1-dodecyl-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:

15 Sep - 3 Oct 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Statistical variation of results, unstable test substance concentrations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Laurylimidazole

IUPAC Name:

Laurylimidazole

Details on test material:

- Name of test material (as cited in study report): MTDID 13999
- Substance type: pure active substance
- Physical state: clear yellowish liquid
- Analytical purity: 99%
- Expiration date of the lot/batch: 23 Sep 2016
- Storage condition of test material: At room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations and controls, at t = 0hr, 24hr, and 72 hrs
- Sampling method: Single 2-mL samples were taken from test chambers. At the end of the exposure period, replicates with algae were pooled at each concentration before sampling
- Sample storage conditions before analysis: in freezer. In a validation study, freezer storage did not affect measured substance concentration

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test substance at 5 mg/L in test medium was stirred for 15 minutes with a magnetic stirrer. This stock solution was then diluted in test medium to make the appropriate test solutions.
- Controls:Blank test medium
- Evidence of undissolved material: Test solutions were clear and colorless

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
.
- Method of cultivation: Stock cultures were maintained in M1 medium (Nederlandse Praktijk Richtlijn no. 6505).
NaNO3, 500 mg/l
K2HPO4∙3H2O, 52 mg/l
MgSO4∙7H2O, 75 mg/l
Na2CO3∙10H2O, 54 mg/l
C6H8O7∙H2O, 6 mg/l
NH4NO3, 330 mg/l
CaCl2∙2H2O, 35 mg/l
C6H5FeO7∙xH2O, 6 mg/l
H3BO3, 2∙9 mg/l
MnCl2∙4H2O, 1.81 mg/l
ZnCl2, 0.11 mg/l
CuSO4∙5H2O, 0.08 mg/l
(NH4)6Mo7O24∙4H2O, 0.018 mg/l

ACCLIMATION
Four days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2 according to OECD 201) at a cell density of 1e+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg/L as CaCO3

Test temperature:

22.3 - 23.3 °C

pH:

7.7 - 8.2

Nominal and measured concentrations:

Nominal: Control, 4.6 µg/L, 10 µg/L, 22 µg/L, 46 µg/L, 100 µg/L
Measured (Time weighted average):

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL all-glass container, 50 mL fill volume
- Agitation: Yes, during incubation the algal cells were kept in suspension by continuous shaking
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 105.5E+04 cells/mL
- No. of vessels per concentration (replicates): 3 (each treatment group, with algae), 1 (intermediate concentration, without algae),
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Standard medium prepared in reverse osmosis purified water
- Ca/mg ratio: 1
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: Continuous
- Light intensity and quality: Fluorescent (TL-D) lamps with a light intensity within the range of 83 to 86 μE/(m²∙s). Test vessels were placed randomly and randomly repositioned every day.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cells were counted using a microscope and a counting chamber to determine inoculum density. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm).
- Appearance: at the end of the test, microscopic examination was done on the test concentration closest to the EC50 to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2x
- Range finding study: yes
- Test concentrations: Control, 0.10 µg/L, 1.0 µg/L, 10 µg/L, 100 µg/L, 1000 µg/L
- Results used to determine the conditions for the definitive study: EC50 between 10 µg/L and 100 µg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate (performed ca. 3 weeks prior to tests)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.57 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI, 4.29 - 7.14 µg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.1 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI, 0.85 - 3.10 µg/L

Details on results:

- Exponential growth in the control: yes (Table 2, 3)
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 10 µg/l (nominal) when compared to the control.
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
- Other: Test substance concentration declined throughout the test. This decline was concentration-dependant, and was less pronounced in the algae-free test chamber (Table 1)

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 1.7 mg/L. Historical range for the reference substance at the contract lab lies between 0.82 and 2.3 mg/L
- Other: Reference substance toxicity assay conducted ca. 3 weeks prior to test substance.

Reported statistics and error estimates:

Growth rate data were normally distributed by Shapiro-Wilk's test, p(W) = 0.685 > 0.05. Variance of growth rate data were not homogeneous by Levene's test, p(F) = 0.021 < 0.05. Welch-t test for Inhomogeneous Variances with Bonferroni-Holm Adjustment was used to assess statistical significance of the results. While all results for ≥2.2 µg/L were biologically significant, the statistical significance could not in most cases be assigned due to the high variability in the experimental data (Table 4). The NOEC could not be determined since the lowest test substance concentration had a statistically significant effect. EC10 and EC50 were determined using probit analysis on growth rate data (Table 3)

