2,6-di-tert-butyl-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:

From July 27th, 2017 to July 31st, 2017

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:

July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): The test item coated fraction of the quartz sand was then incorporated into the remainder of the artificial soil by thoroughly mixing it in 5 minutes.
- Controls: Solvent (acetone) treated, water treated;
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Organic solvent
- Evaporation of vehicle before use: The solvent was allowed to evaporate at room temperature for 2 hours under a fume hood.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Source: Co. Landenberger (72355 Schömberg, Germany) at ECT Oekotoxikologie GmbH since February 1994
- Age at test initiation (mean and range, SD): at least two months, but not more than one year old. Age did not differ among individuals by more than 4 weeks.
- Weight at test initiation (mean and range, SD): between 300 and 600 mg

ACCLIMATION
- Acclimation period: 24 hours
- Acclimation conditions (same as test or not): same as test
- Health during acclimation (any mortality observed): No mortality observed

Study type:

laboratory study

Substrate type:

natural soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

18.2–21.4 ºC

pH:

Soil pH-value (day 0): 6.1–6.2
Soil pH-value (day 56): 6.8–6.9

Moisture:

Soil moisture (day 0): 48.3–51.8% of the WHCmax
Soil moisture (day 56): 58.1–67.3% of the WHCmax

Details on test conditions:

TEST SYSTEM
- Test container (material, size):Bellaplast containers, inert (non-toxic) plastic (Polystyrol). Test vessels had a base area of 11 x 15.5 cm (= 170.5 cm²) and a height of 6 cm. The test vessels were covered with a transparent and perforated lid.
- Amount of soil or substrate: 500 g dw
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group:4 replicates per treatment
- No. of replicates per control: 4
- No. of replicates per vehicle control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate):
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 μm higher than 50%)
0.3–1% Calcium carbonate (CaCO3).
- Organic carbon (%): 2.5
- Maximum water holding capacity (in % dry weigth): 54.4
-Pretreatment of soil: The air-dried peat was shredded in a chaff-cutter. Afterwards, all parts of the artificial soil were mixed homogeneously. The pH value was adjusted to 6±0.5 using calcium carbonate. The 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

OTHER TEST CONDITIONS
- Photoperiod: 16 h light:8 h dark with a constant light intensity at the substrate surface of 400–800 lx.
- Light intensity: 409–556 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality, Biomass, morphology (0, 28d), Reproduction (0, 56d)

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Test concentrations: 6.25, 12.5, 25.0, 50.0, 100 mg test item/kg soil (dw)

Nominal and measured concentrations:

- Test concentrations: 6.25, 12.5, 25.0, 50.0, 100 mg test item/kg soil (dw)

Reference substance (positive control):

yes

Remarks:

Boric acid

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

25 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

50 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

87.9 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: 5.0%
- Total mass of adults at beginning of test:
Control: 368.8 ± 22.1 mg
Solvent Control: 366.3 ± 19.3 4 mg
6.25: 368.4 ± 19.1 mg
12.5: 369.0 ± 17.2 mg
25.0: 369.8 ± 16.3 mg
50.0: 365.5 ± 16.1 mg
100: 366.2 ± 13.3 mg
- Changes in body weight of live adults (% of initial weight) at end of exposure period:
control: 32.6
Solvent Control: 3 31.5
6.25: 28.6
12.5: 35.0
25.0: 33.0
50.0: 29.9
100: 39.6
- No. of offspring produced:
Control: 316.5 ± 20.2
Solvent Control: 344.1 ± 38.1
6.25: 337.5 ± 64.8
12.5: 312.8 ± 56.2
25.0: 337.0 ± 31.1
100: 132.0 ± 24.1
- Morphological abnormalities: None observed
- Behavioral changes: None observed

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: Concerning number of juveniles statistical analysis (William’s t-test, p = 0.05, one-sided) showed a significant difference between the control and the concentrations of 300 and 600 mg boric acid/kg soil (dw).

Reported statistics and error estimates:

Dunnett’s test (2-sided, p≤0.05), Williams t-test (1-sided, p≤0.05), non-linear regression using 3-parameter normal cumulative distribution function (CDF).

