4,5-bis(hydroxymethyl)-2-phenyl-1H-imidazole

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 August 2021 to 09 December 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with the OECD 202 (2004) guideline without deviation under GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 13, 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 18-20 August 2020, Date on certificate: 04 March 2021

Analytical monitoring:

yes

Details on sampling:

Single samples for analysis were taken from the control and the test concentration of 100 mg/L at the start and at the end of the test.
- Samples preparation: Samples were injected in the analytical system after a dilution by 2 with acetonitrile (500 µL of sample + 500 µL of acetonitrile). If the sample concentrations were too high and not included in the concentration range of the calibration, they were diluted appropriately with a test water/acetonitrile (50/50 v/v) solution.

Vehicle:

no

Details on test solutions:

PREPARATION OF STOCK AND TEST SOLUTIONS
- Method: The mixing vessel was a cylindrical glass bottle sealed with a screw cap and fitted with a drain port near the bottom for drawing off the stock solution. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and test water was added. Then 151.39 mg test item was weighed on a weighing boat that afterwards was placed above the mixing vessel and rinsed with test water. The mixing vessel was then carefully filled with the remaining volume of test water to obtain 1 L of stock solution and thereafter was closed immediately. Mixing was initiated with the vortex in the centre extending at least to 20 % of the vessel depth from the top to the bottom of the vessel. After 22 hours of stirring in the dark at approx. 80 °C (set temperature of the magnetic stirrer with hot plate, corresponding to an actual temperature of the stock solution around 50 °C (the test substance was heated to aid solution preparation and homogeneity -based on results of the melting point of the substance this temperature does not lead to decomposition or changes of the substance and therefore will not impact the results (OECD 102, NOEL, 2022, no change up to 110 °C - see Section 4 of IUCLID)), the contents of the vessel were allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were discarded via the drain port. Samples were taken from the filtered stock solution and chemically analysed. Then the filtered stock solution (through 11 µm cellulose filter paper) was diluted with test water as necessary to obtain the required test concentration of 100 mg/L into a 500-mL volumetric flask (based on the analytically confirmed concentration of the stock solution (141.8 mg/L)). After filling and introduction of daphnids the vessels were sealed immediately. The test solution was observed to be clear and colourless (Tyndall effect, checked via laser beam, was negative). The test was carried out without adjustment of the pH.
- Controls: Test water without test substance but treated in the same way as the test substance solution.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Daphnia magna Straus (Cladocera, Crustacea), clone 5, sex Female.
- Age at study initiation (mean and range, SD): < 24 hours old at the test initiation
- Stage and instar at study initiation: not first brood progeny at the test initiation
- Method of breeding: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C/Daphnia/day. The water was changed twice a week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Feeding during test: No feeding.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

250 mg/L (as CaCO3).

Test temperature:

Controlled environment cabinet.
The temperature of the test medium was between 20.2 and 20.9 °C throughout the test (average value: 20.6 °C), and complied with the requirements (20 °C ± 2 °C, constant within 1 °C).

pH:

From 7.84 to 8.12.

Dissolved oxygen:

From 7.94 to 8.58 mg/L. All these dissolved oxygen concentrations correspond to values >= 80% of the air-saturation value.

Salinity:

Not applicable.

Conductivity:

No data.

Nominal and measured concentrations:

- Nominal test concentration: 100 mg/L
- Measured test concentrations: 103.90 and 104.90 mg/L at the start and the end of the test, respectively. See table 6.1.3/2 in "Any other information on results incl. tables".

Details on test conditions:

TEST SYSTEM
- Test vessel: 60-mL glass flasks sealed with assembled screw cap with hole and PTFE/silicone septum. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Type: Closed
- Aeration: No aeration of the test solutions occurred throughout the test.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): Not applicable. A static test was performed (stability of the test item was demonstrated in closed and static conditions during the range-finding test).
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): Not applicable.
- Biomass loading rate: 5 daphnids per vessel each completely filled with test solution and without headspace.
- Introduction of daphnids: Daphnids were introduced into the test vessels each completely filled with test solution and stoppered without headspace immediately after filling the test vessels with test solutions.

TEST MEDIUM / WATER PARAMETERS
Reconstituted water (Elendt M4 medium), as prescribed in OECD TG 202.

OTHER TEST CONDITIONS
- Adjustment of pH: no.
- Photoperiod: 16h light / 8h dark
- Light intensity: no data.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Eventual immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of test vessels.

VEHICLE CONTROL PERFORMED: not applicable

RANGE-FINDING STUDY
- Test concentrations: 0, 0.32, 1.0, 3.2, 10.0, 32.0, 100.0 and 176.4 mg test item/L (= filtered stock solution).
- Results used to determine the conditions for the definitive study: After 24 and 48 hours of exposure, no immobilisation of Daphnia magna was observed in the control and all tested concentrations. Only one daphnid was weakened compared to the others, in the first replicate of the 176.4 mg/L test concentration after 24 hours. Therefore, based on the results of the range-finding test, a limit test was performed at 100 mg/L in order to demonstrate that the test item had no effect on the test organism at this test concentration.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (batch no.: 20l144110; purity: min. 99.8%)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Result based on analytically confirmed nominal concentration.

