Fatty acids, C8-10, C12-18-alkyl esters

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Nov - 29 Nov 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)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solution(s) were prepared following general guidance provided in OECD 23 in order to achieve a water accommodated fraction of the test substance. Generally, each test solution was prepared separately (differential loading) by directly adding test substance to test medium according to the loading of 10, 32, 100, 320, 1000 mg/L. A defined volume of test media was placed into a beaker and a glass sampling tube was suspended in the beaker with the top of the tube sufficiently above the surface of the test medium to avoid overflow from the surrounding surface layer. The test substance was pipetted carefully, on the water surface outside of the glass tubes. The beakers were covered and shaken gently (approx.. 50 RPM, to avoid emulsion formation) for about one day. Afterwards stirring, the required volume of aqueous fraction was drawn off for testing via the glass sampling tube. The aqueous fraction was inspected visually for the presence of any undissolved test substance. The exposure was started after separation of the undissolved material by adding inoculum culture to the WAF at a ratio of 1:100. According to OECD 23 the aqueous fraction of the test solution, after separation of the undissolved material, was a water accommodated fraction (OECD 23 definition) and was considered analogous to nominal concentration.
- Eluate: no
- Differential loading: yes
- Controls: yes, test medium control

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: SAG 86.81
- Source (laboratory, culture collection): Collection of algal cultures in University of Göttingen/Germany
- Age of inoculum (at test initiation): A stock algal culture is maintained continuously at the test facility. Before the exposure an inoculum culture is prepared from the stock culture and incubated for 4 days at 21 – 24 °C (max. temperature difference 2 °C). After this time, the inoculum culture is in exponential growth phase and can be used to initiate the test (study day 0).
- Method of cultivation: same as test

ACCLIMATION
- 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

Test temperature:

22.7 – 23.0 °C

pH:

7.9 - 8.4

Nominal and measured concentrations:

nominal: 0, 10, 32, 100, 320, 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): plugged with gas permeable silicone sponge caps
- Size, headspace, fill volume: 250 mL, headspace: 150 mL, fill volume: 100 mL
- Aeration: continuous shaken
- Initial cells density: 0.5 x 10E+04 cells/mL
- Control end cells density: 80.2 x 10E+04 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: according to guideline
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: Artificial light, type universal white (OSRAM L 25), permanent illumination. To minimize the potential effect of slight variations in illumination, the test vessels were re-arranged daily. Average 7009 lux (within ± 15% variability) at a wave length of 400 - 700 nm

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Algal growth measured as in vivo chlorophyll-a fluorescence (pulsed excitation with light flashes having a wavelength of 430 nm)
- Determination of correlation between fluorescence and cell density: After the end of the exposure the control replicates were mixed and serially diluted by factor 2. The fluorescence of aliquots from the undiluted mixture and the dilutions were measured and in parallel cell density was determined by a direct microscopic count (two counts in a Neubauer haemocytometer). These data were used to derive a linear correlation between fluorescence and cell density.
- Algal morphology: The inoculum culture was observed microscopically at the start of the test to verify normal and healthy cells. At test termination, a pooled sample from each test concentration was examined and any abnormal appearance of the algae noted.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ≤ 3.2
Range finding study
- Results used to determine the conditions for the definitive study: The test concentrations were selected on the basis of a range finding test (experimental conduct in accordance with GLP but without a GLP Status). The results of the 72 hour range finding test were (as nominal concentrations):EfluorescenceC50 > 100 mg/L; ErC50 > 100 mg/L. However there was 14% effect (yield) at 100 mg/L therefore a full concentration range will be performed including concentrations >100 mg/L in order to estimate the EC50.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 1 000 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.:

>= 1 000 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.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 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 (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities observed
- Unusual cell shape: no remarkable observations
- Any stimulation of growth found in any treatment: yes, inevery treatment for growth rate and in test group 32, 100 and 320 investigating yield.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: All of the test solutions were visibly clear and colorless after preparation. There was no visible undissolved test substance and no other remarkable observations through the end of exposure (72 h).

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: The ErC50 (72 h) of the control substance potassium dichromate was 0.78 mg/L (Date of the last control experiment: 25 Sep 2012) which is in the range of 0.71 - 0.97 mg/ as indicated in ISO Guideline 8692.

Reported statistics and error estimates:

SAS statistical software was used for the statistical evaluation of the data.

The 72-hour results of the algal growth inhibition test including effect concentrations based onnominal (loading rate) concentrations are summarized below.

Test Group (Loading rate) [mg/L]	Mean Cell Density (cells/mL)x 10^4 at 72 hours	% Inhibition (as yield)^a	% Inhibition (as growth rate)^a
0 (control)	80.2	0.0	0.0
10	77.7	3.05	-0.83
32	80.2	-0.08	-1.73
100	81.2	-1.32	-0.07
320	80.3	-0.12	-0.63
1000	79.5	0.80	-0.42

a: Negative values indicate a stimulatory effect relative to the control.

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 may have influenced the results.

Validity criteria fulfilled:

yes

Description of key information

No effects on growth rate of Desmodesmus subspicatus up to the water solubility limit (< 0.05 mg/L; OECD 201).

Additional information

One GLP Guideline study according to OECD 201 is available investigating the toxicity of fatty acids, C8-10, C12-18-alkyl esters (CAS 95912-86-0) to aquatic algae (Salinas 2013). Desmodesmus subspicatus was exposed to nominal loading rates of 10, 32, 100, 320 and 1000 mg/L for 72 h (Water Accommodated Fraction). No effects were observed on growth and yield after 72 h of incubation resulting in an EL50 (72 h) > 1000 mg/L and a NOELR (72 h) ≥ 1000 mg/L. Based on the result it can be concluded that fatty acids, C8-10, C12-18-alky esters (CAS 95912-86-0) will not exhibit effects on aquatic algae up to the water solubility limit.