2-heptadecyl-1H-imidazole

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 - 9 Feb 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

1

Reference substance name:

2-heptadecyl-1H-imidazole

EC Number:

245-589-7

EC Name:

2-heptadecyl-1H-imidazole

Cas Number:

23328-87-2

Molecular formula:

C20H38N2

IUPAC Name:

2-heptadecyl-1H-imidazole

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 stock solution was prepared by directly weighing 200 mg in 2000 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). Afterwards the test item had not gone into solution completely. Small, evenly distributed particles were visible within the solution and particles were observed at the bottom of the vessel and on the surface of the solution. Insoluble particles of the test item were separated from the liquid phase by filtration through a filter with 0.22 µm mean pore size which was saturated beforehand. 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 filtrated solution was clear and transparent.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis 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:

22.0 - 23.3 °C

pH:

control: 7.36 - 8.47
treatment: 7.34 - 8.75

Nominal and measured concentrations:

nominal concentrations: 100, 55.6, 30.9, 17.1, 9.53 mg/L and control
measured concentrations: < LOD for all treatment solutions

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 (targeted)
- Control end cells density: 28.39 × 10E+04 cells
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: AAP-Medium (according to Annex 3 of OECD 201)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Intervals of water quality measurement: Measurements of pH-value were performed at t = 0 h and t = 72 h, the temperature was measured continuously and recorded at hours 0, 24, 48 and 72 h.

OTHER TEST CONDITIONS
- Adjustment of pH: pH was adjusted to 7.5 ± 0.1 with NaOH or HCl, if necessary
- Photoperiod: continuous light
- Light intensity and quality: 75.9 µEm-2s-1 (mean)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorescence measurement (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: 1.8
- Range finding study: yes
- Test concentrations in range finding study: Control, 100, 10.0, 1.00, 0.100 and 0.0100 mg/L
- Results used to determine the conditions for the definitive study: After 72 h at termination of the test no concentration response relation and only minor inhibition was observed for the inhibition of growth rate and yield up to and including a nominal test item concentration of 10.0 mg/L. At a nominal test item concentration of 100 mg/L an inhibition of growth rate of 69.9 % and an inhibition of yield of 95.1 % was observed.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate; tested regularly

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

46.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

filtered

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 34.9 - 60.0 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

25.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

filtered

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 20.4 - 31.5 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

17.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

filtered

Basis for effect:

other: growth rate and yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

30.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

filtered

Basis for effect:

other: growth rate and yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

filtered

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 8.83 - 29.7 mg/L

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

11.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

filtered

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 7.24 - 15.5 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): normal for the control and up to and including a nominal test item concentration of 17.1 mg/L.
- Unusual cell shape: At a nominal test item concentration of 30.9 mg/L few deformed cells were observed, at a nominal test item concentration of 55.6 mg/L the number of deformed cells observed increased. No cells were observed a nominal test item concentration of 100 mg/L.

For further 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 NOELR and LOELR were determined by using a multiple comparison method (Jonckheere Terpstra test, left sided, for growth rate, Dunnetts-t-test, left sided, for yield). The EL10, 20, 50-values for growth rate and yield were determined by probit analysis following the Gompertz and normal distribution, respectively. Due to statistical reasons inhibition-values above 100 % were set to 100.

Any other information on results incl. tables

Table 1: Average cell numbers for each sampling time and concentration

Conc.	Average cell numbers [10^4/mL]
[mg/L]	0 h	24 h	48 h	72 h
Control	0.53	1.37	5.41	28.39
9.53	0.53	1.27	5.47	27.66
17.1	0.53	1.02	3.85	20.08
30.9	0.53	1.03	2.24	10.29
55.6	0.53	1.00	1.60	4.56
100	0.53	0.46	0.21	0.16

Table 2: Percentage inhibition of growth rate

Concentration [mg/L]	% Inhibition of growth rate
	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
9.53	5.9	-1.8	0.0
17.1	30.6	14.0	8.3
30.9	31.4	44.1	29.6*
55.6	35.5	57.7	55.1*
100	121.1	141.2	139.2*^1)

* Statistically significant different to the control

1) Value was set to 100 for EC10, 20, 50-calculation

Table 3: Percentage inhibition of yield

Concentration [mg/L]	% Inhibition of yield
	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
9.53	11.9	-1.2	2.6
17.1	41.7	32.0	29.8
30.9	40.5	65.0	65.0*
55.6	44.0	78.1	85.5*
100	108.3	106.6	101.3 *^1)

* Statistically significant different to the control

1) Value was set to 100 for EC10, 20, 50-calculation

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
	0.58	1.81	7.15	39.81	39.23
	0.60	1.66	7.39	36.55	35.95
Control	0.52	1.29	4.50	20.00	19.48
	0.52	1.29	5.54	29.58	29.06
	0.52	1.28	3.85	24.64	24.12
	0.44	0.86	4.00	19.78	19.34
Mean	0.53^1)	1.37	5.41	28.39	27.86
	0.53	1.29	5.80	32.46	31.93
9.53	0.53	1.11	5.20	24.18	23.65
	0.53	1.41	5.41	26.35	25.82
Mean	0.53	1.27	5.47	27.66	27.13
	0.53	1.14	4.60	24.25	23.72
17.1	0.53	0.82	3.15	16.23	15.70
	0.53	1.09	3.80	19.77	19.24
Mean	0.53	1.02	3.85	20.08	19.55
	0.53	1.33	4.11	19.54	19.01
30.9	0.53	1.07	1.42	5.87	5.34
	0.53	0.70	1.19	5.45	4.92
Mean	0.53	1.03	2.24	10.29	9.76
	0.53	1.37	2.30	7.83	7.30
55.6	0.53	0.97	1.85	5.08	4.55
	0.53	0.67	0.64	0.76	0.23
Mean	0.53	1.00	1.60	4.56	4.03
	0.53	0.33	0.22	0.03	-0.50
100	0.53	0.37	0.19	0.24	-0.29
	0.53	0.68	0.21	0.20	-0.33
Mean	0.53	0.46	0.21	0.16	-0.37

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.	factor of 53.57	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%	30%	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%.	5.4%; 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”.