After evaporation of the solvent, the mixture was added to an amount of test substrate representing 3500 g dry mass (adjusted on approx. 20 % WHC) and mixed thoroughly. Subsequently, the test substrate was moistened to a final moisture of 54 – 60 % WHC. The test substrate was prorated to each test container.

The control consists of test substrate adjusted to 60 % of the maximum water holding capacity. As the test item was applied to quartz sand using a solvent, an additional control reflecting solvent-mediated application followed by evaporation is conducted. The solvent control was conducted in eight replicates, complying with the test guideline. The blank control was run in quadruplicate.

Test organisms (species):

Eisenia sp.

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Eisenia andrei
- Source: Regenwurmfarm Tacke, Klosterdiek 61, 46325 Borken, Germany. Specimens used in the test were bred in the laboratory of the Fraunhofer IME.
- Age at test initiation (mean and range, SD): 2 - 12 months old
- Weight at test initiation (mean and range, SD): wet mass between 250 mg and 600 mg.
- Breeding conditions: The breeding conditions followed SOP V3-255/05. Worms were bred in 1:1 mixtures of cow manure and sphagnum peat (dry mass basis) at 20 °C ± 2 °C.

ACCLIMATION
- Acclimation period: The worms were conditioned for 16 days in the artificial soil before use.
- Acclimation conditions (same as test or not): The same food which was also used in the test was given in a sufficient amount.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Remarks:

none

Post exposure observation period:

None

Test temperature:

The incubation temperature was measured continuously with a thermograph. With 19–22 °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 5.9 – 6.2. At test end, pH of the artificial soil was 5.7 – 5.9, independent from test item concentration.

Moisture:

Between 54.0 to 60.0% WHC at test start.
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) and amount of soil: Round glass containers with a volume of 1 – 2 L were used. The containers were filled up with about 550 g dry mass of artificial soil to ensure a moist substrate depth of about 5-6 cm. 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.
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control: 8

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).

OTHER TEST CONDITIONS
- Photoperiod: controlled light/dark cycle of 16 h : 8 h
- Light intensity: The light intensity was measured using an illuminance meter (MINOLTA) with photometric sensor in Lux. With 750 – 783 lx the permitted value of about 400 - 800 lx was kept.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Adult worms were determined after 28 days and 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 28 days 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: yes

TEST CONCENTRATIONS
The nominal concentration in the test containers with test item was 62.5, 125, 250, 500 and 1000 mg ST 01 C 14 per kg dry mass soil. The concentrations were chosen due to a non-GLP acute limit test showing no mortality at 1000 mg/kg.

Nominal and measured concentrations:

- Nominal concentrations: 62.5, 125, 250, 500 and 1000 mg/kg dry mass soil
- Measured concentrations: see table 6.3.1/1 in "Any other information on results incl. tables".

Reference substance (positive control):

yes

Remarks:

Carbendazim

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

62.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

116 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CL: 45 - 185 mg/kg soil dw

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CL: 975 - >1000 mg/kg soil dw

Details on results:

- 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.

- Mortality: The results of survival are presented as mean values in tables 6.3.1/2 in "Any other information on results incl. tables". There is no significant difference between control and solvent control. In addition, no significant difference when compared all treatments with solvent control (Fisher's Exact Binomial Test). In conclusion, the survival rate of Eisenia andrei was obviously not affected by the test substance. The NOECmortality was found to be ≥ 1000 mg test substance/kg dry mass soil (nominal concentration), the highest concentration tested.

- Weight change: The results of weight change are presented in table 6.3.1/3 in "Any other information on results incl. tables". There is no significant difference between control and solvent control. In addition, no significant difference when compared all treatments with control (Welch-t test, one-sided). In conclusion, no effect on weight change due to the test substance was detected. The NOECweight change for the tested species Eisenia andrei was found to be ≥ 1000 mg test substance/kg dry mass soil (nominal concentration).

- Reproduction: The results of reproduction are presented in tables 6.3.1/4 in "Any other information on results incl. tables". There is no significant difference between control and solvent control. However, the nominal concentrations of 125, 250, 500 and 1000 mg/kg are considered significant when compared with control (Williams test, one-sided). In conclusion, the reproduction of Eisenia andrei was significantly affected by the test substance starting at a concentration of 125 mg test substance/kg dry mass soil (nominal concentration). The NOEC was found to be 62.5 mg test substance/kg (nominal concentration). There was a concentration response effect for reproduction. The EC10 was found to be 116 mg (95 % CL: 45 – 185 mg) test substance/kg (nominal concentration).

- Effect concentrations. Table 6.3.1./5 in "Overall remarks" presents the NOECs and EC values of the test substance for weight change, survival and reproduction.

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 in a non GLP test not longer than 12 month ago. In the last reference test, the NOEC for weight change and mortality was found to be ≥ 3 mg/kg. No concentration dependence occurred. EC values could not be calculated. Reproduction was significantly affected by carbendazim. The NOEC was suggested to be 0.38 mg/kg. The EC10 and EC50 were 0.5 mg/kg and 1.1 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.

