Imidazolium compounds, 1-[2-(2-carboxyethoxy)ethyl]-1(or 3)-(2-carboxyethyl)-4,5-dihydro-2-norcoco alkyl

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:

February 4th, 2019 - To be completed after finalization of the study report

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)

Deviations:

yes

Remarks:

The temperature during the exposure period slightly exceeded the recommended range of 21 to 24°C by a maximum of 0.2°C. This deviation is not considered to have an impact on the integrity of the study results.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Solid content: 99.70%.

Analytical monitoring:

yes

Details on sampling:

Samples of stock solution, control solutions and test solutions were taken to determine the actual test item concentrations. Solutions were sampled in duplicate at the start and at the end of exposure. The duplicate samples were kept separately as a reserve. The nominal volume per sample was 10 mL. After sampling and before shipment to the analytical test site, the samples were stabilised by adding acetonitrile directly after sampling (acetonitrile volume: 20% of the water sample volume: 10 mL of sample were mixed with 2 mL of acetonitrile). All samples were stored in amber glass bottles in the dark at a temperature of ≤ -18 °C.

Analysis of the test item concentrations in the samples was performed using an LC-MS/MS method. The content of C12 amphopropionate was used as a quantifier of the test item. Acrylic acid, another constituent of the test item, was also quantified but it was eventually not used for determination of the test item concentrations due to recoveries lower than with C12 amphopropionate.

Vehicle:

no

Details on test solutions:

A stock solution was prepared at nominal concentration of 50.0 mg test item/L by adding 0.0511 g test item to 1000 mL test medium. This stock solution was stirred at ca. 600 rpm for 70 min at ambient temperature in the dark. This stock solution was subsequently used to prepare the test solutions. The required volume of stock solution was pipetted into glass volumetric flasks and diluted/filled-up to the calibration mark with test medium and stirred for 5 minutes on a magnetic stirrer.

The medium was sterilised by sterile filtration (pore size 0.2 µm) before use. All glassware and materials used for testing purposes were sterilised for at least 3 hours at ≥150 °C using a heating furnace or autoclaved for 20 minutes at 120°C, except preparation flasks (volumetric flasks) used for preparation of test solutions.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata.
- Strain: SAG 61.81.
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Albrecht-von-Haller-Institut, Universität Göttingen, Germany.
- Age of the pre-culture: 4 days.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

From 22.0°C to 24.2°C.

pH:

From 7.5 to 8.4.

Nominal and measured concentrations:

Nominal concentrations: control, 0.5, 1.58, 5.0, 15.8 and 50.0 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass Erlenmeyer flasks (300 mL) covered by air-permeable lids.
- Material, size, headspace, fill volume: 100±5 mL.
- Aeration:
- Initial cells density: 3000 per mL.
- Control end cells density: 530800 per mL.

GROWTH MEDIUM
- Standard medium used: yes (OECD medium).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: none.
- Photoperiod: 24h light/0h dark.
- Light intensity and quality: mean 72.9 µE m–2s–1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber at the start of the test; fluorometer after 24, 48 and 72 hours.
- Other: morphological abnormalities.count

TEST CONCENTRATIONS
- Spacing factor for test concentrations: square root of ten.
- Range finding study: nor reported (non-GLP range-finding test).

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.68 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% cI = 7.18-8.21 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.07 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% cI = 0.878-1.27 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The measured concentrations ranged between 90–102% of the nominal concentrations after 0 hour and 98–108% after 72 hours. Therefore, the test item concentrations were within ± 20% based on nominal concentration. No decrease of test item concentrations were observed throughout the exposure period. Thus, the biological results are calculated and reported based on nominal test item concentrations.

In none of the concentration levels up to 1.58 mg test item/L, deformed and/or damaged algal cells were observed during microscopic inspection. At the intermediate concentration level (5.00 mg test item/L) mostly all algae cells were deformed and/or enlarged. At the highest concentration levels (15.8 and 50.0 mg test item/L), no algae were observed.

Results with reference substance (positive control):

The ErC50 value for the toxic reference item, potassium dichromate, was determined as 1.253 mg/L. This value is within the range of the international ring test (ISO (2004). Therefore the results of this reference test are acceptable and the test conditions are reliable.

