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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

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Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-12-03 to 2016-06-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Version / remarks:
(1984)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
- Concentrations: A limit test with a test item concentration of 1000 mg/kg soil dry weight was performed.
- Sampling method: The WHCmax, pH-value and the moisture content of the artificial soil were determined prior to the adaptation of the earthworms. At the start and the end of the test, the pH-value and moisture content of the test medium were analysed in the treatments and the control using mixed samples of all replicates. The live weight of the earthworms was determined individually on the day of application (day 0) and on day 14. The mean weight change was assessed on day 14 after application. Mortality, behaviour and morphological changes of the earthworms were recorded 7 and 14 days after application.

Vehicle:
yes
Remarks:
Acetone
Details on preparation and application of test substrate:
- Method of mixing into soil: The respective test item was weighed out (nominal weight: 3330 mg; actual weight: 3330.3 mg) for the test item concentration, solved in Acetone (2.5 mL per replicate) and mixed thoroughly with quartz sand (10 g per replicate). After complete evaporation of the solvent, the spiked quartz sand was added to the artificial soil. Demineralised water was added to the artificial soil to adjust the humidity of the artificial soil to a moisture of 54 % of the maximum water holding capacity. Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 555 g soil dry weight will be filled into each test vessel.

- Controls: Artificial soil moistened with demineralised water not containing test or reference item was used as a control medium.
- Solvent Control: Artificial soil treated with the solvent, moistened with demineralised water not containing test or reference item was used as a solvent control medium.
- Chemical name of vehicle: Acetone
- Concentration of vehicle in test medium (stock solution and final test solution): The respective test item was weighed out (3330.3 mg) and solved in Acetone (2.5 mL per replicate) and mixed thoroughly with quartz sand (10 g per replicate).
- Evaporation of vehicle before use: Yes. After complete evaporation of the solvent, the spiked quartz sand was added to the artificial soil.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Eisenia fetida (Annelida, Lumbricidae)
- Source: Breeding stock culture maintained at the test facility.
- Breeding: Organisms of the species Eisenia fetida are bred at the test facility in covered plastic vessels containing potting compost at 20 ± 2°C in the dark
- Age at test initiation: aged 2 – 12 months, with clitellum
- Weight at test initiation: live weight of 0.31 to 0.54 g

ACCLIMATION
- Acclimation period: The earthworms were conditioned to artificial soil two days prior to the start of exposure.
- Acclimation conditions: same as test
Study type:
laboratory study
Substrate type:
artificial soil
Remarks:
Components: 10 % Peat, air-dried and finely ground; 20 % Kaolin, kaolinite content > 30 %; 69 % Air-dried quartz sand (sand with > 50 % particles size of 0.05 to 0.2 mm); 0.35 % calcium carbonate (CaCO3) to adjust the pH to 6.0 ± 0.5.
Limit test:
yes
Total exposure duration:
14 d
Test temperature:
Temperature nominal: 20 ± 2°C; actual: 18-22 °C
pH:
pH value nominal 6.0 ± 0.5; actual: 5.33-5.55
Moisture:
The soil moisture content and the WHCmax were determined prior to experimental starting. Two days before earthworm insertion into the test vessels the artificial soil (16 kg) was pre-moistened with 1968 g deionised water to achieve a moisture of 27 % of the WHCmax of the artificial soil. The moistened soil was kept covered to prevent evaporative water losses prior to the start of exposure.

Moisture [%] of dry weight day 0: 26.3-27.0, day 14: 25.3-25.9
Details on test conditions:
TEST SYSTEM
- Test container (material, size): glass vessels, volume 1.5 L
- Amount of soil or substrate: 555 g soil dry weight / test vessel
- No. of organisms per container (treatment): 10 adult earthworms / test vessel
- No. of replicates per treatment group: 6
- No. of replicates per control: 6
- No. of replicates per vehicle control: 6

