3-methyl-1-vinyl-1H-imidazolium chloride

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Feb 2015 to 27 Feb 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): 3-Methyl-1-vinyl-1H-imidazolium chloride
- Appearance: brownish yellow liquid with characteristic smelling
- Batch No.: Mischprobe B 407 / 408 vom 23.08.14 14001329T0 – 14001629T0
- Date of manufacture: August 2014
- Expiry date: August 2015
- Purity: 97.7%
- Composition: 58.3% main component (the test substance is an aqueous solution)
- Water solubility: miscible
- Storage condition of test material: at ambient temperature without exposure to light in a tightly sealed container

Analytical monitoring:

yes

Details on sampling:

Samples at exposure initiation and at exposure termination collected from each test item concentration and the control were analyzed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The amount of the test item of 80.04 mg was weighed into a glass crystalliser and quantitatively transferred into a flask by multiple washing with the AAP medium and filled up to 1000 mL, respectively. The test item concentration of 80 mg/L was visually homogeneous and transparent. Lower test item concentrations were prepared by sequential dilutions with the AAP medium. In order to minimize the variability between replicates, test item concentrations and the control were prepared in flasks of larger capacity.
- Application: To obtain the initial algal density of 1E+04 cells/mL, 400 mL of each test item concentration was inoculated with 1.06 mL of the preculture (3.781E+06 cells/mL), whereas 900 mL of the control was inoculated with 2.38 mL of the pre-culture.
- Controls: AAP medium

The test material as such exists as aqueous solution. The test material concentrations correspond to certain nominal concentrations based on active ingredient. From here on, test concentrations will be expressed solely as active ingredient (a.i.).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: SAG 61.81
- Source: A standard laboratory culture cultivated at the Institute of Industrial Organic Chemistry, Branch Pszczyna, Department of Ecotoxicology, Laboratory of Aquatic Toxicology according to the recommendations specified in the OECD Guideline for the Testing of Chemicals No. 201. The algae, Pseudokirchneriella subcapitata, on agar bevels were obtained from the Culture Collection of Algae at Göttingen University, Germany
- Method of cultivation: The algae were transferred from agar bevels to a fresh AAP medium, in 250 mL Erlenmeyer flasks and incubated at temperature 21 - 24°C under constant illumination (a pre-culture). The algal culture was renewed (transferred to a fresh medium) twice a week under sterile conditions. The pre-culture with cells density of 1E+04 cells per mL was renewed four days before the definitive test.

ACCLIMATION
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.4 - 23.0 °C

pH:

- Start: 7.07 - 7.24
- End: 7.35 - 8.70

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 0.24, 0.76, 2.44, 7.81, 25 and 80 mg/L test substance (based on range-finding test)
- Nominal concentrations based on active ingredient*: 0 (control), 0.14, 0.44, 1.42, 4.55, 14.6, and 46 mg a.i./L
- Measured concentrations (t = 0h): - Measured concentrations (t = 72h):

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass Erlenmeyer flasks with a capacity of 250 mL closed with air permeable stoppers
- Fill volume: 100 mL
- Initial cells density: 1E+04 cells/mL
- No. of vessels per concentration (replicates): 3 (+ 1 replicate without algae used as background for spectrophotometric measurements)
- No. of vessels per control (replicates): 6 (+ 1 replicate without algae used as background for spectrophotometric measurements)
- Incubation: flasks were arranged at random in a thermostatic chamber (ILW400STD incubator with a shaker, Pol-Eko-Aparatura, Poland)
- Agitation: test vessels were continuously mechanically shaken at 90 rounds per minute

GROWTH MEDIUM
- Standard medium used: yes, AAP medium

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH values were measured at exposure initiation, before splitting up into replicates and at exposure termination in pooled replicates

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: constant illumination
- Light intensity and quality: 7618 - 7825 lux, fluorescent lighting (six 24-W light bulbs)

EFFECT PARAMETERS MEASURED: growth rate and yield
- Determination of cell concentrations: The number of algal cells was determined with an indirect method which involves a spectrophotometric measurement of the absorbance of an algal suspension at 670 nm. The absorbance value was then converted into the number of cells using a standard curve. The transmittance was measured in replicates without the algal inoculum. In order to document the standard curve, the control 72 h-old algae culture was used in a series of diluted samples. The algae cells were calculated by microscopic counting in a Burker chamber and absorbance was measured. The standard curve was the regression curve of algae cells number versus absorbance measured. The average specific growth rates and yield were calculated based on the numbers of cells recalculated from absorbance values measured at 24, 48 and 72 h for the cell number based on the standard curve.
- Other: Microscopic observations of algae cell morphology were performed at exposure termination

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2

RANGE-FINDING STUDY
- Test concentrations: 0 (control), 0.01, 0.1, 1.0, 10, and 100 mg/L
- Results used to determine the conditions for the definitive study: In the test item concentrations of 0.01 and 0.1 mg/L no growth rate inhibition was reported as compared to the algae growth in the control. The growth inhibition estimated at 72 hours was 2.8% in the test item concentration of 1.0 mg/L, 14.4% in the test item concentration of 10 mg/L and higher than 100.0% in the test item concentration of 100 mg/L when compared with the control.

