Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts

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:

2 - 13 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23 March 2006, Annex 5 corrected 28 July 2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

20 May 2008, amended 7 December 2015, for the purpose of its adaptation to technical progress, by Commission Regulation (EU) 2016/266

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts

EC Number:

275-085-2

EC Name:

Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts

Cas Number:

70983-43-6

Molecular formula:

see structural formula below, for n=4 C13H24N2O3 HO.Na

IUPAC Name:

Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts

Test material form:

liquid

Remarks:

oily liquid

Details on test material:

Batch No.: 54542L16
Storage at room temperature, protected from light.

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The following test flasks were set up:
- Test solution (Tn); containing test medium, test item and algae (three replicates per test concentration)
- Blank control (Bn); containing test medium and algae (six replicates)
- Since the test item is soluble, the test solutions were prepared by respective dilutions of a stock solution. A stock solution was prepared by adding the test item to sterile-filtered Algal medium (pH adjusted at 8.0 ± 0.2) in sterile glassware and stirred until dissolution. 100 ml of each test solution was added to the sterile test flasks and inoculated with an exponentially growing preculture of algae.
Pure Algal medium, also sterile filtered into sterile test flasks and inoculated with the exponentially growing preculture of algae mentioned above, served as blank controls. For the incubation, the test flasks were then placed on an orbital shaker at a nominal shaking speed of about 130 revolutions per min.
- Evidence of undissolved material: Not relevant. Since the test item is soluble, the test solutions were prepared by respective dilutions of a stock solution.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Test organism: Axenic slope culture of Desmodesmus subspicatus 86.81SAG (Collection of Algal Cultures, Institute of Freshwater Ecology, University of Göttingen, D-37073 Göttingen, Germany
- Culture: 250 ml flask containing 100 ml of sterile OECD medium inoculated with cell material from an axenic slope culture
- Illumination: Continuous (2000–3000 lux) from Osram Fluora L18W77 and Osram Daywhite L18W840 (Osram AG, Winterthur, Switzerland)
- Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
- Control of sensitivity: twice per year, with potassium dichromate

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

not reported

Test temperature:

Min: 22.7; Max: 23.9; Mean: 23.4

pH:

7.6 - 8.0

Dissolved oxygen:

not reported

Salinity:

not reported

Conductivity:

not reported

Nominal and measured concentrations:

The concentrations of Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts in the test media were measured by HPLC analysis at the beginning and after 24, 48 h and 72 h of exposure. The measured concentrations at the beginning of exposure were 111, 33.8, 10.3, 3.31 and 1.18 mg/l and 106, 32.3, 10.2, 3.37 and 1.38 mg/l at the end of the test. However, because of the variable composition (chain lengths C5 – 8) of the test item and the relatively high limit of quantification of the HPLC method (1.07 mg/l), the lowest test concentration could not be measured sensibly. These analyses nonetheless showed that the four highest concentrations were correctly dosed (105 to 111% of nominal) and remained stable over the 72h test period (95 to 102% of the initially measured concentration); hence it is expected that the lowest concentration was also correctly dosed and remained stable.
Therefore, the effective concentrations ErCx/EyCx were assessed based on the nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml flasks, all-glass, with 100 ml of test medium, shaken (130 rpm)
- Test medium: Recommended OECD medium
- Preculture: Exponentially growing culture of Desmodesmus subspicatus
- Illumination: Source: continuous from Osram Fluora L 18W77 and Osram Daywhite L 18W840 (Osram AG, Winterthur, Switzerland)
- Light intensity: the light intensity amounts to about 3000 lux which corresponds to about 40 µE•m-2•s-2
- Homogeneity: the light intensity is maintained within ±15% from the average light intensity over the incubation area. The test vessels are repositioned every day within the incubation area to minimize variation.
- Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
- pH: Initially adjusted to 8 ± 0.2; the pH of the control medium should not increase by more than 1.5 units during the test
- Test type: Static exposure conditions over a period of 72 h
- Starting cell density: 0.5–0.85 µg/ml with respect to dry weight corresponding to about 2–5•103 cells/ml (i.e. OD680 of about 0.005 units)

RANGE FINDING STUDY
Prior to the definitive test a non-GLP range finding test with nominal concentrations of 0.2, 2.0 and 100 mg/l of Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts was performed. The HPLC determinations indicate that the test item concentrations are stable. Based on the results of this non-GLP range finding test the test concentrations for the definitive test were 100, 31.6, 10.0, 3.16 and 1.00 mg/l nominal concentration.

