Ethanol, 2,2'-iminobis-, N-(C13-15-branched and linear alkyl) derivs.

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:

01 Oct - 19 Dec 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Richtlinien für die Prüfung von Pflanzenschutzmitteln im Zulassungsverfahren Part VI, 2-2: „Auswirkungen von Pflanzenschutzmitteln auf die Reproduktion und das Wachstum von Eisenia fetida/Eisenia andrei“, January 1994.

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss Federal Office of Public Health, Bern, Switzerland

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): Since the test item is homogeneously dispersible in water (according to a solubility pre-experiment), the test item was applied to the artificial soil by means of an aqueous application solution. Therefore, an application solution for the highest test item concentration was prepared by mixing 5000 mg of the test item into 1000 mL purified water by intense stirring for 10 minutes. After this treatment, the test item was homogeneously dispersed in the application solution. Aliquots of this application solution were further diluted with purified water to obtain the application solutions of the lower test concentrations. Aliquots of 100 mL of the intensively stirred application solutions were incorporated into the artificial soil (500 g dry weight) of each replicate. The soil was intensely mixed in a laboratory mixer to make sure that the test item was distributed homogeneously in the test medium. Finally, 42.6 mL of purified water was mixed into the soil. Additionally, a control (the same artificial soil substrate treated analogously to the test item vessels, but without test item) and a positive control with the reference item Derosal® was tested in parallel. A single concentration of the positive control was tested: 8.33 mg Derosal® per kg dry soil (corresponding to 5.0 mg carbendazim per kg dry soil). The application solution for the reference item was prepared by mixing 41.7 mg Derosal® homogeneously into 1000 mL purified water for 20 minutes. Then, 100-mL aliquots of this application solution were incorporated into the artificial soil of each replicate (500 g dry weight per replicate) by intense mixing in a laboratory mixer and the moisture content was adjusted.
- Controls: yes, artificial soil substrate treated analogously to the test item vessels, but without test item

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: earthworm
- Source: Obtained from a synchronized culture maintained at Harlan Laboratories. During breeding and keeping, they were fed with suitable food (i.e. horse manure and potatoes).
- Age at test initiation: The worms used were 3-4 months old adults with a clitellum. The age of the worms from the synchronized culture did not differ by more than 4 weeks.
- Weight at test initiation: The individual body wet weight of the test organisms at the start of the test ranged from 307 to 597 mg (the worms were weighed individually at the test start).

ACCLIMATION
- Acclimation period: Acclimatized one day before test initiation.
- Acclimation conditions (same as test or not): same as test

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

20 - 22 °C

pH:

6.0 - 6.1 (start of the test)
5.8 - 6.0 (after 8 weeks)

Moisture:

28 - 29% (start of the test)
35 - 37% (after 8 weeks)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Plastic boxes (length about 16.5 cm, width about 12 cm, height about 6.5 cm, area about 200 cm) were used as test vessels. The test vessels were covered by transparent lids to prevent worms from escaping and to reduce evaporation during the test period. The lids were perforated to allow air exchange.
- Amount of soil or substrate: For each test vessel, 507.4 g of the prepared air dry artificial soil (corresponding to 500 g dry weight) was mixed with 2.8 g calcium carbonate and 5 g food in a laboratory mixer. During mixing, 100 mL of purified water, containing the desired concentration of the test item was added. Finally, 42.6 mL of purified water was added. The amount of 650 g test substrate (corresponding to 500 g dry weight) was filled into each test vessel resulting in a soil layer of approx. 5-6 cm.
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): Sphagnum peat: 10%, Kaolin clay: 20%, Sand: 69%, Calcium carbonate: about 0.6% (for the adjustment to pH 6.0 ± 0.5), Food (dried organic manure): 1%

OTHER TEST CONDITIONS
- Photoperiod: 16 h light / 8 h dark
- Light intensity: 470 - 709 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortality (after 4 weeks), body weight (after 4 weeks), reproduction (after 8 weeks), assessment of food consumption

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Nominal and measured concentrations:

nominal: control, 62.5, 125, 250, 500, 1000 mg/kg soil dw

Reference substance (positive control):

yes

Remarks:

carbendazim

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

125 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

145 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CL: 48 - 227 mg/kg soil dw

