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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:
November 16, 2016 to March 06, 2017.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The earthworm Eisenia fetida (Lumbricidae) was chosen as a representative of the soil fauna. The selection of the test system was based on the requirement of:
- Corrigendum to Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC. Official Journal of the European Union L136/3.
- Commission Regulation (EU) No 640/2012 of 6 July 2012 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) Official Journal of the European Union L193/1.
The study was conducted in compliance with Good Laboratory Practice Principles according to:
 OECD (1998). The OECD Principles of Good Laboratory Practice (as revised in 1997). OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring No. 1. ENV/MC/CHEM(98)17. Organisation for Economic Co-Operation and Development, Paris.
 Chemikaliengesetz in der Fassung der Bekanntmachung vom 28. August 2013 (BGBl. I S. 3498, 3991), das zuletzt durch Artikel 4 Absatz 97 des Gesetzes vom 18. Juli 2016 (BGBI. I S. 1666) geändert worden ist. Anhang 1 (zu § 19a Abs. 1) Grundsätze der Guten Laborpraxis (GLP). [German Chemical Agents Act dated 28 August 2013, Federal Law Gazette, Germany (BGBl) p. 3498, 3991, last amended by Article 4 of 18. Juli 2016 (BGBl. I S. 3498, 3991), Appendix I (rel. to §19a (1), Principles of Good Laboratory Practice].
and according to:
 OECD Guideline No. 222 for the Testing of Chemicals "Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei)" adopted July 29, 2016
 The International Standard ISO 11268-2 Part 2 (2012) "Soil Quality – Effects of Pollutants on Earthworms– Part 2: Determination of Effects on Reproduction to Eisenia fetida/Eisenia andrei ".
Qualifier:
according to guideline
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Version / remarks:
2016
Deviations:
yes
Remarks:
The temperature slightly lower than required for around 3% of the measures may not affect significantly the results. The quartz coated with the item mixed with soil only 5 minutes instead of 10 may not affect significantly the results..
GLP compliance:
yes (incl. QA statement)
Remarks:
Certificates is included in the report document (page 5)
Analytical monitoring:
not required
Vehicle:
yes
Details on preparation and application of test substrate:
The test item was poorly soluble in water; therefore, a volatile organic solvent was used. The stock emulsion and the dosage emulsions were prepared in acetone. All concentrations to be tested were based on the product Rape Oil, oxidized.
The test item was emulsified in a volume of acetone sufficient to prepare a stock emulsion. This stock emulsion was used to produce a range of dosage emulsions for each concentration. An appropriate volume of the stock or respective dosage emulsion was used to soak a portion of the quartz sand of the artificial soil. After evaporation for 2 h of the solvent by placing under a fume hood, the quartz sand was coated by the test item.
The test item coated fraction of the quartz sand was incorporated into the remainder of the artificial soil by thoroughly mixing it in for 5 minutes.
Finally, the treated soil was filled into the test vessels. Per test vessel an amount corresponding to 500 g dw (dry weight) was used.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
The earthworms used in this test, Eisenia fetida (Lumbricidae, Earthworms) have been kept at ECT Oekotoxikologie GmbH since February 1994. The worms for the culture were originally delivered by Co. Landenberger (72355 Schömberg, Germany).
Only adult earthworms (with clitellum) with a fresh weight (FW) between 250 and 600 mg were used. The earthworms were at least two months, but not more than one year old. However the age of individuals did not differ by more than four weeks, for details see section 18.1. Animals which fulfilled these requirements were taken from a synchronised culture. The earthworms selected for the test were acclimatised in artificial soil under test conditions for at least 24 hours before starting the test.
Study type:
laboratory study
Substrate type:
artificial soil
Remarks:
- 5% Sphagnum peat (no visible plant remains; finely ground and air-dried) - 20% Kaolin-Clay (kaolinite content > 30%) - ~74-75% Quartz sand (fine sand content with particles between 50 and 200 microns higher than 50%) - 0.3-1% Calcium carbonate (CaCO3).
Limit test:
no
Total exposure duration:
28 d
Post exposure observation period:
28 days
Test temperature:
Mean 19.8 °C, 17.3 – 21.1 °C
pH:
5.8 - 6.7
Moisture:
28.4 - 65.8 % of dry mass
max 30.3% change between initial and end moisture concentrations.
Details on test conditions:
TEST SYSTEM
- Test container (material, size):
- Amount of soil or substrate:
- 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
- Composition: 5% Sphagnum peat, 20% Kaolin-Clay (kaolinite content > 30%), ~74-75% Quartz sand (fine sand content with particles between 50 and 200 microns higher than 50%), 0.3-1% Calcium carbonate (CaCO3)
- Organic carbon (%): not provided
- Maximum water holding capacity (in % dry weigth): not provided
- CEC: not provided
- Pretreatment of soil: none: not provided
- Storage (condition, duration): not provided
- Stability and homogeneity of test material in the medium: After evaporation the solvent by placing it under a fume hood the coated fractions of the quartz sand were incorporated into the remainders of the artificial soil to prepare the different test item soil concentrations. No information were provided on homogenity of test material in the medium.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark
- Light intensity: with a constant light intensity at the substrate