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition of the artificial substrate: 10 % Peat, air-dried and finely ground; 20 % Kaolin, kaolinite content > 30 %; 69 % Air-dried quartz sand (sand with > 50 % particles size of 0.05 to 0.2 mm); 0.35 % calcium carbonate (CaCO3) to adjust the pH to 6.0 ± 0.5.
- Maximum water holding capacity (in % dry weigth): WHCmax 52.9 g/100 g solid dry weigth
- Pretreatment of soil: The soil moisture content and the WHCmax were determined prior to experimental starting. Two days before earthworm insertion into the test vessels the artificial soil (16 kg) was pre-moistened with 1968 g deionised water to achieve a moisture of 27 % of the WHCmax of the artificial soil.
- Storage (condition, duration): The moistened soil was kept covered to prevent evaporative water losses prior to the start of exposure.
- Stability and homogeneity of test material in the medium: The test medium was thoroughly mixed to ensure a homogenous distribution.

OTHER TEST CONDITIONS
- Photoperiod: 24 h
- Light intensity: 400 - 800 lx

EFFECT PARAMETERS MEASURED: The live weight of the earthworms was determined individually on the day of application (day 0) and on day 14. The mean weight change was assessed on day 14 after application. Mortality, behaviour and morphological changes of the earthworms were recorded 7 and 14 days after application.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test 1000 mg/kg soild dry weight
- Justification for using less concentrations than requested by guideline: A range finding test has been performed with 10, 100 and 1000 mg/kg soil dry weight).

- Range finding study
- Test concentrations: A preliminary non-GLP range finding test was performed with 3 test item concentrations (10, 100 and 1000 mg/kg soil dry weight).
- Results used to determine the conditions for the definitive study: In the definitive study a limit test with 1000 mg/kg soil dry weight has been conducted.
Nominal and measured concentrations:
nominal 1000 mg/kg soil dry weight
Reference substance (positive control):
yes
Remarks:
2-Chloroacetamide
Key result
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Change of body weight, behaviour, pathological symptoms
Key result
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Change of body weight, behaviour, pathological symptoms
Details on results:
- Mortality at end of exposure period: No evident mortality was observed in the control and the limit test item concentration 1000 mg/kg SDW after 14 days of exposure.
- Total mass of adults at beginning of test: Sum 25.6 g (10 earthworms in 6 replicates of the contro). Mean individual weights in the control: 0.43 ± 0.03 g/earthworm
- Changes in body weight of live adults (% of initial weight) at end of exposure period: During the test, the biomass loss of the earthworms was below 20 % in the control .
- Morphological/Behavioural abnormalities: No pathological symptoms nor changes in the earthworm behaviour were observed in the control as well as in the limit test item concentration 1000 mg/kg SDW after 7 and 14 days of exposure.
Results with reference substance (positive control):
The acute toxicity of 2-Chloroacetamide on the earthworm Eisenia fetida fetida (SAVIGNY) was determined over a period of two weeks. The LC50-value was in the range between 20 and 80 mg/kg SDW as recommended by the guideline. The LC50 value was 28.2 mg/kg soil dry weight.

Earthworm Mortality in [%] after 7 and 14 Days of Exposure

 

 

Mortality [%]

 

Test item concentration

[mg product/kg SDW]

Day 7

Day 14

1

2

3

4

5

6

MV

1

2

3

4

5

6

MV

Control

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Solvent control

0

0

10

0

0

0

1.67

0

0

10

0

0

0

1.67

1000

0

0

0

0

0

0

0

0

0

0

0

0

0

0

MV = mean value

Earthworm Behaviour and Pathological Symptoms after 7 and 14 Days of Exposure

Test item concentration [mg product/kg SDW]

Effect

Replicate

1

2

3

4

5

6

Day 7

Control

A)

10/10

10/10

10/10

10/10

10/10

10/10

Solvent control

A)

H)

10/10

-

10/10

-

9/10

1/10

10/10

-

10/10

-

10/10

-

1000

A)

10/10

10/10

10/10

10/10

10/10

10/10

 

Day 14

Control

A)

10/10

10/10

10/10

10/10

10/10

10/10

Solvent control

A)

H)

10/10

-

10/10

-

9/10

1/10

10/10

-

10/10

-

10/10

-

1000

A)

