6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

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:

other: GLP Guideline Study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

- The test item was not sufficiently soluble in water; therefore, a volatile organic solvent was used. The stock solution was prepared in acetone.
- The test item was dissolved in a volume of acetone sufficient to prepare a stock solution. This stock solution was used to produce the various dosage solutions of the test item.
- An appropriate volume of the stock and the dosage solutions respectively were used to soak a portion of the quartz sand of the artificial soil.
- After evaporation of the solvent by placing the application vessels under a fume hood, the quartz sand was coated by the test item.

Composition of this artificial soil (the percentages refer to the dry weight)::
- 5% Sphagnum peat (no visible plant remains; finely ground and air-dried)
- 20% Kaolin-Clay (kaolinite content > 30%)
- ~74% Quartz sand (fine sand content with particles between 50 and 200 microns higher than 50%)
- 0.3-1% Calcium carbonate (CaCO3).
- With respect to the properties of the test item (log POW ≥2) 5% instead of 10% peat was used considering the influence on bioavailability (EPPO 2003).
- air dried peat was shredded in a chaff-cutter
- afterwards, all parts of the artificial soil were mixed homogeneously
- pH value was adjusted to 6 ± 0.5 using calcium carbonate.
- moisture content was adjusted to 40-60% of WHCmax using deionised water.
- The water content of the test substrate was checked weekly by reweighing the test vessels and losses of water were compensated.
- Calcium carbonate was used to get a pH-value of 6 ± 0.5. Determination of the maximum water capacity (WHCmax) of the artificial soil.
- One day before starting the test, the artificial soil (per replicate 500 g dw) was pre-moistened with deionised water (20-40% of WHCmax).

Incubation conditions:
- Temperature: 20 +/- 2 ºC
- Light/dark cycle: 16 h light : 8 h dark with a constant light intensity at the substrate surface of 400 – 800 lx

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Eisenia fetida
- Source: kept at ECT Oekotoxikologie GmbH since February 1994, originally delivered by Co. Landenberger (D-72355 Schömberg).
- Only adult earthworms (with clitellum) with a fresh weight (FW) between 250 and 600 mg were used. The earthworms were 2 – 3 months old, the age of individuals did not differ by more than four weeks.
- Selecting of adult earthworms out of the synchronisation culture in order to acclimatise them for at least 24 h in untreated artificial soil.

ACCLIMATION
- Acclimation period: 24 hours
- Acclimation conditions: acclimatised in artificial soil under test conditions

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

Mean 19.7 °C, 18.2 – 21.0 °C (recommended 20 ± 2 °C)

pH:

- soil pH-value (day 0): 6.0 – 6.2 (recommended 6 ± 0.5)
- soil pH-value (day 56): 7.1

Moisture:

- soil moisture (day 0): 49.0 – 51.4% of the WHCmax, (recommended 40 – 60% of the WHCmax),
- soil moisture (day 56): 62.1 – 68.4% of the WHCmax

Details on test conditions:

- Test vessel: Bellaplast containers (Company Kastelplast, D-55120 Mainz) consiting of inert (non-toxic) plastic (Polystyrol), base area of 11 x 15.5 cm (= 170.5 cm²) and a height of 6 cm, covered with a transparent and perforated lid
- Light/dark cycle: 16 h light : 8 h dark
- Light intensity: 434 - 631 Lx (recommended 400 - 800 Lx)
- Exposure: mixed with artificial soil;
- Control: solvent treated, water treated;
- Test system: 10 Eisenia fetida per test vessel;
- Test design: 5 treatments plus control and solvent control, 4 replicates per treatment, 8 for the solvent control and 4 for the water control;
- Test parameters: Mortality and biomass after 28 days; reproduction after 56 days;
- Endpoints: NOEC/LOECMortality, NOEC/LOECBiomass, NOEC/LOECReproduction, EC50, Reproduction;
- Statistics: Fisher Exact test (p ≤ 0.05; 1-sided); (Williams t-test (biomass: 2-sided, p ≤ 0.05; reproduction: 1-sided, p ≤ 0.05).
- The test item was applied once at the beginning of the test
- The earthworms were introduced into the test vessels, after the treated artificial soil was placed into the test vessels. The test animals were exposed to the test item by dermal and alimentary uptake.

