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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Remarks:
Conducted according to guideline in effect at the time of study conduct
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Remarks:
Conducted according to guideline in effect at the time of study conduct
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The test concentrations of the test item were measured using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS). At the start of the definitive test, two samples (10 mL) were taken from the freshly-prepared control and test media. After 72 hours, the contents of the replicate flasks for each group were pooled and further samples taken for analysis. Additional samples were also taken from a flask containing the test item at 0.00930 and 10.0 mg/L but with no algal cells, in order to obtain information on the extent of adsorption/absorption of the test substance by the algal cells. On each occasion, one of the samples was analysed and the other was stored in a freezer in case further analysis was required.

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
The test substance (10 mg) was added to dilution medium (ca. 900 ml) in a volumetric flask (1 L) and made up to volume with gentle mixing and an overnight slow stir (to avoid foaming) to give test medium with a nominal concentration of 10 mg/L. This solution was then serially diluted to give test media with nominal concentrations of 3.13, 0.977, 0.305, 0.0954, 0.0298 and 0.00930 mg/L.

Dilution Medium = Sterile algal nutrient medium as recommended in Official Journal No. L383A Part C.3 and OECD Procedure 201
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 278/4, SAMS Research Services Ltd., Dunstaffnage Marine Laboratory, Dunbeg, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): 6 days


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21.9 to 22.5°C
pH:
7.96 to 8.35
Nominal and measured concentrations:
Nominal concentrations: 0.00930, 0.0298, 0.0954, 0.305, 0.977, 3.13 and 10.0 mg/L
Mean Measured concentrations: 0.00936, 0.0305, 0.0870, 0.272, 0.950, 3.18 and 9.22 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL conical flasks
- Initial cells density: ca. 1.00 x 10e4 cells/mL.
- Control end cells density: 1.04 x 10e6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 4440 to 8880 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable): 24, 48 and 72 hours
- Determination of cell concentrations: cell densities were measured using a Coulter Z Series Particle Count and Size Analyser
- Other: The presence of any abnormal cells was also noted during screening of each test level.

TEST CONCENTRATIONS
- Range finding study data: The range finding test employed nominal test concentrations of 0.01, 0.1, 1, 10 and 100 mg/L. After 72 hours, algal growth was inhibited by 99% at 10 and 100 mg/L, 58% at 1 mg/L and 15% at 0.1 mg/L, with no inhibition at 0.01 mg/L. Based on these findings, the nominal test concentrations selected for the definitive test were 0.00930, 0.0298, 0.0954, 0.305, 0.977, 3.13 and 10.0 mg/L.
Key result
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
3.26 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits: 3.02, 3.51 mg/L
Key result
Duration:
72 h
Dose descriptor:
other: EbC50
Effect conc.:
1.49 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% confidence limits: 1.10, 1.96 mg/L
Key result
Duration:
72 h
Dose descriptor:
other: EyC50
Effect conc.:
1.36
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% confidence limits: 1.00, 1.78 mg/L
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No microscopic abnormalities of the cells were detected at Day 1. At Day 2 there were low levels of swollen cells (ca. 10%) at 10.0 mg/L (nominal) only. At Day 3, deformed and swollen cells were observed at 0.305 mg/L and higher concentrations. Additionally, at 3.13 and 10.0 mg/L, cell lysis was also observed.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start of the test, the test media were colourless; some foam was observed at 3.13 and 10.0 mg/L (nominal) when the media were mixed.
Reported statistics and error estimates:
The data were compiled in an Excel spreadsheet and analysed using SAS 9.1 , using mean measured concentrations. All 95% confidence intervals for EC50 were calculated using the likelihood ratio method. The EC10 and EC20 (with 95% confidence interval) were also estimated by reparameterising the above formulae. For area under the growth curve, growth rate and yield, Williams’ test was also used to compare each treated group with control unless there was evidence of a nonmonotonic dose-response relationship, in which case Dunnett’s test was used.

Parameter

At 72 hours

Measured

Concentration

mg/L

% Inhibition

 

 

 

Growth rate

Not detectable

0.0

0.00936

0.9

0.0305

1.2

0.0870

1.8

0.272

4.8*

0.950

8.0***

3.18

49.1***

9.22

81.3***

 

 

 

Area under the

growth curve

Not detectable

0.0

0.00936

5.6

0.0305

6.8

0.0870

10.1

0.272

17.6*

0.950

29.3***

3.18

80.8***

9.22

91.3***

 

 

 

Yield

Not detectable

0.0

0.00936

3.9

0.0305

4.0

0.0870

7.7

0.272

20.3*

0.950

31.4***

3.18

90.6***

9.22

98.6***

*p<0.05, **p<0.01, ***p<0.001 using William’s test

The “no observed effect concentration” (NOEC) for growth rate, area under the growth curve and yield was 0.0870 mg/L (measured).

Validity criteria fulfilled:
yes
Conclusions:
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
72-hour ErC50 = 3.26 mg/L
72-hour EbC50 = 1.49 mg/L
72-hour EyC50 = 1.36 mg/L
Executive summary:

The effect of the test substance on the growth of the unicellular green alga Pseudokirchneriella subcapitata was assessed under non-axenic conditions. Triplicate algal cultures, with an initial cell density of ca. 10e4/mL, were exposed to the test substance dissolved in algal nutrient medium at nominal concentrations of 0.00930, 0.0298, 0.0954, 0.305, 0.977, 3.13 and 10.0 mg/L. The test media prepared in OECD medium from an aqueous stock solution, which was made by the direct addition of the test substance to the dilution medium. The test concentrations were measured using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS). Based on an arithmetic mean, overall measured concentrations were 0.00936, 0.0305, 0.0870, 0.272, 0.950, 3.18 and 9.22 mg/L. The cultures were incubated in an orbital incubator under continuous illumination at temperatures ranging from 21.9 to 22.5°C for 72 hours.

No microscopic abnormalities of the cells were detected at Day 1. At Day 2 there were low levels of swollen cells (ca. 10%) at 10.0 mg/L (nominal) only. At Day 3, deformed and swollen cells were observed at 0.305 mg/L and higher concentrations. Additionally, at 3.13 and 10.0 mg/L, cell lysis was also observed. The 72-hour ErC50 (growth rate) of the test substance, EbC50 (area under the growth curve), and EyC50 (yield) were 3.26, 1.49 and 1.36 mg/L based on measured concentrations. The “no observed effect concentration” (NOEC) for growth rate, area under the growth curve and yield was 0.0870 mg/L (measured).

Description of key information

72-hour ErC50 = 3.26 mg/L
72-hour EbC50 = 1.49 mg/L
72-hour EyC50 = 1.36 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
3.26 mg/L

Additional information