Registration Dossier

Ecotoxicological information

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:
27 January 2017 to 24 March 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:
see Principal of method other than guideline
Principles of method if other than guideline:
Food was provided on day -1 (test start) instead of day 1.
At test end, the soil moisture deviated by more than 10 % from the initial value in all treatments.
The room temperature increased to 23 °C for about 7.5 h.
These deviations are considered to have no impact on quality and integrity of the study.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Appearance: Colourless clear liquid
Purity: 93.3%
Batch no.: 1501442030
Specific details on test material used for the study:
Test item TRIGONOX 421
Chemical name 1,1,3,3-Tetramethylbutyl peroxy-2-ethylhexanoate
Product Name TRIGONOX 421
Batch number 1501442030
CAS number 22288-43-3
Content (certified) 93.3 %
Appearance Colourless, clear, liquid
Water solubility At 5 °C immiscible
Relative Density 0.9 at -10 °C
pH Weakly acidic
Expiry date 2017-04-19
Recommended storage Store at -20 °C
Receipt 2016-09-28
Identification parameter Name, batch number, state, color and turbidity
Retention of the test item At least 1 g has been retained on 2016-10-25 and stored at
-20 ± 2 °C.
Storage conditions -20 ± 2 °C, protected from light, in tightly closed original container

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Determination of the test item
Each concentration level and the control were confirmed by analytical verification via HPLC-DAD. The test item TRIGONOX 421 was analysed in the application solutions subsequently to their preparation on day -1. No specific analysis in the soil was carried out as different extraction methods resulting in low recoveries confirmed the strong binding capacity of the test item to soil. Therefore, the measurement of the application solutions was useful to demonstrate an accurate dosing of the test item

Test substrate

Vehicle:
yes
Details on preparation and application of test substrate:
Application
On day -1, the respective test item amount was weighed out for each test item concentration and dissolved in acetone. 2.5 mL stock solution per replicate were mixed thoroughly in a sub-portion of the artificial soil (10 g per replicate). After complete evaporation of the solvent the treated soil was given to the remaining artificial soil. Subsequently, the test medium was mixed thoroughly to ensure a homogenous distribution. After addition and mixing with the necessary food amount, the soil was covered to prevent any evaporation and stored in the dark under test conditions.
On day 0, after further mixing, a soil amount corresponding to about 600 g soil dry weight was filled into test vessels.

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 - 23 °C
The room temperature increased to 23 °C for about 7.5 hours. This deviation is considered to have no impact on quality and integrity of the study.
pH:
Application rate
[mg TRIGONOX 421/kg SDW] pH-value
day 0 day 56
Control 6.38 6.05
Solvent
Control 6.31 5.98
31.25 6.26 5.94
62.5 6.25 6.11
125 6.23 6.00
250 6.16 5.97
500 6.19 6.05


Moisture:
Moisture and WHCmax of the Test Media
Application rate
[mg TRIGONOX 421/kg SDW] Moisture [%] of dry weight WHCmax of
artificial soil
[g/100 g DW]
day 0 day 56
Control 18.5 24.4* 36.6
Solvent Control 18.7 24.6*
31.25 18.5 24.0*
62.5 18.3 24.2*
125 18.4 24.5*
250 18.4 24.6*
500 18.4 24.4*
WHC = Water Holding Capacity
* = The soil moisture deviated by more than 10 % from the initial value.
Details on test conditions:
Experimental Procedure

Test item TRIGONOX 421

Application rates 31.25 – 62.5 – 125 – 250 – 500 mg/kg soil dry weight

Control Artificial soil moistened with demineralised water without test or reference item was used as control medium

Solvent Control
Artificial soil including a sub-portion of artificial soil spiked with acetone and moistened with demineralised water without test item was used as a solvent control.

Test duration 8 weeks

Number of replicates 8 replicates per control and 4 replicates per test item concentration

Number and age of 10 adult earthworms (with clitellum) aged 2-12 months were used
the earthworms per replicate. The difference of age did not deviate by more than 1 month (see “Synchronisation”).