Feeding:
- the adult earthworms were fed with finely ground cow manure (free of growth promoters, nematicides or similar veterinary pharmaceuticals, provided by Jesus Bruderschaft e.V. Gnadenthal, D-65597 Hünfelden).
- an amount of 5 g moistened with 10 mL deionised water was spread on the soil surface of each test container. If food remained uneaten the ration was reduced on demand. After removing the adults on day 28 a further 5 g of food was mixed with the artificial soil of each test vessel.
- first feeding one day after application of the test item and introduction of the adult earthworms, the adult earthworms were fed weekly during the first 4 weeks of the test
- After removing the adults on day 28 a further 5 g of food moistened with 10 mL deionised water was mixed with the artificial soil of each test vessel.

Preparation of the test item stock solution and dosage solution:
- stirring for 3 minutes
- 50 ml of each mixture was spiked on 220 g quartz sand.
- solvent evaporation under a fume hood
- coated quartz sand was incorporated into 1980 g artificial soil (total amount of 2200 g) to prepare the different test item soil concentrations.
- Water was added to adjust the final soil moisture of 40 to 60% of WHCmax.
- The prepared soil was mixed for 5 min.
- The test substrate soil was mixed thoroughly until the test item was homogeneously distributed
- Filling test containers with artificial soil (corresponding to 500 g dw per container).
- Determination of the pH-value and the moisture of the artificial soil once for the concentration and for the controls (samples were taken from one test vessel).

Nominal and measured concentrations:

100, 178, 316, 562 and 1000 mg test item/kg soil (dw)

Reference substance (positive control):

yes

Remarks:

boric acid (150, 300 and 600 mg/kg)

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

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:

other: Biomass

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:

other: Biomass

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Since inhibition of reproduction was < 50% in all treatment levels no EC50 value was calculated and was regarded as > 1000 mg a.s./kg soil (dw).
- Almost the whole food was consumed by the worms in most of the test vessels after 7, 14, 21 and 28 days of the application of the test item. Half of the food was consumed by the worms in one test vessel of 316 mg a.s./kg soil (dw) after 14 and 21 days. After 28 days the worms consumed less than half of the food in that test vessel. Half of the food was consumed by the worms in another test vessel of 316 mg a.s./kg soil (dw) after 28 days
- No other effects on behaviour or morphology of the adult earthworms were observed in both test runs.

Results with reference substance (positive control):

Ten Eisenia fetida (clitellate adults) per replicate were exposed to boric acid for 28 days at nominal concentrations of 150, 300, and 600 mg boric acid/kg artificial soil (dw) at 18.2 – 21.4 °C and 416 – 535 lux. After 28 days of exposure the adult earthworms were removed.
2.5% mortality was observed in the control, no mortality at the concentrations of 150, 300 and 600 mg Boric acid/kg soil (dw
Concerning reproduction the statistical analysis (William’s t-test) showed a significant difference between the control and all concentrations of the reference item tested. The EC50 value was calculated by Probit analysis using Linear Max. Likelihood Regression as 259.1 mg Boric Acid/kg artificial soil (dw) (95% confidence limits = 234.0 – 286.2 mg Boric Acid/kg artificial soil (dw)).
The estimated EC50 value in this study (259.1 mg boric acid /kg soil (dw)) is slightly lower than recommended by the guideline. However, since the calculated EC50 is lower by factor of 1.5 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:

Statistics: Fisher Exact test (p ≤ 0.05; 1-sided); (Williams t-test (biomass: 2-sided, p ≤ 0.05; reproduction: 1-sided, p ≤ 0.05).

Adult mortality (28d)

0% mortality was observed in the control, 2.5% in the solvent control and 0 – 2.5% mortality in all concentrations of the test item tested. Fisher’s Exact Test (1-sided, p ≤ 0.05) showed no significant differences concerning mortality between the solvent control and all test item concentrations tested. Therefore, NOEC_Mortality was considered to be ≥ 1000 mg a.s./kg soil (dw) (the highest concentration of the test item tested). The LOEC_Mortality could not be determined and was regarded as > 1000 mg a.s./kg soil (dw).