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : No

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1,5
- Justification for using less concentrations than requested by guideline: Not provided
- Range finding study: no
- Test concentrations: 200, 300, 450, 670, 1000 mg item/kg dw soil
- Results used to determine the conditions for the definitive study: none
Nominal and measured concentrations:
Control / Solvent control / 200 / 300 / 450 / 670 / 1000 mg test item/kg dw soil
Reference substance (positive control):
yes
Remarks:
Boric acid EC50 = 325.4 mg/kg dw soil
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
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:
other: Biomass
Key result
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:
EC50
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
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:
other: 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:
reproduction
Details on results:
- Mortality at end of exposure period: 2.5% in control, 0% in Solvent control and all tested concentrations
- Total mass of adults at beginning of test (mg fresh weight): Control = 398.0±36.7, solvent control = 392.3±34.0, conc. 200mg test item/kg soil (dw) = 392.7±30.7, conc. 300mg test item/kg soil (dw) = 395.7±26.4, conc. 450mg test item/kg soil (dw) = 393.7±24.7, conc. 670mg test item/kg soil (dw) = 392.1±30.2, conc. 100mg test item/kg soil (dw) = 396.4±25.6
- Changes in body weight of live adults (% of initial weight) at end of exposure period: Control = 68.6, Solvent control = 51.7, conc. 200 = 51.1, conc. 300 = 62.8, conc. 450 = 54,6, conc. 670 = 60.4, conc. 1000 = 60.6
- No. of offspring produced: Control = 392.3 ± 42.4, Solvent control = 393.8 ± 46.3, conc. 200 = 364.0 ± 88.0, conc. 300 = 377.3 ± 50.2, conc. 450 = 358.5 ± 52.4, conc. 670 = 398.5 ± 49.7, conc. 1000 = 400.3 ± 56.0
- No. of unhatched cocoons: none
- Morphological abnormalities: none
- Behavioural abnormalities: none
- Other biological observations: none
Results with reference substance (positive control):
The EC50 value for boric acid tested as the toxic reference in a separate study (ECT study number IRR1602) was calculated by Probit analysis using linear max. likelihood regression as 325.4 mg boric acid/kg artificial soil (dw) (95% confidence limits: 314.6 – 349.7 mg boric acid/kg artificial soil (dw)). The estimated EC50 value is slightly lower than recommended by the guideline. However, since the calculated EC50 is a factor of 1.2 lower than the recommended value of 400 mg Boric acid/ kg soil (dw) acceptable sensitivity of the test system is assumed.
Reported statistics and error estimates:
Biomass Data were checked for normality by the R/s test procedure and for homogeneity by Cochran’s test. Treatment means were compared by using ANOVA followed by Dunnett’s t-test (p ≤ 0.05, two-sided) and tested for statistically significant differences compared to the control (Sachs 1982).
Reproduction: Data were checked for normality by the R/s test procedure and for homogeneity by Cochran’s test. Treatment means were compared by using ANOVA followed by Dunnett’s t-test (p ≤ 0.05, one-sided) and tested for statistically significant differences compared to the control (Sachs 1982).
Validity criteria fulfilled:
yes
Conclusions:
The study, which follows OECD-222 guideline and was conducted under GLP conditions, is reliable and its deviations are minor. The NOECs for mortality, adult biomass and reproduction are above 1000 mg item/kg dw soil.
Mortality.
2.5% mortality was observed in the water control. No mortality was observed in the solvent control and in all concentrations of the test item. Therefore, no further computations were performed to compare mortality between the solvent control and all test item concentrations. As a result, NOECMortality was considered to be ≥ 1000 mg test item/kg soil (dw) (the highest concentration tested). The LOECMortality could not be determined and was regarded as > 1000 mg test item/kg soil (dw).
Adult biomass.
Statistical analysis showed no significant difference (Dunnett’s test, 2-sided; p ≤ 0.05) concerning biomass development of individual adults over 28 days between the solvent control and all concentrations of the test item. Therefore, the NOECBiomass was considered to be ≥ 1000 mg test item/kg soil (dw) (the highest concentration tested). The LOECBiomass could not be determined and was regarded as > 1000 mg test item/kg soil (dw).
Reproduction.
Statistical analysis (Dunnett’s t-test, 1-sided, p ≤ 0.05) showed no significant difference concerning the number of juveniles between the solvent control and all concentrations tested. Therefore, the NOECReproduction was considered to be ≥ 1000 mg test item/kg soil (dw). The
LOECReproduction could not be determined and was regarded as > 1000 mg test item/kg soil (dw). Since inhibition of reproduction was < 50%, the EC50 could not be calculated and was considered to be > 1000 mg test item/kg soil (dw).
Executive summary:

Rape Oil, oxidized Reproduction toxicity to the earthworm Eisenia fetida was tested in artificial soil with 5% peat according to OECD Guideline No. 222 for the Testing of Chemicals "Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei)" adopted July 29, 2016, and under GLP conditions.