Body weight (actual) 0.32 to 0.58 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 (24000 g) was pre-moistened with 2136 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 enable sufficient gas exchange, light input and prevent the test substrate from drying.

Application
On day -1, the respective test item amount was weighed out for each test item concentration and dissolved in acetone. 2.5 mL stock solution per replicate were mixed thoroughly in a sub-portion of the artificial soil (10 g per replicate). After complete evaporation of the solvent the treated soil was given to the remaining artificial soil. Subsequently, the test medium was mixed thoroughly to ensure a homogenous distribution. After addition and mixing with the necessary food amount, the soil was covered to prevent any evaporation and stored in the dark under test conditions.
On day 0, after further mixing, a soil amount corresponding to about 600 g soil dry weight was filled into test vessels.

Temperature 20  2 °C

Photoperiod 16 h
Light intensity 400 - 800 lx

Feeding
The earthworms were fed with air-dried cattle manure weekly during the first 4 weeks as presented in the table below. At day -1 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 (see Table 3).

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, 42, 49 – –

Nominal and measured concentrations:
31.25 – 62.5 – 125 – 250 – 500 mg/kg soil dry weight
Reference substance (positive control):
yes
Remarks:
Carbendazim

Results and discussion

Effect concentrationsopen allclose all
Duration:
8 wk
Dose descriptor:
EC50
Effect conc.:
484 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
8 wk
Dose descriptor:
NOEC
Effect conc.:
250 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
8 wk
Dose descriptor:
EC10
Effect conc.:
134 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
8 wk
Dose descriptor:
LOEC
Effect conc.:
500 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
8 wk
Dose descriptor:
NOEC
Effect conc.:
500 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
8 wk
Dose descriptor:
LOEC
Effect conc.:
> 500 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Mortality at end of exposure period:
No evident earthworm mortality (< 10%) was observed in the controls and all test item concentrations
- Total mass of adults at beginning of test:
At test start, the earthworms had individual weights of 0.32 - 0.58 g
- Changes in body weigth of live adults (% of initial weight) at end of exposure period:
There were no statistically significant differences in earthworm body weights in the treatment rates 31.25 to 500 mg TRIGONOX 421/kg soil dry weight compared to the control
- No. of offspring produced:
The reproduction rate (average number of juveniles produced) was 146 in the pooled control and ranged from 65 to 147 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 31.25 to 250 mg TRIGONOX 421/kg SDW compared to the pooled control. However, at the test item concentration 500 mg TRIGONOX 421/kg SDW the earthworm reproduction was statistically significantly reduced. The EC10/50-values for reproduction was determined to be 134 and 484 mg TRIGONOX 421/kg SDW, respectively.
The coefficient of variation calculated for the reproduction of the control was 24.9 % and thus lower than 30 %.

Results with reference substance (positive control):
Reference Item - Reproduction Rate (Number of Juveniles after 8 Weeks)
Application rate
[mg a.i./kg DW] 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.i. = active ingredient 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:
Adult Mortality The arithmetic mean and NOEC were determined. Since no mortality occurred the LCx – values were not determined.
For details, please refer to section 13.

Biomass, Reproduction
Significant differences in biomass and reproduction were determined in comparison with the pooled control group by Williams Multiple Sequential t-test Procedure.
When running the test, a Shapiro-Wilk’s Test on Normal Distribution, a Levene’s Test on Variance Homogeneity (with Residuals) and a Trend Analysis on Contrasts were done first. The -value (acceptable probability of incorrectly concluding that there is a difference) was  = 0.05. For the endpoint of reproduction, the arithmetic mean and the variance (coefficient of variation) per treatment and control were calculated. The EC10,50 - values for reproduction were calculated using Weibull analysis using linear max. likelihood regression.

