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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:
2015-02-25 to 2015-04-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Study according to OECD guidelines
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Deviations:
no
Principles of method if other than guideline:
N/A
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Soybeanoil, epoxidized is a clear, viscous, colourless liquid with 100% purity.
Batch number 2014234102
Expiry date 2015-12-02
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
N/A

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
At experimental start and experimental completion, pH-value and moisture content of the test medium was determined in every treatment and control from pooled samples of all replicates. The water content of each test container was checked weekly by weighing. The weight loss was replenished with the appropriate amount of demineralised water. The body weight of the adult earthworms was individually determined on day 0 and day 28 for each test container. After the first four weeks the adult earthworms were removed. Mortality and morphological changes of the adult earthworms were recorded for each test container. After an additional four weeks, the number of offspring hatched from the cocoons was counted for each test container.

Test substrate

Vehicle:
no
Details on preparation and application of test substrate:
The respective test item amount was weighed out for each test item concentration and mixed with 40 g quartz sand (10 g per replicate). The spiked quartz sand was mixed with the artificial soil. Additional demineralised water was then added to the artificial soil to adjust the humidity of the artificial soil to a moisture content of about 54 % 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 added into test vessels.

Test organisms

Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
Organisms of the species Eisenia fetida were bred at the test facility in covered plastic vessels containing potting compost. 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. During breeding, the earthworms are fed with dried litter of stinging nettle and porridge oats. After test commencement the earthworms were fed with cattle manure during the first 4 weeks.

Study design

Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
8 wk
Post exposure observation period:
N/A

Test conditions

Test temperature:
Nominal range 18 - 22°C, measured range 17 – 23.5 °C, the room temperature increased to 23.5 °C for about 8 hours and decreased to 17 °C for about 17 hours. These deviations are not considered to have any impact on quality and integrity of the study.

pH:
Start of exposure period (day 0) - 6.90 - 6.95, end of exposure period (day 56) 6.70 - 6.82
Moisture:
Start of exposure period (day 0) - 18.3 - 20.8 % of dry weight, end of exposure period (day 56) - 23.6 - 28.1 % of dry weight
Details on test conditions:
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. There were 8 control replicates and 4 replicates per treatment concentration. Test units 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. Each test chamber contained about 600 g soil dry weight. Transparent and perforated lids enabled sufficient gas exchange, light input and prevented the test substrate from drying. The photoperiod was 16 h with a light intensity of 400 - 800 lx. Throughout the test the replicates were weighed weekly. Evaporated water was added to maintain the soil moisture.

Ten earthworms were added to each replicate, earthworms had an individual bodu weight between 0.31 and 0.51 g at the start of the experimental phase.

Nominal and measured concentrations:
Nominal concentrations were 62.5, 125, 250, 500 and 1000 mg Soybeanoil, epoxidized/kg soil dry weight.
Reference substance (positive control):
yes
Remarks:
Carbendazim

Results and discussion

Effect concentrationsopen allclose all
Duration:
8 wk
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
8 wk
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Key result
Duration:
8 wk
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
After 28 days of exposure in soil, no earthworm mortalities, significant pathological symptoms or changes in the behaviour of adult earthworms were observed in any of the treatments. Moreover, no statistically significant differences in earthworm body weights were observed at all application rates compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 37 in the control and ranged between 39 and 54 in the test item treatment rates. Compared to the control, no statistically significant reduction of reproduction was observed in any of the test item concentrations. The coefficient of variation calculated for the reproduction of the control was 20.2 % and thus lower than 30 %. Overall, the NOEC of the test item concerning mortality, biomass and reproduction was determined to be 1000 mg/kg soil dry weight. The EC50-value for biomass and reproduction was not determined since no significant effects ≥ 50 % occurred.
Results with reference substance (positive control):
Reference Item - Reproduction Rate (Number of Juveniles after 8 Weeks)
Application rate
[mg a.i./kg DW] Mean ± SD CV % of control Statistical Significance
Control 69 ± 16.0 23.1 - -
0.5 63 ± 18.9 29.9 91.3 No
1 45 ± 8.73 19.4 65.2 Yes
2 0.25 ± 0.50 n.d. 0 Yes
4 0 ± 0 n.d. 0 Yes