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.53 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: 8.03 – 9.04 mg a.i./L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.11 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: 4.65 – 5.55 mg a.i./L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14.62 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: 13.77 – 15.49 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

8.77 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: 7.97 – 9.52 mg/L

Details on results:

GROWTH RATE / YIELD
- Exponential growth in the control: yes
- The growth rate and yield observed showed a clear concentration-response relationship, where inhibition (5.8%) intitiated at approximately 4.55 mg a.i./L and increased to 91.1 and 100% inhibition at 14.6 and 46 mg a.i./L, respectively. For details see 'Any other information on results incl. tables'.

MICROSCOPIC EVALUATION
- In all test item concentrations no morphology differences were reported as compared to the algae cells in the control.

Results with reference substance (positive control):

The most recent test, performed between 27.12.2014 - 30.12.2014, determined a 72h-ErC50 of 2.58 mg/L (95% C.L.: 2.49 – 2.67 mg/L). The results from the positive control with 3,5-dichlorophenol were within the normal ranges for this reference item.

Reported statistics and error estimates:

The endpoint values were determined based on the nominal test item concentrations and nominal concentrations of the active ingredient (as the test item is a 58.3% aqueous solution). The calculations and statistical analysis were performed with a commercial software ToxRat Professional (Probit analysis using linear max. likelihood regression for growth rate and Weibull analysis using linear max. likelihood regression for yield).

Table: Growth rate and yield

Nominal test item concentration [mg/L]	Growth Rate				Yield
	0-24 h	24-48 h	48-72 h	0-72 h	72 h
Control	1.834	1.752	1.655	1.747	1.878
	1.709	1.897	1.488	1.698	1.62
	1.709	1.946	1.547	1.734	1.805
	1.834	1.963	1.618	1.805	2.235
	1.565	1.999	1.552	1.705	1.657
	1.891	1.754	1.621	1.755	1.926
mean	1.757	1.885	1.58	1.741	1.854
standard deviation	0.119	0.108	0.062	0.039	0.222
0.24	1.485	2.09	1.611	1.729	1.779
	1.64	2.173	1.668	1.827	2.39
	1.565	2.271	1.672	1.836	2.46
mean	1.563	2.178	1.651	1.797	2.209
standard deviation	0.077	0.091	0.034	0.06	0.375
0.76	1.64	1.996	1.598	1.744	1.863
	1.398	2.197	1.585	1.727	1.768
	1.398	2.091	1.513	1.667	1.477
mean	1.479	2.095	1.565	1.713	1.703
standard deviation	0.139	0.101	0.046	0.04	0.201
2.44	1.485	2.1	1.483	1.69	1.58
	0.946	2.259	1.792	1.666	1.47
	0.946	2.669	1.571	1.729	1.779
mean	1.126	2.343	1.615	1.695	1.609
standard deviation	0.311	0.294	0.159	0.032	0.157
7.81	1.198	2.197	1.631	1.675	1.514
	0.61	2.667	1.749	1.675	1.514
	0.61	2.104	1.994	1.569	1.098
mean	0.806	2.323	1.791	1.64	1.375
standard deviation	0.339	0.302	0.185	0.061	0.24
25	n.d.	0	0.693	0.129	0.005
	0.792	0	n.d.	0.203	0.008
	0.387	n.d.	1.386	0.129	0.005
mean	n.d.	n.d.	n.d.	0.154	0.006
standard deviation	n.d.	n.d.	n.d.	0.043	0.002
80	n.d.	n.d.	n.d.	n.d.	n.d.
	n.d.	n.d.	n.d.	0.033	0.001
	n.d.	n.d.	n.d.	0.033	0.001
mean	n.d.	n.d.	n.d.	n.d.	n.d.
standard deviation	n.d.	n.d.	n.d.	n.d.	n.d.

n.d. - not determined

Table: Inhibition of growth rate and yield of algae

Nominal test item concentration [mg/L]	Nominal active ingredient concentration [mg/L]	% Inhibition at 72 h (growth rate)	% Inhibition at 72 h (yield)
Control	Control	0	0
0.24	0.14	-3.3*	-19.2*
0.76	0.44	1.6	8.1
2.44	1.42	2.6	13.2
7.81	4.55	5.8	25.8
25	14.6	91.1	99.7
80	46	101.1**	100

