N-[3-(dimethylamino)propyl] C6-9 alkyl amides

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:

2018-10-25 to 2018-12-21 with the definitive exposure phase from 2018-10-26 to 2018-12-20

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:

yes

Remarks:

Food was provided on day 0 (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 6 h.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

LC-MS/MS analysis

Details on sampling:

Each concentration level and control were analysed via LC-MS/MS analysis on day 0, day 28 and at test end using pooled samples of all replicates. Additionally, on day of application, spiked quartz sand of the control, solvent control as well as of the test item concentration1000 mg/kg sdw were sampled.

Vehicle:

yes

Remarks:

Acetone, Quartz sand

Details on preparation and application of test substrate:

One day before insertion of the earthworms, the respective test item amount (nominal: 12200 mg/25 mL, actual: 12200.1 mg/25 mL) was weighed out for a stock solution and dissolved in acetone. The stock solution was further diluted with acetone to receive 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). After complete evaporation of the solvent, the spiked quartz sand was added to the artificial soil (2400 g sdw). Demineralised water (248 g) was added to adjust the humidity of the artificial soil to a moisture of 54 % of the WHCmax. Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and was stored covered under test conditions until further use. On day 0, after further mixing, 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: Eisenia fetida (Annelida, Lumbricidae)
Source: Breeding stock culture maintained at the test facility
Age at test initiation: Adult
Weight at test initation (mean and range): mean 0.47 g, range 0.40 - 0.54 g

ACCLIMATION
Acclimation period: 2 days
Acclimation conditions (same as test or not): not; photoperiod 24 hours instead of 16 hours
Health during acclimation (any mortality observed): no mortality observed

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Test temperature:

18 - 23 °C

pH:

Application rate
[mg C-6410/kg SDW]
pH-value
day 0 day 55
Control 5.69 6.28
Solvent Control 5.61 6.20
62.5 5.66 6.26
125 5.65 6.17
250 5.66 6.26
500 5.69 6.43
1000 5.75 6.42

Moisture:

Application rate
[mg C-6410/kg SDW]
Moisture [%] of dry weight
day 0 day 55
Control 18.2 21.7*
Solvent Control 18.5 21.0*
62.5 18.4 20.4*
125 18.0 20.1*
250 17.2 19.4*
500 17.2 19.5*
1000 17.2 19.8*
* = 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 SYSTEM
- Test container (material, size): Test units containing about 610 g soil dry weight were round plastic boxes with an inner diameter of 15.0 cm corresp onding to a bottom surface area of 177 cm2 and a height of 14 cm. Transparent and perforated lids enable sufficient g as exchange, light input and prevent the test substrate from drying.
- Amount of soil or substrate: 610 g sdw per replicate
- No. of organisms per container (treatment): 10 per replicate
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

ARTIFICIAL SOIL
Artificial soil consisted of the following components:
- 5 % peat, air-dried and finely ground
- 20 % kaolin, clay, kaolinite content > 30 %
- 74 % air-dried quartz sand (sand with > 50 % particle size of
0.05 - 0.2 mm)
- 0.18 % 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.


OTHER TEST CONDITIONS
- Photoperiod: 16 h
- Light intensity: 639 ± 88.4 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The body weight of the adult earthworms was determined individually on day 0 and day 28 for each replicate.
After the first four weeks, the adult earthworms were removed. Mortality and morphological changes of the adult earthworms were recorded for each replicate. After a further four weeks, the number of offspring (juveniles) hatched from the cocoons was counted for each test container.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: factor 2
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: 1 - 10 - 100 - 1000 mg/kg soil dry weight
- Results used to determine the conditions for the definitive study:

Range Finding Test (28 days):
Mortality and Change of Live Weight
Concentration Mortality of adult earthworms Change of Live weight *
[mg test item/kg soil dry weight] [%] [%]
Control 0 -9
1 0 -11
10 5 -11
100 0 -8
1000 0 -4
*) negative value = decrease of live weight

Nominal and measured concentrations:

Sampling date Day 0 Day 28 Day 55
Nominal test item
concentration
[mg/kg] C-6410
Meas. conc. [mg/kg] [%] Meas. conc. [mg/kg] [%] Meas. conc. [mg/kg] [%]
1000 1097 110 124 12 11.1 1
500 434 87 64.9 13 7.40* 1
250 253 101 29.3 12 < LOQ
125 120 96 19.2 15 < LOQ
62.5 59.4 95 12.7 20 < LOQ
Solvent Control < LOQ < LOQ < LOQ
Control < LOQ < LOQ < LOQ