Any other information on results incl. tables

Table 2, Individual cell densities in the laurylimidazole algal toxicity test
Time-weighted average concentration (µg/L)	Replicate	Time
		0 h	24 h	48 h	72 h
Control	1	1.0	4.817	22.526	117.14
	2	1.0	4.774	21.474	117.12
	3	1.0	4.412	19.032	95.665
	4	1.0	4.189	19.756	102.41
	5	1.0	4.398	19.299	94.215
	6	1.0	4.313	20.477	106.16
Mean:		1.0	4.5	20.4	105.5
Std.Dev.:		0.0	0.3	1.4	10.0
CV:		0.0	5.7	6.6	9.5
2.2	1	1.0	4.739	17.295	78.109
	2	1.0	4.658	18.246	79.924
	3	1.0	4.340	14.593	54.964
Mean:		1.0	4.6	16.7	71.0
Std.Dev.:		0.0	0.2	1.9	13.9
CV:		0.0	4.6	11.3	19.6
2.4	1	1.0	3.395	8.659	20.231
	2	1.0	4.040	11.202	44.955
	3	1.0	4.689	12.968	48.063
Mean:		1.0	4.0	10.9	37.7
Std.Dev.:		0.0	0.6	2.2	15.3
CV:		0.0	16.0	19.8	40.4
5.9	1	1.0	3.308	8.318	24.817
	2	1.0	4.086	11.922	23.076
	3	1.0	1.675	3.358	6.74
Mean:		1.0	3.0	7.9	18.2
Std.Dev.:		0.0	1.2	4.3	10.0
CV:		0.0	40.7	54.7	54.8
7.0	1	1.0	3.961	10.224	15.424
	2	1.0	3.949	3.906	5.227
	3	1.0	1.000	1.000	1.499
Mean:		1.0	3.0	5.0	7.4
Std.Dev.:		0.0	1.7	4.7	7.2
CV:		0.0	57.4	93.5	97.6
12.6	1	1.0	1.000	1.000	1.000
	2	1.0	1.658	2.560	2.446
	3	1.0	1.000	1.000	1.083
Mean:		1.0	1.2	1.5	1.5
Std.Dev.:		0.0	0.4	0.9	0.8
CV:		0.0	31.2	59.3	53.8

 

 

 

Table 3, Growth rates (1/day) in the laurylimidazole algal toxicity test
Time-weighted average concentration (µg/L)	Replicate	Interval
		0-24 h	24-48 h	48-72 h	0-72 h
Control	1	1.572	1.543	1.649	1.588
	2	1.563	1.504	1.696	1.588
	3	1.484	1.462	1.615	1.520
	4	1.432	1.551	1.646	1.543
	5	1.481	1.479	1.586	1.515
	6	1.462	1.558	1.646	1.555
Mean:		1.499	1.516	1.639	1.551
Std.Dev.:		0.056	0.040	0.037	0.032
CV:		3.800	2.700	2.300	2.000
		CV = 5% for all section-specific control growth rates
2.2	1	1.556	1.295	1.508	1.453
	2	1.539	1.365	1.477	1.460
	3	1.468	1.213	1.326	1.336
Mean:		1.521	1.291	1.437	1.416
Std.Dev.:		0.047	0.076	0.097	0.070
CV:		3.100	5.900	6.800	4.900
2.4	1	1.222	0.936	0.849	1.002
	2	1.396	1.020	1.390	1.269
	3	1.545	1.017	1.310	1.291
Mean:		1.388	0.991	1.183	1.187
Std.Dev.:		0.162	0.048	0.292	0.161
CV:		11.600	4.800	24.700	13.500
5.9	1	1.196	0.922	1.093	1.071
	2	1.408	1.071	0.660	1.046
	3	0.516	0.696	0.697	0.636
Mean:		1.040	0.896	0.817	0.918
Std.Dev.:		0.466	0.189	0.240	0.244
CV:		44.800	21.100	29.400	26.600
7.0	1	1.376	0.948	0.411	0.912
	2	1.373	-0.011	0.291	0.551
	3	0.000	0.000	0.405	0.135
Mean:		0.917	0.312	0.369	0.533
Std.Dev.:		0.794	0.551	0.067	0.389
CV:		86.600	176.200	18.300	73.000
12.6	1	0.000	0.000	0.000	0.000
	2	0.506	0.434	-0.046	0.298
	3	0.000	0.000	0.080	0.027
Mean:		0.169	0.145	0.011	0.108
Std.Dev.:		0.292	0.251	0.063	0.165
CV:		173.200	173.200	556.600	152.400

 

Table 4, Percentage inhibition of growth rate (total test period) during the final test
Test substance concentration (TWA, µg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.551	0.0316	6	0.0
2.2	1.416	0.0699	3	8.7 * ¹
2.4	1.187	0.1605	3	23.5 ²
5.9	0.918	0.2442	3	40.9 ²
7.0	0.533	0.3889	3	65.7 ²
12.6	0.108	0.1650	3	93.0 *
*, effect statistically significant (Welch-t test) 1, effect not biologically significant (<10% effect) 2, effect biologically significant

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 72-h EC50 (growth rate) of laurylimidazole to Pseudokirchneriella subcapitata was 5.57 (95% CI, 4.29 - 7.14) µg/L based on time-weighted average concentration (OECD 201). The 72-hour EC10 was 2.10 µg/L (95%, 0.85 - 3.03 µg/L)

Executive summary:

Toxicity of laurylimidazole to the freshwater alga Pseudokirchneriella subcapitata was assessed according to OECD201. Concentrations between 4.6 µg/L and 100 µg/L were used in the definitive test. Analytically determined concentrations declined throughout the test. Therefore, time-weighted average (TWA) concentrations were used to calculate effect concentration. The 72-hour NOEC (growth rate) could not be determined and was less than the lowest TWA concentration tested (<2.2 µg/L). The 72-hour EC10 was 2.10 µg/L (95%, 0.85 - 3.03 µg/L). The 72-hour EC50 was 5.57 µg/L (95% CI, 4.29 - 7.14 µg/L).

The study was conducted according to internationally accepted test guidelines and in accord with GLP criteria. Test substance concentrations were confirmed analytically. Test substance concentrations were not stable during the test. In addition, the results showed considerable variability. Both difficulties are to be expected given the low concentrations necessary to determine the test substance's toxicity, and its tendency as a surfactant to adsorb to surfaces including the multiplying cells. The study is deemed reliable with restrictions. It is suitable for Risk Assessment, Classification & Labeling, and PBT Analysis.