10/10

10/10

10/10

10/10

10/10

10/10

A) no obvious pathological symptoms or behavioural changes

H) dead earthworm

Validity criteria fulfilled:
yes
Remarks:
The mortality in the control group did not exceed 10 % at the end of the test. The average loss of biomass in the control group did not exceed 20 % at the end of the test.
Conclusions:
No evident mortality (< 10 %) and no pathological symptoms or changes in earthworm behaviour were observed in the control and the limit test item concentration after 14 days of exposure. Therefore, the NOEC was determined to be >=1000 mg/kg soil dry weight. Also in terms of body weight loss, no statistically significant effects were observed in the limit concentration after 14 days of exposure compared to the pooled control. Therefore, the NOEC was determined to be >=1000 mg/kg soil dry weight.
Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Version / remarks:
(2004)
Deviations:
no
Principles of method if other than guideline:
Food was provided on day 0 (test start) instead of day 1 due to good experience with this procedure. At the test end, the soil moisture deviated by more than 10 % from the initial value in all treatments. These deviations are considered to have no impact on quality and integrity of the study.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 62.5, 125, 250, 500, 1000 mg/kg soil dry weight
- Sampling method: Analytical evaluation of the test substance was carried out via LC-MS/MS at day 0 and day 55 in all tested concentration levels and in the control. At day 0 sampling was carried out from the newly prepared test concentrations. At day 55 samples were taken directly from the test vessels. 5.0 g soil were weighed into centrifuge tubes and acetonitrile was added. The samples were shaken for 15 minutes and centrifuged at 10000 g at 20°C for 5 min. The extracts were collected in measuring flasks and 20 mL HPLC water containing 0.2% formic acid was added. The measuring flasks were tapped up with acetonitrile and HPLC water containing 0.1% formic acid.
- Sample storage conditions before analysis: Sampels were stored in an autosampler at room temperature until analysis.
Vehicle:
yes
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): The respective amount for each test substance concentration was weighed out, solved in Acetone and mixed thoroughly with quartz sand. After complete evaporation of the solvent, the spiked quarz sand was added to the artificial soil. Demineralised water was added to the artificial soil to adjust the humidity to a moisture of 54 % of the maximum water holding capacity. The test medium was thoroughly mixed to ensure a homogenous distribution and was filled into the test vessels.
- Controls: Artificial soil with quartz sand moistened with demineralised water not containing test or reference item; Solvent control
- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 2.5 mL per replicate
- Evaporation of vehicle before use: yes
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: earthworm
- Source: breeding stock culture maintained at the test facility
- Age at test initiation: snychronized, with clitellum
- Weight at test initiation: 0.37 to 0.56 g

ACCLIMATION
- Acclimation period: 2 days before test start
- Acclimation conditions (same as test or not): adaptation to artifical soil
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Test temperature:
actual 18.0 - 22.5 °C
pH:
5.79 to 6.45
Moisture:
18.6 % to 24.4 %
Details on test conditions:
TEST SYSTEM
- Test container: round plastic boxes, diameter 15.0 cm, surface area 177 cm2
- Amount of soil or substrate: 610 g soil per replicate
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- No. of replicates per vehicle control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition of artificial soil: 5 % peat, 20 % kaolin, kaolinite content > 30 %, 74 % air-dried quartz sand (sand with > 50 % particle size of 0.05 - 0.2 mm), 0.19 % calcium carbonate
- Maximum water holding capacity: 37.5 g/100 g dw
- Pretreatment of soil: The WHCmax, the moisture content and the pH-value of the artificial soil were determined and adjusted.
- Storage (condition, duration): The moistened soil was kept covered to prevent evaporative water losses prior to the start of exposure.

OTHER TEST CONDITIONS
- Photoperiod: 16 h
- Light intensity: 400-800 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): At experimental starting and experimental completion, the pH-value and moisture content of the test medium were determined in each treatment and the control from pooled samples of all replicates. The water content of each test container was checked weekly by weighing. The weight loss was replenished with the appropriate amount of demineralised water. The body weight of the adult earthworms was determined individually on day 0 and day 28 for each replicate. After the first four weeks, the adult earthworms were removed. Mortality and morphological changes of the adult earthworms were recorded for each replicate. After a further four weeks, the number of offspring hatched from the cocoons was counted for each test container. Test concentrations of test item and the controls were confirmed by analytical verification on day 0 and at test end from pooled samples of all replicates.