Reference substance (positive control):

yes

Remarks:

Carbendazim

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Mortality, behaviour, pathological symptoms, change of body weight

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Mortality, behaviour, pathological symptoms, change of body weight

Key result

Duration:

8 wk

Dose descriptor:

LOEC

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

125 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

8 wk

Dose descriptor:

EC10

Effect conc.:

45.7 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CI 10.8 - 193

Key result

Duration:

8 wk

Dose descriptor:

other: EC 20

Effect conc.:

89.6 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CI 21.7 - 377

Key result

Duration:

8 wk

Dose descriptor:

EC50

Effect conc.:

325 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CI 53.3 - > 1000

Details on results:

- Mortality at end of exposure period:
No evident earthworm mortality (< 10%) was observed in the controls and all test item concentrations


- Changes in body weight of live adults (% of initial weight) at end of exposure period:
At test start, the earthworms had individual weights of 0.40 - 0.54 g.

Body Weight Changes of the Adult Earthworms
Application rate
[mg C-6410/kg SDW]
Replicate Mean body weights per replicate Mean body weight change of earthworms Per replicate
Test start 28 days Mean ± SD Sig.
[g] [g] [g] [%] [g] [%]
Control 1 0.47 0.55 0.08 17.0 0.05 ± 0.02 9.66 ± 3.96 –
2 0.46 0.52 0.06 13.0
3 0.47 0.51 0.04 8.5
4 0.46 0.51 0.05 10.9
5 0.47 0.51 0.04 8.5
6 0.46 0.48 0.02 4.3
7 0.47 0.50 0.03 6.4
8 0.46 0.50 0.04 8.7
Solvent Control 1 0.46 0.52 0.06 13.0 0.06 ± 0.01 12.4 ± 3.07 –
2 0.47 0.53 0.06 12.8
3 0.46 0.54 0.08 17.4
4 0.47 0.51 0.04 8.5
5 0.46 0.53 0.07 15.2
6 0.47 0.51 0.04 8.5
7 0.46 0.51 0.05 10.9
8 0.47 0.53 0.06 12.8
Pooled Control - 0.05 ± 0.02 11.0 ± 3.70 -
62.5 1 0.47 0.53 0.06 12.8 0.08 ± 0.02 17.2 ± 3.91 No
2 0.46 0.55 0.09 19.6
3 0.47 0.57 0.10 21.3
4 0.46 0.53 0.07 15.2
125 1 0.47 0.53 0.06 12.8 0.05 ± 0.02 10.8 ± 3.96 No
2 0.46 0.53 0.07 15.2
3 0.47 0.51 0.04 8.5
4 0.46 0.49 0.03 6.5
250 1 0.47 0.55 0.08 17.0 0.05 ± 0.02 11.3 ± 3.94 No
2 0.46 0.50 0.04 8.7
3 0.47 0.52 0.05 10.6
4 0.46 0.50 0.04 8.7
500 1 0.46 0.54 0.08 17.4 0.08 ± 0.02 17.2 ± 4.74 No
2 0.47 0.56 0.09 19.1
3 0.46 0.56 0.10 21.7
4 0.47 0.52 0.05 10.6
1000 1 0.47 0.56 0.09 19.1 0.09 ± 0.01 18.8 ± 2.14 No
2 0.46 0.56 0.10 21.7
3 0.47 0.55 0.08 17.0
4 0.46 0.54 0.08 17.4
SD = Standard deviation
Sig. = Statistical Significance (Dunnett’s Multiple t-test Procedure, α = 0.05) compared to pooled control


- No. of offspring produced:
The reproduction rate (average number of juveniles produced) was 75 in the pooled control and ranged from 12 to 59 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rate 62.5 and
125 mg C-6410/kg soil dry weight compared to the pooled control. However, at the test item concentrations 250 to 1000 mg C-6410/kg soil dry weight the earthworm reproduction was statistically significantly reduced


- Morphological/behavioural abnormalities:
No significant pathological symptoms or changes in the behaviour of adult earthworms were observed in the controls and all test item concentrations

