Hexanamide, N-(2-ethylhexyl)-3,5,5-trimethyl-

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:

2015-05-21 to 2015-07-17

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))

Deviations:

yes

Remarks:

Food was provided on day 0 (test start) instead of day 1 due to good experience with this procedure. At the test end, the soil moisture deviated by more than 10 % from the initial value in all treatments

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

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 with quartz sand (10 g per replicate). The treated quartz sand was given to the artificial soil. Additional demineralised water was added to the artificial soil to adjust the humidity of the artificial soil to a moisture of 54 % of the WHCmax.
Subsequently, the test medium was mixed thoroughly to ensure a homogenous distribution and about 600 g SDW were filled into test vessels.

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: 18 - 22 °C

pH:

Application rate
[mg/kg SDW] pH-value
[g/100 g DW]
day 0 day 56
Control 6.87 6.37
Solvent Control 6.74 6.44
22 6.73 6.25
46 6.66 6.38
100 6.77 6.35
220 6.66 6.23
460 6.71 6.18

Moisture:

Application rate
[mg/kg SDW] Moisture [%] of dry weight WHCmax of artificial soil
[g/100 g DW]
day 0 day 56
Control 19.4 24.2* 38.6
Solvent Control 19.8 24.2*
22 19.6 24.9*
46 18.3 22.9*
100 18.7 23.8*
220 18.4 23.5*
460 18.4 24.2*

Details on test conditions:

Test duration
8 weeks

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

Number and age of the earthworms
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 (see “Synchronisation”).

Body weight (actual)
0.30 to 0.52 g

Adaptation
Earthworms were adapted to artificial soil 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)
- 0.24 % calcium carbonate (CaCO3) to achieve a pH of 6.0  0.5

The maximum water holding capacity, the moisture content and the pH of the artificial soil were determined and adjusted. Two days before test start the artificial soil (24 kg) was pre-moistened with 2256 g demineralised water to achieve a moisture content corresponding to 27 % of the maximum water capacity 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 SDW 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 enabled sufficient gas exchange, light input and prevented drying of the test substrate.

Application
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 with quartz sand (10 g per replicate). The treated quartz sand was given to the artificial soil. Additional demineralised water was added to the artificial soil to adjust the humidity of the artificial soil to a moisture of 54 % of the WHCmax.
Subsequently, the test medium was mixed thoroughly to ensure a homogenous distribution and about 600 g SDW were 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 during the first 4 weeks. 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 at weekly intervals. Water was added to compensate for individual evaporation losses and to maintain the soil moisture, which should not deviate by more than 10 % of its initial value at the end of the test. Additional moistening was performed in case the soil appeared too dry.

Nominal and measured concentrations:

22-46-100-220-460 mg Antimussol ZJ/kg SDW

Reference substance (positive control):

yes

Remarks:

Carbendazim

Duration:

8 wk

Dose descriptor:

EC50

Effect conc.:

76.9 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 55.3-106.5

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

22 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 significant mortality of adult earthworms was observed in the controls or all treatment rates after 28 days of exposure to the test item.
- Total mass of adults at beginning of test:
At test start the earthworms had individual weights in the recommended range of 0.30 - 0.52 g.
- Changes in body weigth of live adults (% of initial weight) at end of exposure period:
The biomass of the adult earthworms increased throughout the first 28 days in all application rates with statistically significant differences in the test item rates 100 mg/kg SDW and above (Williams Sequential t-test, α = 0.05) compared to the pooled control. The EC50-value of biomass was not determined since no reduction of biomass exceeding 50 % occurred.
- No. of offspring produced:
The mean numbers of juveniles in the control and the solvent control were 56 and 67, respectively. The average number of juveniles in the treated groups ranged from 0 to 54 . Compared to the pooled control, the reproduction of the earthworms was statistically significantly reduced at treatment rates 46 mg/kg SDW and above. The EC50-value was determined to be 76.9 mg/kg SDW. The coefficient of variation calculated for the reproduction of the control was 24.3 % for the control and 18.3 % for the solvent control 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 Sig.
Control 1 55 69 ± 16.0 23.1 _ _
2 67
3 66
4 69
5 61
6 99
7 51
8 86
0.5 1 47 63 ± 18.9 29.9 91.3 No
2 88
3 67
4 50
1 1 55 45 ± 8.73 19.4 65.2 Yes
2 37
3 49
4 38
2 1 1 0.25 ± 0.50 n.d. 0 Yes
2 0
3 0
4 0
4 1 0 0 ± 0 n.d. 0 Yes
2 0
3 0
4 0
Rep. = Replicate a.i. = active ingredient SD = Standard deviation CV = Coefficient of variation
n.d. = not defined
Sig. = Statistical Significance (ANOVA. α = 0.05) compared to control

According to OECD 222, significant effects of Carbendazim should be observed between 1 and 5 mg a.i./kg SDW.

