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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:
25 Jun - 23 Aug 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: test substance not visibly present in chambers, no analytical monitoring, but test chambers sealed to prevent volatilization losses.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
study period reduced to 48 hours because of sealed test bottle
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MTDID 9580
- Substance type: pure active substance
- Physical state: liquid
- Analytical purity: 99%
- Expiration date of the lot/batch: 31 Dec 2007
- Stability under test conditions: Not completely soluble in test medium at a loading rate of 100 mg/L. Reacts slowly with oxygen, e.g. in the presence of moisture, resulting in acid fluorides, carboxylic acids and HF. Exposure to air was kept at a minimum during the preparation procedures, since the test substance was indicated to be volatile.
- Storage condition of test material: At room temperature in the dark under nitrogen

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Exposure to air was kept at a minimum during the preparation procedures, since MV 31 was indicated to be volatile. An aqueous mixture, prepared by direct addition to the test vessels at a loading rate of 100 mg/L, was stirred for 15 minutes. After stirring, the mixture was observed to be clear and colourless. The mixture was tested as such together with a control.

- Controls: Blank only
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Propagation was in M1 medium. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C. Illumination was
60 to 120 µE/m²/s when measured in the photosynthetically effective wavelength range of 400
to 700 nm.
ACCLIMATION
- Acclimation period: 4 days. Cells from the algal stock culture were inoculated in culture medium at a cell density of 1E+04 cells/ml. The cell density was measured immediately before use.
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: not specified

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
Duration of the test was reduced because test chambers were closed.

Test conditions

Hardness:
24 mg CaCO3/L
Test temperature:
22.5 - 23.0 °C
pH:
7.1 -9.0
Dissolved oxygen:
N/A
Salinity:
N/A
Nominal and measured concentrations:
Nominal concentrations only, blank and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 100 mL glass vessels with screw cap and septum, containing 75 mL medium and 1.5 mL inoculum
- Aeration: no
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 31.80E+04 cell/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, adjusted M2. NaHCO3 content increased to 100 mg/L, pH adjusted to 7.0
- Source/preparation of dilution water: Adjusted M2 was prepared with Milli-Q.
- Culture medium different from test medium: Propagation was in M1 medium. Inoculum was prepared by a 4-day preculture of a small number of cells in adjusted M2.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity and quality: 76 - 83 µE/m²∙s using 30 W "Cool While" TLD lamps

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cells were counted at the beginning of the test using a microscope and a counting chamber. At 24 and 48 hours, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer. Samples for counting were taken by syringe without opening the test vessels.
-Appearance: Microscopic observation at end of test to verify a normal and healthy appearance of the inoculum culture, and to observe for any abnormal appearance of the algae.

PRELIMINARY TEST:
- A 72-hour algae test was initially conducted but repeated due to performance of controls. The preliminary report indicates that the stock culture was passaged in standard M2 (50 mg/L NaHCO3, pH 8.1) prior to inoculation into adjusted M2 medium, with NaHCO at 100 µg/L [sic]. In the preliminary experiment, undissolved MV 31 was present in test chambers during the experiment. Variability in section-by-section specific growth rates for control replicates was unacceptibly high even with the exclusion of the 48-72 hour section (see Table A), and controls were no longer in exponential phase by 48 hours. However, the average of section-specific growth rates were not significantly different between the control and test replicates by Student's T-test (calculated for this study summary). The test was repeated with a 48-hour study duration to obtain better performance in controls.
Reference substance (positive control):
yes
Remarks:
potassium dichromate, ca. 10 weeks prior to test.