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels:
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 64 65 ± 14.0 21.5 _ _
2 58
3 55
4 47
5 80
6 78
7 55
8 85
0.5 1 48 56 ± 10.9 19.4 86.2 No
2 72
3 51
4 53
1 1 38 57 ± 14.1 24.7 87.7 No
2 58
3 60
4 72
2 1 35 38 ± 10.3 27.1 58.5 Yes
2 53
3 29
4 36
4 1 16 15 ± 2.2 14.8 23.1 Yes
2 13
3 14
4 18
Rep. = Replicate a.s. = active substance SD = Standard deviation CV = Coefficient of variation
*) = Statistically significantly reduced compared to control (Williams Multiple Sequential t-test Procedure)

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, LOEC and NOEC were determined. Since no mortality occurred the LCx – values were not determined.

Reproduction
Since no statistical difference was determined between the control and the solvent control, both controls were pooled for further evaluation.
- Shapiro-Wilk’s Test on Normal Distribution
- Levene’s Test on Variance Homogeneity (with Residuals)
- Dunnett’s Multiple t-test Procedure
The alpha value (acceptable probability of incorrectly concluding that there is a difference) was alpha = 0.05. For the endpoint of reproduction, the arithmetic mean and the variance (coefficient of variation) per treatment and control were calculated. The EC10, 20, 50 -values for reproduction were calculated using 3-param.normal CDF.

Biomass
Since no statistical difference was determined between the control and the solvent control, both controls were pooled for further evaluation.
- Shapiro-Wilk’s Test on Normal Distribution
- Levene’s Test on Variance Homogeneity (with Residuals)
- Dunnett’s Multiple t-test Procedure
The alpha-value (acceptable probability of incorrectly concluding that there is a difference) was alpha = 0.05. For the endpoint of biomass, the arithmetic mean and the variance (coefficient of variation) per treatment and control were calculated.

Application rate	Replicate
[mgC-6410/kg SDW]	1	2	3	4	5	6	7	8	Mean
Control	0	40	0	0	0	0	0	0	5
Solvent Control	0	0	0	0	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	0	0	0					0
1000	0	10	0	0					2.5

Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Application rate       Replicate

[mg C-6410/kg  SDW]

Observation parameter       1       2       3       4       5       6       7       8

Control       A

H       10/10

-       6/10

4/10       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

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       10/10       10/10       10/10       10/10       

1000       A

H       10/10

-       9/10

1/10       10/10

-       10/10

-       

A = no obvious pathological symptoms       H = earthworm dead

Table 6:         Body Weight Changes of the Adult Earthworms   

Application rate [mg C-6410/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	0.55	0.08	17.0	0.05 ± 0.02	9.66 ± 3.96	–
	2	0.46	0.52	0.06	13.0
	3	0.47	0.51	0.04	8.5
	4	0.46	0.51	0.05	10.9
	5	0.47	0.51	0.04	8.5
	6	0.46	0.48	0.02	4.3
	7	0.47	0.50	0.03	6.4
	8	0.46	0.50	0.04	8.7
Solvent Control	1	0.46	0.52	0.06	13.0	0.06 ± 0.01	12.4 ± 3.07	–
	2	0.47	0.53	0.06	12.8
	3	0.46	0.54	0.08	17.4
	4	0.47	0.51	0.04	8.5
	5	0.46	0.53	0.07	15.2
	6	0.47	0.51	0.04	8.5
	7	0.46	0.51	0.05	10.9
	8	0.47	0.53	0.06	12.8
Pooled Control	-					0.05 ± 0.02	11.0 ± 3.70	-
62.5	1	0.47	0.53	0.06	12.8	0.08 ± 0.02	17.2 ± 3.91	No
	2	0.46	0.55	0.09	19.6
	3	0.47	0.57	0.10	21.3
	4	0.46	0.53	0.07	15.2
125	1	0.47	0.53	0.06	12.8	0.05 ± 0.02	10.8 ± 3.96	No
	2	0.46	0.53	0.07	15.2
	3	0.47	0.51	0.04	8.5
	4	0.46	0.49	0.03	6.5
250	1	0.47	0.55	0.08	17.0	0.05 ± 0.02	11.3 ± 3.94	No
	2	0.46	0.50	0.04	8.7
	3	0.47	0.52	0.05	10.6
	4	0.46	0.50	0.04	8.7