EFFECT PARAMETERS MEASURED
- Cell density: Cell concentrations were determined every 24 h using a spectrophotometer at 680nm wavelength (Shimadzu UV 1800, Shimadzu Schweiz GmbH, Römerstr. 3, CH-4153 Reinach). Aliquots of 5 ml were removed from each test flask under sterile conditions. Cell concentrations at time 0 h were only determined in the blank controls.

Reference substance (positive control):

not required

Remarks:

Control of sensitivity: None – the culture is re-purchased annually

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

16.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

60.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.93 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

19.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: yes (growth rate and yield)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

With respect to the endpoint growth rate, following effects were observed as compared to the blank controls: 71% at 100 mg/l, 24% at 31.6 mg/l, 6% at 10.0 mg/l and 3% at 3.16 mg/l. No significant effects were observed at 1.00 mg/l as compared to the blank controls.
Based on these effects and the nominal concentrations, the median effect concentration with respect to growth rate (ErC50/0–3 days) of the test item to Desmodesmus subspicatus was calculated to be 60.7 mg/l (95% confidence limits: 56.6–65.2 mg/l).
The low effect concentration with respect to growth rate ErC10 was calculated to be 16.9 mg/l (95% confidence limits: 14.3–19.5 mg/l).
The no observed effect concentration (NOErC) with respect to growth rate was determined to be 1.00 mg/l.

With respect to the endpoint yield (biomass production), following effects were observed as compared to the blank controls: 97% at 100 mg/l, 67% at 31.6 mg/l, 23% at 10.0 mg/l and 14% at 3.16 mg/l. No significant effects were observed at 1.00 mg/l as compared to the blank controls.
Based on these effects and the nominal concentrations, the median effect concentration with respect to yield (EyC50/0–3 days) of the test item to Desmodesmus subspicatus was calculated to be 19.8 mg/l (95% confidence limits: 17.3–22.7 mg/l).
The low effect concentration with respect to yield EyC10 was calculated to be 4.93 mg/l (95% confidence limits: 3.48–6.34 mg/l).
The no observed effect concentration (NOEyC) with respect to yield was determined to be 1.00 mg/l.

Reported statistics and error estimates:

Determination of the effect concentrations (ErCx and EyCx)
The effect concentrations (ErC10, ErC50 and EyC10, EyC50) were calculated with ToxRat® Standard Version 3.2.1 (ToxRat® Solutions GmbH, Alsdorf, Germany). For the calculation of the ECx and associated confidence intervals, inhibitions below 0% and inhibitions above 100% were set to 0.1% and 100%, respectively.

Determination of the no observed effect concentrations (NOErC and NOEyC)
The statistical significance of differences between effects observed for control and test item treatments was determined by Dunnett’s test or Student-t test using the ToxRat® Standard Version 3.2.1 (ToxRat® Solutions GmbH, Alsdorf, Germany).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

In the control cultures: average specific growth rate of 1.52 (>0.92)/coefficient of variation of average specific growth rates: 1.7% (<=7%)/ mean coefficient of variation for section-by-section specific growth rates 33.6% (<=35%).

