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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)
Adequacy of study:
key study
Justification for type of information:
Refer to analogue justification provided in IUCLID section 13.
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:
(WAF)
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Source: CAS 36078-10-1, Dako, 2013, D. subspicatus, 72 h
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, 72 h
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Source: CAS 3234-85-3, Evonik, 2012, D. subcapitata, 72 h
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Source: CAS 3234-85-3, Evonik, 2012, D. subcapitata, 72 h
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1.465 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Source: CAS 3234-85-3, Evonik, 2012, D. subcapitata, 72 h
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 1.465 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Source: CAS 3234-85-3, Evonik, 2012, D. subcapitata, 72 h
Conclusions:
No toxic effects on algae (OECD 201) up to the limit of water solubility (< 0.02 mg/L and < 0.05 mg/L, respectively).
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Sep - 13 Sep 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:
Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: WAF of nominal 100 mg/L and test culture
- Sampling method: Samples of test media were taken directly from the filtered WAF and from the test culture after addition of the algae at test start (0 h). At test end (72 h) the samples were taken from the replicates and pooled. Samplings of the 'OECD growth medium' were done by the staff of the analytical laboratory using a 'research 5000' pipette. The aqueous samples of 5.0 mL were pipetted directly into the extraction (centrifuge) tubes; the pipette tips were rinsed multiple and the surface was wiped with a lint free paper towel prior taking the analytical sub-sample. To assess possible wall effects on the glass surface of the test vessels (250 mL conical glass flasks), a sub-sample of the prepared “stock WAF” and of the control WAF were filtered directly into an extraction tube; the volume was determined by differential weighing.
- Sample storage conditions before analysis: The samples were processed and measured directly after sampling without any time delay.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Due to the very low water solubility of the test item a Water Accommodated Fraction (WAF) of the test item will be prepared according to the OECD guidance No. 23. Before weighing the test item, which was delivered in pellets, was crushed using a mortar. 200.0 mg test item was transferred into a 2 L sterile brown glass flask (acetone washed and sterilised) that has a bottom draining port. Sterile growth medium (2 L) and an acetone washed, sterilised star shaped stirring bar were added to the bottle and stirred slowly to avoid bubble and foam formation for about 66 h at room temperature (about 20 °C). Another flask was filled with growth medium only for the controls replicates and stirred in the same way. All work was done under sterile conditions. The test solution (WAF) was filtered by using a 0.2 µm filter (e.g. Sartolab 150v, PES-membrane, Sartorius Stedim Biotech S.A., Aubagne Cedex, France) to separate insoluble parts from the aqueous phase.
- Eluate: no
- Differential loading: yes
- Controls: yes, test medium control
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: 61.81 SAG
- Source: SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Göttingen, Germany
- Method of cultivation: The stock cultures are maintained fulfilling the criteria of the OECD guideline. Prior to testing a pre-culture was established in standard OECD growth medium to obtain exponentially-growing algae for the test.

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
20 °C
pH:
8.03 - 8.18 (control)
8.07 - 8.55 (100 mg/L WAF)
Nominal and measured concentrations:
nominal: 0, 100 mg/L (WAF)
measured: < LOQ, 0.63 - 0.82 µg/L (0 h), 0.48 - 4.92 µg/L (72 h)
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: covered with air-permeable silicone-sponge caps
- Material, size: glass, 250 mL
- Aeration: continuously shaken on an orbital laboratory shaker (100-150 rpm)
- Initial cells density: approx. 10000 cells/mL
- Control end cells density: 102118E+04 cells/mL
- No. of vessels per concentration (replicates): 8
- No. of vessels per control (replicates): 8

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified water processed using an ELGA "PURELAB" system

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 7038 - 7399 lux (OSRAM "day light")

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The cell concentrations were determined in the inoculum culture prior to the addition to the test vessels at test start and after 24, 48 and 72 h in the test cultures. The cell density was measured using an electronic particle counter (CASY 1 TT, Roche Diagnostics GmbH).

