Reaction mass of 2-ethyl-4-methyl-1H-imidazole-1-propiononitrile and 2-ethyl-5-methyl-1H-imidazole-1-propiononitrile

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 - 06 Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 Apr 2004

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: "Daphnia sp., Acute Immobilization Test" in "Testing Methods for New Chemical Substances", Japan

Version / remarks:

March 31, 2011, No. 0331-7, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; March 29, 2011, No. 5, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 110331009, Environmental Policy Bureau, Ministry of the Environment, Japan; latest revision, December 21, 2015, No. 1221-1, Pharmaceutical and Food Safety Bureau, Ministry ofHealth, Labour and Welfare; December 9, 2015, No. 1, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 1512211, Environmental Policy Bureau, Ministry of the Environment, Japan

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-ethyl-4-methyl-1H-imidazole-1-propiononitrile and 2-ethyl-5-methyl-1H-imidazole-1-propiononitrile

EC Number:

947-727-8

Cas Number:

568591-00-4

Molecular formula:

C9H13N3

IUPAC Name:

Reaction mass of 2-ethyl-4-methyl-1H-imidazole-1-propiononitrile and 2-ethyl-5-methyl-1H-imidazole-1-propiononitrile

Sampling and analysis

Analytical monitoring:

yes

Remarks:

HPLC

Details on sampling:

- Concentrations: All test concentrations and the control at the start (0 h) and end (48 h) of exposure.
- Sampling method: At the start of exposure, samples were taken from a separate replicate. At the end of exposure, samples were taken from the middle layer of the test solution of every test vessel. The sample volume was 10 mL.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution with a nominal concentration of 100 mg/L was prepared by mixing and stirring 0.2 g test item into 2000 mL dilution water. Required volumes of the stock solution were diluted with dilution water to prepare the test solutions. For the 100 mg/L level, the stock solution was divided into each test vessel for test solution.
- Differential loading: No
- Controls: Dilution water without test item
- Evidence of undissolved material: The test solutions in all exposure levels and the control were colorless and clear at the start and end of exposure.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Clone A
- Source: University of Sheffield, UK (09 Jul 1990)
- Age of daphnids at test start: < 24 h old daphnids. The parent animals used for the test were from the same lot, were 24 d old and had a survival rate of 100%.
- Breeding conditions: Water: dechlorinated tap water; Water temperature: 20 ± 1 °C; Photoperiod: 16 h light/8 h dark; Feeding: 0.1 - 0.2 mg C (organic carbon content) Chlorella vulgaris per day per daphnid.
- Feeding during test: No feeding during test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

32 mg/L (Ca and Mg, dilution water)

Test temperature:

19.8 - 19.9 °C

pH:

7.7 - 8.6

Dissolved oxygen:

8.8 - 9.0 mg/L

Nominal and measured concentrations:

Control, 9.53, 17.1, 30.9, 55.6, and 100 mg/L (nominal)
< 0.100, 9.91, 17.7, 31.9, 57.4, and 106 mg/L (geometric mean measured 0 - 72 h)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beakers filled with 100 mL test solution and covered with a transparent plastic lid.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water
- Total organic carbon: < 0.5 mg/L
- Metals: > LOD
- Pesticides: < LOD
- Chlorine: < 0.02 mg/L
- Alkalinity: 36 mg/L
- Ca/mg ratio: 9.1 mg/L Ca, 2.2 mg/L Mg
- Conductivity: 13 mS/m
- Culture medium different from test medium: Culture medium same as test medium
- Intervals of water quality measurement: Dissolved oxygen, pH and temperature were measured at the start and end of exposure.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light/ 8 h dark
- Light intensity: Room light

EFFECT PARAMETERS MEASURED:
- Immobilization and symptoms: after 24 and 48 h

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10.0, and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Yes, 70% immobility observed after 48 h in the 100 mg/L treatment level. Observed symptoms: immobilization and reduced activity.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (tested periodically)

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

99.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits

Remarks:

84.6 - 143 mg/L

Details on results:

- Behavioural abnormalities: None in the control. After 48 h, reduced activity was observed at 30.9 mg/L (2 individuals), 55.6 mg/L (16 individuals), and 100 mg/L (6 individuals). Immobilization was observed at 55.6 mg/L (1 individual) and 100 mg/L (10 individuals).
- Mortality of control: 0%
- Other adverse effects control: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test solutions in all exposure levels and the control were colorless and clear at the start and end of exposure.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the effect value was within the stipulated range (mean ± 2SD: 0.1 - 0.35 mg/L, n = 102) to background data in the testing facility.
- Relevant effect levels: EC50 (48 h) = 0.12 mg/L potassium dichromate
- Period of reference test: 18 - 20 Oct 2017

Reported statistics and error estimates:

Probit analysis

Any other information on results incl. tables

VALIDITY OF STUDY

The study fulfilled the validity criteria of the guideline (Table 1).

 

Table 1: Validity criteria for OECD 202.

Criterion from the guideline	Outcome	Validity criterion fulfilled
In the control, including the control containing the solubilising agent, not more than 10% of the daphnids should have been immobilized.	0% immobilization in the control.	Yes
The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.	Dissolved oxygen concentrations in the control and all treatment levels were 9.0 mg/L at the end of exposure.	Yes

 

ANALYTICAL RESULTS

At the start of exposure, the measured test item concentrations were 9.77 – 103 mg/L (101 – 103% of the nominal concentrations). At the end of exposure, the measured test item concentrations were 10.0 – 108 mg/L (105 – 108% of the nominal concentrations). Hence, the measured test item concentrations were within ± 20% of the nominal concentrations (Table 2).

Table 2. Measured test item concentrations in the test solutions.

nominal concentration [mg/L]	measured concentration [mg/L] (percentage of measured concentration versus the nominal concentration)
	At test start	At test end	Geometric mean
Control	n.d.	n.d.	--
9.53	9.77 (103)	10.0 (105)	9.91 (104)
17.1	17.5 (102)	18.0 (105)	17.7 (104)
30.9	31.4 (101)	32.4 (105)	31.9 (103)
55.6	56.5 (102)	58.3 (105)	57.4 (103)
100	103 (103)	108 (108)	106 (106)

n.d.: < 0.10 mg/L

BIOLOGICAL RESULTS

After 48 h, the minimum concentration causing 100% immobility was > 100 mg/L because it was not obtained from the concentration range of the definitive study. The maximum concentration causing no immobility after 48 h was 30.9 mg/L (Table 3).

Table 3. Immobilization.

nominal concentration [mg/L]	vessel	24 h		48 h
		number of immobilized daphnids/total daphnids	immobility [%]	number of immobilized daphnids/total daphnids	immobility [%]
Control	A	0/5	0	0/5	0
	B	0/5		0/5
	C	0/5		0/5
	D	0/5		0/5
9.53	A	0/5	0	0/5	0
	B	0/5		0/5
	C	0/5		0/5
	D	0/5		0/5
17.1	A	0/5	0	0/5	0
	B	0/5		0/5
	C	0/5		0/5
	D	0/5		0/5
30.9	A	0/5	0	0/5	0
	B	0/5		0/5
	C	0/5		0/5
	D	0/5		0/5
55.6	A	1/5	5	1/5	5
	B	0/5		0/5
	C	0/5		0/5
	D	0/5		0/5
100	A	1/5	35	1/5	50
	B	1/5		3/5
	C	2/5		3/5
	D	3/5		3/5

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.