Reported statistics and error estimates:

For each concentration, the percent mortality, the percent loss/increase in biomass of the adults, and the number of offspring produced in the test was determined. All statistical tests (depending on normal distribution and homogeneity of variances the STUDENT-t test, Fisher`s Exact Binomial Test, Williams Multiple Sequential t-test, or Welch t-test for the NOEC calculation; ECx calculation) were performed with the computer software ToxRat Professional by ToxRat® Solutions GmbH. For the ECx calculation a probit-analysis assuming log-normal distribution of the values was applied.

Table 6.3.1/1: Measured concentrations of the test substance in soil (mg/kg) and percentage recovery (%) of the dose introduced into the test vessel at test start and test end

Nominal concentrations (mg test item/kg dry mass soil)
Test item concentration	Solvent control	62.5 mg/kg	125 mg/kg	250 mg/kg	500 mg/kg	1000 mg/kg
Measured concentrations (mg/kg dry mass soil)
Test start	6.09 mg/kg	64.4 mg/kg 103.1%	123.6 mg/kg 98.9%	247.8 mg/kg 99.1%	485.4 mg/kg 97.1%	996.5 mg/kg 99.6%
Test end	<LOQ	25.2 mg/kg 40.3%	39.0 mg/kg 31.2%	106.3 mg/kg 42.5%	208.1 mg/kg 41.6 %	568.8 mg/kg 56.9%

The initial measured test item concentrations in the soil at test start corresponded to 103.1 %, 98.9 %, 99.1 %, 97.1 % and 99.6 % of the nominal dose introduced into the test vessels in the treatments with 62.5, 125, 250, 500 and 1000 mg test item per kg dry soil

nominal concentration, respectively. The measured test item concentration of 6.09 mg/kg in the solvent control at test start must be due to contamination in the sampling or eluation process, since control vessels were filled and prepared first at test start. The value is in the LOQ range of 1.94 – 6.12 mg/kg calculated for the respective measurement series. However, since biological endpoints do not vary between solvent control and the lowest treatment level, the contamination is assumed to be without influence on the evaluation and the integrity of the study. At the end of the test measured substance concentrations in the soil corresponded to 40.3 %, 31.2 %, 42.5 %, 41.6 % and 56.9 % of the nominal dose introduced into the test vessels. Measurements in the treatment level 250 mg/kg showed a continuous decrease with time. According to the guideline, nominal concentrations were applied for evaluation of the biological endpoints.

Table 6.3.1/2: Living worms (sum of all replicates) and survival (mean values, %) after 28 days

Nominal test item concentration	Control	Solvent control	62.5 mg/kg	125 mg/kg	250 mg/kg	500 mg/kg	1000 mg/kg
Living worms (individuals)	40	79	37	36	36	35	40
Survival (%)	100	99	93	90	90	88	100

Introduced worms at test start: 80 (solvent control), 40 (all other treatments).

Table 6.3.1/3: Weight change at test end

Nominal test item concentration	Control	Solvent control	62.5 mg/kg	125 mg/kg	250 mg/kg	500 mg/kg	1000 mg/kg
Weight change compared to the start value (%)	65	66	54	65	64	64	72
Weight change compared to control (%)	-1	/	-8	0	-2	-1	3

Table 6.3.1/4: Number of juveniles at the end of the 56 days study period (mean values and coefficient of variation, CV) and reproduction inhibition (mean values, %)

Nominal test item concentration	Control	Solvent control	62.5 mg/kg	125 mg/kg	250 mg/kg	500 mg/kg	1000 mg/kg
Juveniles (CV)	107.3 (15.0)	109.8  (9.8)	101.3 (10.9)	95.8 (13.8)	92.0 (8.2)	80.0 (20.3)	60.8 (6.2)
Reproduction inhibition	2%	/	8%	13%*	16%*	27%*	45%*

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

Validity criteria fulfilled:

yes

Conclusions:

The test substance has chronic effects on Eisenia andrei under the chosen test conditions. The relevant NOEC for reproduction was found to be nominal 62.5 mg test substance/kg dry soil. The EC10 reproduction was found to be nominal 116 mg (95 % CL: 45 – 185 mg) test substance/kg.

Executive summary:

At the Fraunhofer Institute for Molecular Biology and Applied Ecology the influence of the test substance on mortality, biomass, and reproduction of adult earthworms of the species Eisenia andrei was investigated according to the OECD 222 guideline, with GLP compliance. For this, the worms were placed in a defined artificial soil substrate containing the test item in different concentrations (62.5, 125, 250, 500 and 1000 mg/kg dry mass soil; a concentration of 1000 mg/kg met the criteria of the most OECD Guidelines dealing with terrestrial test organisms concerning highest test concentration). A single application of test item was made at the beginning of the test. Effects on mortality and biomass were determined after 28 days, the effects on reproduction were determined after 56 days. Eight replicates were prepared for the solvent control and four for each concentration. According to the guideline, evaluation was done on the basis of nominal test item concentrations. However, to verify test item concentration in the test matrix, concentrations in the artificial soil in the course of the study were measured. The initial measured test item concentrations in the soil were between 97 % and 103 % of nominal concentrations. The test item concentrations measured after 28 days were between 31 % and 56 % of nominal concentrations. Measurements in a representative treatment level (250 mg/kg) showed a continous decrease with time.

