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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 Oct - 4 Oct 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Remarks:
Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Karlsruhe, Germany
Analytical monitoring:
yes
Details on sampling:
- Sampling method: At the start (0 h) and at the end of the experimental phase (72 h) samples (10 mL) were taken from the highest, the middle and the lowest concentrations out of each replicate for chemical analysis and pooled in a glass beaker before transferred into brown glass vials (4 mL). At the end additional samples were taken from the test vessels with algae in the same way. Algae were separated by filtration (0.45 µm CA filter) to avoid disturbance during the analytical process.
- Sample storage conditions before analysis: The samples were stored in 4 mL brown glass vials which were filled up to the brim to avoid oxidative processes during storage. Samples were stored for a maximum period of 9 days in the refrigerator at 5 °C ± 3 K before analysis was performed. The cooled samples were sent via over-night express parcel service in an isolated transport box containing cooling elements and arrived at the next morning in good condition at the analytical laboratory.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Due to the low water solubility of the test item, the study was performed with WAF ("water accommodated fraction") prepared with algal growth medium according to OECD 201. The test item is a mobile liquid and therefore difficult to weight. Therefore it was decided to pipette the test item into the 2000 mL beaker containing algal medium. According to the density indication from the sponsor of 0.8529 g/cm³ the amount to be added was converted into volume. The suspension was stirred for 48 hours at 20 - 21 °C in the dark. For stirring a magnetic stirrer with a 2 cm stir bar was used. After stirring was stopped, the suspension was allowed to float and sediment for a period of 70 minutes. After stopping the stirring in all WAFs oily drops were floating on the surface. After one hour sedimentation and flotation period no particles or oily droplets could be observed in suspension which could affect the organisms physically. The WAFs itself were clear and were therefore not filtered to obtain the water soluble fractions (WSF). The test solutions were taken from the middle of the suspension in the beakers using a glass tube and transferred into the test vessels. The loading rates (LR) for the WAFs were chosen 1.11-fold higher than the nominal loading rates intended in the test solutions so as to take into account the dilution by the algal inoculum suspension (10%). 45 mL test solution was transferred into each test vessel (100 mL wide necked Erlenmeyer flask) and 5 mL of the algae inoculum suspension (0.8 E+05 algae mL) were added to obtain an algae starting concentration of 0.8 E+04 algae/mL. For the controls, 5 mL algae inoculum suspension was added to 45 mL algal medium. This medium was stirred in the same way as the WAF. Three replicates have been prepared for the test vessels and six for the control vessels.
- Eluate: no
- Differential loading: yes
- Controls: yes, growth medium control
- Evidence of undissolved material: no
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: CHODAT SAG No. 86.81
- Source (laboratory, culture collection): Obtained from the German Environmental Agency (UBA), Berlin-Marienfelde and cultivated in suspension culture (strain cultured at laboratory since June 2012).
- Method of cultivation: Twice a week the stock suspension is diluted into fresh Holm-Hansen medium under axenic conditions to keep it in exponential growth.

