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Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22.04.2021 - 30.04.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test substance: Santalol oil
- Test substance No.: 20/0281-1
- Batch identification: 2007VD01SALraw0
- Cas-No.: 2576531-09-2
- Purity: 100% UVCB
- Content: (Z)-alpha-santalol: 29.5 - 30.2%; (Z)-beta-santalol: 19.7 - 19.1%
- Homogeneity: Given
- Storage stability. Expiry Date: 24.08.2021

Analytical monitoring:

yes

Details on sampling:

Sampling was performed at the start of the exposure )0h) from the uninoculated (without algae) replicate 7 of each test group.
At the end of the exposure (72h), samples were taken from the combined inoculated replicates from each test group.
After 72h, samples were also taken from the additional uninoculated (without algae) replicates from each test group.
All collected samples were stored at approx. -20°C until transported to the Analytical Laboratory for analysis.

Vehicle:

no

Details on test solutions:

TEST SUBSTANCE PREPARATION

The test substance is poorly soluble in water. Therefore, the test solutions were prepared following general guidance provided in OECD 23 to achieve a water accommodated fraction (WAF) of the test substance.
Each test solution was prepared separately (differential loading) by directly adding test substance to test medium.
Glass aspirator bottles (5-L) with a bottom side-outlet attached to a stopcock were filled with 5.8-L test media. For each test solution the test substance was pipetted carefully on the water surface, respectively. The bottles were closed and the solutions stirred on a magnetic plate for approx. 96 hours. The stirring was slow (approx. 150 rpm) so that no vortex was formed. The undissolved test substance should remain on the surface and the required volume of the saturated solution (approx. 2.5 mL) was removed from the bottom of the bottles and inspected visually for the presence of any undissolved test substance by observing the scattering of a laser light through the test solution (Tyndall effect).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Name: Pseudokirchneriella subcapitata KORSHIKOV (SAG 61.81)
- Source (laboratory, culture collection): SAG (Collection of algal cultures in Göttingen, Germany)
- Age of inoculum (at test initiation): 4 days

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.1 - 22.3 °C

pH:

7.3 - 7.4 (at test start); 7.7 - 10.0 (after 72 h)

Nominal and measured concentrations:

Nominal test concentration [mg/L]: 0 (control), 0.32, 1.0, 3.2, 10.0, 32.0 and 100.0, as loading rate, based on test substance density without a correction for purity or composition;
Mean measured concentration (geometric mean) [mg/L]: 0 (control), 0.13, 0.47, 1.93, 4.28, 3.97 and 4.81, based on test substance density without a correction for purity or composition

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (nominal volume 250 mL) closed with glass plugs (the test substance is potentially volatile. Therefore, closed test vessels were used to and maintain stable exposure conditions)
- Test volume: approx. 300 mL (Flasks are completely filled, no headspace)
- Aeration: no
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 0.3 x 10^4 cells/mL
- Control end cells density: 96.1 cells x 10000/mL
- No. of vessels per concentration (replicates): 3 (+1 additional replicate per test group for initial concentration control analysis)
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: -

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:
- Photoperiod: no
- Light intensity and quality: 5431 - 5890 lux (mean: 5660 lux) at a wavelength of 400 - 700 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Algal growth measured as in vivo chlorophyll-a fluorescence (pulsed excitation with light flashes having a wavelength of 430 nm), daily observations

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorphenol

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

8.11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

13.89 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

38.92 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

3.56 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.47 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.93 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no remarkable observations in 72 hours for all test concentrations (no algal cells observed at 100 mg/L)
- Unusual cell shape: -
- Colour differences: -
- Flocculation: -
- Adherence to test vessels: -
- Aggregation of algal cells: -
- Other: -
- Any stimulation of growth found in any treatment: -
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The measured concentrations deviated markedly from the loading rate.
The algae were exposed to all soluble components of the test substance at the given loading rates under testing conditions. The test substance is a UVCB and as such every constituent contributes to a different degree to overall solubility, depending on its own individual solubility and its mass fraction in the test substance. Thus, for an evaluation of the results the effect concentrations should be expressed based on the nominal loading rates and were considered to be an accurate representation of exposure levels throughout the experiment.

