1-ethyl-3-methyl-3H-imidazolium dicyanidoamide(1-)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Sampling was conducted in the definitive test at the beginning, and after 24, 48 and 72 h of exposure.

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTIONS
Since EMIM Dicyanamid is a water soluble substance, the test concentrations were prepared by dilution of a 100 mg/l stock solution of the test material in aerated algal medium. The test solutions were filtered prior to the addition to the test vessels (0.45 μm cellulose acetate membrane sterile syringe filter, VWR International).

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Common name: Desmodesmus subspicatus
- Strain: SAG 86.81
- Source (laboratory, culture collection): Collection of algal cultures in University of Göttingen/Germany
- Age of inoculum (at test initiation): The axenic slope culture is yearly re-purchased. Potassium dichromate is used to control the sensitivity of the culture twice a year.
- Method of cultivation: 250 ml flask containing 100 ml of sterile OECD medium inoculated with cell material from axenic slope cultures; Continuous illumination (2000–3000 lux) from Osram Fluora L18W77 and Osram Daywhite L18W840 (Osram AG, Winterthur, Switzerland); 22 ± 2 °C, thermo-controlled room

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.8 - 22.7°C

pH:

7.2 - 7.8

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 1.25, 2.50, 5.00, 10.0 and 20.0 mg/l (based on a range-finding test)
- Measured concentrations (t=0h): 0.994, 1.36, 2.43, 4.31 and 9.47 mg/L
- Geometric mean concentrations (t=0 to t=72h): 0.832, 1.30, 2.54, 4.53 and 8.79 mg/L (84, 96, 105, 105 and 93% of the initial concentration, respectively).

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL flasks
- Material, size, headspace, fill volume: 250 mL Erlenmeyer flasks, test volume 100 mL
- Shaking rate: continuous (appr. 130 rpm)
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution: One application at test start
- Initial cells density: 0.5x10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per test group for estimating initial concentration control analsis only (replicates): 3
- No. of vessels per blank control for estimating initial concentration: 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium prepared according to OECD Guidleine for Testing of Chemicals, No. 201 Algal growth inhibition test, Mar 2006, Annex 5 corrected July 2011
- pH-value: The pH of the control medium should not increase by more than 1.5 units during the test. Determined in the medium batch which is used for the preparation and dilution of the stock solution
- Starting cell density: 0.50–0.85 μg/ml with respect to dry weight corresponding to about 2–5•103 cells/ml (i.e. OD680 of about 0.005 units)

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: Source: continuous from Osram Fluora L 18W77 and Osram Daywhite L 18W840 (Osram AG, Winterthur, Switzerland);
- Light intensity and quality: Light intensity (target): the light intensity amounts to about 3000 lux which corresponds to about 40 μE•m-2•s-2 according to the OECD TG no. 2011; Homogeneity (target): the light intensity is maintained within ±15% from the average light intensity over the incubation area

EFFECT PARAMETERS MEASURED
- Observation: Cell concentrations were determined every 24 h using a spectrophotometer (680 nm wavelength). Aliquots of 5 ml were removed from each test flask under sterile conditions. Cell concentrations at time 0 h were only determined in the control vessels
- Chemical analysis: The chemical analysis of EMIM Dicyanamid was performed with a combined approach. The cation (1H-imidazolium, 1-ethyl-3-methyl-) was measured by HPLC. Additionally, EMIM Dicyanamid was measured by dissolved organic carbon (DOC) determinations. Both analysis were conducted in the definitive test at the beginning, and after 24, 48 and 72 h of exposure.

- HPLC: The analysis was performed using a Dionex System (pump P680 LPG and autosampler ASI-100) with UV-detection (UV-Detector UVD170U) at 210 nm. The column used was an UltraSep ES PHARM RP 18 (SEPSERV Separation Service, DE-10587 Berlin) at 25°C. The mobile phase consisted of acetonitrile (A) and a sodium 1-heptanesulfonate buffer at pH 2.4 (B). The buffer was prepared by dissolving 700 μl phosphoric acid (85%) and 1.1 g of sodium 1-heptanesulfonate monohydrate in 1000 ml of water. A gradient with a flow rate of 1 ml/min was applied: 0 min. A: 5% B: 95%; 10 min. A: 30% B: 70%; 15 min. A: 30% B: 70%. Standard solutions were prepared in 5% acetonitrile and 95% H3PO4 (0.01 M). The quantification limit of the analytical method (LOQ) was 4 mg/l with respect to EMIM Dicyanamid (lowest standard solution tested). The above HPLC method was provided by Sigma-Aldrich Chemie GmbH, CH-9471 Buchs/Switzerland) and consequently adapted and validated by the test facility.

- DOC analysis: DOC was determined with a Shimadzu 5050A TOC-Analyzer (Shimadzu Schweiz GmbH, Römerstr. 3, CH-4153 Reinach) using the NPOC-mode. For each determination, 3 single injections were performed. The samples from the individual test vessels were filtrated (0.45 μm) and acidified to pH <2. Prior to analysis, the samples were sparged with CO2-free high purity air for 5 min to remove inorganic carbon. The quantification limit of the analytical method (LOQ) was 0.5 mg C/l (lowest standard solution tested).

TEST CONCENTRATIONS
Prior to the definitive test, a non-GLP range finding with nominal concentrations of EMIM Dicyanamid of 1.0, 10 and 100 mg/l was performed. Based on the results of this non-GLP range finding test, the nominal concentrations reported under 'Nominal and measured concentrations' were selected.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 12.6-17.9

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.73 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 2.22-5.03

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.79 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: cation

Basis for effect:

growth rate

Remarks on result:

other: 5.85-8.20

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.99 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: cation

Basis for effect:

growth rate

Remarks on result:

other: 1.24-2.60

Description of key information

ErC50 = 14.7 mg/L (Desmodesmus subspicatus)

ErC10 = 3.73 mg/L (Desmodesmus subspicatus)

Additional information

The toxicity towards freshwater algae was determined in a study according to OECD TG 201 and in compliance with GLP criteria (BMG, 2013). In this study, exponentially growing freshwater alga (Desmodesmus subspicatus) were exposed to nominal test substance concentrations of 0 (control), 1.25, 2.50, 5.00, 10.0 and 20.0 mg/L for 72 hours under static conditions. Test concentrations were analytically verified and determined to be 0 (control), 0.886, 2.07, 4.71, 9.53 and 19.3 mg/L (geometric mean). At the start of the test and after 24, 48 and 72 hours exposure duration, the algae were scored for number of cells. The 72-h EC50 and EC10 values for growth rate of the test substance are determined at 14.7 mg/L and 3.73 mg/L for the test substance, respectively.