The results of the reference test indicate that the response of earthworms to carbendazim is acceptable according to the test guideline (OECD 222).
Reported statistics and error estimates:
Significant differences in biomass and reproduction were determined in comparison to the control group using Williams Multiple Sequential t-test and Welch t-test. For the endpoint of reproduction, the arithmetic mean and the variance (coefficient of variation) per treatment and control were calculated. Calculations were carried out using software Excel, MICROSOFT and ToxRat Professional, TOXRAT SOLUTIONS GMBH.

Any other information on results incl. tables

 

Table 1:  Mortality of Adult Earthworms in [%] after 28 Days of Exposure

Application rate [mg/kg dry soil weight]

Mean

Control

0

62.5

0

125

0

250

0

500

0

1000

0

 

Table 2:  Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Application rate

Replicate

[mg/kg dry soil weight]

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

62.5

A

10/10

10/10

10/10

10/10

125

A

D / G

10/10

-

10/10

-

10/10

-

9/10

1/10

250

A

10/10

10/10

10/10

10/10

500

A

10/10

10/10

10/10

10/10

1000

A

10/10

10/10

10/10

10/10

 

 

Table 3: Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate

 [mg/kg dry soil weight]

Replicate

Number of juveniles

Mean±SD

CV [%]

[%]

of control

Sig.

Control

1

35

37 ± 7.46

20.2

-

-

2

31

3

51

4

33

5

45

6

31

7

31

8

35

62.5

1

56

39 ± 11.8

30.2

105

No

2

31

3

31

4

39

125

1

42

42 ± 6.02

14.3

114

No

2

36

3

39

4

50

250

1

46

53 ± 6.16

11.6

143

No

2

61

3

52

4

53

500

1

60

44 ± 15.2

34.6

119

No

2

54

3

30

4

32

1000

1

38

54 ± 17.8

32.9

146

No

2

79

3

46

4

54

SD = Standard deviation        CV = Coefficient of variation    

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

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of Soybeanoil, epoxidized to the earthworm species Eisenia fetida was determined in a static 8 week test. The mortality, biomass and reproduction NOECs of soybeanoil, epoxidised concerning were determined to be 1000 mg/kg soil dry weight. The EC50 values for biomass and reproduction were not determined since no significant effects ≥ 50 % occurred. The reproduction of the earthworms was not statistically significantly reduced in any of the treatments compared to the control.
Executive summary:

The toxicity of Soybeanoil, epoxidized to the earthworm species Eisenia fetida was determined in a static 8 week test according to OECD Guideline 222 (2004). Nominal exposure concentrations were 62.5, 125, 250, 500 and 1000 mg Soybeanoil, epoxidized/kg soil dry weight. Each test concentration was replicated 4 times with 8 control replicates, each replicate contained ten earthworms. After 28 days of exposure in soil, no earthworm mortalities, no significant pathological symptoms or changes in the behaviour of adult earthworms were observed in any of the treatment concentrations. Moreover, no statistically significant differences in earthworm body weights were observed in any treatment compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 37 in the control and was 39, 42, 53, 44 and 54 in the 62.5, 125, 250, 500 and 1000 mg/kg dry weight soil treatment rates. Compared to the control, no statistically significant reduction of reproduction was observed in any of the soybeanoil, epoxidized concentrations. The mortality, biomass and reproduction NOECs of soybeanoil, epoxidized concerning were determined to be 1000 mg/kg soil dry weight. The EC50 values for biomass and reproduction were not determined since no significant effects ≥ 50 % occurred. All validity criteria recommended by the test guideline were fulfilled.