2,4-di-tert-butylphenol

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study following OECD guideline 222

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Lot No.: 1437
Article number: 6084
Purity: 99.94%

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, vehicle control, and test substance dosing solutions
- Sample storage conditions before analysis: All solutions were stored refrigerated when not in use.

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): Carrier sand.
- Controls: A 10 mL aliquot of neat acetone was added to carrier sand and used to prepare a vehicle control soil.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone.
- Evaporation of vehicle before use: Yes.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Eisenia fetida (earthworm)
- Source: Vicker’s Farms, Orlando, Florida
- Age at test initiation: approximately 5 months of age with a developed clitellum
- Weight at test initiation (range): 460-567 mg wet weight

ACCLIMATION
- Acclimation period: yes
- Acclimation conditions (same as test or not): same
- Health during acclimation (any mortality observed): no mortalities observed

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Remarks:

None

Post exposure observation period:

None

Test temperature:

20 ± 1°C

pH:

Soil pH (day 0): 5.83 to 6.04
Soil pH (day 56): 5.21 to 5.91

Moisture:

Soil Moisture (day 0): 19.7 to 23.2%
Soil Moisture (day 56): 20.6 to 23.7%
Soil Moisture at 50% WHC: 22.5%

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Glass jars, approximately 21 cm in height and 12 cm in diameter with a volume of approximately 1.8 L.
- Amount of soil or substrate: 600 g
- 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: 4

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 70% silica sand (approximately 85% less than 200 µm grain size), 20% kaolin clay (96 to 99% kaolin), and 10% sphagnum peat based on dry weight equivalents
- Maximum water holding capacity (in % dry weigth): 75.9 mL per 100 grams dry soil
- Pretreatment of soil: CaCO3 was added to the soil to adjust the pH to fall within the 6.5 +/- 0.5 range
- Storage (condition, duration): Not applicable
- Stability and homogeneity of test material in the medium: Method validation for the recovery of 2,4-di-tert-butylphenol in acetone was performed.

OTHER TEST CONDITIONS
- Photoperiod: 16-hr light to 8-hr dark
- Light intensity: 708 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Weight, mortality and reproduction

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study
- Test concentrations: 0 (control), 0 (vehicle control), 0.0010, 0.010, 0.10, 1.0, and 10 mg/kg dry soil
- Results used to determine the conditions for the definitive study: All live earthworms were normal in appearance and behavior. Average of 21, 17, 31, 22, 33, 25, and 36 cocoons per replicate for the control, vehicle control, 0.0010, 0.010, 0.10, 1.0, and 10 mg/kg test substance treatments, respectively.

Nominal and measured concentrations:

Nominal:
0 (control), 0 (vehicle control), 3.0, 6.0, 12, 23, 45, 90, 180, and 360 mg/kg

Reference substance (positive control):

yes

Remarks:

Carbendazim (Lot No. 3372000, received from Chem Service, West Chester, Pennsylvania)

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

12 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

23 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

90 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

180 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Mortality:
After 28 days of exposure to 2,4-di-tert-butylphenol, percent survival of the adult worms was 95, 90, 83, 90, 88, 90, 93, 88, 5, and 0% in the control, vehicle control, 3.0, 6.0, 12, 23, 45, 90, 180, and 360 mg/kg test treatments, respectively
- Body weigth:
The control worms lost an average of 0.1717 g per replicate or lost 3% in replicate mass during the 28-day exposure. The vehicle control worms gained an average of 0.0777 g per replicate or 2% in replicate mass during the 28-day exposure. The mean replicate weight gain of the surviving treatment animals in the 3.0 to 90 mg/kg treatments ranged from 0.2749 to 0.6487 grams or from 6 to 13%. The worms exposed to 120 mg/kg treatment soils lost an average of 2.7237 g per replicate or lost 48% in replicate mass during the 28-day exposure. Due to 100% mortality, there was no weight data for the 360 mg/kg test treatment at test termination
- No. of offspring produced: The average reproduction values were 92, 86, 71, 68, 75, 44, 46, 2, 0, and 0 juveniles for nominal soil concentrations of 0 (control), 0 (vehicle control), 3.0, 6.0, 12, 23, 45, 90, 180, and 360 mg/kg, respectively.

Results with reference substance (positive control):

A toxic standard test performed with the worm lot used in this study was performed from 15 April to 10 June 2015 at nominal concentrations of 0 (control), 0.25, 0.50, 1.0, 2.0, and 4.0 mg carbendazim/kg test treatments. After 28 days of exposure to carbendazim, percent survival of the adult worms was 90 in the control and ranged from 70 to 85% in the carbendazim test treatments, respectively. After 56 days of exposure, the average reproduction values were 75, 87, 64, 37, 23, and 1 juveniles for nominal concentrations of 0 (control), 0.25, 0.50, 1.0, 2.0, and 4.0 mg carbendazim/kg dry soil. The NOEC and EC50 values for reproduction were 0.50 and 1.1 mg carbendazim/kg dry soil, respectively. These values are consistent with the guideline expectations that there will be an effect of this reference toxicant at a range of concentrations between 1.0 and 5.0 mg carbendazim/kg dry soil.

