Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-enenitrile

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-02 to 2018-01-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentration levels and the controls were analytically verified at day 0 and 28. Additionally, the treated quartz sand was analysed at day 0.
- Sampling method: The samples were analysed with a GC-MS method
- Sample storage conditions before analysis: all samples were stored at room temperature

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

The respective test item amount (2440 mg, actual: 2440.1 mg) was weighed out for a stock solution and solved in acetone (100 mL). The stock solution was further diluted to achieve the respective spiking solutions. The appropriate amount of spiking solution (2.5 mL per replicate) was mixed thoroughly with quartz sand (10 g per replicate). Additionally 40 g quartz sand per test item concentration and solvent control were treated likewise and used for analytical measurements. After complete evaporation of the solvent, the spiked quartz sand was added to the artificial soil. Demineralised water was added to the artificial soil 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 610 g soil dry weight were filled into each test vessel.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: earthworm
- Age at test initiation: adult earthworms (with clitellum) aged 2 - 12 months
- Weight at test initiation: 0.37 to 0.54 g

ACCLIMATION
- Acclimation period: one day prior to test start
- Acclimation conditions: same as test
- Health during acclimation, any mortality observed: no

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Test temperature:

20 +/- 2 °C

pH:

6.32

Moisture:

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.

Details on test conditions:

TEST SYSTEM
- Test container: Test units, containing about 610 g soil dry weight, were glass vessels (volume 1.5 L, used for preserving). One transparent and perforated lid and one additional transparent lid enabled sufficient gas exchange and light input thereby preventing the test substrate from drying.
- No. of organisms per container: 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- No. of replicates per vehicle control: 8

OTHER TEST CONDITIONS
- Photoperiod: 16 h
- Light intensity: 400 - 800 lx

EFFECT PARAMETERS MEASURED:
A) no obvious pathological symptoms
B) no reaction to touching
C) no negative phototactical reaction
D) spontaneous segmentation and separation
E) spasmodic winding
F) yellow excretion from the oral aperture
G) ulcer and skin bleedings
H) dead earthworms

VEHICLE CONTROL PERFORMED: yes

Nominal and measured concentrations:

Nominal: 0.78; 1.56; 3.125; 6.25; 12.5; 25; 50; 100 mg/kg soil dry weight
measured at start of exposure: 202; 104; 119; 105; 109; 111; 119; 105 % respectively
measured 28 days after exposure:

Reference substance (positive control):

yes

Remarks:

Carbendazim

Key result

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, biomass, and reproduction

Remarks on result:

other: no significant effects occurred at the highest concentration tested

Details on results:

Please refer to respective tables under "any other information on results including tables"

Results with reference substance (positive control):

Results with reference substance valid: yes

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

Application rate	Replicate
Mg test item/kg soil dry weight	1	2	3	4	5	6	7	8	Mean
Control	0	0	0	0	10	0	0	0	1.215
Solvent Control	0	0	0	0	0	0	0	0	0
0.78	0	0	0	0	-				0
1.56	0	0	0	0					2.5
3.125	0	0	0	10					0
6.25	0	0	0	0					0
12.5	0	0	0	0					0
25	0	0	0	0					0
50	0	10	0	0					2.5
100	0	0	0	0					0

 

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

Application rate	Replicate
Mg test item/kg soil dry weight	Observation parameter	1	2	3	4	5	6	7	8
Control	A H	10/10 -	10/10 -	10/10 -	10/10 -	9/10 1/10	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
0.78	A	10/10	10/10	10/10	10/10	-
1.56	A	10/10	10/10	10/10	10/10
3.125	A H	10/10 -	10/10 -	10/10 -	9/10 1/10
6.25	A	10/10	10/10	10/10	10/10
12.5	A	10/10	10/10	10/10	10/10
25	A	10/10	10/10	10/10	10/10
50	A H	10/10 -	9/10 1/10	10/10 -	10/10 -
100	A	10/10	10/10	10/10	10/10

A = no obvious pathological symptoms; H = earthworm dead

 

 

 

 

 

 

Table 3 Mean Body Weight Changes of the Adult Earthworms

 

Application rate Mg test item/kg soil dry weight	Mean body weight change of earthworms (Test Start – 28 days +/- SD	In percent	Statistical significance (Dunnett´s Multiple t-test Procedure, alpha = 0.05 compared to solvent control
Control	0.05 +/- 0.04	12.1 +/- 8.98	-
Solvent Control	0.11 +/- 0.03	23.9 +/- 6.68	-
0.78	0.09 +/- 0.01	20.3 +/- 2.83	No
1.56	0.12 +/- 0.02	25.9 +/- 4.87	No
3.125	0.09 +/- 0.05	20.3 +/- 10.4	No
6.25	0.14 +/- 0.02	30.9 +/- 3.31	No
12.5	0.12 +/- 0.03	25.9 +/- 7.29	No
25	0.12 +/- 0.03	25.9 +/- 6.76	No
50	0.09 +/- 0.03	20.8 +/- 6.79	No
100	0.12 +/- 0.03	27.0 +/- 6.93	No

 

 

