2,6-di-tert-butylphenol

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 March 2017 to 03 May 2017

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Food was provided on day 0 due to good experience with this procedure. At test end, the soil moisture deviated by > 10 % from the initial value in all treatments.

GLP compliance:

yes

Test material

Test material information

1

Reference substance name:

2,6-di-tert-butylphenol

EC Number:

204-884-0

EC Name:

2,6-di-tert-butylphenol

Cas Number:

128-39-2

Molecular formula:

C14H22O

IUPAC Name:

2,6-di-tert-butylphenol

Test material form:

solid

Details on test material:

- State of aggregation: White to yellow, solid
- Water solubility: The water solubility of 2,6-di-tert-butylphenol is 4.11 mg/L at 25 °C at pH 7 determined with a method equivalent or similar to OECD Guideline 105 (Water Solubility)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Analytical evaluation of the test item 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY was carried out via GC-MS/MS for the various concentrations of 4.90 to 300 mg/kg artificial soil dry weight and the controls at test start and test end.

Test substrate

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

The test item was treated with a mortar. The respective test item amount was weighed out for each test item concentration, solved in Acetone (2.5 mL per replicate) and mixed thoroughly with quartz sand (10 g per replicate). After complete evaporation of the solvent, the spiked quartz sand was added to the artificial soil. Demineralised water was added to adjust the humidity of the artificial soil to a moisture of 40 – 60 % of the maximum water holding capacity. Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 600 g soil dry weight was filled into each test vessel.

Test organisms

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Test system
Eisenia fetida (Annelida, Lumbricidae)
Reason for the selection
Eisenia fetida is suitable for this kind of study and is one of the recommended species according to the guideline.
Source
Breeding stock culture maintained at the test facility
Breeding
Organisms of the species Eisenia fetida are bred at the test facility in covered plastic vessels containing potting compost.
Synchronisation of the earthworm population
Earthworms of homogeneous age and body weight were used in the definitive test. Synchronisation of the population was achieved by placing adult earthworms into breeding boxes and removing the adults after 4 weeks. Offspring from the remaining cocoons reached the adult age after 2 months at the earliest.
Feeding
During breeding, the earthworms are fed with a litter of dried stinging nettle leaves and porridge oats. A sufficient amount of the food was provided depending on the feeding rate and the density of the earthworm population in the vessels.

Study design

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Test conditions

Test temperature:

Nominal: 20 +/- 2 °C
Actual: 18 - 22 °C

pH:

Application rate
[mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY/kg SDW]
pH-value
day 0 day 56
Control 6.35 6.13
Solvent Control 6.26 6.09
4.90 6.25 6.12
8.82 6.24 5.92
15.88 6.24 6.12
28.58 6.17 6.18
51.44 6.20 6.05
92.59 6.20 6.18
166.67 6.26 5.82
300 6.08 6.02

Moisture:

Application rate
[mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY/kg SDW]
Moisture [%] of dry weight
day 0 day 56
Control 19.7 25.4
Solvent Control 19.6 23.9
4.90 19.5 23.5
8.82 19.1 23.5
15.88 19.1 23.6
28.58 19.5 24.9
51.44 19.7 24.3
92.59 19.3 25.1
166.67 19.4 25.5
300 19.7 23.5
The soil moisture deviated by more than 10 % from the initial value. This deviation is considered to have no impact on quality and integrity of the study.

Details on test conditions:

