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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance.
Adequacy of study:
key study
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Water Accommodated Fraction (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:
Water Accommodated Fraction (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:
Water Accommodated Fraction (WAF)
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Source: CAS 93803-87-3, Croda, 1998, S. capricornutum
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Water Accommodated Fraction (WAF)
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Source: CAS 93803-87-3, Croda, 1998, S. capricornutum
Conclusions:
No toxicity to aquatic algae was recorded up to the limit of water solubility (OECD 201, D. subspicatus/S. capricornutum, 72 h).
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
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
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:
experimental study
Adequacy of study:
key study
Study period:
21 Apr - 24 Apr 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The highest test concentration was slightly turbid which probably has affected the outcome of this study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Two solutions were prepared at nominal concentrations of 10 and 100 mL respectively in order to achieve maximum exposure concentrations. These solutions were stirred for approx. 21 h, stabilized for 4 h and separated using a separation funnel afterwards. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control
- Evidence of undissolved material: The separated solutions were slightly turbid.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: CCAP 278/4
- Source: Laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from pure culture on agar. The suspensions were continuously aerated and exposed to light (4000-9000 lux) in a climate room at a temperature of 23 ± 2 °C.

ACCLIMATION
- Acclimation period: 4 d before the start of the test, cells from the algal stock culture were inoculated in a culture medium at a cell density of 2x10^4 cells/mL.
- Culturing media and conditions (same as test or not): same as test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
20 - 22 °C
pH:
8.3 - 8.5
Nominal and measured concentrations:
nominal: 0, 10 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size: all-glass, 100 mL
- Aeration: continuously shaking
- Initial cells density: 1x10^4 cells/mL
- Control end cells density: 47.5 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Other: One vessel of each test concentration without algae.

GROWTH MEDIUM
- Standard medium used: yes (according to ISO guideline)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water purified by reverse osmosis and then passed over activated carbon and ion-exchange cartridges.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity and quality: TLD-lamps of 18 Watt yielding 6000 - 6500 lux.

EFFECT PARAMETERS MEASURED: growth rate (every 24 h)
- Determination of cell concentrations: At the beginning of the test, cells were counted by microscope using a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Lambda Spectrophotometer (Perkin-Elmer, Illinois, USA), with a cuvette of 5 cm path-length. Algal medium was used as blank and the extra replicates as background for the treated solutions.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10x
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accommodated Fraction (WAF)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accommodated Fraction (WAF)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Test solution at nominal 100 mg/L was slightly turbid.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 1.7 mg/L based on growth rate reduction (95% CL: 1.1 - 2.8 mg/L)

Cell growth was inhibited significantly (31.2%) at nominal 100 mg/L, while cell growth was not affected at nominal 10 mg/L. This significant effect is probably related to the turbid test solution which adsorbed a fraction of light needed for algal cell growth. Since both test concentrations are above water solubility it can be concluded that both the EC 50 and the NOEC exceed the maximum possible water solubility of the test substance.

Table 1: Percentage reduction of growth rate at different time intervals.

Concentration of test substance:

Cell growth inhibition (0-72 h):

Growth rate [cells/mL/h-1]

Area (mean)

Inhibition [%]

mean µ1

Reduction [%]

blank

894.41

--

0.05360

--

10 mg/L

855.12

4.4

0.05204

2.9

100 mg/L

615.50

31.2

0.05140

4.1

Description of key information

No effects up to the limit of water solubility (OECD 201, D. subspicatus/S. capricornutum, 72 h); read-across

Key value for chemical safety assessment

Additional information

There is no study available assessing the toxicity of the target substance 2-octyldodecyl 12-[(1-oxooctadecyl)oxy]octadecanoate (CAS 90052-75-8) to algae. Therefore, read-across to the two structurally related source substances Lauryl Oleate (dodecyl oleate, CAS 36078-10-1) and 2-octyldodecyl isooctadecanoate (CAS 93803-87-3) was conducted in accordance with Regulation (EC) No 1907/2006 Annex XI, 1.5. Both source substances are characterized by similar fatty acid chain lengths as well as alcohol components and are therefore considered suitable representatives for the assessment of the toxicity of the target substance to algae. A detailed justification of the analogue approach is provided in IUCLID Section 13.

Both available studies with the suitable source substances were conducted according to the OECD guideline 201 and GLP.

In the available study assessing the toxicity of the source substance Lauryl Oleate (dodecyl oleate, CAS 36078-10-1), Desmodesmus subspicatus was exposed to five water accommodated fractions with nominal loading levels up to 100 mg/L (Dako, 2013). Measured values of test item concentrations in the test solutions resulted in concentrations below the Limit of Quantification (< 0.005 mg/L) for all test concentrations.

After 72 h, no effects on the growth rate and yield were recorded, resulting in a NOErLR (72 h) of ≥ 100 mg/L and an ErL50 (72 h) of > 100 mg/L. Thus, it is concluded that the source substance Lauryl Oleate (dodecyl oleate, CAS 36078-10-1) does not cause toxic effects on aquatic algae up to the limit of water solubility.

In the second study with the source substance 2-octyldodecyl isooctadecanoate (CAS 93803-87-3) Selenastrum capricornutum was exposed to two water accommodated fractions with nominal loading levels of 10 and 100 mg/L test item for 72 h under static conditions (Croda, 1998). A chemical analysis of the test substance concentrations in the test vessels was not possible since no applicable method could be established due to the very low water solubility of the test item.

After 72 h, an inhibition of cell growth by 31.2% was observed in the WAF test solution with a nominal loading level of 100 mg/L, whereas no inhibition was observed in the WAF test solution of 10 mg/L. This effect was related to insoluble particles which might have adsorbed the light required for algal cell growth. Since both test concentrations are above the water solubility it can be concluded that both the ErC50 and the NOErC exceed the maximum possible water solubility of the test substance, resulting in an EL50 (72 h) of > 100 mg/L and a NOELR (72 h) of ≥ 100 mg/L.

Based on this result it is concluded that 2-octyldodecyl isooctadecanoate (CAS 93803-87-3) does not cause toxic effects on aquatic algae up to the limit of water solubility.

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 the target substance 2-octyldodecyl 12-[(1-oxooctadecyl)oxy]octadecanoate (CAS 90052-75-8) does not cause toxic effects to algae up to the limit of water solubility.