ACCLIMATION
- Culturing media and conditions (same as test or not): Holm-Hansen medium was used for cultivation. Algal growth medium according to OECD 201 was used to prepare the pre-culture three days before initiation of the study and for the study.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23.0 - 23.2 °C
pH:
7.9 - 8.0
Nominal and measured concentrations:
nominal: control, 3.57, 8.22, 18.90, 43.48 and 100 mg/L
measured: < LOQ (0.005 mg/L) in all test vessels
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks, wide necked
- Type: sealed with a sterile cellulose stopper
- Material, size, headspace, fill volume: glass, 100 mL, headspace: 50 mL, fill volume: 50 mL
- Aeration: shaken
- Initial cells density: 0.07E+05 cells/mL
- Control end cells density: 9.09E+05 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, according to guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water
- Culture medium different from test medium: yes, Holm-Hansen medium was used for cultivation. Algal growth medium according to OECD 201 was used to prepare the pre-culture three days before initiation of the study and for the study.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 120 µE/m² s ± 5.7% (PAR)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: To determine the conversion factor between the measured surrogate parameter chlorophyll fluorescence and the biomass, as required according to the OECD guideline, a correlation between fluorescence and cell count was provided. For this purpose six dilutions with the nominal cell counts of 0.05 x 10E+05, 0.1 x 10E+05, 0.5 x 10E+05, 1 x 10E+05, 5 x 10E+05 and 10 x 10E+05 were prepared by diluting the pre-culture used for the test with algal medium. For all dilutions the cell count was determined with the Coulter Counter Z2, and the chlorophyll fluorescence was determined in the same way as in the test. Both values were correlated and the equation describing the curve was calculated. The correlation allows calculation the cell count from the measured surrogate parameter chlorophyll fluorescence.
- Chlorophyll measurement: For measuring the chlorophyll fluorescence of the algae in each test vessel 200 µl test medium was transferred into a 96-well micro-plate. The fluorescence was measured with the microplate reader TECAN infinite F200 at an excitation wave length of 465 nm and an emission wave length of 670 nm. Each measurement was conducted in duplicate. If the variation coefficient was > 10% the measurement was repeated. Algal growth medium was measured as blank. The measured values were corrected for the blank values obtained with the algal medium. The dimensionless values for the fluorescence are a measure for the biomass (OECD 201).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.3
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(WAF)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(WAF)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(WAF)
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(WAF)
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control: yes
- Unusual cell shape: not observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Any stimulation of growth found in any treatment: Several loading rates tested resulted in a slight increase of algal growth.
Results with reference substance (positive control):
A quality check takes place at regular intervals using a concentration range of potassium dichromate (Sigma, Steinheim, Germany, Lot No. 074K0617, from November, 22nd, 2005) in order to assess the validity of the test system. The last quality check was performed in June 2012. The ErC50 was 0.94 mg/l (0.84 – 1.05 mg/L 95%-CI) and the EyC50 (Yield) was 0.49 mg/l (0.47 – 0.51 mg/L 95%-CI).
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to the analogue approach justification document provided in section 13.
Reason / purpose:
read-across source
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(WAF)
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Source: CAS 36078-10-1, Dako, 2013, D. subspicatus
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(WAF)
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Source: CAS 36078-10-1, Dako, 2013, D. subspicatus
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(WAF)
Basis for effect:
other: yield
Remarks on result:
other:
Remarks:
Source: CAS 36078-10-1, Dako, 2013, D. subspicatus
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(WAF)
Basis for effect:
other: yield
Remarks on result:
other:
Remarks:
Source: CAS 36078-10-1, Dako, 2013, D. subspicatus
Conclusions:
No short-term or long-term effects to aquatic microalgae were observed up to the limit of water solubility (OECD 201, D. subspicatus, RA to CAS 36078-10-1).

Description of key information

No effects up to the limit of water solubility (OECD 201, D. subspicatus); read-across

Key value for chemical safety assessment

Additional information

There is no study available investigating the toxicity of Fatty acids, C16 and C18-22 - unsatd., C16-18 and C18 unsatd. alkyl ester (CAS 90990-29-7) to aquatic algae. Therefore, a read-across to the structurally related source substance dodecyl oleate (CAS 36078-10-1) was conducted in accordance with Regulation (EC) No 1907/2006 Annex XI, 1.5.

The source substance is characterized by fatty acids and fatty alcohols of similar chain lengths as the target substance, and is therefore considered a suitable representative for the assessment of the toxicity of the target substance to aquatic algae. A detailed analogue approach justification is provided in IUCLID section 13 of the technical dossier.

 

The key study with the source substance was performed according to OECD 201 (GLP) with the green algae Desmodesmus subspicatus. Five water accommodated fractions with nominal loading rates of up to 100 mg/L were tested. Measured values resulted in concentrations below the limit of quantification (< 0.005 mg/L) for all test concentrations. No effects on the growth rate and yield were observed after an exposure of 72 h, resulting in a NOErLR (72 h) of ≥ 100 mg/L and an ErL50 (72 h) of > 100 mg/L.

 

Based on the structural and chemical similarity of the target and source substances, the target substance is expected to exhibit a similar ecotoxicological profile. Therefore, it can be concluded that Fatty acids, C16 and C18-22 - unsatd., C16-18 and C18 unsatd. alkyl ester (CAS 90990-29-7) will not exhibit short-term or long-term toxic effects to aquatic algae up to the limit of water solubility.