Table 1. Effects on Mortality, Biomass and Reproduction of Eisenia fetida.

Concentration [mg test item/kg soil (dw)]	Adult mortality [%]	Biomass [Mean change in % of the initial weight per replicate]	Number of Juveniles [% of Solvent Control]
Control	0.0	32.6	92.0
Solvent Control	0.0	31.5	-
6.25	0.0	28.6	98.1
12.5	0.0	35.0	90.9
25.0	0.0	33.0	97.9
50.0	0.0	29.9	85.8*
100	5.0	39.6	38.4*
EC50 [and 95% Cl]	-	-	87.9 [76.0 - 101.7]
NOEC	≥ 100	≥ 100	25.0
LOEC	> 100	> 100	50.0

*Statistically significant different to Solvent Control (Williams t-test, p = 0.05, one-sided)

-not applicable

Table 2. Number of dead earthworms after 28 days (n = 80 for solvent control; n = 40 for control and concentrations). Data are given as absolute values and in percent of the total number (mortality).

Concentration [mg test item/kg soil (dw)]	Number of dead adult worms	Mortality [%]
	Day 28	Day 28
Control	0	0.0
Solvent Control	0	0.0
6.25	0	0.0
12.5	0	0.0
25.0	0	0.0
50.0	0	0.0
100	2	5.0

Table 3. Average live weight (mg) per surviving adult earthworm at the beginning of the test and after 28 days (n = 80 for solvent control; n = 40 for control and concentrations). Data are given as absolute value and in mean percent of the initial weight change per replicate.

Concentration [mg test item/kg soil (dw)]	Biomass (mg fresh weight)		Mean change in % of the initial weight per replicate
	Day 0	Day 28	Day 28
Control	368.8 ± 22.1	488.2 ± 23.2	32.6
Solvent Control	366.3 ± 19.3	480.8 ± 20.4	31.5
6.25	368.4 ± 19.1	472.7 ± 9.0	28.6
12.5	369.0 ± 17.2	497.6 ± 11.3	35.0
25.0	369.8 ± 16.3	491.5 ± 28.9	33.0
50.0	365.5 ± 16.1	474.2 ± 6.7	29.9
100	366.2 ± 13.3	510.7 ± 11.0	39.6

Table 4. Reproduction (given as mean number of juveniles ± standard deviation and as % of the solvenr control) in the treated test vessels

Concentration [mg test item/kg soil (dw)]	Number of Juveniles per Treatment (mean ± sd)	Number of Juveniles [% of Solvent Control]
Control	316.5 ± 20.2	92.0
Solvent Control	344.1 ± 38.1	-
6.25	337.5 ± 64.8	98.1
12.5	312.8 ± 56.2	90.9
25.0	337.0 ± 31.1	97.9
50.0	295.3 ± 37.2	85.8*
100	132.0 ± 24.1	38.4*

Table 5. Physical parameters of artificial soil at the beginning and the end of the Definitive Test (the moisture is given in % of the dry weight and in % of the WHCmax (54.4%) of the artificial soil).

Concentration [mg test item/kg soil (dw)]	pH		Start		Moisture	End
	Start	End	% of dry mass	% of WHCmax	% of dry mass	% of WHCmax	% change of initial moisture
Control	6.1	6.8	28.2	51.8	34.9	64.2	23.9
Solvent Control	6.1	6.8	26.7	49.1	34.1	62.7	27.7
100	6.1	6.8	27.2	50.0	31.6	58.1	14.8
50	6.1	6.8	27.1	49.8	33.7	61.9	24.3
25	6.1	6.8	27.5	50.6	36.6	67.3	34.6
12.5	6.2	6.9	26.3	48.3	34.6	63.6	31.7
6.25	6.2	6.9	26.5	48.7	35.2	64.7	32.9

