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

Toxicity to terrestrial plants

Link to relevant study record(s)

Reference

Endpoint:

toxicity to terrestrial plants: 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 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Only the two stock solutions were tested for their nominal test item concentration:
A quantity of 40,905.6 mg of test item was made up with acetone to a total volume of 100 mL in a volumetric flask. The resulting solution (nominal test item concentration 409 mg/mL) was stirred for 5 minutes to completely dissolve the test item. The solution was used as stock solution.
For the test with A. cepa (second run), a quantity of 9092 mg of the test item was made up with acetone to a total volume of 25 mL in a volumetric flask. The nominal concentration of this stock solution was 363.6 mg/mL. The solution was stirred for 5 minutes to dissolve the test item.

Vehicle:

yes

Remarks:

sand

Details on preparation and application of test substrate:

Test soil:
Standard soil LUFA Type 2.3, a sandy loam, was purchased from the Landwirtschaftliche Untersuchungs- und Forschungsanstalt (LUFA), D-67346 Speyer, Germany. LUFA soil Type 2.3 was collected from an uncultivated field at Offenbach, Rhineland-Palatinate, Germany, and sieved (<= 2 mm). The field had neither received any organic fertilisers nor plant protection substances during the last four years before sampling. The soil was stored at ECT GmbH at ambient temperature until usage.
The solution of a nominal test item concentration of 409 mg/mL was used as stock solution. Aliquots of the stock solution were diluted with acetone to achieve further four test solutions in a geometric series, resulting in nominal test item concentrations ranging from 25.57 mg/mL to 204.53 mg/mL, respectively. For one treatment, the stock solution was used without further dilution. The test solutions were applied at a volume of 40 mL to 162 g of quartz sand. After complete passive evaporation of the solvent at ambient conditions the spiked sand was mixed thoroughly into 16.2 kg soil dry weight equivalent by stirring for 5 minutes.
For the second run with A. cepa a quantity a stock solution of a nominal test item concentration of 363.6 mg/mL was used. The solution was stirred for 5 minutes to dissolve the test item. Aliquots of the stock solution were diluted with acetone to achieve further four test solutions in a geometric series, resulting in test item concentrations ranging from 22.7 mg/mL to 181.8 mg/mL. For one treatment the stock solution was used without further dilution. The test solutions were applied at a volume of 5 mL to 18 g of quartz sand. After complete passive evaporation of the solvent at ambient conditions the spiked sand was mixed thoroughly into 1.8 kg soil dry weight equivalent by stirring for 5 minutes.

Species:

Allium cepa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

Family: Amaryllidaceae
Source of supply: Enza Zaden Deutschland GmbH & Co. KG, Dannstadt-Schauernheim, Germany.

Species:

Avena sativa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

Family: Poaceae
Source of supply: Nordsaat Saatzucht GmbH, Langenstein, Germany

Species:

Brassica napus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Family: Brassicaceae
Source of supply: Lufa Speyer, Speyer, Germany.

Species:

Cucumis sativus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Family: Cucurbitaceae
Source of supply: Enza Zaden Deutschland GmbH & Co. KG, Dannstadt-Schauernheim, Germany.

Species:

Pisum sativum

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Family: Fabaceae
Source of supply: Enza Zaden Deutschland GmbH & Co. KG, Dannstadt-Schauernheim, Germany.

Species:

Lycopersicon esculentum

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Family: Solanaceae
Source of supply: Enza Zaden Deutschland GmbH & Co. KG, Dannstadt-Schauernheim, Germany.

Test type:

seedling emergence and seedling growth test

Study type:

laboratory study

Substrate type:

natural soil

Limit test:

no

Total exposure duration:

17 d

Test temperature:

Air temperature: 21.5-35.3°C (mean = 26.9°C)
for repeated test: Air temperature: 22.9-25.0°C (mean = 23.8°C)

pH:

5.7 ± 0.6

Moisture:

Rel. humidity: [%]: 23.0-70.4 (mean = 37.9 %)
for repeated test: Rel. humidity: [%]: 38.3-73.8 (mean = 56.2 %)

Details on test conditions:

