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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Nov 2017 - 13 Feb 2018

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Adopted 23 March 2006, Annex 5, corrected 28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstal für Umwelt, Messungen und Naturschutz, Baden-Württemberg, Germany (10 Dec 2015)

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-MS/MS

Details on sampling:

- Concentrations: All test item concentrations and the control at test start (0 h, fresh media), after 24 h (aged media) and at test end (72 h, aged media).
- Sampling method: Samples of 10 mL were taken from fresh test solutions at test start (0 h) and from aged test solutions after 24 and 72 h. For each sampling, a retain sample was also taken.
- Sample storage conditions before analysis: Deep freezer (≤ - 18 °C). Samples are regarded as stable if the samples are deep frozen for up to 30 d. The maximum storage length in this study was 15 d.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution with a nominal test item concentration of 1000 mg/L test item was prepared by homogenising the test item in test medium (AAP medium) by shaking.
- Differential loading: No, lower test solutions were prepared by dilution of the stock solution with test medium.
- Controls: Untreated test medium
- Evidence of undissolved material: The stock solution was clear and transparent.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Single cell green alga
- Strain: SAG 61.81
- Source: Stock cultures are regularly ordered from a commercial supplier (MBM Sciencebridge GmbH, Göttingen, Germany)
- Age of inoculum (at test initiation): 3 - 4 d (pre-culture in exponential growth phase)
- Method of cultivation: Sterile, liquid stock cultures grown semi-continuously in 100 mL Erlenmeyer flasks under continuous illumination (4440 - 8880 lux/ 60 - 120 µEm^-2 * s^-1), at 21 - 24 °C and continuous rotation shaking at 105 rpm for CO2 supply. Old medium is periodically replaced by fresh mineral solution.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23.0 °C (control)
22.5 - 23.1 °C (treatments)

pH:

7.31 - 7.95 (control)
7.27 - 7.98 (treatments)

Nominal and measured concentrations:

Control, 9.53, 17.1, 30.9, 55.6, and 100 mg/L (nominal)
< LOD, 9.50, 18.2, 30.3, 62.3, and 93.0 mg/L (measured at 0 h)
< LOD, 10.4, 17.5, 31.0, 60.0, and 96.1 mg/L (measured at 72 h)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks with aluminium caps filled with 50 mL test solution
- Initial cell density: 0.51E+04 cells/mL
- Control end cell density: 24.28E+04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes (AAP medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: AAP medium according to Annex 3 of OECD guideline 201
- pH of test medium: 7.5 ± 0.1, adjusted with NaOH or HCl, if necessary
- Culture medium different from test medium: Culture medium same as test medium.
- Intervals of water quality measurement: The pH was recorded at test start (0 h) and at test end (72 h). Temperature was recorded continuously and recorded daily. Light intensity was measured at test start.

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Mean light intensity: 75.9 µEm^(-2)s^(-1)
- Other: The test vessels were placed in a temperature controlled incubator with continuous agitation and uniform lighting.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Fluorescence microplate reader (Infinite 200Pro, emission wavelength of 670 nm) at 0, 24, 48, and 72 h
- Morphological appearance: Microscopy, at the end of the test (72 h).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.798
- Range finding study: Yes
- Test concentrations (range-finding test): Control, 0.01, 0.10, 1.00, 10.0, and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes, no inhibition of growth rate or yield was observed after 72 h up to and including 10.0 mg/L. At 100 mg/L an inhibition of growth rate of 69.7% and an inhibition of yield of 96.5% was observed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

27.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 22.5 - 31.8 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

65 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 59.7 - 70.5 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

15.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other:

Remarks:

yield

Remarks on result:

other: 95% confidence limits: 12.8 - 17.6 mg/L

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

34.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other:

Remarks:

yield

Remarks on result:

other: 95% confidence limits: 31.0 - 37.4 mg/L

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

17.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

30.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: Normal morphology was observed at test end up to and including the nominal test item concentration of 55.6 mg/L. No cells were observed at 100 mg/L (nominal).
- Any observations that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50 (72 h): 0.971 mg/L potassium dichromate
- Date of reference test: 15 - 25 Jan 2018

Reported statistics and error estimates:

Statistical analyses were performed by SAS (2002 - 2010). The following tests were applied: Shapiro-Wilk Test (normality of data), Levene Test (homogeneity of variance). The NOEC and LOEC were determined by multiple comparison (Jonckheere Terpstra test, left sided, for growth rate and yield). The ECx values for growth rate and yield were determined by Probit Analysis following Gompertz and logisitc distribution, respectively.

Any other information on results incl. tables

VALIDITY CRITERIA

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

Table 1: Validity criteria for OECD 201.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	Cell numbers in the controls increased by a factor of 47.61, corresponding to a growth rate of 1.28712/d.	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	The mean coefficient of variation for the section-by-section specific growth rates in the controls was 25%.	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata.	The coefficient of variation of average growth rate in replicate control cultures was 2.6%.	Yes

 

ANALYTICAL RESULTS

The measured initial concentrations ranged from 93 – 112% of nominal (Table 2). The measured end concentrations sampled at test end (72 h) were 96% - 109% of nominal. Therefore, effect values were based on the nominal test item concentrations.

Table 2. Analytical results

Test item nominal [mg/L]	test item, nominal [mg/L]	Sampling	test item found
			[mg/L]	% of nominal
Control	0	0 h fresh	< LOD	-
		24 h aged	< LOD	-
		72 h aged	< LOD	-
9.53	9.53	0 h fresh	9.50	100
		24 h aged	9.67	101
		72 h aged	10.4	109
17.1	17.1	0 h fresh	18.2	106
		24 h aged	18.5	108
		72 h aged	17.5	102
30.9	30.9	0 h fresh	30.3	98
		24 h aged	31.0	100
		72 h aged	31.0	100
55.6	55.6	0 h fresh	62.3	112
		24 h aged	61.4	110
		72 h aged	60.0	108
100	100	0 h fresh	93.0	93
		24 h aged	99.2	99
		72 h aged	96.1	96

 - = not calculated; LOD = 0.0300 mg/L; LOQ = 0.100 mg/L

 

BIOLOGICAL RESULTS

After 72 h a dose-response curve for inhibition of growth rate and yield was observed. The inhibition of growth rate peaked at 84.7% at a nominal test item concentration of 100 mg/L. The inhibition of yield peaked at 98.1% at a nominal test item concentration of 100 mg/L. Significant inhibitory effects were determined for growth rate and yield at nominal test item concentrations of 30.9 mg/L and above (Table 3 and Table 4). The overall LOEC was 30.9 mg/L and the corresponding NOEC was 17.1 mg/L.

Table 3. Percentage inhibition of growth rate.

Conc.	% Inhibition of growth rate
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
9.53	-16.3	-0.6	1.3
17.1	-27.0	-0.9	2.8
30.9	21.0	24.5	16.8*
55.6	-2.1	26.2	33.4*
100	71.0	83.4	84.7*

* Statistically significant different to the control

 

Table 4: Percentage of inhibition of yield

Conc.	% Inhibition of yield
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
9.53	-26.3	-1.2	4.8
17.1	-45.0	-1.7	10.3
30.9	18.8	45.4	46.5*
55.6	-2.5	50.4	74.0*
100	78.8	94.2	98.1*

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

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