Table 5. Number of juveniles after 56 days

Concentration [mg test item/kg soil (dw)]	Number of Juveniles	Number of Juveniles per Treatment (mean ± sd)
Control A	332
Control B	308	316.5 ± 20.2
Control C	292
Control D	334
Solvent Control A	353
Solvent Control B	397	344.1 ± 38.1
Solvent Control C	357
Solvent Control D	367
Solvent Control E	292
Solvent Control F	350
Solvent Control G	354
Solvent Control H	283
6.25 A	425
6.25 B	292	337.5 ± 64.8
6.25 C	285
6.25 D	348
12.5 A	279
12.5 B	386	312.8 ± 56.2
12.5 C	326
12.5 D	260
25.0 A	327
25.0 B	383	337.0 ± 31.1
25.0 C	315
25.0 D	323
50.0 A	332
50.0 B	297	295.3 ± 37.2
50.0 C	308
50.0 D	244
100 A	162
100 B	103	132.0 ± 24.1
100 C	132
100 D	131

Validity criteria fulfilled:

yes

Remarks:

Mortality in the solvent control (required: ≤ 10%): 0.0%. Mean number of juveniles per replicate in the solvent control (standard deviation)(required: ≥30): 344.1±38.1. Coefficient of variation of juveniles in the solvent control (required: ≤30%): 11.1%

Conclusions:

The EC50 for reproduction for the test item was determined to be 87.9 mg test item/kg soil (dw).

Executive summary:

A toxicity test to the soil organism Eisenia fetida was performed following OECD 222 and under GLP. Eisenia fetida were introduced in test vessels filled with 500 g dw of artificial soil. The test item had been previously mixed with artificial soil. The test item was used to soak a portion of the quartz sand of the artificial soil. The two highest concentrations of the mixture were pestled in a mortar, then the test item coated fraction of the quartz sand was incorporated into the remainder of the artificial soil by thoroughly mixing it in 5 minutes. 5 treatments plus a control (4 replicates) and solvent control (8 replicates) were performed. The test item was sparingly soluble in water; therefore, a volatile organic solvent was used. The stock solution was prepared in acetone and dilutions were performed to obtain the following concentrations: 6.25, 12.5, 25.0, 50, 100 mg test item/kg soil (dw). NOEC/LOEC for the effects of the test item on the reproduction (56 days after application), mortality and the biomass development (28 days after application) were determined. All validity criteria were fulfilled. The NOEC for mortality and biomass was ≥ 100 mg test item/kg soil dw while the LOEC was > 100 mg test item/kg soil dw. The NOEC for reproduction was determined to be 25.0 mg test item/kg soil dw while the LOEC was 50 mg test item/kg soil dw. The EC50 for reproduction for the test item after 56  days was determined to be 87.9 mg test item/kg soil (dw) (95% confidence limits = 76.0 – 101.7 mg test item/kg soil (dw)).

Description of key information

Key study. Method according to OECD 222, GLP study. The EC50 for reproduction for the test item days was determined to be 87.9 mg test item/kg soil (dw)

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

87.9 mg/kg soil dw

Additional information

A toxicity test to the soil organism Eisenia fetida was performed following OECD 222 and under GLP. Eisenia fetida were introduced in test vessels filled with 500 g dw of artificial soil mixed with the test item.5 treatments plus a control (4 replicates) and solvent control (8 replicates) were performed. The test item was sparingly soluble in water; therefore, a volatile organic solvent was used. The stock solution was prepared in acetone and dilutions were performed to obtain the following concentrations: 6.25, 12.5, 25.0, 50, 100 mg test item/kg soil (dw). NOEC/LOEC for the effects of the test item on the reproduction (56 days after application), mortality and the biomass development (28 days after application) were determined. All validity criteria were fulfilled. The NOEC for mortality and biomass was ≥ 100 mg test item/kg soil dw while the LOEC was > 100 mg test item/kg soil dw. The NOEC for reproduction was determined to be 25.0 mg test item/kg soil dw while the LOEC was 50 mg test item/kg soil dw. The EC50 for reproduction for the test item after 56  days was determined to be 87.9 mg test item/kg soil (dw) (95% confidence limits = 76.0 – 101.7 mg test item/kg soil (dw)).