Software
The data presented in the tables of the report were computer-generated and have been rounded for presentation. Thus, manual
re-calculation of the data based on the results presented in this report may result in minor deviations from these figures.
Calculations were carried out using software
- Excel, MICROSOFT
- ToxRat Professional, TOXRAT SOLUTIONS GMBH

Any other information on results incl. tables

  Adult mortality

No evident earthworm mortality (< 10%) was observed in the controls and all test item concentrations

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

Application rate

Replicate

[mg TRIGONOX 421/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

31.25

0

0

0

0

-

0

62.5

0

0

0

0

0

125

0

0

0

0

0

250

0

0

0

0

0

500

0

10

0

0

2.5

 

 Pathological Symptoms and Changes in Behaviour

No significant pathological symptoms or changes in the behaviour of adult earthworms were observed in the controls and all test item concentrations

 

Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Application rate

Replicate

[mg TRIGONOX 421/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

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

31.25

A

10/10

10/10

10/10

10/10

-

62.5

A

10/10

10/10

10/10

10/10

125

A

10/10

10/10

10/10

10/10

250

A

10/10

10/10

10/10

10/10

500

A

H

10/10

-

9/10

1/10

10/10

-

10/10

-

  A = no obvious pathological symptoms      H = earthworm dead

 Live Weight

At test start, the earthworms had individual weights of 0.32 - 0.58 g. There were no statistically significant differences in earthworm body weights in the treatment rates 31.25 to 500 mg TRIGONOX 421/kg soil dry weight compared to the control.

 Body Weight Changes of the Adult Earthworms   

Application rate

[mg TRIGONOX 421/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

0.43

0.53

0.10

23.3

0.10 ± 0.02

22.2 ± 5.28

2

0.43

0.53

0.10

23.3

3

0.43

0.54

0.11

25.6

4

0.43

0.49

0.06

14.0

5

0.44

0.58

0.14

31.8

6

0.44

0.53

0.09

20.5

7

0.44

0.52

0.08

18.2

8

0.44

0.53

0.09

20.5

Solvent Control

1

0.43

0.50

0.07

16.3

0.10 ± 0.02

22.1 ± 4.34

2

0.43

0.52

0.09

20.9

3

0.44

0.52

0.08

18.2

4

0.44

0.56

0.12

27.3

5

0.43

0.51

0.08

18.6

6

0.43

0.55

0.12

27.9

7

0.44

0.54

0.10

22.7

8

0.44

0.55

0.11

25.0

Pooled Control

-

0.10± 0.02

22.1 ± 4.67

-

31.25

1

0.44

0.54

0.10

22.7

0.10 ± 0.03

23.6 ± 6.73

No

2

0.44

0.54

0.10

22.7

3

0.43

0.57

0.14

32.6

4

0.43

0.50

0.07

16.3

62.5

1

0.44

0.53

0.09

20.5

0.13 ± 0.04

30.6 ± 9.03

No

2

0.44

0.56

0.12

27.3

3

0.43

0.61

0.18

41.9

4

0.43

0.57

0.14

32.6

125

1

0.44

0.52

0.08

18.2

0.10 ± 0.03

22.5 ± 6.87

No

2

0.44

0.52

0.08

18.2

3

0.43

0.52

0.09

20.9

4

0.43

0.57

0.14

32.6

Body Weight Changes of the Adult Earthworms (continued)

 

Application rate

[mg TRIGONOX 421/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]

[%]

250

1

0.44

0.56

0.12

27.3

0.11 ± 0.03

25.3 ± 6.51

No

2

0.44

0.58

0.14

31.8

3

0.43

0.50

0.07

16.3

4

0.43

0.54

0.11

25.6

500

1

0.43

0.56

0.13

30.2

0.13 ± 0.03

29.4 ± 6.08

No

2

0.44

0.56

0.12

27.3

3

0.43

0.59

0.16

37.2

4

0.44

0.54

0.10

22.7

SD = Standard deviation                                                                       

Sig. = Statistical Significance (Williams Multiple Sequential t-test Procedure,α= 0.05) compared to control

Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate

 [mg TRIGONOX 421/kg dry soil weight]

Replicate

Number of juveniles

Mean±SD

CV              [%]

[%]

of pooled control

Sig.