Details on results:

See tables 6.1.3/1 and 6.1.3/2 in "Any other information on results incl. tables".

After 24 and 48 hours of exposure, no immobilisation of Daphnia magna was observed in the control and the test concentration of 100 mg/L, confirming the observations of the range-finding test where results showed no immobilisation at any of the tested concentrations from 1 to 100 mg/L.
Based on these results, the 24 and 48-hour EC50 values were therefore > 100 mg test item/L.

Chemical analysis revealed that test item levels found were stable, with losses of test item < 20%. Moreover, geometric means of measured concentrations were within ± 20% of the nominal concentrations. Therefore, since the concentrations of the test item were satisfactorily maintained within ± 20% of the initial and nominal concentrations throughout the test, the evaluation of the effects on Daphnia magna can be based on analytically confirmed nominal values.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: On February 2, 2021 (KD21-001; most recent test), the 24h-EC50 was 1.278 mg/L. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD TG 202 (expected 24h-EC50: 0.6 to 2.1 mg/L) at this time.

Reported statistics and error estimates:

No statistical analysis was performed. Effective concentrations were determined directly from the raw data.

Table 6.1.3/1: Acute immobilisation of daphnids after 24 and 48 hours in the final test

 

Analytically confirmed nominal concentration (mg test item/L)	Replicate	Number of daphnids exposed	Immobilisation at 24 h		Immobilisation at 48 h
			Number	Total %	Number	Total %
Control	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
100	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0

 

Table 6.1.3/2: Concentration of the test item (mg/L) in test water - Final test:

 

Nominal concentration (mg/L)	Measured concentration (mg/L)		Relative loss to initial value (t=0 h - t=48 h) (%)	Geometric mean of measured concentrations
	Start (t=0 h)	End (t=48 h)		mg/L	% nominal
Control	Abs.	Abs.	N.A.	N.A.	N.A.
100	103.90	104.90	-1	104.40	104

N.A.: not applicable

% = Percent of expected nominal concentration in test item.

Abs.= Absence: concentrations below the LOQ (1.00 mg/L) and the LOD (0.30 mg/L).

Validity criteria fulfilled:

yes

Remarks:

In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress. Dissolved oxygen concentration at the end of the test was ≥ 3 mg/L in controls and test vessels.

Conclusions:

The toxic effect of the test substance to the freshwater invertebrate Daphnia magna was investigated in a closed static test. Under the experimental conditions and based upon analytically confirmed nominal concentrations, the 48-hour EC50 value was determined to be higher than 100 mg/L.

Executive summary:

This study was performed to assess the acute toxicity of test substance to Daphnia magna, according to OECD Test Guideline 202 and EU Method C.2, with GLP compliance.

A limit test was performed following the results of a range-finding test. Twenty daphnids (four replicates, five daphnids per replicate) were exposed to the test substance at a nominal concentration of 100 mg/L and to a control. The immobilisation of the daphnids was determined in a closed static 48-hour test by visual observation after 24 and 48 hours. Samples taken from the control and the test concentration of 100 mg/L were analysed at the start and the end of the test in order to determine if concentrations of the test item were maintained.

The test item levels were found to be relatively stable throughout the test (within ± 20% of the initial and nominal concentrations throughout the test). Thus, the evaluation of the effects on daphnids was based on analytically confirmed nominal concentrations.

After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the test concentration of 100 mg/L.

In conclusion, under the experimental conditions and based upon analytically confirmed nominal concentrations, the 48-hour EC50 value was determined to be higher than 100 mg/L.

Description of key information

48h-EC50 (Daphnia magna) > 100 mg/L; OECD TG 202 and EU Method C.2; N. DELPIT (2021)

Additional information

One key study for the assessment of ready biodegradability is available for the test item. This study was performed to assess the acute toxicity of test substance to Daphnia magna, according to OECD Test Guideline 202 and EU Method C.2, with GLP compliance.

A limit test was performed following the results of a range-finding test. Twenty daphnids (four replicates, five daphnids per replicate) were exposed to the test substance at a nominal concentration of 100 mg/L and to a control. The immobilisation of the daphnids was determined in a closed static 48-hour test by visual observation after 24 and 48 hours. Samples taken from the control and the test concentration of 100 mg/L were analysed at the start and the end of the test in order to determine if concentrations of the test item were maintained.  Preparation of the test item required moderate heating (80 °C) to ensure homogenity and dissolution in a practicable and timely manner, the heating did not effect the test item and therefore, nor the results. Based on the results of the melting point of the substance this temperature does not lead to decomposition or changes of the substance and therefore will not impact the results (OECD 102, NOEL, 2022, no change in test item upto 110 °C - see Section 4 of IUCLID)).

The test item concentrations were stable throughout the test (within ± 20% of the initial and nominal concentrations throughout the test). Thus, the evaluation of the effects on daphnids was based on analytically confirmed nominal concentrations.

After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the test concentration of 100 mg/L.

In conclusion, the 48-hour EC50 value was determined to be higher than 100 mg/L.