- Results with reference substance valid? Yes

Reported statistics and error estimates:

Earthworm Reproduction

STUDENT t-test of control and solvent control
Normality check was passed (Shapiro-Wilk´s; p > 0.01).
Variance homogeneity check (F-test) was passed (p > 0.01).
There is no statistically significant difference between control and solvent control.


EC50 determination
Point estimates from the 3-param. normal CDF
Point estimates from the 3-param. normal CDF with offspring number at 8 wks: Selected effective concentrations (ECx) of the test item; cl: confidence limit
Toxicity Metric EC10 EC50
Value [mg/kg] 19.973 76.869
lower 95%-cl 15.274 55.284
upper 95%-cl 26.119 106.459
n.d.: not determined due to mathematical reasons
The confidence limits of the EC10 used as a parameter were computed by means of the standard error of parameter b1; confidence limits for the remaining ECx were estimated by Monte-Carlo simulation using the parameter errors obtained from the inverse Hessian matrix (1000 runs).

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	10	0	0	0	#	0	0	1.43
Solvent Control	0	0	0	0	0	0	0	0	0
22		0		10		0		0		–					2.50
46		0		0		0		10						2.50
100		0		0		0		0						0
220		10		0		10		0						5.00
460		0		0		0		20						5.00

# = replicate was not taken into account for analyses.

Body Weight Changes of the Adult Earthworms      

Application rate [mg/kg dry soil weight]	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	0.38	0.54	0.16	42.1	0.17 ± 0.03	44.4 ± 8.99	–
	2	0.38	0.62	0.24	63.2
	3	0.38	0.56	0.18	47.4
	4	0.39	0.54	0.15	38.5
	5	0.39	0.56	0.17	43.6
	6	#
	7	0.38	0.52	0.14	36.8
	8	0.38	0.53	0.15	39.5
Solvent Control	1	0.39	0.58	0.19	48.7	0.17 ± 0.03	43.1 ± 8.41	–
	2	0.38	0.57	0.19	50.0
	3	0.38	0.54	0.16	42.1
	4	0.38	0.50	0.12	31.6
	5	0.39	0.57	0.18	46.2
	6	0.38	0.53	0.15	39.5
	7	0.38	0.50	0.12	31.6
	8	0.39	0.52	0.13	33.3
22	1	0.39	0.51	0.12	30.8	0.13 ± 0.02	33.2 ± 5.75	No
	2	0.39	0.52	0.13	33.3
	3	0.40	0.51	0.11	27.5
	4	0.39	0.55	0.16	41.0
46	1	0.39	0.52	0.13	33.3	0.16 ± 0.02	40.3 ± 5.41	No
	2	0.39	0.57	0.18	46.2
	3	0.38	0.54	0.16	42.1
	4	0.38	0.53	0.15	39.5
100	1	0.38	0.49	0.11	28.9	0.11 ± 0.01	29.2 ± 2.13	Yes
	2	0.39	0.51	0.12	30.8
	3	0.39	0.51	0.12	30.8
	4	0.38	0.48	0.10	26.3
220	1	0.38	0.52	0.14	36.8	0.13 ± 0.01	32.5 ± 3.41	Yes
	2	0.38	0.49	0.11	28.9
	3	0.39	0.51	0.12	30.8
	4	0.39	0.52	0.13	33.3
460	1	0.38	0.46	0.08	21.1	0.10 ± 0.02	24.7 ± 5.13	Yes
	2	0.38	0.50	0.12	31.6
	3	0.39	0.49	0.10	25.6
	4	0.39	0.47	0.08	20.5

SD = Standard deviation                                # = replicate was not taken into account for analyses.