Methodology:
Seeds of two monocotyledonous species, Avena sativa (oat) and Allium cepa (onion), as well as four dicotyledonous species, Brassica napus (rape), Cucumis sativus (cucumber), Pisum sativum (pea) and Solanum lycopersicum syn. Lycopersicon esculentum (tomato) were planted in a natural sandy loam soil (standard soil LUFA Sp2.3) immediately after test item application and left to grow under controlled conditions for 14 to 17 days following 50 % emergence of the control plants. Five test item concentrations were tested: 62.5, 125, 250, 500 and 1000 mg/kg soil dry weight.
The test item was dissolved in acetone and spiked onto quartz sand which was mixed thoroughly into the soil after complete evaporation of the solvent. Soil for growing control plants was amended either with non-spiked sand (control) or with acetone spiked sand (solvent control).
Soils were supplied with water or nutrient solution by bottom watering. The test was performed in growth chambers equipped with artificial lighting (202 to 345 µE m-2 s-1, for 16 hours per day) and air conditioning (air temperature ranging from 21.5 to 35.3°C (22.9 to 25.0°C for Allium cepa)). The pots were placed randomly at the beginning of the test and were re-arranged at minimum weekly intervals. On day 7 after 50% emergence of control, seedlings were evaluated visually. At test end, seedlings were counted, evaluated visually, and harvest to determine shoot length and shoot fresh weight.

Nominal and measured concentrations:

Nominal concentration: 62.5, 125, 250, 500 and 1000 mg/kg soil dry weight

Reference substance (positive control):

no

Species:

Allium cepa

Duration:

17 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: shoot length

Species:

Allium cepa

Duration:

17 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: shoot fresh weight

Species:

Allium cepa

Duration:

17 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Species:

Avena sativa

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:

other: shoot fresh weight

Species:

Avena sativa

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:

other: shoot length

Species:

Avena sativa

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:

seedling emergence

Species:

Brassica napus

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:

other: shoot fresh weight

Species:

Brassica napus

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:

other: shoot length

Species:

Brassica napus

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:

seedling emergence

Species:

Cucumis sativus

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:

other: shoot length

Species:

Cucumis sativus

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:

other: shoot fresh weight

Species:

Cucumis sativus

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:

seedling emergence

Species:

Pisum sativum

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:

other: shoot length

Species:

Pisum sativum

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:

other: shoot fresh weight

Species:

Pisum sativum

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:

seedling emergence

Species:

Lycopersicon esculentum

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:

other: Shoot length

Species:

Lycopersicon esculentum

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:

other: shoot fresh weight

Species:

Lycopersicon esculentum

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:

seedling emergence

Details on results:

No effects on shoot fresh weight, shoot length and seedling emergence were observed in any of the test species at any of the tested test item concentrations up to and including 1000 mg/kg soil dry weight.
Seedling emergence and survival:
Day 0 of the test was defined as the day by which at least 50% of the seedlings in the untreated control pots of a particular species had emerged. Visual detrimental effects were assessed on day 7 and at the end of the test, i.e. earliest on day 14, latest on day 17. Shoot length and fresh weight were evaluated only at the end of the test.

Reported statistics and error estimates:

Statistical analysis:
Emergence and survival data were checked by the Fisher's exact binominal test with Bonferroni correction to determine the NOEC and LOEC. Significance level was alpha = 0.05, one sided greater .
Probit Analysis was used to determine the effective concentrations EC10, EC20 and EC50.
Normal distribution and homogeneity of variances were checked by the Kolmogorov-Smirnov Test and Levene’s test, respectively.
NOEC and LOEC were determined by applying either the Dunnett’s Multiple t-Test or the Welch t-Test (in case of inhomogeneous variances). Significance level was alpha = 0.05, one-sided smaller.
Statistical evaluations were performed using the ToxRat Pro software Version 2.10 (ToxRat Solutions GmbH, 2010).

Nominal test item concentrations [mg/kg soil], mean shoot length [mm] and mean shoot fresh weight [g] at the end of the test.

