Registration Dossier

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:
07 Jul - 07 Aug 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: guideline study under GLP, analytical confirmation of test substance concentration, unstable test substance concentrations in media.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD series on testing and assessment number 23
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MTDID 15655
- Physical state: yellowish viscous liquid
- Analytical purity: 91.4%
- Expiration date of the lot/batch: 27 August 2017
- Storage condition of test material: at room temperature

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test concentrations and controls, at t = 0hr, 24hr, and 72 hrs
- Sampling method: Duplicate 2-mL samples were taken. At t = 0 hr, samples were taken from stock solutions. At t = 24 hours, duplicate 2-mL samples were taken from a flask set up for this purpose. At t = 72 hr, replicates were pooled at each concentration before taking samples.
- Sample storage conditions before analysis: in freezer.

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solutions of test substance dissolved in acetone at 1.5 g/L, 150 mg/L, and 15 mg/L were prepared. 100 µL of stock solution was added to 1 L of test medium and stirred for ten minutes to make the test solutions.
- Controls: solvent control (blank test medium with 100 µL acetone per liter), blank control. Cell growth data for both controls were compared and no statistical difference was found. The controls were combined for purposes of calculation, and the growth media were pooled before analysis
- Evidence of undissolved material: Test solution was clear and colorless

In a preliminary test, solutions were made at loading rates of 1.0, 10 and 100 mg/L (far in excess of the projected water solubility limit of ca 150 µg/L). After two day's stirring, the solutions were allowed to settle for three hours, after which the water soluble fractions (WSFs) were collected. All WSF solutions made were clear and colorless. In other testing, the test substance formed stable emulsions that could not be broken by e.g. centrifugation.

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
Three 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:
yes
Total exposure duration:
72 h
Remarks on exposure duration:
combined rangefinding/limit test

Test conditions

Hardness:
24 mg/L as CaCO3
Test temperature:
21.7 - 22.6 °C
pH:
8.0 - 8.1
Nominal and measured concentrations:
Nominal: Control, 150 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: 147.4E+04 cells/mL
- No. of vessels per concentration (replicates): 6 (a parallel rangefinding test had triplicate vessels with 1.5 µg/L or 15 µg/L test substance in M2 medium). An additional flask was set up for the 24-hr concentration measurement. Algae-free test flasks were also assembled.
- No. of vessels per control (replicates): 6 each for solvent and medium blanks. An additional flask was set up for the 24-hr concentration measurement.

GROWTH MEDIUM
- Standard medium used: yes, M2 medium per OECD TG 201

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Standard medium (M2) 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 61 to 72 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 initial inoculum density and to determine cell density at 48 h and 72 h. Cell densities at 24 h, and during the preliminary rangfinding experiment, 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
- Range finding study: yes, two range finding studies conducted. The final study was conducted as a combined limit/range-finding test.
- Test concentrations: For initial range-finding test, control and WSFs created at 1.0, 10 and 100 mg/L loading rate. For parallel range-finding test, additional nominal concentrations of 1.5 µg/L and 15 µg/L were done.
- Results used to determine the conditions for the definitive study: No. Loading rates for initial test were far in excess of water solubility limit. Test substance forms stable emulsions which were assumed not to be removed during test solution preparation, as evidenced by the 100 mg/L solution still being visibly turbid.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 22 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: No significant effect at highest concentration tested
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
22 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: No significant effect at highest concentration tested
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 highest concentration when compared to the control.
- Any stimulation of growth found in any treatment: no
- Effect concentrations exceeding solubility of substance in test medium: yes
- Other: Test substance concentration declined throughout the test in both biological and abiotic test vessels (Table 1). Test substance could not be detected in biological test vessels within 24 hours of test initiation.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 1.3 mg/L (growth rate). 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 36 days prior to test substance.
Reported statistics and error estimates:
Growth rate data were normally distributed by Shapiro-Wilk's test, p(W) = 0.372 > 0.01. Variance of growth rate data were homogeneous by Levene's test, p(F) = 0.742 > 0.01. The NOEC was determined by Dunnett's multiple t-test. No significant effect on growth rate was found at the highest concentration tested (t = -0.83 < -2.24, 20 df)

Any other information on results incl. tables

Table 2, Individual cell densities in the Z-acetate algal toxicity test

Time-weighted average concentration

Replicate

Time

0 h

24 h

48 h

72 h

Medium control (<LOD)