Test item 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY
Preliminary Study
A preliminary non-GLP range finding test was performed with 3 test item concentrations (each with 2 replicates) without the addition of food.
Application rates
300 – 166.67 – 92.59 – 51.44 – 28.58 – 15.88 – 8.82 – 4.90 mg/kg soil dry weight (Factor 1.8)
Control
Artificial soil moistened with demineralised water without test or reference item was used as control medium
Solvent Control
Artificial soil, spiked with acetone and moistened with demineralised water, without test or reference item will be used as solvent control medium.
Test duration
8 weeks
Number of replicates
8 replicates per control and 4 replicates per test item concentration
Number and age of the earthworms
10 adult earthworms (with clitellum) aged 2-12 months were used per replicate. The difference of age did not deviate by more than 1 month.
Body weight (actual)
0.40 to 0.57 g
Adaptation
Earthworms were adapted to the artificial soil to be used for the test two days prior to test start.
Homogeneity of groups
The worms were washed with demineralised water and dried gently on a paper towel. The homogeneity of the population was checked by weighing the earthworms. After confirmation of homogeneity, groups of 10 earthworms (chosen by a randomised procedure) were weighed and introduced into each test vessel.
Test medium Artificial soil consisted of the following components:
- 5 % peat, air-dried and finely ground
- 20 % kaolin, kaolinite content > 30 %
- 74 % air-dried quartz sand (sand with > 50 % particle size of 0.05 - 0.2 mm)
- 0.20 % calcium carbonate (CaCO3) to achieve a pH of 6.0 +/- 0.5
The WHCmax, the moisture content and the pH-value of the artificial soil were determined and adjusted. Two days before test start the artificial soil (31400 g) was pre-moistened with 2889 g demineralised water to achieve a moisture content corresponding to 27 % of the WHCmax of the artificial soil. The moistened soil was kept covered to prevent evaporative water losses prior to the start of exposure.
Test container
Test units containing about 600 g soil dry weight were round plastic boxes with an inner diameter of 15.0 cm corresponding to a bottom surface area of 177 cm2 and a height of 14 cm. Transparent and perforated lids will enable sufficient gas exchange, light input and prevent the test substrate from drying.
Application
The test item was treated with a mortar. The respective test item amount was weighed out for each test item concentration, solved in Acetone (2.5 mL per replicate) and mixed thoroughly with quartz sand (10 g per replicate). After complete evaporation of the solvent, the spiked quartz sand was added to the artificial soil. Demineralised water was added to adjust the humidity of the artificial soil to a moisture of 40 – 60 % of the maximum water holding capacity. Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 600 g soil dry weight was filled into each test vessel.
Temperature
20 +/- 2 °C
Photoperiod
16 h
Light intensity
400 - 800 lx
Feeding
The earthworms were fed with air-dried, non treated (medicated) cattle manure weekly during the first 4 weeks. At test start and at day 28 the food was mixed carefully into the soil, on the other days the food was placed onto the soil surface. No further feeding took place during the remaining 4 weeks of the test.
The cattle manure was delivered by LEHR- UND FORSCHUNGSGUT RUTHE, Schäferberg 1, 31157 Sarstedt, Germany.
Moistening
Throughout the test the replicates were weighed weekly. Evaporated water was replenished by adding demineralised water to maintain the soil moisture which should not deviate by more than 10% of the initial value at the end of the test. Additional moistening was performed in case the soil appeared too dry.
Feeding and Additional Moistening During the Test
Day Amount of food [g]
Cattle manure Amount of demineralised water
[mL]
0 5 –
7, 14, 21, 28 5 10
35, 41, 49 – –

Nominal and measured concentrations:

Nominal:
4.9 – 8.82 – 15.88 – 28.58 – 51.44 – 92.59 – 166.67 - 300 mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY/kg SDW (Factor 1.8)
The recovery rates at day 0 were between 40 and 74% of the nominal concentrations and at test end between < LOQ and 2.1%. Control samples were < LOQ.

Reference substance (positive control):

yes

Remarks:

CARBENDAZIM: 0.5 - 1 - 2 - 4 mg a.s./kg SDW The test is conducted once within twelve months. The most recent test was carried out from 09 February to 06 April 2017 (Study no. RBR71702).

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

8 wk

Dose descriptor:

LOEC

Effect conc.:

166.67 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 2

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

92.59 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 3

Duration:

8 wk

Dose descriptor:

LC10

Effect conc.:

106 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95 % confidence interval

Remarks:

81.2 - 126

Effect concentrations 4

Duration:

8 wk

Dose descriptor:

other: LC20

Effect conc.:

134 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95 % confidence interval

Remarks:

110 - 155

Effect concentrations 5

Duration:

8 wk

Dose descriptor:

LC50

Effect conc.:

208 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95 % confidence interval

Remarks:

181 - 244

Effect concentrations 6

Duration:

8 wk

Dose descriptor:

LOEC

Effect conc.:

300 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Effect concentrations 7

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

166.67 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Effect concentrations 8

Duration:

8 wk

Dose descriptor:

LOEC

Effect conc.:

92.59 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 9

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

51.44 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 10

Duration:

8 wk

Dose descriptor:

EC10

Effect conc.:

90.6 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95 % confidence interval

Remarks:

73.1 - 112

Effect concentrations 11

Duration:

8 wk

Dose descriptor:

other: EC20

Effect conc.:

102 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95 % confidence interval

Remarks:

82.4 - 125

Effect concentrations 12

Duration:

8 wk

Dose descriptor:

EC50

Effect conc.:

127 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95 % confidence interval

Remarks:

99.7 - 163

Details on results:

- Mortality at end of exposure period:
No evident earthworm mortalities (< 10%) nor pathological symptoms or changes in the behaviour of adult earthworms were observed in the control groups and the test item concentrations 4.90 to 92.59 mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY/kg soil dry weight. However, at the test item concentrations 166.67 and 300 mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY/kg soil dry weight 45% and 70% mortality occurred.
The EC10/20/50-value for mortality was determined to be 106, 134 and 208 mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY/kg SDW, respectively.

- Total mass of adults at beginning of test:
At test start, the earthworms had individual weights of 0.40 - 0.57 g.
- Changes in body weight of live adults (% of initial weight) at end of exposure period:
There were no statistically significant differences in earthworm body weights in the test item concentrations 4.90 to 166.67 mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY/kg soil dry weight. However, at the test item concentration 300 mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY/kg soil dry weight the body weight gain was statistically significantly reduced. Due to increased mortality in the test item concentrations 166.67 and 300 mg/kg soil dry weight the evaluation of the body weight gain in these concentrations is considered to be less meaningful. Therefore no ECx – values were determined.

- No. of offspring produced:
The reproduction rate (average number of juveniles produced) was 137 in the pooled control and ranged from 0 to 145 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 4.90 to 51.44 mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY//kg soil dry weight compared to the pooled control. However, at the test item concentration 92.59 to 300 mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY//kg soil dry weight the earthworm reproduction was statistically significantly reduced. The EC10/20/50-value for reproduction was determined to be 90.6, 102 and 127 mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY/kg SDW, respectively

Results with reference substance (positive control):

Reference Item - Reproduction Rate (Number of Juveniles after 8 Weeks)
Application rate
[mg a.s./kg
soil dry weight] Rep. Number of juveniles Mean ± SD CV % of control Significance*
Control 1 170 152 ± 26.0 17.1 _ _
2 141
3 125
4 123
5 199
6 167
7 136
8 156
0.5 1 182 153 ± 25.6 16.7 101 No
2 167
3 132
4 131
1 1 128 145 ± 26.7 18.4 95.4 No
2 154
3 119
4 178
2 1 10 6 ± 3.65 60.8 3.95 Yes
2 8
3 2
4 4
4 1 0 0 ± 0.0 - 0.0 Yes
2 0
3 0
4 0
Rep. = Replicate a.s. = active substance SD = Standard deviation CV = Coefficient of variation
Sig. = Statistically significantly reduced compared to control (Multiple Sequential-rejective Welsh-t-test After
Bonferroni-Holm )

According to OECD 222 significant effects of Carbendazim should be observed between 1 and 5 mg a.s./kg soil DW.

Reported statistics and error estimates:

For each endpoint, solvent control was statistically compared to the control. Since no statistically significant deviations occurred, both controls were pooled for further calculations. Comparison of the results only versus solvent control is not preferred by the authorities.
Adult Mortality
The arithmetic mean and the NOEC were determined. Significant differences of mortality were determined by Step-down Rao-Scott-Cochran-Armitage Test Procedure. When running the test, a Qualitative Trend Analysis by Contrasts and a Tarone’s Test Procedure were done first. The alpha-value was 0.05. For the endpoint of mortality, the arithmetic mean and the coefficient of variation per treatment and control were calculated. The LC10,20,50-values for mortality were calculated using Probit analysis (linear max. likelihood regression).
Biomass, Reproduction
Significant differences in reproduction were determined by Multiple Sequential-rejective Welsh-t-test after Bonferroni-Holm.
When running the tests, a Shapiro-Wilk’s Test on Normal Distribution and a Levene’s Test on Variance Homogeneity were done first. The alpha-value was 0.05. For the endpoint of reproduction, the arithmetic mean and the coefficient of variation per treatment and control were calculated. The EC10,20,50-values for reproduction were calculated using sigmoidal dose response.
Significant differences in biomass were determined using Dunnett’s Method and ANOVA. When running the tests, a Shapiro-Wilk’s Test on Normal Distribution and an Equal Variance Test were done first. Due to increased mortality in the test item concentrations 166.67 and 300 mg/kg soil dry weight the body weight gain in these concentrations is considered to be less meaningful. Therefore no ECx-values were determined.
Software
- Excel, MICROSOFT
- ToxRat Professional, TOXRAT SOLUTIONS GMBH
- Sigma Plot, SPSS INC.

Any other information on results incl. tables

 Adult Mortality

No evident earthworm mortalities (< 10%) nor pathological symptoms or changes in the behaviour of adult earthworms were observed in the control groups and the test item concentrations 4.90 to 92.59 mg 2,6-DI-TERT-BUTYLPHENOL HIGHPURITY/kg soil dry weight. However, at the test item concentrations 166.67 and 300 mg 2,6-DI-TERT-BUTYLPHENOL HIGHPURITY/kg soil dry weight 45% and 70% mortality occurred.

The EC_10/20/50-value for mortality was determined to be 106, 134 and 208 mg 2,6-DI-TERT-BUTYLPHENOL HIGHPURITY/kg SDW, respectively.

 

Mortality of Adult Earthworms in [%] after 28 Days of Exposure

Application rate	Replicate
[mg2,6-DI-TERT-BUTYLPHENOL HIGHPURITY/ kg SDW]	1	2	3	4	5	6	7	8	Mean
Control	0	0	0	0	0	0	0	0	0
Solvent Control	0	0	0	0	0	0	0	0	0
4.90	0	0	0	0	–				0
8.82		0		0						0		0		0
15.88		0		0						0		0		0
28.58		0		0						0		0		0
51.44		0		0						0		0		0
92.59		0		0						0		10		2.5
166.67		40		50						70		20		45
300		40		40						10		10		70

 

Pathological Symptoms and Changes in Behaviour

No significant pathological symptoms or changes in the behaviour of adult earthworms were observed in the controls and the test item concentrations 4.90 to 92.59 mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY/kg SDW . However, at the test item concentrations 166.67 and 300 mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY/kg SDW mortality increased.

Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Application rate	Replicate
[mg2,6-DI-TERT-BUTYLPHENOL HIGHPURITY/ kg SDW]	Observation parameter	1	2	3	4	5	6	7	8
Control	A	10/10	10/10	10/10	10/10	10/10	10/10	10/10	10/10
Solvent Control	A H	10/10 -	10/10 -	10/10 -	10/10 -	9/10 1/10	10/10 -	10/10 -	10/10 -
4.90	A	10/10	10/10	10/10	10/10	-
8.82	A	10/10	10/10	10/10	10/10
15.88	A	10/10	10/10	10/10	10/10
28.58	A	10/10	10/10	10/10	10/10
51.44	A	10/10	10/10	10/10	10/10
92.59	A H	10/10 -	10/10 -	10/10 -	9/10 1/10
166.67	A D H	5/10 1/10 4/10	3/10 2/10 5/10	3/10 - 7/10	8/10 - 2/10
300	A H	6/10 4/10	6/10 4/10	- 10/10	- 10/10

A = no obvious pathological symptoms            D = spontaneous segmentation and separation              H = earthworm dead

 Live Weight

At test start, the earthworms had individual weights of 0.40 - 0.57 g. There were no statistically

significant differences in earthworm body weights in the test item concentrations 4.90 to 166.67

mg 2,6-DI-TERT-BUTYLPHENOL HIGHPURITY/kg soil dry weight. However, at the test item

concentration 300 mg 2,6-DI-TERT-BUTYLPHENOL HIGHPURITY/kg soil dry weightthe body

weight gain was statistically significantly reduced (Table 7).Due to increased mortality in the test

item concentrations 166.67 and 300 mg/kg soil dry weight the evaluation of the body weight gain

in these concentrations is considered to be less meaningful. Therefore no EC_x– values were determined.  

Body Weight Changes of the Adult Earthworms        

Application rate [mg2,6-DI-TERT-BUTYLPHENOL HIGHPURITY/kg SDW]	Replicate	Mean body weights per replicate		Mean body weight change of earthworms
		Test start	28 days	Per replicate		Mean ± SD		Sig.
		[g]	[g]	[g]	[%]	[g]	[%]
Control	1	0.47	0.52	0.05	10.6	0.06 ± 0.02	13.2 ± 4.78	–
	2	0.46	0.50	0.04	8.7
	3	0.46	0.52	0.06	13.0
	4	0.46	0.52	0.06	13.0
	5	0.46	0.50	0.04	8.7
	6	0.46	0.51	0.05	10.9
	7	0.46	0.56	0.10	21.7
	8	0.47	0.56	0.09	19.1
Solvent Control	1	0.47	0.56	0.09	19.1	0.07 ± 0.02	14.9 ± 4.40	–
	2	0.46	0.51	0.05	10.9
	3	0.46	0.54	0.08	17.4
	4	0.46	0.54	0.08	17.4
	5	0.47	0.50	0.03	6.4
	6	0.46	0.54	0.08	17.4
	7	0.46	0.54	0.08	17.4
	8	0.47	0.53	0.06	12.8
Pooled Control	-					0.07± 0.02	14.0 ± 4.51	-
4.90	1	0.46	0.51	0.05	10.9	0.07 ± 0.02	14.0 ± 3.92	No
	2	0.47	0.56	0.09	19.1
	3	0.46	0.51	0.05	10.9
	4	0.47	0.54	0.07	14.9
8.82	1	0.46	0.50	0.04	8.7	0.06 ± 0.02	12.8 ± 3.82	No
	2	0.47	0.52	0.05	10.6
	3	0.47	0.55	0.08	17.0
	4	0.47	0.54	0.07	14.9
15.88	1	0.47	0.52	0.05	10.6	0.06 ± 0.01	12.3 ± 2.08	No
	2	0.46	0.52	0.06	13.0
	3	0.47	0.52	0.05	10.6
	4	0.47	0.54	0.07	14.9
28.58	1	0.46	0.56	0.10	21.7	0.08 ± 0.04	17.9 ± 8.85	No
	2	0.46	0.52	0.06	13.0
	3	0.47	0.51	0.04	8.5
	4	0.46	0.59	0.13	28.3

51.44	1	0.45	0.50	0.05	11.1	0.09 ± 0.03	18.6 ± 5.36	No
	2	0.46	0.55	0.09	19.6
	3	0.46	0.57	0.11	23.9
	4	0.46	0.55	0.09	19.6
92.59	1	0.47	0.54	0.07	14.9	0.06 ± 0.02	13.0 ± 4.30	No
	2	0.46	0.53	0.07	15.2
	3	0.46	0.49	0.03	6.5
	4	0.46	0.53	0.07	15.2
166.67	1	0.45	0.53	0.08	17.8	0.04 ± 0.04	9.38 ± 7.73	No
	2	0.46	0.46	0.00	0.0
	3	0.45	0.48	0.03	6.7
	4	0.46	0.52	0.06	13.0
300	1	0.46	0.39	-0.07	-15.2	-0.06 ± 0.01	-13.3 ± 2.69	Yes
	2	0.44	0.39	-0.05	-11.4
	3	0.45	n.d.	n.d.	n.d.
	4	0.46	n.d.	n.d.	n.d.

SD = Standard deviation                                                                          

Sig. = Statistical Significance (Dunnett’s Test,α= 0.05, ANOVA) compared to control

n.d. = not determinable due to 100% mortality

  

 Reproduction Rate

The reproduction rate (average number of juveniles produced) was 137 in the pooled control and ranged from 0 to 145 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 4.90 to 51.44 mg 2,6-DI-TERT-BUTYLPHENOL HIGHPURITY//kg soil dry weight compared to the pooled control. However, at the test item concentration 92.59 to 300 mg 2,6-DI-TERT-BUTYLPHENOL HIGHPURITY/kg soil dry weight the earthworm reproduction was statistically significantly reduced. The EC_10/20/50-value for reproduction was determined to be 90.6, 102 and 127 mg 2,6-DI-TERT-BUTYLPHENOL HIGHPURITY/kg SDW, respectively.

The coefficient of variation calculated for the reproduction of the pooled control was 13.1 % and thus lower than 30 %.

 

Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate [mg2,6-DI-TERT-BUTYLPHENOL HIGHPURITY/kg SDW]	Replicate	Number of juveniles	Mean±SD	CV [%]	[%] of pooled control	Sig.
Control	1	129	131 ± 18.9	14.5	-	-
	2	161
	3	141
	4	120
	5	129
	6	125
	7	98
	8	147
Solvent Control 31.	1	148	142 ± 16.2 165 ± 13.8	11.4 8.35	- 111	- No
	2	133
	3	130
	4	116
	5	166
	6	142
	7	143
	8	160
Pooled Control	-	-	137 ± 17.9	13.1	-	-
4.90	1	152	131 ± 25.4	19.4	94.9	No
	2	96
	3	127
	4	147
8.82	1	149	125 ± 17.5	14.0	90.6	No
	2	116
	3	124
	4	109
15.88	1	133	145 ± 15.6	10.8	105	No
	2	167
	3	135
	4	144
28.58	1	148	116 ± 28.9	24.9	84.1	No
	2	85
	3	132
	4	100

51.44	1	142	120 ± 26.5	22.1	87.0	No
	2	143
	3	106
	4	90
92.59	1	108	117 ± 7.77	6.64	84.8	No
	2	126
	3	113
	4	119
166.67	1	16	20 ± 8.27	41.4	14.5	Yes
	2	29
	3	10
	4	23
300	1	0	0 ± 0	0.0	0.0	Yes
	2	0
	3	0
	4	0

SD   =   Standard deviation CV = Coefficient of variation

Sig.  =   StatisticalSignificance (Multiple Sequential-rejective Welsh-t-test after Bonferroni-Holm)compared to the
               pooled control

 

 

Measured Exposure Concentrations during the Definitive Test

Analytical evaluation of the test item 2,6-DI-TERT-BUTYLPHENOL HIGH PURITYwas carried out via GC-MS/MS for the various concentrations of 4.90 to 300 mg/kg artificial soil dry weight and the controls at test start and test end.

The recovery rates at day 0 were between 40 and 74% of the nominal concentrations and at test end between < LOQ and 2.1%. Control samples were < LOQ.

 Measured Concentrationsand Percent of Nominal of 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY

Sampling date	2017-03-03 start of exposure		2017-04-27/28 end of exposure
Start of analysis	2017-03-03		2017-04-28
Test item nominal concentration [mg/kg SDW]	2,6-DI-TERT-BUTYLPHENOL HIGH PURITY
	Meas. amount [mg/kg SDW]	[%]	Meas. amount [mg/kg SDW]	[%]
300	221	74	5.78	1.9
166.67	115	69	3.48	2.1
92.59	46.8	51	1.20	1.3
51.44	27.7^1)	54	0.800^2)	1.6
28.58	14.3	50	< LOQ	-
15.88	7.07	45	< LOQ	-
8.82	3.62	41	< LOQ	-
4.90	1.98	40	< LOQ	-
Solvent control	< LOQ		< LOQ
Control	< LOQ		< LOQ

 

1)                     = Analysed with ASE extraction with acetonitrile on 2017-03-13, measured on 2017-03-15

2)                     = Measured amount > LCL but < LOQ, only for information

Meas.amount = Measuredamount, enrichment and dilution factor taken into account

%                     = Percent of the nominal concentration of the test item

LOQ                =Limit of quantification of the analytical method (1.26 mg test item/kg SDW)

LCL                  = Lowest calibration level (5 µg test item/L)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY did not induce evident earthworm mortalities (< 10%) nor pathological symptoms or changes in the behaviour of adult earthworms in the control groups and the test item concentrations 4.90 to 92.59 mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY/kg soil dry weight. However, at the test item concentrations 166.67 and 300 mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY/kg soil dry weight 45% and 70% mortality occurred, respectively. There were no statistically significant differences in earthworm body weights in the test item concentrations 4.90 to 166.67 mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY/kg soil dry weight. However, at the test item concentrations 300 mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY/kg soil dry weight the body weight gain was statistically significantly reduced. After a further four weeks, the reproduction rate (average number of juveniles produced) was 137 in the pooled control and ranged from 0 to 145 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 4.90 to 51.44 mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY//kg soil dry weight compared to the pooled control. However, at the test item concentration 92.59 to 300 mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY//kg soil dry weight the earthworm reproduction was statistically significantly reduced.