Reported statistics and error estimates:

Base estimates of EC10 and EC50 values and their 95% confidence limits were calculated using the trimmed Spearman-Karber method and probit method. When the p-value for Goodness of Fit was > 0.05 and there was no other evidence of questionable convergence, the probit method was selected for reporting. When this criterion was not achieved, the trimmed Spearman-Karber method was selected for reporting.
The survival and reproduction NOEC values were estimated using a one-way analysis of variance (ANOVA) procedure and a one-tailed Dunnett's and Williams’ test. Prior to the Dunnett's and Williams’ tests, a Shapiro-Wilk’s and the Levene’s tests were performed to determine data normality and homogeneity of the treatment variances. The survival data did not met the criterion for normality and homogeneity of variance, therefore a non-parametric analysis was performed on the survival data. The reproductive data met the criterion for normality and homogeneity of variance, therefore a parametric analysis was performed on the reproductive data.
SAS software (version 9.3 for Windows)

Reproduction during 56 -Day exposure

Nominal Soil Concentration (mg/kg)	Replicate	Number of SurvivingAdult Worms	Number ofJuvenile Worms	AverageReproduction
Control(0)	A/B/C/D	8/10/10/10	73/76/122/96	92 (% CV = 25)
Vehicle Control (0)	A/B/C/D	10/10/8/8	86/87/65/104	86 (% CV = 19)
Pooled Control	-	-	-	89 (%CV = 21)
3.0	A/B/C/D	7/9/8/9	37/60/117/70	71 (% CV = 47)
6.0	A/B/C/D	7/9/10/10	83/74/89/27	68 (% CV = 41)
12	A/B/C/D	10/7/9/9	71/34/77/117	75 (% CV = 45)
23	A/B/C/D	8/9/10/9	44/0/62/69	44 * (% CV = 71)
45	A/B/C/D	9/8/10/10	71/60/17/34	46 * (% CV = 54)
90	A/B/C/D	9899	0/0/5/3	2 * (% CV = 122)
120	A/B/C/D	1/0/0/1	0/0/0/0	0 a (% CV = NA)
360	A/B/C/D	0/0/0/0	0/0/0/0	0 a (% CV = NA)

a The reproduction data from this treatment level was not analyzed with the mean comparisons tests due to statistically significant survival effects at this treatment.

* Statistically significant reduction (p ≤ 0.05) of total number of young as compared to the pooled control (Dunnett’s and Williams’ test for significance).

Validity criteria fulfilled:

yes

Conclusions:

The terrestrial toxicity of 2,4-di-tert-butylphenol towards soil macroorganisms was assessed in a Survival and Reproduction Test with the Earthworm, Eisenia fetida. The NOEC and LOEC values after 56 days of exposure for reproduction were 12 and 23 mg/kg, respectively.

Executive summary:

The terrestrial toxicity of 2,4-di-tert-butylphenol towards soil macroorganisms was assessed in a Survival and Reproduction Test with the Earthworm, Eisenia fetida. The study was conducted at ABC Laboratories, Inc. 7200 E. ABC Lane, Columbia, Missouri 65202. The objective of this test was to determine chronic effect of the test substance on the reproductive output of the earthworm in soil with low total organic carbon content.

Test Species: Eisenia fetida, clitellate adults (460 to 567 mg wet weight at initiation for the treatment means) Length of Study:56 days Artificial Soil Characterization:70% silica sand, 20% clay, 10% peat moss, inherent moisture 4.07% Calculated

The nominal Soil Concentrations: 0 (control), 0 (vehicle control), 3.0, 6.0, 12, 23, 45, 90, 180, and 360 mg/kg Environmental Conditions: Temperature:20 ± 1°C Soil pH (day 0):5.83 to 6.04 Soil pH (day 56):5.21 to 5.91 Soil Moisture (day 0):19.7 to 23.2% Soil Moisture (day 56):20.6 to 23.7% Soil Moisture at 50% WHC:22.5% Photoperiod:16-hr light to 8-hr dark Light Intensity:708 lux

The survival of the control adult animals was 95%, which met the acceptability of the test protocol and guidance document. Each control replicate containing 10 adult worms after the first 28 days of exposure produced more than the minimum acceptability criterion of 30 juvenile worms and the %CV value for the control reproduction was less than the maximum acceptability of 30% as specified by the protocol and the test guideline. The NOEC and EC50 values for reproduction from the same lot of worms used in the definitive test were 0.50 and 1.1 mg carbendazim/kg dry soil, respectively. These values are consistent with the guideline expectations that there will be an effect of this reference toxicant at a range of concentrations between 1.0 and 5.0 mg carbendazim/kg dry soil. Therefore the data generated from the definitive testing should be considered as an accurate estimation of the potential toxicological response of the test substance.

The EC10 for survival was 103 mg/kg, with 95% confidence limits of 43.4 to 128 mg/kg. The EC50 for survival was 132 mg/kg, with 95% confidence limits of 83.0 to 152 mg/kg. The NOEC and LOEC values for survival were 90 and 180 mg/kg, respectively. The EC10 for reproduction was 9.68 mg/kg, with 95% confidence limits of 1.96 to 17.4 mg/kg. The EC50 for reproduction was 31.8 mg/kg, with 95% confidence limits of 21.9 to 41.8 mg/kg. The NOEC and LOEC values for reproduction were 12 and 23 mg/kg, respectively.

Description of key information

The terrestrial toxicity of 2,4-di-tert-butylphenol towards soil macroorganisms was assessed in a Survival and Reproduction Test with the Earthworm, Eisenia fetida. The NOEC and LOEC values after 56 days of exposure for reproduction were 12 and 23 mg/kg, respectively.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:

12 mg/kg soil dw

Additional information

The terrestrial toxicity of 2,4-di-tert-butylphenol towards soil macro organisms was assessed in a Survival and Reproduction Test with the Earthworm, Eisenia fetida according to OECD test guideline 222. The NOEC and LOEC values after 56 days of exposure for reproduction are 12 and 23 mg/kg, respectively, and hence the NOEC is used for PNECsoil derivation.