SD = Standard deviation                                                                      

Sig. = Statistical Significance (Dunnett’s Multiple t-test Procedure,α= 0.05) compared to pooled control

Table 6:         Body Weight Changes of the Adult Earthworms (continued)

 

Application rate [mgC-6410/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]	[%]
500	1	0.46	0.54	0.08	17.4	0.08 ± 0.02	17.2 ± 4.74	No
	2	0.47	0.56	0.09	19.1
	3	0.46	0.56	0.10	21.7
	4	0.47	0.52	0.05	10.6
1000	1	0.47	0.56	0.09	19.1	0.09 ± 0.01	18.8 ± 2.14	No
	2	0.46	0.56	0.10	21.7
	3	0.47	0.55	0.08	17.0
	4	0.46	0.54	0.08	17.4

SD = Standard deviation                                                                         

Sig. = Statistical Significance (Dunnett’s Multiple t-test Procedure,α= 0.05) compared to pooled control

Table7: Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate [mgC-6410/kg dry soil weight]	Replicate	Number of juveniles	Mean±SD	CV              [%]	[%] of pooled control	Sig.
Control	1	53	72 ± 21.0	29.2	-	-
	2	47
	3	56
	4	83
	5	89
	6	108
	7	62
	8	78
Solvent Control	1	53	79 ± 22.4 165 ± 13.8	28.3 8.35	- 111	- No
	2	90
	3	52
	4	82
	5	104
	6	54
	7	97
	8	99
Pooled Control	-	-	75 ± 21.3	28.4	-	-
62.5	1	54	59 ± 11.3	19.1	78.7	No
	2	75
	3	49
	4	59
125	1	37	55 ± 20.0	36.4	73.3	No
	2	39
	3	70
	4	75
250	1	28	47 ± 20.4	43.3	62.7	Yes
	2	31
	3	62
	4	67
500	1	30	32 ± 16.4	51.3	42.7	Yes
	2	11
	3	34
	4	51
1000	1	22	12 ± 7.68	64.0	16.0	Yes
	2	9
	3	14
	4	4

SD = Standard deviation       CV = Coefficient of variation      

Sig. = Statistical Significance (Dunnett’s Multiple t-test Procedure,α= 0.05) compared to the pooled control

Table 8:         Measured Concentrations and Percent of Nominal concentration

ofC-6410in quartz sand on day -1

 

Sampling date		Day -1
Nominal concentration of the test item		C-6410
[mg/kg soil dry weight]	[mg/kg quartz sand]^1)	Meas. conc. [mg/kg quartz sand]	[%]
1000	61000	48865	80
Solvent Control		< LOQ
Control		< LOQ

 

¹⁾                                      = based on 40 g quartz sand in 2440 g artificial soil

Meas. conc.      = measured concentration of the test item

%                        = Percent of the nominal concentration of the test item

LOQ                   =Limit of quantification of the analytical method (10 mg test item/kg)

 

Table 9:         Measured Concentrationsand Percent of Nominal concentration ofC-6410

during the definitive test

 

Sampling date	Day 0		Day 28		Day55
Nominal test item concentration [mg/kg]	C-6410
	Meas. conc. [mg/kg]	[%]	Meas. conc. [mg/kg]	[%]	Meas. conc. [mg/kg]	[%]
1000	1097	110	124	12	11.1	1
500	434	87	64.9	13	7.40^*	1
250	253	101	29.3	12	< LOQ
125	120	96	19.2	15	< LOQ
62.5	59.4	95	12.7	20	< LOQ
Solvent Control	< LOQ		< LOQ		< LOQ
Control	< LOQ		< LOQ		< LOQ

 

Meas. conc.      = measured concentration of the test item

%                        = Percent of the nominal concentration of the test item

LOQ                   =Limit of quantification of the analytical method (10 mg test item/kg)

*                          = < LOQ but > 70% LOQ

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, C-6410 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 all test item concentrations compared to the pooled control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 75 ± 21.3 in the pooled control and ranged from 12 to 59 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 62.5 and 125 mg C-6410/kg soil dry weight compared to the pooled control. However, at the test item concentrations 250 to 1000 mg
C-6410/kg soil dry weight the earthworm reproduction was statistically significantly reduced.

