2-phenylimidazole

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 Nov 2017 - 30 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 23 March 2006, Annex 5 corrected: 28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Karlsruhe, Germany

Test material

Test material information

Constituent 1

Reference substance name:

2-phenylimidazole

EC Number:

211-581-7

EC Name:

2-phenylimidazole

Cas Number:

670-96-2

Molecular formula:

C9H8N2

IUPAC Name:

2-phenyl-1H-imidazole

Test material form:

solid: flakes

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Analytical samples were taken and analysed in the main test from control and all test item concentrations at 0 hours (initial value) from fresh test solutions and after 24 hours and 72 hours from aged test solutions.
- Sample storage conditions before analysis: All samples were stored deep frozen until they were transferred to the analytical laboratory.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated stock solution was prepared by directly weighing 100 mg in 1000 mL test medium. This stock solution was stirred in the dark at room temperature for 24 h (based on OECD Series on Testing and Assessment No. 23). Subsequently the necessary volumes for the test were withdrawn via a Teflon tube from the medium level of the stock solution. The test item solutions were made by diluting the stock solution with test medium without algae to give the required nominal test item concentrations. Algae were added to each solution individually resulting in target cell densities of 0.5 × 10E+04 cells per mL in each solution. Approximately 50 mL of the prepared solutions were transferred to each test vessel.
- Controls: blank control without test material
- Eluate: no
- Differential loading: no
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The solution was clear and transparent.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: freshwater green algae
- Strain: SAG 61.81
- Source: MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany
- Age of inoculum (at test initiation): 3-4 days
- Method of cultivation: The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Conditions of cultivation: Illumination: continuously (approx. 4440 - 8880 lux at cell culture level or 60 - 120 µEm-2s-1); Temperature: 21 - 24 °C; Culture flasks: 100 mL Erlenmeyer flasks; CO2 supply by shaking on a rotating shaker, approximately 105 rpm

ACCLIMATION
- Acclimation period: 3-4 days
- Culturing media and conditions: same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.7 - 22.9 °C

pH:

control: 7.40 - 8.63
treatment: 7.29 - 8.50

Nominal and measured concentrations:

nominal: 100, 31.3, 9.77, 3.05, 0.954 mg/L and control
measured ranges: 110-111, 34.2-35.5, 10.7-11.2, 3.20-3.25, 1.01-1.04 and < LOD

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (100 mL) with aluminium caps were filled up with ~ 50 mL test solution
- Initial cells density: 0.5 × 10E+04 cells per mL (targeted)
- Control end cells density: 42.66 × 10E+04 cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Aeration: continously shaken at 105 rpm

GROWTH MEDIUM
- Standard medium used: AAP-Medium (according to Annex 3 of OECD 201)
- pH of the test medium: 7.5 ± 0.1.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: yes, according to guideline
- Intervals of water quality measurements: The pH was recorded at test start and test end. Temperature was measured continuously over the whole test period and recorded daily.

OTHER TEST CONDITIONS
- Photoperiod: continuous light
- Light intensity and quality: 88.8 µE m-2 s-1 (mean)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Fluorescence measurement; The fluorescence measurements were performed with a fluorescence microplate reader (infinite 200Pro) with an emission wavelength of 670 nm and evaluated with Tecan i-control (Software for Tecan Readers Tecan i-control, 1.11.1.0). Additionally, the morphological appearance of the algae cells was observed microscopically at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: yes
- Test concentrations in range finding study: 100, 10.0, 1.00, 0.100 and 0.0100 mg/L, control
- Results used to determine the conditions for the definitive study: After 72 h at termination of the test no inhibition of growth rate or yield was observed for test item concentrations up to and including 0.100 mg/L. At higher test item concentrations the inhibition of growth rate peaked in 126.9 % at a nominal test item concentration of 100 mg/L and the inhibition of yield peaked in 101.6 % at a nominal test item concentration of 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate; tested regularly

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.86 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 1.36-2.40 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.74 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 7.33-10.4 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.582 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 0.383-0.788 mg/L

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 2.02-2.93 mg/L

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.954 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The cells were considered normal for the control and up to and including a nominal test item concentration of 9.77 mg/L. No cells were observed at 31.3 and 100 mg/L nominal test item concentration.

