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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

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:

There was one deviation from the guidelines concerning some pH values. This deviation did not affect the integrity of the study.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

March 23, 2006

Deviations:

yes

Remarks:

There was one deviation from the guideline concerning some pH values. This deviation did not affect the integrity of the study.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

May 30, 2008

Deviations:

yes

Remarks:

There was one deviation from the guideline concerning some pH values. This deviation did not affect the integrity of the study.

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 (from a replicate of each treatment with algae dedicated exclusively to chemical analyses) 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 150.86 mg test item were 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 contents of the vessel were allowed to stand undisturbed for at least 1 hour before use (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 in test item unto 110 °C - see Section 4 of IUCLID)). 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 11µm cellulose filter paper) was diluted with test water as necessary and fixed amount of inoculum (5.103 cells/mL per vessel) to obtain the required test concentration of 100 mg/L in the test vessels (based on the analytically confirmed concentration of the stock solution (141.0 mg/L)) that were immediately sealed after filling with a minimum headspace.
- Controls: Test water without test substance but treated in the same way as the test substance solutions.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): At the start of the test, the test solution in test vessels was observed to be clear and colourless (Tyndall effect, checked via laser beam, was negative).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19 - 75005 PARIS, bred in the Laboratoires des Pyrénées et des Landes under standardised conditions, according to the test guidelines.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (= test water) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2 °C.

ACCLIMATION
4 days before the start of the test, cells from the algal stock culture were inoculated in test water at a cell density of 1.10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

350 mg/L (as NaHCO3). To insure a satisfactory CO2 supply for the algal growth in a closed system.

Test temperature:

Controlled environment cabinet. The temperature in the incubator was situated between 22.0 and 23.6 °C throughout the test (average value: 23.5 °C), and complied with the requirements (23 °C ± 2 °C, constant within 2 °C).

pH:

From 8.20 to 10.13 (see "Overall remarks" for further detail).

Dissolved oxygen:

No data.

Salinity:

Not applicable.

Conductivity:

No data.

Nominal and measured concentrations:

- Nominal test concentration: 100 mg/L
- Measured test concentrations: 106.70 and 91.70 mg/L at the start and the end of the test, respectively. See table 6.1.5/4 in "Any other information on results incl. tables".

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass closed flasks with ground glass stopper, completely filled with test solution with minimum headspace. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Type (delete if not applicable): Closed
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): Not applicable.
- Initial cells density: An initial cell density of 5.103 cells/mL using the exponentially growing pre-culture.
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable.
- Test environment: Controlled environment cabinet (23 °C ± 2 °C); vessels were distributed randomly in the incubator and redistributed over the test at t=24h and t=48h. During incubation, the algal cells were kept in suspension by continuous magnetic stirring (250 rpm).

GROWTH MEDIUM
- Standard medium used: yes.

TEST MEDIUM / WATER PARAMETERS
Original medium from OECD TG 201. Since the test was performed in sealed conditions, additional sodium bicarbonate was added to test water to insure a satisfactory CO2 supply for the algal growth (for all treatments and inoculum suspension): 7 mL of NaHCO3 were added to the sterilised water during test water reconstitution (instead of 1 mL) to obtain a final concentration of 350 mg/L.

OTHER TEST CONDITIONS
- Sterile test conditions: not reported.
- Adjustment of pH: The pH of the test solutions was not adjusted.
- Photoperiod: Continuous illumination.
- Light intensity: 4 440-8 880 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable (limit test).
- Range finding study: Yes
- Test concentrations in the range-finding tests: 0, 1.0, 3.2, 10, 32 and 100 mg/L (first range-finding test) and 0, 1.0, 3.2 and 100 mg/L (second range-finding test).
- Results used to determine the conditions for the definitive study: Two range-finding test were performed. In the first test, the cell counts at 1.0 and 3.2 mg/L were surprisingly low compared to the higher test concentrations. A technical error in preparing the test medium for these concentrations was suspected. Therefore, another test was carried out (second test). Based on the results of the range-finding tests, a limit test was performed at 100 mg/L in order to demonstrate that EC50 was higher than 100 mg/L.

Reference substance (positive control):

yes

Remarks:

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

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Result based on analytically confirmed nominal concentration.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: Result based on analytically confirmed nominal concentration.

Details on results:

See "Growth curve" in Illustration, and Tables 6.1.5/1, 6.1.5/2, 6.1.5/3 and 6.1.5/4 in "Any other information on results incl. tables".

After 72 hours of exposure, the inhibition of algal growth rate and yield were 5.7 % and 25.9 %, respectively. Based on these results, the 72-hour ErC50 and EyC50 was therefore > 100 mg/L.

Chemical analysis revealed that test item levels 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 can therefore be based on analytically confirmed nominal values.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: On February 9, 2021 (KA21-001; most recent test), the 72h-EC50 was 0.759 mg/L for the parameter growth rate. Hence, the sensitivity of this batch of Pseudokirchneriella subcapitata was consistent with the level proposed by the ISO 8692 (expected 72h-ErC50: 0.65 to 1.73 mg/L).

Reported statistics and error estimates:

The software ToxRat® Professional was used to perform statistical analyses.