 

Treatment code	C	SC	T1	T2	T3	T4	T5
Test item [mg/kg soil dw]	0.0	0.0	62.5	125	250	500	1000
	Shoot length [mm]
A. cepa	176	196	204	203	226	208	208
A. sativa	339	331	346	323	364	325	335
B. napus	172	173	177	174	171	169	163
C. sativus	135	135	124	122	123	118	120
P. sativum	146	149	168	151	164	161	158
S. lycopersicum	164	163	158	159	155	180	161
	Shoot fresh weight [g]
A. cepa	0.320	0.445	0.455	0.483	0.559	0.461	0.457
A. sativa	0.942	0.836	0.978	0.927	1.005	0.881	0.909
B. napus	3.218	3.060	2.981	3.078	2.760	2.807	2.563
C. sativus	2.949	2.733	2.805	2.519	2.496	2.516	2.587
P. sativum	1.816	1.684	2.144	1.731	1.931	2.008	1.945
S. lycopersicum	1.907	2.080	1.749	1.751	1.723	2.072	1.602

C = Control; SC = solvent control; T1 to T5 = test item treated

Seedling emergence

Plant species	Total no of seeds introduced	Seedling emergence until day 7 (later ones were not considered)
A. cepa	24	19
A. sativa	24	20
B. napus	21	19
C. sativus	21	20
P. sativum	21	21
S. lycopersicum	21	17

Validity criteria fulfilled:

yes

Remarks:

The validity criteria were fulfilled as seedling emergence in the control was ≥ 70% and less than 10% of the control plants died. No phytotoxic effects were observed in the controls.

Conclusions:

The effect of the test item 6,6’-di-tert-butyl-2,2’-methylenedi-p-cresol (Vulkanox BKF) on the emergence and growth of six species of higher terrestrial plants was tested under laboratory conditions.
No statistically significant adverse effects of the test item were observed with any of the end points in any of the six test species at any of the tested concentrations up to and including 1000 mg/kg soil dry weight, which was the highest tested concentration.
Therefore the lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC) were >1000 mg/kg soil dry weight and ≥1000 mg/kg soil dry weight, respectively.

Executive summary:

Seeds of two monocotyledonous species, Avena sativa (oat) and Allium cepa (onion), as well as four dicotyledonous species, Brassica napus (rape), Cucumis sativus (cucumber), Pisum sativum (pea) and Solanum lycopersicum (tomato) were planted in a natural sandy loam soil immediately after test item application and left to grow under controlled conditions for 14 to 17 days following 50 % emergence of the control plants. Five test item concentrations were tested: 62.5, 125, 250, 500 and 1000 mg/kg soil dry weight. The test item was dissolved in acetone and spiked onto quartz sand which was mixed thoroughly into the soil after complete evaporation of the solvent. Soil for growing control plants was amended either with non-spiked sand (control) or with acetone spiked sand (solvent control). Soils were supplied with water or nutrient solution by bottom watering. The test was performed in growth chambers equipped with artificial lighting (202 to 345 µE m-2 s-1, for 16 hours per day) and air conditioning (air temperature ranging from 21.5 to 35.3°C (22.9 to 25.0°C for Allium cepa)). The pots were placed randomly at the beginning of the test and were re-arranged at minimum weekly intervals. On day 7 after 50% emergence of control, seedlings were evaluated visually. At test end, seedlings were counted, evaluated visually, and harvest to determine shoot length and shoot fresh weight.

The effect of the test item 6,6’-di-tert-butyl-2,2’-methylenedi-p-cresol (Vulkanox BKF) on the emergence and growth of six species of higher terrestrial plants was tested under laboratory conditions. No statistically significant adverse effects of the test item were observed with any of the end points in any of the six test species at any of the tested concentrations up to and including 1000 mg/kg soil dry weight, which was the highest tested concentration. Therefore the lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC) were >1000 mg/kg soil dry weight and ≥1000 mg/kg soil dry weight, respectively.

Description of key information

For the long-term study of terrestrial plants according to the OECD-Guideline 208 for the test item Vulkanox BKF a NOEC of >=1000 mg/kg soil dw was found for the shoot fresh weight, the shoot length and seedling emergence as there was no effect to be seen for all the treated specimen. For all species and treatments there were no significant differences between the controls and the treated samples.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for terrestrial plants:

1 000 mg/kg soil dw