Nonanamide, N,N-dimethyl-

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:

from 2018-04-19 to 2018-06-14

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:

see field Principles of method if other than guideline

Deviations:

yes

Principles of method if other than guideline:

Food was provided on day 0 (test start) instead of day 1 due to good experience with this procedure.
At test end, the soil moisture deviated by more than 10 % from the initial value in all treatments.
These deviations are considered to have no impact on quality and integrity of the study.

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

Application
The respective test item amount (1200,0 mg) was weighed out for a stock solution and dispersed in demineralised water (1000 mL). The stock solution was further diluted with demineralised water to receive the respective application solutions (200 mL). The application solutions and additional demineralised water (42 g) was added to the artificial soil (2400 g soil dry weight) to adjust the humidity of the artificial soil to a moisture of 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 filled into each test vessel.

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 type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Test temperature:

nominal: 20 +/- 2°C
actual: 17 - 22 °C
The room temperature decreased to minimum 17 °C for about 8 hours. Since adapting of the soil to a change of the room temperature is very slow , this deviation is considered to have no impact on quality and integrity of the study.

pH:

pH-Values of the Test Media
Application rate
[mg/kg SDW] pH-value
day 0 day 56
Control 6.40 6.44
0.78 6.55 6.68
1.56 6.39 6.53
3.13 6.52 6.61
6.25 6.57 6.50
12.5 6.36 6.59
25 6.34 6.61
50 6.31 6.69
100 6.37 6.51

Moisture:

Moisture and WHCmax of the Test Media
Application rate
[mg/kg SDW] Moisture [%] of dry weight WHCmax of artificial soil
[g/100 g DW]
day 0 day 56
Control 17.8 22.4* 33.5
0.78 17.2 23.5*
1.56 17.5 21.8*
3.13 17.2 21.2*
6.25 16.9 20.6*
12.5 16.9 22.1*
25 16.6 21.7*
50 16.8 21.3*
100 16.6 22.0*

Details on test conditions:

Experimental Procedure

Test item Genagen PA

Application rates
Based on the results of a preliminary study (see Annex I) six test item concentrations were tested:
100 – 50 – 25 – 12.5 – 6.25 – 3.125 – 1.56 – 0.78 mg/kg soil dry weight (Factor 2)

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

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

Body weight (actual) 0.38 to 0.53 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)
- 0.18 % calcium carbonate (CaCO3) to achieve a pH of 6.0  0.5

The maximum water holding capacity (WHCmax), the moisture content and the pH-value of the soil were determined. Two days before test start the artificial soil (27000 g) was pre-moistened with 1890 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
The respective test item amount (1200,0 mg) was weighed out for a stock solution and dispersed in demineralised water (1000 mL). The stock solution was further diluted with demineralised water to receive the respective application solutions (200 mL). The application solutions and additional demineralised water (42 g) was added to the artificial soil (2400 g soil dry weight) to adjust the humidity of the artificial soil to a moisture of 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 filled into each test vessel.

Temperature 20  2 °C

Photoperiod 16 h
Light intensity 400 - 800 lx

Feeding (procedure of adding the food during the study)
The earthworms were fed with cattle manure weekly during the first 4 weeks, as presented in Table 3. 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.
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 5 5
14, 20, 28 5 10
35, 42, 49 - -

Nominal and measured concentrations:

Based on the results of a preliminary study six test item concentrations were tested:
100 – 50 – 25 – 12.5 – 6.25 – 3.125 – 1.56 – 0.78 mg/kg soil dry weight (Factor 2)

Reference substance (positive control):

yes

Remarks:

Carbendazim

Duration:

8 wk

Dose descriptor:

EC50

Effect conc.:

10.4 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% confidence interval

Remarks:

(2.91 to > 100)

Duration:

8 wk

Dose descriptor:

other: EC20

Effect conc.:

0.79 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 955 confidence interval

Remarks:

(< 0.78 to 2.52)

Duration:

8 wk

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, biomass

Duration:

8 wk

Dose descriptor:

LOEC

Effect conc.:

12.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

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

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

6.25 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality at end of exposure period:
No earthworm mortalities of adult earthworms were observed in the control and all test item concentrations.
- Total mass of adults at beginning of test:
At test start, the earthworms had individual weights of 0.38 - 0.53 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 at any of the test item concentrations compared to the control
- No. of offspring produced:
The reproduction rate (average number of juveniles produced) was 67 in the control and ranged from 24 to 60 at the test item treatment rates. There was no statistically significant difference in earthworm reproduction in the treatment rates 0.78 to 6.25 mg/kg soil dry weight compared to the control. The finding at 3.13 mg/kg is regarded to be of no significance due to a missing dose-response relationship. However, at the test item concentrations 12.5 to 100 mg/kg soil dry weight the earthworm reproduction was statistically significantly reduced. The coefficient of variation calculated for the reproduction of the control was 10.7 % 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
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.i. = active ingredient 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.i./kg soil DW.

Reported statistics and error estimates:

NOEC/LOEC-values
Adult Mortality Since no mortality was observed, no further computations have been performed.

Biomass - Shapiro-Wilk’s Test on Normal Distribution
- Levene’s Test on Variance Homogeneity (with Residuals)
- Trend Analysis by Contrasts (Monotonicity of Concentration/Response)
- Dunnett’s Multiple t-test Procedure

Reproduction - Dunnett’s Multiple Comparison Test

The -value (acceptable probability of incorrectly concluding that there is a difference) is  = 0.05. For details please refer to chapter 13.

EC-values
Reproduction The EC20/50 – values for reproduction were determined running Sigmoidal dose-response.

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
- Sigma Plot, SPSS INC.
- ToxRat Professional Version 3.2.1 , TOXRAT SOLUTIONS GMBH
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.

  Adult mortality

No earthworm mortalities of adult earthworms were observed in the control and all test item concentrations.

 

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

Application rate	Replicate
[mg/kg SDW]	1	2	3	4	5	6	7	8	Mean
Control	0	0	0	0	0	0	0	0	0
0.78	0	0	0	0	-				0
1.56	0	0	0	0					0
3.13	0	0	0	0					0
6.25		0		0						0		0		0
12.5		0		0						0		0		0
25		0		0						0		0		0
50		0		0						0		0		0
100		0		0						0		0		0

 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/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
0.78	A	10/10	10/10	10/10	10/10	-
1.56	A	10/10	10/10	10/10	10/10
3.13	A	10/10	10/10	10/10	10/10
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	10/10	10/10	10/10	10/10
100	A	10/10	10/10	10/10	10/10

  A = no obvious pathological symptoms      

 Live Weight

At test start, the earthworms had individual weights of 0.38 - 0.53 g. There were no statistically significant differences in earthworm body weights at any of the test item concentrations compared to the control.