*calculated inhibition values are lower than 0.0%, what means that the algae cell density at observation during exposure is

higher than the algae cell density in the control

** calculated inhibition values are higher than 100.0%, what means that the algae cell density at observation during exposure is

lower than the initial algae cell density

Table: Endpoint values of growth rate based on nominal test item concentrations

Endpoint value [mg/L]	Time of exposure
	24 h	48 h	72 h
ErC50	5.12 (3.11 – 8.49)	12.80 (11.13 – 15.12)	14.62 (13.77 – 15.49)
ErC20	0.99 (0.34 – 1.80)	8.70 (7.31)	10.46 (9.64 – 11.22)
ErC10	0.42 (0.10 – 0.91)	7.11 (5.70 – 8.30)	8.77 (7.97 – 9.52)

( - ) - 95% confidence interval

The endpoint values were calculated with a probit method

Table: Endpoint values of yield based on nominal test item concentrations

Endpoint value [mg/L]	Time of exposure
	24 h	48 h	72 h
EyC50	2.23 (1.24 – 3.60)	9.10 (6.87 – 12.19)	11.22 (9.15 – 14.71)
EyC20	< 0.24	4.10 (2.04 – 5.66)	6.30 (4.24 – 7.84)
EyC10	< 0.24	2.416 (0.82 – 3.80)	4.30 (2.29 – 5.76)

( - ) - 95% confidence interval

The endpoint values were calculated with Weibull method

Table: Endpoint values of growth rate based on nominal concentrations of the active ingredient

Endpoint value [mg/L]	Time of exposure
	24 h	48 h	72 h
ErC50	2.98 (1.80 - 4.94)	7.46 (6.49 – 8.82)	8.53 (8.03 – 9.04)
ErC20	0.58 (0.20 – 1.05)	5.07 (4.26 – 5.83)	6.10 (5.62 – 6.54)
ErC10	0.25 (0.06 – 0.53)	4.14 (3.32 – 4.84)	5.11 (4.65 – 5.55)

( - ) - 95% confidence interval

The endpoint values were calculated with a probit method

Table: Endpoint values of yield based on nominal concentrations of the active ingredient

Endpoint value [mg/L]	Time of exposure
	24 h	48 h	72 h
EyC50	1.30 (0.72 – 2.09)	5.30 (4.01 – 7.11)	6.54 (5.33 – 8.58)
EyC20	< 0.14	2.38 (1.19 – 3.30)	3.67 (2.47 – 4.57)
EyC10	n.d.	1.41 (0.48 – 2.21)	2.50 (1.33 – 3.35)

( - ) - 95% confidence interval

n.d. – not determined

The endpoint values were calculated with Weibull method

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on materials and methods incl. tables'.

Description of key information

The 72-h ErC50 and 72-h ErC10 are 8.53 mg/L and 5.11 mg/L (based on active ingredient), respectively, in freshwater algae (P. subcapitata).

Additional information

The toxicity towards freshwater algae was determined in a study according to OECD TG 201 and in compliance with GLP criteria. In this study, exponentially growing freshwater algae (Pseudokirchneriella subcapitata) were exposed to nominal concentrations of 0 (control), 0.14, 0.44, 1.42, 4.55, 14.6, and 46 mg a.i./L (corresponding to nominal test substance concentrations of 0 (control), 0.24, 0.76, 2.44, 7.81, 25 and 80 mg/L) for 72 hours under static conditions. Analytical confirmation of nominal test concentrations showed that all test concentrations remained well within ±20% of nominal concentrations throughout the test. Therefore, effect concentrations are expressed as nominal. At the start of the test and after 24, 48 and 72 hours exposure duration, cell densities were determined and the algae were inspected microscopically for abnormalities. At the end of the test the percentage inhibition of yield and average specific growth rate were calculated. No abnormal cells were observed and the cell numbers recorded indicate a clear concentration-response relationship, where inhibition was observed from a concentration of 4.55 mg a.i./L up to 91.1 and 100% inhibition at 14.6 and 46 mg a.i./L, respectively. Based on these findings, the 72-h EC50 values for growth rate and yield were determined at 8.53 mg a.i./L and 6.54 mg a.i./L (corresponding to 14.62 mg/L and 11.22 mg/L based on test substance), respectively. The 72-h EC10 for growth rate and yield were determined at 5.11 mg a.i./L and 2.50 mg a.i./L (corresponding to 8.77 mg/L and 4.30 mg/L based on test substance), respectively.