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1, 10 and 100 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1.465 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 1.465 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1.465 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 1.465 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: ErC50: 2.91 mg/L (95% confidence limit: 2.35 - 3.36 mg/L)

Table 1: Cell number (x 10E+04 to equal cells/mL) dependent on loading of the test item and time (CV: coefficient of variation). Cell number at test start: 10000 cells/mL.

 

24 h

48 h

72 h

Loading [mg/L]

Control

100 mg/L

Control

100 mg/L

Control

100 mg/L

 

4,54

3,318

22,240

18,290

93,550

83,230

 

4,636

3,045

24,920

21,740

129,700

93,110

 

4,265

4,083

24,250

23,990

117,200

117,800

 

3,856

3,623

22,740

24,600

114,000

118,700

 

3,763

3,238

21,270

23,310

98,780

81,500

 

4,303

2,551

25,080

23,350

114,500

107,000

 

4,43

3,176

26,380

20,760

121,500

103,700

 

4,698

3,906

27,360

24,070

140,800

111,900

Replicates

8

8

8

8

8

8

Mean

4,311

3,368

24,280

22,514

116,254

102,118

Std.Dev.

0

0

2

2

15,31

14,67

CV

8,0

14,6

8,6

9,5

13,2

14,4

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).

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

Since there is no study available assessing the toxicity of the target substance hexadecyl palmitate (CAS 540-10-3) to aquatic algae, read-across to the structurally related source substances tetradecanoic acid, tetradecyl ester (CAS 3234-85-3) and lauryl oleate (dodecyl oleate, CAS 36078-10-1) was conducted in accordance with Regulation (EC) No 1907/2006 Annex XI, 1.5.

Both source substances are characterized by a fatty acid as well as fatty alcohol of similar chain lengths as the target substance. The target substance hexadecyl palmitate (CAS 540-10-3) is an ester of a fatty acid and fatty alcohol of carbon atom chain lengths 16 each. The source substance tetradecanoic acid, tetradecyl ester (CAS 3234-85-3) is an ester of a C14 fatty acid and C14 fatty alcohol. The source substance dodecyl oleate (CAS 36078-10-1) is an ester of a C18 fatty acid and a C12 fatty alcohol. Therefore, the source substances are considered suitable representatives for the assessment of the acute toxicity of the target substance to aquatic algae. A detailed read-across justification is provided in in the analogue justification in IUCLID section 13.

The available studies were conducted according to the OECD guideline 201 and GLP.

In the study assessing tetradecyl myristate (CAS 3234-85-3), a limit test was conducted with a single treatment consisting of a water accommodated fraction (WAF) of a nominal limit loading rate of 100 mg/L to which P. subcapitata was exposed for 72 h (Evonik, 2012). GC/EI-MS analysis resulted in a measured concentration of 0.142 µg/L (arithmetic mean) over the time of exposure. After 72 h, no effects on the growth of algae were observed, resulting in an EL50 of > 100 mg/L (nominal) and > 1.465 µg/L (arithmetic mean of measured concentration), respectively. The derived NOELR was ≥ 100 mg/L (nominal) and ≥ 1.465 µg/L (arithmetic mean measured concentration). 

In the available study with the source substancedodecyl oleate (CAS 36078-10-1) a static test was conducted, in which D. subspicatus was exposed to five WAFs with nominal loading rates ranging from 3.57 to 100 mg/L for 72 h (Dako, 2013). Analytical measurements resulted in test concentrations below the limit of quantification (< 0.005 mg/L) for all treatments. After 72 h of exposure, no effects on the growth rate were observed, 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 as the source substances. Therefore, it can be concluded that hexadecyl palmitate (CAS 540-10-3) will not exhibit toxic effects on aquatic algae up to the limit of water solubility (< 0.846 µg/L).