Reaction mass of N-[(5-methyl-1H-pyrazol-1-yl)methyl]acetamide AND N-[(3-methyl-1H-pyrazol-1-yl)methyl]acetamide

Toxicity to terrestrial arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to terrestrial arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-01-30 - 2013-04-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 232 (Collembolan Reproduction Test in Soil)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Application method:

soil

Specific details on test material used for the study:

Chemical name: N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)
Analysed purity: 99.8 %

Analytical monitoring:

no

Vehicle:

yes

Remarks:

deionised water

Details on preparation and application of test substrate:

An exactly weighed amount of the test item was dissolved in deionised water to make a stock solution, without addition of solubility mediators, immediately before application. This stock solution was diluted (with deionised water such that each dispersion contained the amount of test item in 25 mL required to dose 250 g soil dry weight (at 15 % dry weight water content). Each test item dispersion (25 mL) was thoroughly mixed with the artificial soil using a laboratory mixer, achieving a final water content of 40-60 % of WHC.
The mixing equipment was cleaned with a brush following application of each test item concentration and with cleaning agents after the last mixing. Cleaning agents used are e.g. Mucasol (alkaline cleaning agent for laboratory equipment), water, deionised water and spirit for glass equipment.

Test organisms (species):

Folsomia candida

Animal group:

Collembola (soil-dwelling springtail)

Details on test organisms:

Test organism:
collembolan Folsomia candida (Willem)
Origin of the test organisms: originally purchased from “Biologische Bundesanstalt (BBA)”, Berlin-Dahlem in May 2000
Origin of the test organisms reared in the laboratory of the test facility used in the test: under ambient laboratory conditions Holding and breeding under laboratory conditions
Breeding medium:
mixture of plaster of Paris, activated charcoal and water (8:1:9)
Breeding conditions: - breeding container: plastic vessel of Bellaplast with a transparent lid (9.5 cm x 9.5 cm x 6 cm) ;
- moistening automatically by means of an absorbent wick - aeration in combination with feeding; - food source: granulated dry yeast, supplied twice a week; - light : dark cycle = 16 h : 8 h (diffuse artificial light); - temperature: nominally 20±2 °C Age of collembolans used in the test: 9-12 days (juvenile collembolans)
Test conditions
Test vessel:
glass container (approximately 150 mL) covered with a glass lid
Number of collembolans/ test vessel (= replicate): 10
Number of replicates/ control group: 8 ( + 2 replicates not loaded with collembolans for measurement purposes)
Number of replicates/ treated group: 4 ( + 2 replicates not loaded with collembolans for measurement purposes)
Number of collembolans/ control group: 80
Number of collembolans/ treated group: 40
Feeding regime:
approximately 2 mg granulated dry yeast at the start of the testand after 14 days
The test vessels were set up at random in the test room.
Artificial soil substrate
Composition of artificial soil: - 5 % sphagnum peat; origin: Torfwerk Moorkultur Ramsloh,
26683 Saterland, Germany, classified according to DIN 11540 (as close to pH 5.5-6.0 as possible, no visible plant remained,
finely ground, dried to measured moisture content)
- 20 % kaolin clay (kaolinite content > 30 %);
type: Kaolin W, origin: ERBSLÖH Lohrheim GmbH, 65558 Lohrheim, Germany
- 0.3 % calcium carbonate; origin: MERCK KGaA, 64271 Darmstadt, Germany
- 74.7 % industrial quartz sand; type: Millisil W3,
origin: Quarzwerke GmbH, 50207 Frechen, Germany
(fine sand is dominant with more than 50 % of the particles between 50 and 200 μm)
- deionised water

Study type:

laboratory study

Limit test:

no

Total exposure duration:

28 d

Test temperature:

19.1 – 20.7 ºC

pH (if soil or dung study):

guideline requirement: 6.0 ± 0.5
(determination according to DIN ISO 10390 (2005))
test initiation: 6.25 – 6.30
test completion : 6.12 – 6.22

Humidity:

guideline requirement: 40 – 60 % of WHC
(determination according to DIN ISO 11465 (1996))
test initiation: 24.9 – 25.1 % (equivalent to 58.5 – 58.9 % of WHC)
test completion : 24.5 – 24.7 % (equivalent to 57.5 – 58.0 % of WHC)

Photoperiod and lighting:

source: artificial light (LumiLux L58W)
intensity: 570 lx
duration: light : dark = 16 h : 8 h

Nominal and measured concentrations:

15.63, 31.25, 62.5, 125, 250, 500, 1000 mg test item/kg soil dry weight
(not corrected for purity, spacing factor: 2.0)

Reference substance (positive control):

yes

Remarks:

boric acid (100 % analysed)

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

900 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

616 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

28 d

Dose descriptor:

LC10

Effect conc.:

> 500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

28 d

Dose descriptor:

LC10

Effect conc.:

325 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Results with reference substance (positive control):

To verify the sensitivity of the test system the reference item boric acid is routinely tested at concentrations of 44, 67, 100, 150 and 225 mg/kg soil dry weight. The collembolans of the reference test were from the same source culture as those used in the definitive test.
In the most recent study (BioChem project No. R 12 10 48 003 S, dated May 24, 2012) the EC50 was determined to be 104 mg/kg soil dry weight. The LC50 was determined to be 199 mg/kg soil dry weight. The NOEC for mortality and for reproduction was determined to be 100 and 44 mg/kg soil dry weight, respectively.