 Body Weight Changes of the Adult Earthworms   

Application rate [mg/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	0.44	0.54	0.10	22.7	0.13 ± 0.02	28.1 ± 5.28	–
	2	0.45	0.56	0.11	24.4
	3	0.44	0.59	0.15	34.1
	4	0.45	0.59	0.14	31.1
	5	0.44	0.58	0.14	31.8
	6	0.45	0.54	0.09	20.0
	7	0.44	0.56	0.12	27.3
	8	0.45	0.60	0.15	33.3
0.78	1	0.45	0.60	0.15	33.3	0.14 ± 0.02	30.9 ± 3.44	No
	2	0.44	0.57	0.13	29.5
	3	0.45	0.57	0.12	26.7
	4	0.44	0.59	0.15	34.1
1.56	1	0.45	0.55	0.10	22.2	0.10 ± 0.01	23.1 ± 2.94	No
	2	0.44	0.56	0.12	27.3
	3	0.45	0.55	0.10	22.2
	4	0.44	0.53	0.09	20.5
3.13	1	0.45	0.57	0.12	26.7	0.12 ± 0.03	25.9 ± 5.64	No
	2	0.44	0.58	0.14	31.8
	3	0.45	0.57	0.12	26.7
	4	0.44	0.52	0.08	18.2
6.25	1	0.45	0.56	0.11	24.4	0.12 ± 0.02	27.0 ± 5.02	No
	2	0.44	0.59	0.15	34.1
	3	0.45	0.57	0.12	26.7
	4	0.44	0.54	0.10	22.7
12.5	1	0.45	0.55	0.10	22.2	0.11 ± 0.02	24.2 ± 3.34	No
	2	0.44	0.56	0.12	27.3
	3	0.45	0.57	0.12	26.7
	4	0.44	0.53	0.09	20.5
25	1	0.45	0.54	0.09	20.0	0.10 ± 0.02	23.0 ± 4.86	No
	2	0.44	0.52	0.08	18.2
	3	0.45	0.58	0.13	28.9
	4	0.44	0.55	0.11	25.0
50	1	0.44	0.58	0.14	31.8	0.14 ± 0.03	30.9 ± 5.56	No
	2	0.45	0.62	0.17	37.8
	3	0.44	0.57	0.13	29.5
	4	0.45	0.56	0.11	24.4
100	1	0.44	0.57	0.13	29.5	0.11 ± 0.03	23.7 ± 6.99	No
	2	0.45	0.54	0.09	20.0
	3	0.44	0.57	0.13	29.5
	4	0.45	0.52	0.07	15.6

SD = Standard deviation                                                                        

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

Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate  [mg/kg SDW]	Replicate	Number of juveniles	Mean±SD	CV              [%]	[%] of control	Sig.
Control	1	70	67 ± 7.14	10.7	-	-
	2	66
	3	61
	4	76
	5	59
	6	74
	7	58
	8	73
0.78	1	18	50 ± 40.5	81.0	74.6	No
	2	105
	3	55
	4	21
1.56	1	35	60 ± 17.4	29.0	89.6	No
	2	67
	3	63
	4	75
3.13	1	22	34 ± 13.3	39.1	50.8	No^#
	2	22
	3	46
	4	44
6.25	1	48	42 ± 9.22	22.0	62.7	No
	2	29
	3	49
	4	41
12.5	1	20	25 ± 9.57	38.3	37.3	Yes
	2	16
	3	24
	4	38
25	1	22	26 ± 7.30	28.1	38.8	Yes
	2	18
	3	34
	4	30
50	1	20	36 ± 12.5	34.6	53.7	Yes
	2	46
	3	46
	4	33
100	1	31	24 ± 9.91	41.3	35.8	Yes
	2	34
	3	19
	4	13

SD = Standard deviation        CV = Coefficient of variation 

Sig. = Statistical Significance (Dunnett’s Multiple Comparison Test,α= 0.05) compared to the control

#) = The finding at 3.13 mg/kg is regarded to be of no significance due to a missing dose-response relationship

The reproduction rate (average number of juveniles produced) was 67 in the control and ranged from 24 to 60 at the test item treatment rates. There was no statistically significant difference in earthworm reproduction in the treatment rates 0.78 to 6.25 mg/kg soil dry weight compared to the control. The finding at 3.13 mg/kg is regarded to be of no significance due to a missing dose-response relationship. However, at the test item concentrations 12.5 to 100 mg/kg soil dry weight the earthworm reproduction was statistically significantly reduced.The coefficient of variation calculated for the reproduction of the control was 10.7 % and thus lower than 30 %.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, Genagen PA did not induce earthworm mortalities or pathological symptoms or changes in the behaviour of adult earthworms at any of the test item concentrations. There were no statistically significant differences in earthworm body weights in any test item concentrations compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 67 in the control and ranged from 24 to 60 in the test item treatment replicates. There was no statistically significant difference in earthworm reproduction in the treatment rates 0.78 to 6.25 mg/kg SDW compared to the control. However, at the test item concentrations 12.5 to 100 mg/kg SDW the earthworm reproduction was statistically significantly reduced. The NOEC concerning mortality and biomass was determined to be ≥ 100 mg/kg SDW. The NOEC concerning reproduction was determined to be 6.25 mg/kg SDW. The EC20/50 – values for reproduction were determined.
All validity criteria recommended by the test guidelines were fulfilled.