Reported statistics and error estimates:

The data were evaluated by the Shapiro-Wilk’s Test for normal distribution, and by Levene's Test for homogeneity of variances. The Williams Multiple Sequential t-test was applied to find out whether there were significant differences between the growth of algae in the controls and the algae exposed to the test item concentration. Weibull analyses using linear max. likelihood regression were used to determine the concentration-response function. The statistical software package ToxRat 3.3.0 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) was used for these calculations. The statistical analysis was performed using nominal concentrations.

A clear concentration-response relationship was observed for both biological parameters growth rate and yield during the exposure period. The resulting endpoints are presented in the table below based on nominal concentrations expressed in mg test item/L.

	Endpoints [mg test item/L]
Parameter	EC10	EC20	EC50	NOEC	LOEC
Yield (95% cI)	0.182 (0.139–0.228)	0.428 (0.356–0.500)	1.56 (1.43–1.69)	<0.500	≤0.500
Growth Rate (95% cI)	1.07 (0.878–1.27)	2.35 (2.05–2.64)	7.68 (7.18–8.21)	<0.500	≤0.500

All validity criteria were fulfilled as required by the study plan:

Required:	Found:
Mean biomass increase in the control cultures:factor of at least 16 within the 72-hour test period	176.9
Mean coefficient of variation for section-by-section specific growth rates in the control cultures:up to 35%	22.4%
Coefficient of variation of average specific growth rates during test period in replicate control cultures:up to 7%	1.6%

 The study is therefore considered to be valid.

Validity criteria fulfilled:

yes

Conclusions:

A 72h-EC50 value of 7.68 mg/L (95% cI = 7.18-8.21 mg/L) was determined for the parameter "inhibition of growth rate" based on nominal concentrations (analytically confirmed). A 72h-EC10 value of 1.07 mg/L (95% cI = 0.878-1.27 mg/L) was determined for the parameter "inhibition of growth rate" based on nominal concentrations (analytically confirmed).

Executive summary:

The toxicity of the test item to freshwater green algae Raphidocelis subcapitata was investigated in a GLP-compliant study performed in accordance with OECD Guideline No. 201. A series of concentrations of the test item in OECD Medium was prepared and the test organisms were exposed to these treatments, as well as to controls without the test item, for a period of 72 hours under static conditions. A clear concentration-response relationship was observed for both biological parameters growth rate and yield during the exposure period. A 72h-EC50 value of 7.68 mg/L (95% cI = 7.18-8.21 mg/L) was determined for the parameter "inhibition of growth rate" based on nominal concentrations (analytically confirmed). A 72h-EC10 value of 1.07 mg/L (95% cI = 0.878-1.27 mg/L) was determined for the parameter "inhibition of growth rate" based on nominal concentrations (analytically confirmed).

Description of key information

The toxicity of the test item to freshwater green algae Raphidocelis subcapitata was investigated in a GLP-compliant study (Gilbert & Lang, 2019) performed in accordance with OECD Guideline No. 201. A series of concentrations of the test item in OECD Medium was prepared and the test organisms were exposed to these treatments, as well as to controls without the test item, for a period of 72 hours under static conditions. A clear concentration-response relationship was observed for both biological parameters growth rate and yield during the exposure period. A 72h-EC50 value of 7.68 mg/L (95% cI = 7.18-8.21 mg/L) was determined for the parameter "inhibition of growth rate" based on nominal concentrations (analytically confirmed). A 72h-EC10 value of 1.07 mg/L (95% cI = 0.878-1.27 mg/L) was determined for the parameter "inhibition of growth rate" based on nominal concentrations (analytically confirmed).

Key value for chemical safety assessment

EC50 for freshwater algae:

7.68 mg/L

EC10 or NOEC for freshwater algae:

1.07 mg/L

Additional information

The toxicity of the test item to freshwater green algae Raphidocelis subcapitata was investigated in one GLP-compliant study (Gilbert & Lang, 2019) performed in accordance with standard methods, with minor deviations. The study is considered as reliable (Klimisch 1) and is selected as a key study for the endpoint.