Effects of P70/05/S-5 on mortality of parental collembolans

Treatment group	Control	15.63	mg test item/kg soild.w. 31.25|62.5|125|250				500	1000
Replicate	Number of surviving parental collembolans per replicate (4 weeks after test initiation)
1	10	9	9	9	10	10	10	4
2	9	10	10	10	10	10	10	4
3	10	10	9	9	9	9	9	3
4	10	10	10	10	10	10	10	5
5	10
6	10
7	9
8	10
Mean	9.8	9.8	9.5	9.5	9.8	9.8	9.8	4.0
±SD	0.5	0.5	0.6	0.6	0.5	0.5	0.5	0.8
cv %	4.7	5.1	6.1	6.1	5.1	5.1	5.1	20.4
Mortality (%)	2.5	2.5	5.0	5.0	2.5	2.5	2.5	60.0*

Effects of P70/05/S-5 on number of juvenile collembolans

Treatment group	Control	15.63	mg test item/kg soild.w. 31.25 1 62.5 1 125|250				500	1000
Replicate	Number of juveniles per replicate(4weeks after test initiation)
1	501	441	504	542	581	427	501	93
2	478	587	715	518	563	585	365	112
3	561	452	582	644	632	572	358	49
4	732	735	499	492	505	545	336	99
5	550
6	554
7	669
8	551
Mean	575	554	575	549	570	532	390*	88*
±SD	84.7	137.9	100.8	66.5	52.4	72.1	75.0	27.3
cv %	14.7	24.9	17.5	12.1	9.2	13.5	19.2	31.0
% Reduction of reproduction compared to control	-	4	0	4	1	7	32	85

Validity criteria fulfilled:

yes

Conclusions:

In a 28-day Folsomia candida reproduction study, in which collembolans were exposed to P70/05/S-5, the LC50 was calculated to be 900 mg test item/kg soil dry weight. The NOEC for mortality and reproduction was determined to be 500 and 250 mg test item/kg soil d.w., respectively. The EC50 (based on reproduction) for number of juveniles was calculated to be 616 mg test item/kg soil dry weight.

Executive summary:

The test item caused 2.5, 5.0, 5.0, 2.5, 2.5, 2.5 and 60.0 % parental mortality at test concentrations of 15.63, 31.25, 62.5, 125, 250, 500 and 1000 mg test item/kg soil d.w., respectively. 2.5 % parental mortality was observed in the control. Statistically significant effects (Fisher’s Exact Binomial Test with Bonferroni Correction, α = 0.05, one-sided greater) on parental mortality compared to the control group were recorded at a concentration of 1000 mg test item/kg d.w. (Table 3). Differences between the behaviour of the collembolans in the control and the test item treatment groups could not be observed.

The mean number of juveniles counted 28 days after introduction of the parental collembolans into the test vessels was 575 in the control and 554, 575, 549, 570, 532, 390 and 88 at test concentrations of 15.63, 31.25, 62.5, 125, 250, 500 and 1000 mg test item/kg soil d.w. (Table 4). Statistically significant effects (Williams-t-test, α = 0.05, one-sided smaller) on number of juveniles compared to the control group were recorded at concentrations of 500 and 1000 mg test item/kg d.w.

Therefore, the No-Observed-Effect-Concentration (NOEC) for mortality and reproduction was determined to be 500 and 250 mg test item/kg soil d.w., respectively. The LC50 was calculated to be 900 mg test item/kg soil dry weight. The EC50 (based on reproduction) for number of juveniles was calculated to be 616 mg test item/kg soil dry weight.

All validity criteria for the study were met.

		mg test item/kg soil dry weight
		Mortality	Reproduction
	NOEC	500	250
	LC10/EC10	> 500	325
95	%confidence limits	-	270 to 392
	LC20/EC20	> 500	405
95	%confidence limits	-	352 to 466
	LC50/EC50	900	616
95	%confidence limits	742 to 1090	562 to 676

Description of key information

In a 28-day Folsomia candida reproduction study, in which collembolans were exposed to P70/05/S-5, the LC50 was calculated to be 900 mg test item/kg soil dry weight. The NOEC for mortality and reproduction was determined to be 500 and 250 mg test item/kg soil d.w., respectively. The EC50 (based on reproduction) for number of juveniles was calculated to be 616 mg test item/kg soil dry weight.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil dwelling arthropods:

250 mg/kg soil dw