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/kg SDW]	Replicate	Number of juveniles	Mean±SD	CV              [%]	[%] of control	Sig.
Control	1	74	56 ± 13.6	24.3	-	-
	2	68
	3	37
	4	62
	5	41
	6	#
	7	53
	8	59
Solvent Control	1	58	67 ± 12.2	18.3	-	-
	2	67
	3	53
	4	79
	5	59
	6	87
	7	57
	8	75
22	1	59	54 ± 11.9	22.1	96	No
	2	41
	3	68
	4	48
46	1	14	39 ± 16.8	43.0	70	Yes
	2	50
	3	48
	4	43
100	1	40	32 ± 10.2	31.9	57	Yes
	2	32
	3	37
	4	17
220	1	8	6 ± 2.63	43.8	11	Yes
	2	3
	3	4
	4	8
460	1	0	0 ± 0.50	-	0	Yes
	2	0
	3	0
	4	1

SD = Standard deviation        CV = Coefficient of variation      

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

# = replicate was not taken into account for analyses.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the test item did not induce significant mortality, pathological symptoms or changes of the behaviour of adult earthworms after 28 days of exposure in all tested concentrations. The adult earthworm biomass increased in the control and in all application rates. Thereby, statistically significant differences were observed for the treatments 100 mg/kg SDW and above. After eight weeks of exposure the earthworm reproduction was statistically significantly reduced at the test item concentrations 46 mg/kg SDW and above.

Overall, the NOEC of the test item concerning mortality, biomass and reproduction was determined to be 22 mg/ kg SDW. The EC50-value for reproduction was determined to be 76.9 mg/kg SDW. The EC50-value for biomass was not determined since no significant effects ≥ 50 % occurred.

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 (2004) fromMay 21^stto July 17^th, 2015 with the definitive exposure phase from May 21^stto July 15^thor16^th, 2015 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany.

 

The study was conducted under static conditions over 8 weeks with the test item concentrations 22 – 46 – 100 – 220 – 460 mg/kg SDW. Each application rate was mixed into artificial soil containing 5 % peat. A control using untreated artificial soil was tested under the same conditions as the test item treatments. Additionally, a solvent control containing acetone-spiked quartz sand was prepared. 80 test organisms were inserted into 8 control replicates for both controls and 40 test organisms were divided into 4 replicates for each treatment (10 earthworms per replicate). They had an individual body weight between 0.30 and 0.52 g at experimental starting.

After 28 days of exposure in soil, slight earthworm mortalities but no significant pathological symptoms or changes in the behaviour of adult earthworms were observed in the treatments. Statistically significant differences in earthworm body weights were observed at the treatments 100 mg/kg SDW and above. After a further four weeks, the reproduction rate (average number of juveniles produced) was 56 in the control, 67 in the solvent control and ranged between 0 and 54 in the test item treatment rates. Compared to the control, a statistically significant reduction of reproduction was observed in the test item concentrations 46 mg/kg SDW and above. Overall, the NOEC of the test item concerning mortality, biomass and reproduction was determined to be 22 mg/kg SDW. The EC₅₀-value of reproduction was determined to be 76.9 mg/kg SDW. No EC₅₀ -value for biomass was calculated since no significant effects ≥ 50 % occurred.

 

All validity criteria recommended by the test guidelines were fulfilled.

 Summary of All Observed Effects in theApplication rates

Effects	test item
Application rates [mg/kg SDW]	22	46	100	220	460
Mortality of adult earthworms	No	No	No	No	No
Body Weight Gain	No	No	Yes	Yes	Yes
Reduced reproduction rate (Number of juveniles)	No	Yes	Yes	Yes	Yes
Summary of all effects	No	Yes	Yes	Yes	Yes

  NOEC, LOEC and EC-values

Endpoint	[mg/kg SDW]
LOECmortality	> 460
NOECmortality	≥460
LOECbiomass	100
NOECbiomass	46
LOECreproduction	46
NOECreproduction	22
EC50 reproduction (with 95 % confidence interval)	76.9 (55.3 – 106.5)

 

Description of key information

Under the conditions of this study, the test item did not induce significant mortality, pathological symptoms or changes of the behaviour of adult earthworms after 28 days of exposure in all tested concentrations. The adult earthworm biomass increased in the control and in all application rates. Thereby, statistically significant differences were observed for the treatments 100 mg/kg SDW and above. After eight weeks of exposure the earthworm reproduction was statistically significantly reduced at the test item concentrations 46 mg/kg SDW and above.

Overall, the NOEC of the test item concerning mortality, biomass and reproduction was determined to be 22 mg/ kg SDW. The EC50-value for reproduction was determined to be 76.9 mg/kg SDW. The EC50-value for biomass was not determined since no significant effects ≥ 50 % occurred.

Key value for chemical safety assessment

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

22 mg/kg soil dw