1

1.0

4.265

11.750

45.000

2

1.0

4.197

10.750

48.750

3

1.0

4.974

9.750

46.250

4

1.0

3.792

7.500

49.500

5

1.0

4.377

14.250

51.750

6

1.0

4.763

12.000

62.000

Mean:

1.0

4.4

11.0

50.5

Std.Dev.:

0.0

0.4

2.3

6.1

CV:

0.0

9.6

20.7

12.1

Solvent control (<LOD)

1

1.0

3.705

7.750

43.250

2

1.0

4.081

11.250

47.750

3

1.0

4.248

12.000

42.750

4

1.0

4.153

13.000

52.250

5

1.0

4.039

12.250

53.000

6

1.0

4.248

14.330

55.830

Mean:

1.0

4.1

11.8

49.1

Std.Dev.:

0.0

0.2

2.2

5.4

CV:

0.0

5.0

18.9

11.0

22 µg/L

1

1.0

3.689

11.000

42.250

2

1.0

4.322

18.250

41.000

3

1.0

4.143

9.000

45.750

4

1.0

4.510

12.330

51.670

5

0.0

4.233

13.000

55.250

6

0.0

4.098

12.750

48.500

Mean:

1.0

4.2

12.7

47.4

Std.Dev.:

0.0

0.3

3.1

5.5

CV:

0.0

6.6

24.3

11.6

 

Table 3, Growth rates (1/day) in the Z-acetate algal toxicity test

Time-weighted average concentration

Replicate

Interval

0-24 h

24-48 h

48-72 h

0-72 h

Medium control (<LOD)

1

1.45

1.013

1.343

1.269

2

1.434

0.941

1.512

1.296

3

1.604

0.673

1.557

1.278

4

1.333

0.682

1.887

1.301

5

1.476

1.18

1.29

1.315

6

1.561

0.924

1.642

1.376

Mean:

 

1.477

0.902

1.538

1.306

Std.Dev.:

 

0.0964

0.1963

0.2161

0.0381

CV:

 

6.5

21.8

14

2.9

  

Solvent control (<LOD)

1

1.31

0.738

1.719

1.256

2

1.406

1.014

1.446

1.289

3

1.446

1.038

1.27

1.252

4

1.424

1.141

1.391

1.319

5

1.369

1.11

1.465

1.323

6

1.446

1.216

1.36

1.341

Mean:

1.405

1.043

1.442

1.296

Std.Dev.:

0.0509

0.166

0.1524

0.0372

CV:

3.6

15.9

10.6

2.9

CV = 2.9% for all section-specific control growth rates

22 µg/L

1

1.305

1.093

1.346

1.248

2

1.464

1.44

0.809

1.238

3

1.421

0.776

1.626

1.274

4

1.506

1.006

1.433

1.315

5

1.443

1.122

1.447

1.337

6

1.41

1.135

1.336

1.294

Mean:

 

1.425

1.095

1.333

1.284

Std.Dev.:

 

0.0678

0.2152

0.2768

0.0385

CV:

 

4.8

19.6

20.8

3

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 72-h NOEC (growth rate) of Z-acetate to Pseudokirchneriella subcapitata was >22 µg/L based on time-weighted average concentration (OECD 201), in a limit test done with an initial concentration near the expected solubility limit. No effect was seen during the test.
Executive summary:

Toxicity of Z-acetate to the freshwater alga Pseudokirchneriella subcapitata was assessed in a combined limit/range-finding test conducted according to OECD 201. The test substance was not soluble at the limit of 100 mg/L and forms stable emulsions that are not removed by e.g. centrifugation. Instead, stock solutions were made at 10,000-fold concentration in acetone and diluted 100 µL to 1L in test medium. Maximum concentration tested was 150 µg/L (nominal), which was near the expected water solubility limit for this chemical, with a ten-fold concentration series included below this level. The test substance could not be detected within 24 h after test initiation. Therefore, time-weighted average (TWA) concentrations were used to calculate effect concentration. No effect was observed in the test. The 72-hour NOEC (growth rate) was 0.22 µg/L (TWA). The 72-hour EC50 was >0.22 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. This instability is to be expected given the low water solubility of the test subtance and its tendency 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.