Conclusions:

The acute toxicity of Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts (CAS no. 70983-43-6) to Desmodesmus subspicatus was determined in a 72 hour static test according to OECD guideline 201. With respect to the endpoint growth rate, following effects were observed as compared to the blank controls: 71% at 100 mg/l, 24% at 31.6 mg/l, 6% at 10.0 mg/l and 3% at 3.16 mg/l. No significant effects were observed at 1.00 mg/l as compared to the blank controls).
Based on these effects and the nominal concentrations, the median effect concentration with respect to growth rate (ErC50/0–3 days) of the test item to Desmodesmus subspicatus was calculated to be 60.7 mg/l (95% confidence limits: 56.6–65.2 mg/l).
The low effect concentration with respect to growth rate ErC10 was calculated to be 16.9 mg/l (95% confidence limits: 14.3–19.5 mg/l).
The no observed effect concentration (NOErC) with respect to growth rate was determined to be 1.00 mg/l.
With respect to the endpoint yield (biomass production), following effects were observed as compared to the blank controls: 97% at 100 mg/l, 67% at 31.6 mg/l, 23% at 10.0 mg/l and 14% at 3.16 mg/l. No significant effects were observed at 1.00 mg/l as compared to the blank controls.
Based on these effects and the nominal concentrations, the median effect concentration with respect to yield (EyC50/0–3 days) of the test item to Desmodesmus subspicatus was calculated to be 19.8 mg/l (95% confidence limits: 17.3–22.7 mg/l).
The low effect concentration with respect to yield EyC10 was calculated to be 4.93 mg/l (95% confidence limits: 3.48–6.34 mg/l).
The no observed effect concentration (NOEyC) with respect to yield was determined to be 1.00 mg/l.
The results of the test can be considered reliable without restriction.

Executive summary:

The growth inhibitory effects of the substance Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts (CAS no. 70983-43-6) to the green alga Desmodesmus subspicatus were investigated according to test guideline OECD 201, over a period of 72 h.

The test item is an oily liquid, has a high solubility (>=368g/l at pH 10 and at 22°C) and is 100% pure. Consequently, the test media were prepared by respective dilutions of a stock solution.

The investigated nominal concentrations were 100, 31.6, 10.0, 3.16 and 1.00 mg/l.

Three parallel test vessels were used for each test concentration of the test item and six vessels for the blank controls.

The concentrations of the test item in the test media were measured by HPLC analysis at the beginning and after 24, 48 h and 72 h of exposure. Because of the variable composition (chain lengths C5 – 8) of the test item and the relatively high limit of quantification of the HPLC method (1.07 mg/l), the lowest test concentration could not be measured sensibly. However, these analyses revealed that the four highest concentrations were correctly dosed (105 to 111% of nominal) and remained stable over the 72h test period (95 to 102% of the initially measured concentration); hence it is expected that the lowest concentration was also correctly dosed and remained stable. Therefore, the effective concentrations ErCx/EyCx were assessed based on the nominal concentrations.

With respect to the endpoint growth rate, following effects were observed as compared to the blank controls: 71% at 100 mg/l, 24% at 31.6 mg/l, 6% at 10.0 mg/l and 3% at 3.16 mg/l. No significant effects were observed at 1.00 mg/l as compared to the blank controls.

With respect to the endpoint yield (biomass production), following effects were observed as compared to the blank controls: 97% at 100 mg/l, 67% at 31.6 mg/l, 23% at 10.0 mg/l and 14% at 3.16 mg/l. No significant effects were observed at 1.00 mg/l as compared to the blank controls.

The results of the growth rate (r) and yield (y) inhibition of the test item to the green alga Desmodesmus subspicatus are summarized in the following table showing EC values and 95% confidence limits (cl) based on nominal concentrations:

Parameter, expressed as Measured concentration (mg/l)	ErC10	ErC50	NOErC
Estimate	16.9	60.7	1.00
Lower 95%-cl	14.3	56.6	-
Upper 95%-cl	19.5	65.2	-
Parameter, expressed as Measured concentration (mg/l)	EyC10	EyC50	NOEyC
Estimate	4.93	19.8	1.00
Lower 95%-cl	3.48	17.3	-
Upper 95%-cl	6.34	22.7	-

cl: confidence limit

The 72 h ErC50 value of Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts (CAS no. 70983-43-6) on the green alga Desmodesmus subspicatus was calculated to be 60.7 mg/l. This value is based on the nominal concentrations.

All validity criteria were fulfilled.