Two minor deviations are noted: 1) The temperature was below 18 °C for 44 measuring points (i.e. maximum 44 hours, temperature was logged once per hour during the test) out of 1345 measuring points (minimum 17.3 °C, recommended range 18 – 22 °C); 2) The test item coated fraction of the quartz sand was incorporated into the remainder of the artificial soil by thoroughly mixing it in for approx. 5 minutes instead of 10 minutes. However, these minor deviations are considered to have had no impact on the outcome and validity of this study.

Test was designed with 5 treatments plus water and solvent control, 4 replicates per treatment and for the water control, 8 for the solvent control; The tested concentration were 200, 300, 450, 670 and 1000 mg test item/kg soil dry weight (dw); because of its low solubility, the substance was incorporated into the soil by coating quartz grains. Soil composition was 5% Sphagnum peat (no visible plant remains; finely ground and air-dried), 20% Kaolin-Clay (kaolinite content > 30%), ~74-75% Quartz sand (fine sand content with particles between 50 and 200 microns higher than 50%), and 0.3-1% Calcium carbonate (CaCO3).

Test conditions were 17.3 – 21.1 °C, 16: 8 h light cycle with a constant light intensity at the substrate surface of 438 - 650 lx, Soil pH-value (day 0 / day 56): 5.8 /6.5 -6.7, soil moisture (day 0 / day 56): 49.7 – 54.9% / 60.7 – 65.8%.

The mortality was tested after 28days, and the reproduction after 56days. The exposure period was from November 22, 2016 to January 17, 2017. All validity criteria were fulfilled: Mortality of the adults in the solvent control = 0%, Mean number of juveniles in the solvent control = 393.8, CV for the number of juveniles in the solvent control = 11.7.

Results were as follows:

2.5% mortality was observed in the water control. No mortality was observed in the solvent control and in all concentrations of the test item tested. Therefore, no statistical analysis was performed and the NOECMortality was considered to be ≥ 1000 mg test item/kg soil (dw) (the highest concentration tested). The LOECMortality could not be determined and was regarded as > 1000 mg test item/kg soil (dw).

Statistical analysis showed no significant difference (Dunnett’s test, 2-sided; p ≤ 0.05) concerning biomass development of individual adults over 28 days between the solvent control and all concentrations of the test item. Therefore, the NOECBiomass was considered to be ≥ 1000 mg test item/kg soil (dw) (the highest concentration tested). The LOECBiomass could not be determined and was regarded as > 1000 mg test item/kg soil (dw).

Statistical analysis (Dunnett’s t-test; 1-sided, p ≤ 0.05) showed no significant difference concerning the number of juveniles between the solvent control and all concentrations tested. Therefore, the NOECReproduction was considered to be ≥ 1000 mg test item/kg soil (dw). The LOECReproduction could not be determined and was regarded as > 1000 mg test item/kg soil (dw).

Since inhibition of reproduction was < 50%, the EC50 could not be calculated and was considered to be > 1000 mg test item/kg soil (dw).

Description of key information

Earthworm Eisenia fetida was not affected by all the tested concentrations of the item.

In consequence NOECs and LOECs for mortality or reproduction (56 d) were concluded at the maximum tested concentration, >= 1000 and >1000 mg/kg soil dw, respectively.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
1 000 mg/kg soil dw
Long-term EC10, LC10 or NOEC for soil macroorganisms:
1 000 mg/kg soil dw

Additional information

A toxicity study on one earthworm species was performed according to OECD TG 222 and in compliance with GLP.

The mortality, growth and reproduction of Eisenia fetida (Annelid) in artificial soil (5% Sphagnum peat, 20% Kaolin-Clay, 74-75% Quartz sand, 0.3-1% Calcium carbonate).were followed for 28 days while exposed to 200, 300, 450, 670 and 1000 mg test item/kg soil dry weight.

No mortality was observed in the solvent control and in all concentrations of the test item tested. Therefore, no statistical analysis was performed and the NOEC-Mortality and LOEC-Mortality were concluded as ≥ 1000 mg test item/kg soil (dw) and > 1000 mg test item/kg soil (dw), respectively.

Statistical analysis showed no significant difference (Dunnett’s test, 2-sided; p ≤ 0.05) concerning biomass development of individual adults. Therefore, NOEC-Biomass was considered to be ≥ 1000 mg test item/kg soil (dw) (the highest concentration tested). The LOEC-Biomass could not be determined and was regarded as > 1000 mg test item/kg soil (dw).

Statistical analysis (Dunnett’s t-test; 1-sided, p ≤ 0.05) showed no significant difference concerning the number of juveniles between the solvent control and all concentrations tested. Therefore, the NOEC-Reproduction was considered to be ≥ 1000 mg test item/kg soil (dw) and the LOEC-Reproduction could not be determined and was regarded as > 1000 mg test item/kg soil (dw).

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