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Ecotoxicological information

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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
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

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):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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 concentrationsopen allclose all
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
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.