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Diss Factsheets

Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

Administrative data

toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Version / remarks:
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Pentaerythritol tetrakis(3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate)
EC Number:
EC Name:
Pentaerythritol tetrakis(3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate)
Cas Number:
Molecular formula:
3-{[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoyl]oxy}-2,2-bis({[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoyl]oxy}methyl)propyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate
Test material form:

Sampling and analysis

Analytical monitoring:

Test substrate

Details on preparation and application of test substrate:
Due to the low solubility, the test substance was mixed with quartz sand for application. (required amount of test substance + 25g quartz sand)
The required amounts of artificial soil were weighed into a mixing tray. The test substance / quartz sand mixtures were added and thoroughly mixed with the soil. Thereafter, the water content of the soil was adjusted to about 50% WHCmax using deionized water and was mixed once again. The test mixtures were distributed to the test units.

Test organisms

Test organisms (species):
Eisenia fetida
Animal group:
Details on test organisms:
- Common name: earthworm
- Source: BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
- Age at test initiation (mean and range, SD): adult worms with clitellum, ca. 11 months,
- Weight at test initiation (mean and range, SD): 300 - 600mg fresh weight

- in horse manure

- Acclimation period: through overnight under permanent light and without feeding.
- Acclimation conditions (same as test or not): under test conditions, plastic box, filled with ca 750g (dw) test substrate

Study design

Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
Total exposure duration:
56 d

Test conditions

Test temperature:
6.0 - 6.7 (control), 5.9 - 6.7 (test concentrations)
control 48.7-58.7% WHCmax, test concentrations 48.3-60.8% WHCmax.
A slightly higher value of water content above the required criteria of 50 ±10% WHCmax was observed, probably due to the excessive spraying of deionised water during the moisturisation of food. However, an effect to the test results can be excluded.
Details on test conditions:
- Test container (material, size): Bellaplast plastic containers (inert and non-toxic plastic), bottom area: 15.5*11*6 cm (L * W *H), covered with tranparent perforated lids
- Amount of soil or substrate: 677g wwt (moistened), 550g dwt, layer of about 4 cm and a corresponding surface area of about 170 cm2
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- Worm weight at start of exposure: 300-600mg
- Introduction of worms: Acclimatized worms were washed with tap water in a fine meshed sieve and gently dried on a paper towel. Ten worms were randomly selected and placed on the surface of the test substrate in each test container. The body weight of the worms was determined
per animal before introduction of the worms. Only worms without morphological defects were used in the test.

- Composition (if artificial substrate):
-- quartz sand 74%
-- kaolin clay 21%
-- sphagnum peat 5% With respect to the adsorptive properties of the test item (log Kow >2), 5% of peat instead of 10% were used, considering the influence on bioavailability (OECD 222 and EPPO 2003).
-- CaCO3 0.3%
- Organic carbon (%): 5%
- Water holding capacity (dry weigth): 46.2g/100g (mean value of 2 single detrminations)

- food: finely ground cow manure (free of growth promoters, nematicides or similar veterinary pharmaceuticals, source: Jesus Bruderschaft e.V. Gnadenthal, Huenfelden, Germany)
Initial food supply was given one day after the introduction of worms into test substrate with the test substance. Thereafter, the adult worms were fed weekly during the initial 4 weeks of the test. An amount of 5 g finely ground cow manure moistened with about 5-6 mL deionized water was sprayed on the test substrate surface of each test container. If food remained unconsumed, the ration was reduced on demand. After removing the adults on day 28, further 5 g of food moistened with about 5-6 mL deionized water was mixed with the test substrate of each test vessel.

- Illumination: Light/dark cycle of 16:8 hours, over the exposure period
- Light intensity: start of exposure: 539 Lux, (mean of 5 single measurements: 709, 521, 467, 506, 494 [Lux]), start of adaption 722Lux
-Temperature: In a separate vessel, filled with about 750 g test substrate (dry weight), around two times per week during the exposure time with an electronic thermometer. The water content of this vessel was adjusted initially (on day 0) to the water content of test vessels.

EFFECT PARAMETERS MEASURED AND OBSERVATIONS (with observation intervals if applicable) :
- consumption of feed
- survival of adults (28d) (Worms were classified as dead when they did not respond to a gentle mechanical stimulus to the front end)
- biomass (start of exposure + 28d)
- number of juveniles (56d)
- abnormalties and changes in morphology and behaviour

- Spacing factor for test concentrations: 2
- Test concentrations: 62.5, 125, 250, 500, 1000 mg/kg dw
Nominal and measured concentrations:
0 (control), 62.5, 125, 250, 500 and 1000 mg/kg Dw as nominal concentrations
Reference substance (positive control):
Carbendazim CAS 10605-21-7

Results and discussion

Effect concentrationsopen allclose all
Key result
56 d
Dose descriptor:
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
28 d
Dose descriptor:
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
28 d
Dose descriptor:
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
other: biomass change
Details on results:
Validity criteria:
Mean mortality in the control should be ≤ 10 %. The reproduction in the controls should be at least 30 juveniles per container and the coefficient of variance of reproduction in the control should be ≤ 30 %.
All validity criteria were met.
After 28 days, no adult worm in the control assay was dead. Between 300-400 juveniles (mean value: 361) were counted in the test units of the control assay. The mortality of the adult worms in the control was <10 % and the coefficient of variation concerning to the offspring juvenile worms in the control assay was 6.4 %.

No changes in behaviour and morphology or other abnormalities were observed in this test. No effects on food consumption were observed.

The results in this study are consistent with all validity criteria and the test is valid according to the test guideline of this study. No deviations from the test guidelines or other incidents occurred during the reported test, which may have influenced the results.
Results with reference substance (positive control):
non-GLP project 68E0902/00G026 (start of exposure 16 January 2018)
ECx values [mg/kg soil (dw)], reproduction by counting of juvenile worms, conf. limits 95%
EC10: 0.80 LCL: 0.68 UCL: 0.95
EC50: 1.19 LCL: 1.03 UCL: 1.37
These observed effects show a high sensitivity of the test system and the derived EC50 value is within the expected range from the test guideline (1-5 mg/kg carbendazim/kg soil (dw)) and hence acceptable sensitivity of the test system is assured.

Reported statistics and error estimates:
SAS System computer software
changes in body weight: Williams trend test (statistical unit: test container) one sided and not significant, therefore Dunnett's test was carried out
number of juveniles (offsprings): the Jonkheere-Terpstra trend test (statistical unit: test container). one-sided and not significant, therefore Wilcoxon-test was conducted.
The trend tests showed no significant result in both parameters: change in bodyweights as well as in the number of offsprings.
No ECx calculations were performed, because there was no dose response relationship.

Applicant's summary and conclusion

Validity criteria fulfilled: