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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

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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:
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

Reference
Name:
Unnamed
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 concentrationsopen allclose all
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
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
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
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
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
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
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
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
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
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
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
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 EC10/20/50-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 ECx– 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 EC10/20/50-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

     27.71)

54

0.8002)

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)