Adult biomass (28 d)

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

Reproduction (56 d)

Statistical analysis (Williams test; 1-sided, p ≤ 0.05) showed no significant difference concerning the number of juveniles between the solvent control and all concentrations of the test item tested. Therefore, the NOEC_Reproduction was determined as ≥ 1000 mg a.s./kg soil (dw) (the highest concentration of the test item tested). The LOEC_Reproduction could not be determined and was regarded as > 1000 mg a.s./kg soil (dw).

Validity criteria fulfilled:

yes

Remarks:

(Mortality of the adults in the solvent control ≤ 10%; Mean number of juveniles in the solvent control ≥ 30; CV for the number of juveniles in the solvent control ≤ 30%)

Conclusions:

With the test item Vulkanox BKF a reproduction toxicity to the earthworm Eisenia fetida was made accordeing to OECD Guideline No. 222. Since inhibition of reproduction was < 50% in all treatment levels no EC50 value was calculated and was regarded as > 1000 mg a.s./kg soil (dw). The NOECMortality was considered to be ≥ 1000 mg test item/kg soil (dw). Since the LOECMortality could not be determined it was regarded as > 1000 mg test item/kg soil (dw). NOECBiomass was determined as ≥ 1000 mg test item/kg soil (dw) and the LOECBiomass could not be determined and was considered to be > 1000 mg a.s./kg soil (dw). The NOECReproduction was determined as ≥ 1000 mg test item/kg soil (dw) and the LOECReproduction could not be determined and was regarded as > 1000 mg a.s./kg soil (dw)

Executive summary:

The study was to determine a NOEC/LOEC and an EC50 for the effects of Vulkanox BKF on the reproduction (56 days after application), mortality and the biomass development (28 days after application) of the earthworm Eisenia fetida (Lumbricidae) by dermal and alimentary uptake using a standardised artificial soil. The test item was mixed with the substrate. Only adult earthworms (with clitellum) with a fresh weight (FW) between 250 and 600 mg were used and tested in an artificial soil. The earthworms were introduced into the test vessels, after the treated artificial soil was placed into the test vessels. The test animals were exposed to the test item by dermal and alimentary uptake.

0% mortality was observed in the control, 2.5% in the solvent control and 0 – 2.5% mortality in all concentrations of the test item tested. Fisher’s Exact Test (1-sided, p ≤ 0.05) showed no significant differences concerning mortality between the solvent control and all test item concentrations tested. Therefore, NOECMortality was considered to be ≥ 1000 mg a.s./kg soil (dw) (the highest concentration of the test item tested). The LOECMortality could not be determined and was regarded as > 1000 mg a.s./kg soil (dw).

Statistical analysis showed no significant difference (Williams 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 tested. Therefore, the NOECBiomass was determined as ≥ 1000 mg a.s./kg soil (dw) (the highest concentration of the test item tested). The LOECBiomass could not be determined and was considered to be > 1000 mg a.s./kg soil (dw).

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

Since inhibition of reproduction was < 50% in all treatment levels no EC50 value was calculated and was regarded as > 1000 mg a.s./kg soil (dw).

Description of key information

With the test item Vulkanox BKF a reproduction toxicity to the earthworm Eisenia fetida was made according to OECD Guideline No. 222. Since inhibition of reproduction was < 50% in all treatment levels no EC50 value was calculated and was regarded as > 1000 mg a.s./kg soil (dw). The NOECMortality was considered to be ≥ 1000 mg test item/kg soil (dw). Since the LOECMortality could not be determined it was regarded as > 1000 mg test item/kg soil (dw). NOECBiomass was determined as ≥ 1000 mg test item/kg soil (dw) and the LOECBiomass could not be determined and was considered to be > 1000 mg a.s./kg soil (dw). The NOECReproduction was determined as ≥ 1000 mg test item/kg soil (dw) and the LOECReproduction could not be determined and was regarded as > 1000 mg a.s./kg soil (dw)

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

should read: ≥(EC50, NOEC)