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

536 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CL: 398 - 770 mg/kg soil dw

Details on results:

- Mortality at end of exposure period: 0% in all treatment and the control
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: The mean body weight of the adult worms in the control had increased during the 4-week exposure period on average by 97 mg per worm (20% of the mean initial weight). At all test concentrations of Atmer 163, the mean increase in body weight was between 20% and 23% and statistically not significantly different than in the control (results of a Dunnett t-test, one-sided smaller, a = 0.05). The results for the growth of the test organisms correspond to the observation that no prominent difference in food consumption was determined between the control and all treatments up to and including the highest test concentration of 1000 mg/kg soil dw.
- No. of offspring produced: see table 1 "Any other information on results incl. tables"

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: The reproduction rate of Eisenia fetida in the positive control was nearly completely inhibited (1% of the control value). Therefore, the results of the positive control demonstrated satisfactory test conditions.

Reported statistics and error estimates:

Multiple Dunnett t-test after a one-way analysis of variance (ANOVA) [biomass]
Multiple Williams t-test after a one-way analysis of variance (ANOVA) [reproduction]

Table 1: Reproduction of the earthworms

Treatment [mg/kg soil dw]	Vessel No.	Surviving adults	Juvenile worms per vessel					Juvenile worms per surviving adult					Statistical evaluation*
			#	Mean	SD	CV %	% of control	#	Mean	SD	CV %	% of control
Control	1	10	128	120	13.7	11.4	100	12.8	12.0	1.4	11.4	100	--
	2	10	110					11.0
	3	10	94					9.4
	4	10	131					13.1
	5	10	124					12.4
	6	10	138					13.8
	7	10	122					12.2
	8	10	116					11.6
62.5	1	10	151	143	9.5	6.6	119	15.1	14.3	0.9	6.6	119	n.s.
	2	10	149					14.9
	3	10	130					13.0
	4	10	142					14.2
125	1	10	86	110	29.3	26.6	91	8.6	11.0	2.9	26.6	91	n.s.
	2	10	118					11.8
	3	10	88					8.8
	4	10	148					14.8
250	1	10	93	88	9.2	10.5	73	9.3	8.8	0.9	10.5	73	s.
	2	10	80					8.0
	3	10	98					9.8
	4	10	80					8.0
500	1	10	63	72	18.8	26.3	60	6.3	7.2	1.9	26.3	60	s.
	2	10	92					9.2
	3	10	82					8.2
	4	10	50					5.0
1000	1	10	25	28	21.1	76.7	23	2.5	2.8	2.1	76.7	23	s.
	2	10	12					1.2
	3	10	58					5.8
	4	10	15					1.5
Reference subtsance, 8.33 mg/kg soil dw	1	10	2	1	1.0	76.6	1.0	0.2	0.1	0.1	76.6	1.0	--
	2	10	0					0.0
	3	10	1					0.1
	4	10	2					0.2

*: Statistical comparison of the mean reproduction rate (per vessel) with the control.

n.s.: Mean value statistically not significantly smaller than the control (Williams t-test, one-sided smaller,α= 0.05)

s.: Mean value statistically significantly smaller than the control (Williams t-test, one-sided smaller,α= 0.05)

#: Number of juvenile worms

Description of key information

NOEC (56 d): 125 mg/kg soil dw for Eisenia fetida based on reproduction (OECD 222) 

Key value for chemical safety assessment

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

125 mg/kg soil dw

Additional information

One study is available investigating the effects of Atmer 163 (CAS No. 97925-95-6) on reproduction of Eisenia fetida. The study was performed according to OECD 222 (GLP; Eisner, 2013) using artificial soil. Nominal test concentration up to 1000 mg/kg soil dw were tested. After four weeks mortality and biomass were determined. No impact on both parameters was found. After an additional four weeks the reproduction rate (number of juveniles) was determined. The reproduction of E. fetida was significantly inhibited at concentrations≥250 mg/kg soil dw, resulting in a NOEC (56 d) of 125 mg/kg soil dw.