Results with reference substance (positive control):

In order to verify that the algal cultures are responding normally to toxic stress, tests with a reference substance (3,5 Dichlorphenol) are conducted. Reference substance tests are conducted according to OECD 201 guidelines and in accordance with GLP, but without a GLP status.

The ErC50 of the reference substance 3,5 Dichlorphenol should be in the range: ErC50 = 0.9 – 4.4 mg/L after 72 hours.
The ErC50 (72 h) of the control substance 3,5 Dichlorphenol was 2.81 mg/L (Date of the last control experiment: 09 Mar 2021, project number: 60E0308/02E017).

72-hour results of the algal growth inhibition test
Loading rate [mg/L]	Mean cell density (cells/mL) x 10^4 at 72 hours	Mean yield at 72 hours	Mean specific growth rate at 72 hours
0 (control)	96.1	95.9	2.1
0.32	93.5	93.4	2.1
1	95.4	95.2	2.1
3.2	72.1	71.7	1.9
10.0	36.8	36.6	1.8
32	10.1	9.9	1.3
100	0.6	0.4	0.4

 

Effect concentrations [mg/L based on loading rates after 72 hours
Algal yield [mg/L]		Algal growth rate [mg/L]
EyL10	1.65	EyL10	8.11
EyL20	2.73	EyL20	13.89
EyL50	7.12	EyL50	38.92
NOEyLR	1	NOEyLR	1
LOEyLR	3.2	LOEyLR	3.2

 

Effect concentrations [mg/L based on mean measured concentrations after 72 hours
Algal yield [mg/L]		Algal growth rate [mg/L]
EyC10	1.43	EyC10	3.21
EyC20	1.78	EyC20	3.56
EyC50	2.68	EyC50	4.45
NOEyC	0.47	NOEyC	0.47
LOEyC	1.93	LOEyC	1.93

 

Validity criteria fulfilled:

yes

Conclusions:

The ErL50 of Santalol oil for growth rate and based on loading rates was 38.92 mg/L. The NOErLR was 1.0 mg/L.

Executive summary:

In a 72-hour algal growth study, cultures of Pseudokirchneriella subcapitata were exposed to Santalol oil at loading rates of 0 (control), 0.32, 1.0, 3.2, 10.0, 32.0, and 100 mg/L under static conditions in accordance with the OECD 201 guideline. After 72-hours and based on loading rates, the EL50 for algal growth rate was 38.92 mg/L and the NOEC for algal growth rate was 1.0 mg/L. The measured concentrations deviated markedly from the loading rate.

The algae were exposed to all soluble components of the test substance at the given loading rates under testing conditions. The test substance is a UVCB and as such every constituent contributes to a different degree to overall solubility, depending on its own individual solubility and its mass fraction in the test substance. Thus, for an evaluation of the results the effect concentrations should be expressed based on the nominal loading rates and were considered to be an accurate representation of exposure levels throughout the experiment.

The results in this study are consistent with all validity criteria and the test is valid according to the guidelines of this study. No deviations from test guidelines or other incidents occurred during the course of the reported test which my have influenced the results.

Description of key information

In a 72-hour algal growth study, cultures of Pseudokirchneriella subcapitata were exposed to Santalol oil at loading rates of 0 (control), 0.32, 1.0, 3.2, 10.0, 32.0, and 100 mg/L under static conditions in accordance with the OECD 201 guideline. After 72-hours and based on loading rates, the EL50 for algal growth rate was 38.92 mg/L and the NOEC for algal growth rate was 1.0 mg/L. The measured concentrations deviated markedly from the loading rate.

The algae were exposed to all soluble components of the test substance at the given loading rates under testing conditions. The test substance is a UVCB and as such every constituent contributes to a different degree to overall solubility, depending on its own individual solubility and its mass fraction in the test substance. Thus, for an evaluation of the results the effect concentrations should be expressed based on the nominal loading rates and were considered to be an accurate representation of exposure levels throughout the experiment.

The results in this study are consistent with all validity criteria and the test is valid according to the guidelines of this study. No deviations from test guidelines or other incidents occurred during the course of the reported test which my have influenced the results.

Key value for chemical safety assessment

EC50 for freshwater algae:

38.92 mg/L

EC10 or NOEC for freshwater algae:

1 mg/L