Executive summary:

Effects of GenagenPA on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222 (2016) from 2017-09-20 to 2018-06-14 with the definitive exposure time from 2018-04-19 to 2018-06-14 at the test facility in 31157 Sarstedt, Germany.

 

The study was conducted under static conditions over 8 weeks with the test item concentrations 0.78 – 1.56 – 3.13 – 6.25 – 12.5 – 25 – 50 – 100 mg/kg soil dry weight (SDW). Each application ratewas mixed into artificial soil containing 5 % peat. A control using untreated artificial soil was tested under the same conditions as the test item treatments. For the control, 80 test organisms were inserted into 8 replicates. 40 test organisms were divided into 4 replicates for each treatment (10 earthworms per replicate). They had an individual body weight between 0.38 and 0.53 g at experimental starting.

 

After 28 days of exposure in soil, neither earthworm mortalities nor pathological symptoms or changes in the behaviour of adult earthworms were observed in the controls or any of the test item concentrations.There were no statistically significant differences in earthworm body weights in any test item concentrations compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 67 in the control and ranged from 24 to 60 in the test item treatment replicates. There was no statistically significant difference in earthworm reproduction in the treatment rates 0.78 to 6.25 mg/kg SDW compared to the control. However, at the test item concentrations 12.5 to 100 mg/kg SDWthe earthworm reproduction was statistically significantly reduced.

All validity criteria recommended by the test guidelines were fulfilled.

Summary of All Observed Effects in the Application rates

Effects	Genagen PA
Application rates [mg/kg SDW]	0.78	1.56	3.13	6.25	12.5	25	50	100
Mortality of adult earthworms	No	No	No	No	No	No	No	No
Reduced Body Weight Gain	No	No	No	No	No	No	No	No
Reduced reproduction rate (Number of juveniles)	No	No	No	No	Yes	Yes	Yes	Yes
Summary of all effects	No	No	No	No	Yes	Yes	Yes	Yes

 

 

NOEC, LOEC and EC-values

Endpoint	[mg/kg SDW]
LOECmortality, biomass	> 100
LOECreproduction	12.5
NOECmortality, biomass	≥ 100
NOECreproduction	6.25
EC20reproduction(95 % confidence interval) EC50reproduction(95 %confidence interval)	0.79 ( < 0.78 to 2.52) 10.4 ( 2.91 to > 100)

Description of key information

Under the conditions of this study, Genagen PA did not induce earthworm mortalities or pathological symptoms or changes in the behaviour of adult earthworms at any of the test item concentrations. There were no statistically significant differences in earthworm body weights in any test item concentrations compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 67 in the control and ranged from 24 to 60 in the test item treatment replicates. There was no statistically significant difference in earthworm reproduction in the treatment rates 0.78 to 6.25 mg/kg SDW compared to the control. However, at the test item concentrations 12.5 to 100 mg/kg SDW the earthworm reproduction was statistically significantly reduced. The NOEC concerning mortality and biomass was determined to be ≥ 100 mg/kg SDW. The NOEC concerning reproduction was determined to be 6.25 mg/kg SDW. The EC20/50 – values for reproduction were determined.
All validity criteria recommended by the test guidelines were fulfilled.

Key value for chemical safety assessment

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

6.25 mg/kg soil dw