Executive summary:

Effects of 2,6-DI-TERT-BUTYLPHENOL HIGHPURITYon mortality, biomass and the reproductive potential of the earthworm speciesEisenia fetida(Annelida, Lumbricidae) were determined according to OECD 222 (2016) from 03 March 2017 to 03 May 2017 with the definitive exposure phase from 03 March 2017 to 27/28 April 2017 at the test facility in 31157 Sarstedt, Germany.

The study was conducted under static conditions over 8 weeks with the test item concentrations 4.9 – 8.82 – 15.88 – 28.58 – 51.44 – 92.59 – 166.67 - 300mg 2,6-DI-TERT-BUTYLPHENOL HIGHPURITY/kg SDW. Each application rate was mixed into artificial soil containing 5 % peat. A control including untreated artificial soil and a solvent control including artificial soil treated with the solvent was tested under the same conditions as the test item treatments. 80 test organisms were inserted into 8 control and solvent control replicates, each, and 40 test organisms were divided into 4 replicates for each treatment (10 earthworms per replicate). They had an individual body weight between 0.40 and 0.57 g at experimental starting.

 Each concentration level and control were analysed via GC-MS analysis subsequently to their preparation on day 0 and at test end.The recovery rates at day 0 were between 40 and 74% of the nominal concentrations and at test end between < LOQ and 2.1 %. Control samples were < LOQ.

The method was validated according to SANCO/3029/99 rev.4 (2000) prior to the start of exposure.

After 28 days of exposure in soil, neither evident earthworm mortalities (< 10%) nor pathological symptoms or changes in the behaviour of adult earthworms were observed in the control groups and the test item concentrations 4.90 to 92.59 mg 2,6-DI-TERT-BUTYLPHENOL HIGHPURITY/kg soil dry weight. However, at the test item concentrations 166.67 and 300 mg 2,6-DI-TERT-BUTYLPHENOL HIGHPURITY/kg soil dry weight 45% and 70% mortality occurred, respectively.There were no statistically significant differences in earthworm body weights in the test item concentrations 4.90 to 166.67 mg2,6-DI-TERT-BUTYLPHENOL HIGHPURITY/kg soil dry weight. However, at the test item concentrations 300 mg2,6-DI-TERT-BUTYLPHENOL HIGHPURITY/kg soil dry weightthe body weight gain was statistically significantly reduced. After a further four weeks, the reproduction rate (average number of juveniles produced) was 137 in the pooled control and ranged from 0 to 145 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 4.90 to 51.44 mg 2,6-DI-TERT-BUTYLPHENOL HIGHPURITY//kg soil dry weight compared to the pooled control. However, at the test item concentration 92.59 to 300 mg 2,6-DI-TERT-BUTYLPHENOL HIGHPURITY//kg soil dry weight the earthworm reproduction was statistically significantly reduced.

All validity criteria recommended by the test guidelines were fulfilled.

Summary of All Observed Effects in theApplication rates

Effects	2,6-DI-TERT-BUTYLPHENOL HIGHPURITY
Application rates [mg/kg SDW]	4.90	8.82	15.88	28.58	51.44	92.59	166.67	300
Mortality of adult earthworms	No	No	No	No	No	No	Yes	Yes
Body Weight Gain	No	No	No	No	No	No	No	Yes
Reduced reproduction rate (Number of juveniles)	No	No	No	No	No	Yes	Yes	Yes
Summary of all effects	No	No	No	No	No	Yes	Yes	Yes

 

NOEC, LOEC and EC-values

Endpoint	[mg2,6-DI-TERT-BUTYLPHENOL HIGHPURITY/ kg SDW]
LOECmortality	166.67
NOECmortality	92.59
LOECbiomass	300
NOECbiomass	166.67
LOECreproduction	92.59
NOECreproduction	51.44
LC-valuesmortality (95 % Confidence Interval)	LC10: 106 (81.2 - 126) LC20: 134 (110 - 155) LC50: 208 (181 - 244)
EC-valuesreproduction (95 % Confidence Interval)	EC10: 90.6 (73.1 - 112) EC20: 102 (82.4 - 125) EC50: 127 (99.7 - 163)