VEHICLE CONTROL PERFORMED: yes
Nominal and measured concentrations:
62.5, 125, 250, 500, 1000 mg/kg soil dry weight
Reference substance (positive control):
yes
Remarks:
carbendazim
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:
mortality
Remarks:
biomass
Duration:
56 d
Dose descriptor:
LOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
biomass
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
Duration:
56 d
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Mortality at end of exposure period: no evident earthworm mortality (< 10 %) in the controls and all test item concentrations
- Changes in body weight of live adults (% of initial weight) at end of exposure period: pooled control: 24.4 ±5.17 %
- No. of offspring produced: pooled control 141 ± 18.4 mean number of juveniles
- Morphological abnormalities: no significant pathological symptoms
- Behavioural abnormalities: no changes in the behaviour

The measured concentrations of the test item at the start of the exposure were within the range of 95 to 109% of the nominal values and demonstrate the right preparation of the tested concentrations. At the end of the exposure, the measured concentrations were between 3 and 7%.
Results with reference substance (positive control):
- Results with reference substance valid:
- Relevant effect levels: test concentrations of 0.5, 1, 2, and 4 mg/kg soil dry weight were tested.
- Other: The test is conducted once within twelve months. According to the guideline significant effects with the reference substance should be observed between 1 and 5 mg a.s./kg soil dw. Significant effects were observed at 2 and 4 mg/kg sdw.
Reported statistics and error estimates:
Significant differences in biomass and reproduction were determined in comparison with the pooled control group by probit analysis. When running the test, a Shapiro-Wilk Normality Test and a Levene’s Equal Variance Test were done first. The a-value (acceptable probability of incorrectly concluding that there is a difference) was a = 0.05. For the endpoint of reproduction, the arithmetic mean and the variance (coefficient of variation) per treatment and control were calculated.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, Di(iso)-pentylterephthalate did not induce evident earthworm mortalities (< 10 %) nor significant pathological symptoms or changes in the behaviour of adult earthworms in the controls and all test item concentrations There were no statistically significant differences in earthworm body weights in all test item concentrations compared to the pooled control. There were no statistically significant differences in earthworm reproduction in the treatment rates 62.5 to 1000 mg/kg soil dry weight compared to the pooled control. Overall, the NOEC of the test item concerning mortality, biomass and reproduction was determined to be 1000 mg test item/kg SDW.

Description of key information

In a short-term toxicity study with Eisenia fetida (earthworm), the NOEC was determined to be ≥ 1000 mg/kg soil dry weight. The NOEC concerning mortality, biomass and reproduction has been determined in a long-term test with Eisenia fetida to be 1000 mg/kg sdw.

Key value for chemical safety assessment

Additional information

A short-term limit toxicity test with Eisenia fetida has been conducted to test the acute toxicity of the test substance according to the OECD guideline No. 207 in compliance with GLP. No significant mortality (<10%) and no pathological symptoms or changes in earthworm behaviour were observed in the control and the limit test substance concentration after 14 days of exposure. Also in terms of body weight loss, no statistically significant effects were observed in the limit concentration after 14 days of exposure compared to the pooled control. Therefore, the NOEC was determined to be >=1000 mg/kg soil dry weight.

The effects of the test substance on mortality, biomass and the reproductive potential of earthworms has also been tested in compliance with GLP in a long-term (8 weeks) test according to guideline OECD 222. Eisenia fetida has been exposed to five different test concentrations up to 1000 mg/kg soil dry weight in artifical soil. After 28 days of exposure in soil, neither evident mortalities (<10%) nor significant pathological symptoms or changes in the behaviour of adult earthworms has been observed (NOEC >=1000 mg/kg sdw). There were no statistically significant differences in earthworm body weight and reproduction in the treatment rates 62.5 to 1000 mg/kg soil dry weight compared to the pooled control.