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

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 January 2016 to 15 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

2010

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

May, 2008

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): EMIM Dicyanamid
- Test substance No.: 12/0071-2
- Batch identification: 0013141983
- Identity: Confirmed by 1H-NMR, 13C-NMR and IR spectroscopy.
- Purity: 99.81 area-% (HPLC, 210 nm); 100.0 area-% (HPLC, 225 nm)
- Homogeneity: Homogeneous
- Expiry date: 19 Feb 2017
- Date of production: 20 Feb 2015
- Physical state/appearance: liquid/colorless to brown, clear
- Storage conditions: Ambient at room temperature, under nitrogen
- Storage stability: stable

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test substance was added in the required amounts according to the test concentrations directly to the test vessels with 234 mL deionized water. Aliquots of the reference substance stock solution were dosed to the test vessels and made up with deionized water to a volume of 234 mL. 16 mL synthetic medium were dosed to each test vessel with test substance and reference substance afterwards. To prepare the blank control assays 234 mL of deionized water and 16 mL synthetic medium were mixed.


REFERENCE SUBSTANCE PREPARATIONS
1000.0 mg of the reference substance was added to about 800 mL of deionized water and stirred at room temperature until the reference substance was completely dissolved. The pH value of the stock solution was measured and adjusted to 7.0 with 1 M sodium hydroxide solution. Following this the stock solution was made up to 1L with deionized water. The stock solution appeared yellowish/ clear and no undissolved reference substance was visible.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Origin: aeration tank of the wastewater treatment plant of Mannheim, Germany
- Collection of the test system: 13 Jan 2016
- Arrival in the test facility: 13 Jan 2016

After arrival of the activated sludge suspension in the test facility the suspension was sieved with a fine woven mesh (mesh size about 1 mm). This suspension was pre-aerated over night at room temperature. At the next day the sludge suspension was washed once with drinking water and the suspension was adjusted to 3 g/L Dw.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20.2 - 20.8 °C

pH:

7.5 - 8.1

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 62.5, 125, 250, 500 and 1000 mg/L
- Nominal concentrations of the positive control: 1, 10 and 100 mg/L

Details on test conditions:

PREPARATION OF THE TEST AND TEST START
Prior to the test start the pH-values in all test vessels were checked and adjusted. After addition of 250 mL of inoculum suspension (3 g/L DW) the incubation was started by aeration of the test vessels with pressured air. The vessels for the blank control assays were prepared to the same procedure without addition of test- or reference substance. After 3 hours incubation at 20 ± 2°C the mixtures in the test vessels were placed subsequently for oxygen measuring. The content of oxygen at the start of the measurements was > 7 mg/L. The total oxygen consumption was measured in the sequence BC1-3 and RS1, RS2-5, RS6 and TS1-3, TS4-7, TS8-11 and TS12-15. The oxygen consumption of the blank control BC4-6 were measured at last. No abiotic control was tested.

TEST SYSTEM
- Test vessels: Glas-beakers (nominal volume 1L)
- Test volume: 500 mL
- Aeration: Yes
- Test replicates: 6 replicates for the control; 3 replicates for each test substance concentration; 2 replicates for each reference substance concentration
- Sludge concentration in the test vessel: 1.5 g/L

TEST MEDIUM / WATER PARAMETERS
- Synthetic medium: 16 mL/test vessel of 100-fold concentrated OECD medium

EFFECT PARAMETERS MEASURED: oxygen consumption was measured after 3 hours of exposure, for a period of about 8 to 10 minutes

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

310 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% C.L.: 279.3 - 347.0 mg/L

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% C.L.: 12.5 - 24.6 mg/L

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

47 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% C.L.: 37.4 - 59.3 mg/L

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

At the lowest test concentration of 62.5 mg/L, 22% inhibition of the oxygen consumption was observed. This inhibition increased in a dose-dependent manner up to to 70% inhibition at the highest tested concentration of 1000 mg/L. See 'Any other information on results incl. tables'.