Results and discussion

Effect concentrations
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: growth inhibition ca 50% at 0 - 24 hours, recovered during 24-48 hours
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: No abnormalities observed
- Any stimulation of growth found in any treatment: no (see Table 1)
- Effect concentrations exceeding solubility of substance in test medium: yes
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 1.9 mg/L (potassium dichromate)
- Other: The historical ranges for growth rate reduction is between 0.82 and 2.3 mg/L.
Reported statistics and error estimates:
No statistics were reported because 0-48 hour results were < 10% different. A T-test done for this study summary indicates that 0-48 hour growth rates were not significantly different (P=0.178)

Any other information on results incl. tables

Table 1, Cell counts and growth rates in the algae test

Nominal conc. MV 31

Cell counts

(x 1E+04 cells/mL) at time:

Growth rate (µ) and reduction (%) at given intervals:

0-48 h

0-24 h

24-48 h

0 (hr)

24 (hr)

48 (hr)

µ

(%)

µ

(%)

µ

(%)

control

1.00

7.33

33.31

0.07304

0.08300

0.06308

1.00

7.37

33.10

0.07291

0.08323

0.06259

1.00

6.97

30.58

0.07126

0.08090

0.06161

1.00

7.49

29.36

0.07041

0.08390

0.05692

1.00

6.78

31.07

0.07159

0.07975

0.06343

1.00

6.96

33.40

0.07309

0.08084

0.06535

average

1.0

7.2

31.8

0.07205

2%*

0.08194

2%*

0.06216

5%*

*Mean CV of growth rates.  Section specific growth rate mean CV was 19%

100 mg/L

1.00

2.78

35.52

0.07438

-3

0.04260

48

0.10615

-71

1.00

2.98

33.68

0.07327

-2

0.04550

44

0.10104

-63

1.00

2.49

32.10

0.07227

0

0.03801

54

0.10652

-71

1.00

2.90

32.91

0.07279

-1

0.04436

46

0.10121

-63

1.00

3.12

32.14

0.07229

0

0.04741

42

0.09718

-56

1.00

2.44

32.11

0.07227

0

0.03717

55

0.10738

-73

average

1.0

2.8

33.1

0.07288

-1.2

0.04251

48.1

0.10325

-66.1

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
exponential growth rate in controls, CV <35% in section-specific growth rates, CV <7% in average growth rates.
Conclusions:
The 48-h EC50 (growth rate) for MV 31 to P. subcapitata is > 100 mg/L
Executive summary:

The toxicity of MV 31 to the green algae, Pseudokirchneriella subcapitata, was assessed in a 48-hour limit test conducted according to the OECD 201 method. Test containers were sealed to avoid volatilization losses of test material, and growth medium was amended with 100 mg/L NaHCO3 to provide sufficient carbon dioxide and buffering to maintain exponential growth. An initial limit test at 100 mg/L nominal concentration indicated no statistically significant differences between test and controls in section-by section growth rate, however the control growth did did not meet validity criteria for the test method. A second limit test was therefore completed. Exponential growth was observed during the entire period of exposure in the control vessel and the validity criteria were met in the second test. Results of the tests are reported with respect to a nominal loading of 100 mg/L, as analytical monitoring was not done. Test solutions were clear and colorless throughout the test despite low water solubility of MV 31. Growth rates at the end of the test were not significantly different between controls and test samples. The EC50 is > 100 mg/L (nominal), which is far in excess of the water solubility limit.

The study was performed in accordance with internationally-accepted test guidelines and Good Laboratory Practice (GLP) standards. No analytical monitoring was performed and the test substance, although loaded at a rate well above its water solubility limit, was not visible in the test solutions. Test chambers were sealed throughout the experiment to prevent loss of material. Also, in the initial limit test, MV 31 was visibly present in test chambers and section-by-section specific growth rates were not significantly different. Therefore, this study is reliable with restrictions and the results are suitable for purposes of Risk Assessment, Classification & Labeling, and PBT Analysis.

Results Synopsis

Test Type: static (based on data obtained using OECD201 methodology)

48-hr ErC50: >100 mg/L (nominal concentration), 95% CI not calculable.

48-hr NOEC = 100 mg/L (nominal concentration, growth rate).