No increased mortality and no significant influence on weight change were found due to nominal 1000 mg test substance/kg dry soil, which was the highest concentration tested. However, reproduction was significantly affected starting at nominal 125 mg test substance/kg dry soil. The NOEC for reproduction was found to be nominal 62.5 mg/kg dry soil. The EC10 was found to be 116 mg (95 % CL: 45 – 185 mg)/kg (nominal concentration).

In conclusion, the test substance has chronic effects on Eisenia andrei under the chosen test conditions. The relevant NOEC for reproduction was found to be nominal 62.5 mg test substance/kg dry soil. The EC10 reproduction was found to be nominal 116 mg (95 % CL: 45 – 185 mg) test substance/kg.

Description of key information

- Acute toxicity test, OECD Guideline 207, non-GLP, Klimisch score 2: 48h-EC50 (Eisenia fetida) > 4.0 mg/cm²

- Chronic toxicity test, OECD Guideline 222, GLP, Klimisch score 1: 56d-EC10 (Eisenia andrei) = 116 mg/kg soil dw (95 % CL: 45–185 mg/kg soil dw)

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:

116 mg/kg soil dw

Additional information

To assess the toxicity of the registered substance to earthworm, two reliable key studies are available.

The first study (Harlan, 2011) was performed on the registered substance, according to OECD guideline 207 but without GLP statement, to assess the acute toxicity of the substance to earthworm Eisenia fetida, during 2 days. Only the filter paper contact test was performed. For this purpose, the filter paper was treated with the substance. Adult worms were exposed to a series of concentrations (0.25, 0.5, 1.0, 2.0 and 4.0 mg/cm²) under defined conditions. Additionally, a solvent control was tested in parallel. In each treatment and the solvent control, 10 worms were tested with one worm per test unit. The temperature during the test was comprised between 18.9 and 20°C and the test was performed in the dark. The mortality and symptoms of toxicity of the exposed test organisms were assessed after 48 hours of exposure. No analytical monitoring was performed and all results were related to nominal concentrations of the test item. No mortality was observed in the solvent control at the end of the test and for all test concentrations used. Therefore, the 48h-EC50 was greater than 4.0 mg/cm² and the 48h-NOEC was greater or equal to 4.0 mg/cm². It is not possible to extrapolate from mg/cm² to mg/kg soil dw but 4.0 mg/cm² is considered to an extremely high exposure concentration.

The second study (Fraunhofer, 2015) was performed on the registered substance, according to OECD Guideline 222 with GLP compliance, to assess the chronic toxicity of the substance to earthworm Eisenia andrei, during 56 days. For this, the worms were placed in a defined artificial soil substrate containing the test item in different concentrations (62.5, 125, 250, 500 and 1000 mg/kg dry mass soil). A single application of test item was made at the beginning of the test. Effects on mortality and biomass were determined after 28 days, the effects on reproduction were determined after 56 days. Eight replicates were prepared for the solvent control and four for each concentration. According to the guideline, evaluation was done on the basis of nominal test item concentrations. However, to verify test item concentration in the test matrix, concentrations in the artificial soil in the course of the study were measured. The initial measured test item concentrations in the soil were between 97 % and 103 % of nominal concentrations. The test item concentrations measured after 28 days were between 31 % and 56 % of nominal concentrations. Measurements in a representative treatment level (250 mg/kg) showed a continous decrease with time. No increased mortality and no significant influence on weight change were found due to nominal 1000 mg test substance/kg dry soil, which was the highest concentration tested. However, reproduction was significantly affected starting at nominal 125 mg test substance/kg dry soil. The NOEC for reproduction was found to be nominal 62.5 mg/kg dry soil and the EC10 was found to be 116 mg (95 % CL: 45 – 185 mg)/kg (nominal concentration). In conclusion, the test substance has chronic effects on Eisenia andrei under the chosen test conditions. The relevant NOEC and EC10 values for reproduction were found to be nominal 62.5 mg test substance/kg dry soil and 116 mg (95 % CL: 45 – 185 mg) test substance/kg, respectively.

NOECs are increasingly contested in ecotoxicology because the NOEC is determined as the concentration directly below the LOEC (Lowest Observed Effect Concentration) and depend upon the choice made by the Study Director/Study Monitor and not a statistically interpolated value. Thus, a NOEC may occur at concentrations considerably lower than the point at which no biological effect occurs. The choice of the concentrations tested is therefore a limiting factor for the NOEC determination. EC10 values were considered to be the most conservative and regulatorarily relevent statistical estimation. Therefore, the relevant chronic endpoint value is the EC10 value determined at 116 mg/kg dry soil (95% CL: 45 - 185 mg/kg).