Executive summary:

Effects of C-6410 on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222 (2016) from 2018-10-25 to 2018-12-21 with the definitive exposure phase from 2018-10-26 to 2018-12-20 at the test facility in 31157 Sarstedt, Germany.

The study was conducted under static conditions over 8 weeks with the test item concentrations 62.5 – 125 – 250 – 500 - 1000 mg C-6410/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. 40 test organisms were divided into 4 replicates for each test item treatment (10 earthworms per replicate). They had an individual body weight between 0.40 and 0.54 g at experimental starting.

Each concentration level and control were analysed via LC-MS/MS analysis on day 0, day 28 and at test end using pooled samples of all replicates. Additionally, on day of application, spiked quartz sand of the control, solvent control as well as of the test item concentration1000 mg/kg sdw were sampled.

The analytical results are presented in part 6.1.4. 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 all test item concentrations compared to the pooled control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 75 in the pooled control and ranged from 12 to 59 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rate 62.5 and

125 mg C-6410/kg soil dry weight compared to the pooled control. However, at the test item concentrations 250 to 1000 mg C-6410/kg soil dry weight the earthworm reproduction was statistically significantly reduced.

All validity criteria recommended by the test guidelines were fulfilled.

Table1:               Summary of All Observed Effects in the Application rates

Effects	C-6410
Application rates [mg/kg SDW]	62.5	125	250	500	1000
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	Yes	Yes	Yes
Summary of all effects	No	No	Yes	Yes	Yes

 

Table2:                NOEC, LOEC and EC-values

Endpoint	[mgC-6410/kg SDW]
LOECmortality, biomass	> 1000
NOECmortality, biomass	1000
LOECreproduction	250
NOECreproduction	125
EC-valuesreproduction (95 % Confidence Interval)	EC10: 45.7 (10.8 - 193) EC20: 89.6 (21.7 - 377) EC50: 325 (53.3 - > 1000)

 

Description of key information

After 28 days of exposure to C-6410 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 all test item concentrations compared to the pooled control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 75 in the pooled control and ranged from 12 to 59 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rate 62.5 and 125 mg C-6410/kg soil dry weight compared to the pooled control. However, at the test item concentrations 250 to 1000 mg C-6410/kg soil dry weight the earthworm reproduction was statistically significantly reduced. The 56d NOEC is therefore 125 mg/kg dw. The EC50 for adult mortality is > 1000 mg/kg dw.

Key value for chemical safety assessment

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

125 mg/kg soil dw

Additional information

Effects of C-6410 on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222 (2016) from 2018-10-25 to 2018-12-21 with the definitive exposure phase from 2018-10-26 to 2018-12-20.

 

The study was conducted under static conditions over 8 weeks with the test item concentrations 62.5 – 125 – 250 – 500 - 1000mg C-6410/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. 40 test organisms were divided into 4 replicates for each test item treatment (10 earthworms per replicate). They had an individual body weight between 0.40 and 0.54 g at experimental starting.

 

Each concentration level and control were analysed via LC-MS/MS analysis on day 0, day 28 and at test end using pooled samples of all replicates. Additionally, on day of application, spiked quartz sand of the control, solvent control as well as of the test item concentration 1000 mg/kg sdw were sampled. 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 all test item concentrations compared to the pooled control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 75 in the pooled control and ranged from 12 to 59 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rate 62.5 and 125 mg C-6410/kg soil dry weight compared to the pooled control. However, at the test item concentrations 250 to 1000 mg C-6410/kg soil dry weight the earthworm reproduction was statistically significantly reduced. For all effect values please refer to Table 1.

 

All validity criteria recommended by the test guidelines were fulfilled.

Table1:               Summary of All Observed Effects in the Application rates

Effects	C-6410
Application rates [mg/kg SDW]	62.5	125	250	500	1000
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	Yes	Yes	Yes
Summary of all effects	No	No	Yes	Yes	Yes

 

Table2:                NOEC, LOEC and EC-values

Endpoint	[mgC-6410/kg SDW]
LOECmortality, biomass	> 1000
NOECmortality, biomass	1000
LOECreproduction	250
NOECreproduction	125
EC-valuesreproduction (95 % Confidence Interval)	EC10: 45.7 (10.8 - 193) EC20: 89.6 (21.7 - 377) EC50: 325 (53.3 - > 1000)