Table 4 Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate Mg test item/kg soil dry weight	Mean +/- SD	CV [%]	[%] of pooled control	Statistical significance (Dunnett´s Multiple t-test Procedure, alpha = 0.05 compared to solvent control
Control	133 +/- 26.2	19.7	-	-
Solvent Control	139 +/-16.7	12.0	-	-
Pooled control	136 +/- 21.4	15.7	-	-
0.78	155 +/- 35.6	23.0	114	No
1.56	101 +/- 42.4	42.0	74.3	No
3.125	145 +/-8.18	5.64	107	No
6.25	149 +/- 14.3	9.57	110	No
12.5	151 +/- 44.6	29.5	111	No
25	132 +/- 23.8	18.0	97.1	No
50	119 +/-55.2	46.4	87.5	No
100	131 +/- 31.2	23.8	96.3	No

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the test item 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 at all test item concentrations compared to the solvent control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 136 in the pooled control and ranged from 101 to 155 in the test item treatment rates. There was no statistically significant difference in earthworm reproduction in all treatment rates compared to the pooled control. The NOEC concerning mortality, biomass and reproduction was determined to be > 100 mg/kg soil dry weight. Since no significant effects occurred no LC/ECX - values were determined.
All validity criteria recommended by the test guideline were fulfilled.

Executive summary:

Effects of the test item on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222 (2016). The study was conducted under static conditions over 8 weeks with the test item concentrations 0.78 - 1.56 - 3.125 - 6.25 - 12.5 - 25 - 50 - 100 mg/kg soil dry weight (SDW). Each application rate was mixed into artificial soil containing 5 % peat. A control using untreated artificial soil as well as a solvent control using artificial soil spiked with the solvent was tested under the same conditions as the test item treatments. For the controls 80 test organisms were inserted into 8 replicates, each. 40 test organisms were divided into 4 replicates for each treatment (10 earthworms per replicate). They had an individual body weight between 0.37 and 0.54 g at experimental starting.

Each concentration level, control and solvent control were analysed via GC-MS analysis subsequently to their preparation on day 0 and after 28 days. The recovery rates at day 0 were between 104 and 119 % of nominal concentrations with one exceptional value of 202 % measured in the lowest test item concentration. After 28 days recovery rates were between < LOQ and 9 %. Control and solvent control samples were < LOQ.

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

Since no statistically significant differences concerning mortality after four weeks and reproduction after eight weeks of exposure in soil were observed between the control and the solvent control, the two controls were pooled for these parameters.

Neither evident earthworm mortalities (< 10 %) nor pathological symptoms or changes in the behaviour of adult earthworms were observed in the controls and all test item concentrations. Since a statistically significant difference concerning biomass after four weeks of exposure in soil was observed between the control and the solvent control, the solvent control was used for further evaluation. There were no statistically significant differences in earthworm body weights at all test item concentrations compared to the solvent control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 136 in the pooled control and ranged from 101 to 155 in the test item treatment rates. There was no statistically significant difference in earthworm reproduction in all treatment rates compared to the pooled control. The NOEC concerning mortality, biomass and reproduction was determined to be > 100 mg/kg soil dry weight. Since no significant effects occurred no LC/ECX - values were determined. All validity criteria recommended by the test guideline were fulfilled.

Description of key information

Under the conditions of a OECD 222 test, the test item did not induce evident earthworm mortalities (< 10 %) nor pathological symptoms or changes in the behaviour of adult earthworms in all test item concentrations. The NOEC concerning mortality, biomass and reproduction was determined to be > 100 mg/kg soil dry weight. Since no significant effects occurred no LC/ECX - values were determined.

Key value for chemical safety assessment

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

100 mg/kg soil dw

Additional information

Effects of the test item on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222 (2016). The study was conducted under static conditions over 8 weeks with the test item concentrations 0.78 - 1.56 - 3.125 - 6.25 - 12.5 - 25 - 50 - 100 mg/kg soil dry weight (SDW). Each application rate was mixed into artificial soil containing 5 % peat. A control using untreated artificial soil as well as a solvent control using artificial soil spiked with the solvent was tested under the same conditions as the test item treatments. For the controls 80 test organisms were inserted into 8 replicates, each. 40 test organisms were divided into 4 replicates for each treatment (10 earthworms per replicate). They had an individual body weight between 0.37 and 0.54 g at experimental starting.

Each concentration level, control and solvent control were analysed via GC-MS analysis subsequently to their preparation on day 0 and after 28 days. The recovery rates at day 0 were between 104 and 119 % of nominal concentrations with one exceptional value of 202 % measured in the lowest test item concentration. After 28 days recovery rates were between < LOQ and 9 %. Control and solvent control samples were < LOQ.

The method was validated according to SANCO/3029/99 rev.4 (2000) prior to the start of exposure. Since no statistically significant differences concerning mortality after four weeks and reproduction after eight weeks of exposure in soil were observed between the control and the solvent control, the two controls were pooled for these parameters. Neither evident earthworm mortalities (< 10 %) nor pathological symptoms or changes in the behaviour of adult earthworms were observed in the controls and all test item concentrations. Since a statistically significant difference concerning biomass after four weeks of exposure in soil was observed between the control and the solvent control, the solvent control was used for further evaluation. There were no statistically significant differences in earthworm body weights at all test item concentrations compared to the solvent control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 136 in the pooled control and ranged from 101 to 155 in the test item treatment rates. There was no statistically significant difference in earthworm reproduction in all treatment rates compared to the pooled control. The NOEC concerning mortality, biomass and reproduction was determined to be > 100 mg/kg soil dry weight. Since no significant effects occurred no LC/ECX - values were determined. All validity criteria recommended by the test guideline were fulfilled.