Results with reference substance (positive control):

The 3-h EC50 of the reference substance was 6.9 mg/L

Reported statistics and error estimates:

The consumption rates were used for the determination of the ECx by the probit method based on Finney [Finney, D.J., Probit Analysis; Cambr. Univ. Press, 3rd ed., 1971] with the software TOXRAT Professional 2.10 [Ratte, M.: ToxRat Professional 2.10 (2001-2010). ToxRat Solutions GmbH, Naheweg 15, 52477 Alsdorf, Germany]. The effect concentrations were given with an accuracy of 2 significant digits.

Consumption rate and inhibitory effects of the test substance and reference

Sample	Concentration (mg/L)	O2 consumption rate [mg/Lxh]	Inhibition (%)
Blank	0	27	-
Reference 1	1	22	17
Reference 2	10	12	57
Reference 3	100	2	92
Test conc. 1	62.5	21	22
Test conc. 2	125	17	37
Test conc. 3	250	15	44
Test conc. 4	500	11	58
Test conc. 5	1000	8	70

Validity criteria fulfilled:

yes

Reference 2

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: DIN 38412-L34; Luminescence inhibition assay with Vibrio fischeri (Bestimmung der Hemmwirkung von Flüssigproben auf die Lichtemission von Vibrio fischeri).

Version / remarks:

1991

GLP compliance:

no

Remarks:

Scientific research

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test material (as cited in publication): 1-ethyl-3-methylimidazolium dicyanimide (IM12 [N(CN)2])
- Source: Merck KGaA (Darmstadt, Germany)
- Purity: ≥ 97%

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Vibrio fisheri

Details on inoculum:

- Source: Dr. Lange GmbH (Düsseldorf, Germany).
- Preparation of test organisms: The freeze-dried bacteria were rehydrated and preincubated (in the thermostat LUMIStherm) for 15 minutes at 15 °C with bacteria solution.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

30 min

Salinity:

The medium included 2% sodium chloride

Nominal and measured concentrations:

Multiple concentrations, although the exact concentrations are not specified.

Details on test conditions:

TEST SYSTEM
- Fill volume: 1 mL
- Replicates per concentration: 3
- Replicates per control: 4
- Other: the test was conducted twice.

EFFECT PARAMETERS MEASURED: luminescence (measured with LUMIStox 300) at test start and after 30 minutes (at test end).
- Toxicity: The relative toxicity of the test substance was expressed as the percentage of luminescence compared to the controls.

Reference substance (positive control):

no

Duration:

30 min

Dose descriptor:

EC50

Effect conc.:

1 772 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: luminescence inhibition

Remarks on result:

other: corresponds to 10000 µM (97.5% CI: 9400 - 11000 µM)

Remarks:

Effect value has been corrected by the assessor

Reported statistics and error estimates:

Dose-response curve parameters and plots were done using the drfit package (version 0.05-92) for the R language and environment for statistical computing.

Validity criteria fulfilled:

not specified

Description of key information

The 3-h EC50 value and EC10 values are 310 and 18 mg/L respectively, as observed in a respiration inhibition study with activated sludge of a predominantly domestic sewage.

Additional information

The toxicity of the substance to microorganisms was investigated in an activated sludge respiration inhibition study according to OECD TG 209 and in compliance with GLP criteria (BASF, 2016). In this study non-adapted activated sludge from a predominantly domestic sewage was exposed to nominal test substance concentrations of 0 (control), 62.5, 125, 250, 500 and 1000 mg/L for 3 hours. Test concentrations were not analytically verified. The effects on microorganisms was determined by measuring total respiration rates and comparing these to an unexposed control. Inhibition of oxygen consumption increased in a dose-dependent manner from 22% inhibition at 62.5 mg/L up to 70% inhibition at 1000 mg/L Based on these findings, the 3-h EC50 and EC10 values were determined at 310 mg/L and 18 mg/L, respectively.

Secondly, the toxicity of the substance to marine bacteria (Vibrio fischeri) was investigated in a test according to DIN 38412-L34 (Steudte et al, 2012). After preincubation, the bacteria in solution were incubated with different concentrations of the test substance (and a control) for 30 minutes. At study initiation (before addition of the test substance) and at study termination, luminescence was measured. The relative toxicity of the test substance was expressed as the percentage of luminescence compared to the controls. The 30-min EC50 value based on luminescence inhibition was determined to be 10000 µM (97.5% CI: 9400 - 11000 µM), which corresponds to 1772 mg/L.