Control

1

120

137 ± 34.0

24.8

-

-

2

175

3

169

4

182

5

102

6

115

7

97

8

138

Solvent Control

31.3

1

115

155 ± 38.8

165 ± 13.8

25.0

8.35

-

111

-

No

2

104

3

136

4

138

5

215

6

162

7

197

8

171

Pooled Control

-

-

146 ± 36.4

24.9

-

-

31.25

1

149

129 ± 19.4

15.1

88.4

No

2

126

3

136

4

103

62.5

1

81

105 ± 27.7

26.4

71.9

No

2

80

3

127

4

130

125

1

156

147 ± 34.2

23.3

101

No

2

124

3

191

4

116

250

1

115

123 ± 26.7

21.7

84.3

No

2

99

3

161

4

116

500

1

63

65 ± 30.0

46.1

44.5

Yes

2

34

3

58

4

106

SD = Standard deviation        CV = Coefficient of variation      

Sig. = Statistical Significance (Williams Multiple Sequential t-Test,α= 0.05) compared to the pooled control

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, TRIGONOX 421 did not induce evident earthworm mortalities (< 10%) nor pathological symptoms or changes in the behaviour of adult earthworms in all test item concentrations. There were no statistically significant differences in earthworm body weights in the test item concentrations 31.25 to 500 mg TRIGONOX 421/kg soil dry weight compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 146 in the pooled control and ranged from 65 to 147 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 31.25 to 250 mg TRIGONOX 421/kg soil dry weight compared to the pooled control. However, at the test item concentration 500 mg TRIGONOX 421/kg soil dry weight the earthworm reproduction was statistically significantly reduced.
Executive summary:

Effects of TRIGONOX 421 on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222 (2016) from 27 January 2017 to 24 March 2017 with the definitive exposure phase from 27 January 2017 to 23/24 March 2017 at the test facility in 31157 Sarstedt, Germany.

 

The study was conducted under static conditions over 8 weeks with the test item concentrations 31.25 – 62.5 – 125 – 250 – 500 mg TRIGONOX 421/kg SDW. Each application rate was mixed into artificial soil containing 5 % peat. A control and a solvent control including untreated artificial soil was tested under the same conditions as the test item treatments. 80 test organisms were inserted into 8 control and 8 solvent control replicates each and 40 test organisms were divided into 4 replicates for each test item treatment (10 earthworms per replicate). They had an individual body weight between 0.32 and 0.58 g at experimental starting.

 

Each concentration level and control were analysed via HPLC-DAD analysis subsequently to their preparation on day -1.

The measured concentrations of the test item stock solutions were within the range of 94 to 99% of the nominal values, demonstrating the right preparation of the tested concentrations. 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, solvent control or all test item concentrations. There were no statistically significant differences in earthworm body weights in the test item concentrations 31.25 to 500 mg TRIGONOX 421/kg soil dry weight compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 146 in the pooled control and ranged from 65 to 147 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 31.25 to 250 mg TRIGONOX 421/kg soil dry weight compared to the pooled control. However, at the test item concentration 500 mg TRIGONOX 421/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

TRIGONOX 421

Application rates
[mg TRIGONOX 421/kg
 SDW]

31.25

62.5

125

250

500

Mortality of adult earthworms

No

No

No

No

No

Body Weight Gain

No

No

No

No

No

Reduced reproduction rate
(Number of juveniles)

No

No

No

No

Yes

Summary of all effects

No

No

No

No

Yes

 

NOEC, LOEC and EC-values

Endpoint

[mg TRIGONOX 421/kg SDW]

LOECmortality, biomass

>500

NOECmortality, biomass

500

LOECreproduction

500

NOECreproduction

250

EC-valuesreproduction

(95 % Confidence Interval)

 

EC10: 134 (not determinable)

EC50: 484 (not determinable)