Table 6.1.5/1: Algal cell densities during the final test (expressed as density of algal cells/mL x10^4)

	Replicate	Analytically confirmed nominal concentration (mg test item/L)
		Control	100
t=24 h	1	6.4	4.0
	2	6.8	4.4
	3	4.4	2.8
	4	4.0	4.0
	5	3.6	2.8
	6	6.0	3.6
	Mean	5.2	3.6
	Std. Dev.	1.36	0.67
t=48 h	1	33.6	25.2
	2	34.8	20.0
	3	36.0	26.0
	4	26.0	24.8
	5	32.0	26.0
	6	28.0	26.0
	Mean	31.7	24.7
	Std. Dev.	3.95	2.34
t=72 h	1	124.0	107.2
	2	112.0	76.4
	3	116.0	91.2
	4	107.6	67.2
	5	107.2	80.0
	6	93.2	68.0
	Mean	110.0	81.7
	Std. Dev.	10.31	15.29

At test start 5000 algal cells/mL were incubated; 6 replicates of the controls and 6 replicates of the limit test concentration.

Std. Dev.: standard deviation.

 

Table 6.1.5/2: Yield of algal cells during the final test

	Replicate	Analytically confirmed nominal concentration (mg test item/L)
		Control	100
t=24 h	1	5.9	3.5
	2	6.3	3.9
	3	3.9	2.3
	4	3.5	3.5
	5	3.1	2.3
	6	5.5	3.1
	Mean	4.7	3.1
	Std. Dev.	1.36	0.67
	% Red.	-	34.0
t=48 h	1	33.1	24.7
	2	34.3	19.5
	3	35.5	25.5
	4	25.5	24.3
	5	31.5	25.5
	6	27.5	25.5
	Mean	31.2	24.2
	Std. Dev.	3.95	2.34
	% Red.	-	22.6
t=72 h	1	123.5	106.7
	2	111.5	75.9
	3	115.5	90.7
	4	107.1	66.7
	5	106.7	79.5
	6	92.7	67.5
	Mean	109.5	81.2
	Std. Dev.	10.31	15.29
	% Red.	-	25.9

Values were extracted from the computer program ToxRat.

% Red.: %Reduction in yield relative to the control determined by ToxRat.

 

Table 6.1.5/3: Mean specific growth rate in P. subcapitata during the final test

	Replicate	Analytically confirmed nominal concentration (mg test item/L)
		Control	100
t=0 h - t=24 h	1	2.549	2.079
	2	2.610	2.175
	3	2.175	1.723
	4	2.079	2.079
	5	1.974	1.723
	6	2.485	1.974
	Mean	2.312	1.959
	Std. Dev.	0.2692	0.1936
	% Inh.	-	15.3
t=0 h - t=48 h	1	2.104	1.960
	2	2.121	1.844
	3	2.138	1.976
	4	1.976	1.952
	5	2.079	1.976
	6	2.013	1.976
	Mean	2.072	1.947
	Std. Dev.	0.0644	0.0513
	% Inh.	-	6.0
t=0 h - t=72 h	1	1.838	1.789
	2	1.804	1.676
	3	1.816	1.735
	4	1.791	1.634
	5	1.789	1.692
	6	1.743	1.638
	Mean	1.797	1.694
	Std. Dev.	0.0320	0.0599
	% Inh.	-	5.7

Values were extracted from the computer program ToxRat.

% Inh.: %Inhibition of growth rate relative to the control determined by ToxRat.

 

Table 6.1.5/4: Concentrations of the test item in test water - Final test

Nominal concentration (mg/L)	Measured concentration (mg/L)		Relative loss to initial value (t=0 h - t=72 h) (%)	Geometric mean of measured concentrations
	Start (t=0 h)	End (t=72 h)		mg/L	% nominal
Control	Abs.	Abs.	N.A.	N.A.	N.A.
100	106.70	91.70	14	98.92	99

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:

Cell density in the control: 220-fold increase within 72h. The mean CoV for section-by-section specific growth rates in the control was 32.0 % in 72h and the CoV of average specific growth rates during the whole test period in replicate control was 1.8%.

Conclusions:

The toxic effect of test substance to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test. Under the experimental conditions and based upon analytically confirmed nominal concentrations, the 72-hour ErC50 for growth rate was determined to be higher than 100 mg/L.

Executive summary:

The study was performed to assess the test substance for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata, according to OECD Test Guideline 201 and EU Method C.3, with GLP compliance. 

A limit test was performed following the results of a range-finding test. Algal cells were exposed to the test substance at a nominal concentration of 100 mg/L and to a control. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. 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 concentration throughout the test). Thus, the evaluation of the effects was based on analytically confirmed nominal concentrations.

After 72 hours of exposure, the inhibition of algal growth rate was 5.7% at the test concentration of 100 mg/L.

In conclusion, under the experimental conditions and based on analytically confirmed nominal concentrations, the 72-hour ErC₅₀ was determined to be higher than 100 mg/L.

Description of key information

72h-ErC50 (Pseudokirchneriella subcapitata) > 100 mg/L; OECD TG 201 and EU Method C.3; N. DELPIT (2021)

Additional information

One study was available on the toxicity of the test item to green algae.  The study was performed to assess the test substance for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata, according to OECD Test Guideline 201 and EU Method C.3, with GLP compliance. 

A limit test was performed following the results of a range-finding test. Algal cells were exposed to the test substance at a nominal concentration of 100 mg/L and to a control. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. 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)).

Also, although the pH level in the control varied by more than 1.5 units at the end of the test (1.86 units of difference) this was not considered to have affected the integrity of the study. The cause of the pH increases in the controls and test concentrations was certainly due to the substantial algal growth in conjunction with closed conditions used in the test, despite the additional sodium bicarbonate. All validity criteria were still met regardless of this deviation. 

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

After 72 hours of exposure, the inhibition of algal growth rate was 5.7% at the test concentration of 100 mg/L.

In conclusion, under the experimental conditions the 72-hour ErC₅₀ was determined to be higher than 100 mg/L.