For more details on biological results, please refer to section "Any other information on results incl. tables"

Results with reference substance (positive control):

EC50 (growth rate) = 1.23 mg/L (95% CI: 1.11 - 1.36 mg/L)
EC50 (yield) = 0.685 mg/L (95% CI: 0.610 - 0.764 mg/L)

Reported statistics and error estimates:

The statistical evaluation for the 72 hours period was performed for growth rate and yield using SAS® (2002–2010). A test for normality of the data was performed by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was evaluated by calculating the Levene Test. The NOEC and LOEC were determined by using a multiple comparison method (Jonckheere Terpstra test, left sided, for growth rate and for yield). The EC10, 20, 50-values for growth rate and yield were determined by probit analysis following the normal and logistic distribution, respectively. Due to statistical reasons inhibition-values above 100 % were set to 100.

Any other information on results incl. tables

Table 1: Biological results from the main test

Conc.	Average cell numbers [10^4/mL]
[mg/L]	0 h	24 h	48 h	72 h
Control	0.59	1.87	8.65	42.66
0.954	0.59	2.00	7.42	33.42
3.05	0.59	1.62	5.53	19.98
9.77	0.59	1.11	2.54	4.86
31.3	0.59	0.73	0.93	1.18
100	0.59	0.50	0.47	0.11

Table 2: Percentage inhibition of growth rate

Conc.	% Inhibition of growth rate
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
0.954	-4.9	5.8	5.5
3.05	13.1	16.8	17.7 *
9.77	45.8	45.7	50.7 *
31.3	81.5	83.2	83.8 *
100	115.6	108.2	140.1 *^1)

¹⁾Value was set to 100 for EC_{10, 20, 50}-calculation

* Statistically significant different to the control

Table 3: Percentage inhibition of yield

Conc.	% Inhibition of yield
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
0.954	-9.3	15.4	22.0
3.05	20.2	38.8	53.9 *
9.77	59.7	75.8	89.9 *
31.3	89.1	95.8	98.6 *
100	107.0	101.5	101.1 *^1)

 

¹⁾Value was set to 100 for EC_{10, 20, 50}-calculation

* Statistically significant different to the control

Table 4: Main test: Individual cell numbers

Conc.	Cell numbers [10^4/mL]				Yield
[mg/L]	0 h	24 h	48 h	72 h	0 h – 72 h
	0.60	1.87	9.39	49.49	48.89
	0.61	1.85	8.89	45.06	44.45
Control	0.56	2.07	9.28	41.57	41.01
	0.64	1.82	9.16	45.77	45.13
	0.54	1.87	8.30	45.25	44.71
	0.57	1.76	6.90	28.79	28.22
Mean	0.59^1)	1.87	8.65	42.66	42.07
	0.59	2.13	6.99	33.91	33.32
0.954	0.59	1.92	7.70	33.25	32.66
	0.59	1.94	7.58	33.10	32.51
Mean	0.59	2.00	7.42	33.42	32.83
	0.59	1.52	5.49	19.17	18.58
3.05	0.59	1.69	5.21	17.95	17.36
	0.59	1.65	5.88	22.83	22.24
Mean	0.59	1.62	5.53	19.98	19.39
	0.59	1.02	2.55	5.01	4.42
9.77	0.59	1.19	2.50	4.87	4.28
	0.59	1.12	2.57	4.71	4.12
Mean	0.59	1.11	2.54	4.86	4.27
	0.59	0.81	0.95	1.09	0.50
31.3	0.59	0.70	0.82	1.21	0.62
	0.59	0.69	1.02	1.24	0.65
Mean	0.59	0.73	0.93	1.18	0.59
	0.59	0.63	0.49	0.10	-0.49
100	0.59	0.45	0.47	0.10	-0.49
	0.59	0.42	0.46	0.12	-0.47
Mean	0.59	0.50	0.47	0.11	-0.48

¹⁾The mean cell density of all control replicates is used as initial cell density for all treatment groups

Table 5: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	72.31	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	16%	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	4.3%; did not exceed 7% for the whole test period	yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.