Glycerides, rape-oil mono-, di- and tri-, hydrogenated

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Aug - 24 Aug 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)

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document No. 23 on Aquatic Toxicity of Difficult Substances and Mixtures, OECD 2000

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

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 (especially for difficult test substances)
- Method: The test substance is a mixture that is poorly soluble in water. Therefore the test solutions were prepared following general guidance provided in OECD 23 in order to achieve a water accommodated fraction (WAF) of the test substance. Generally, each test solution was prepared separately (differential loading) by directly adding test substance to test medium. A defined volume of test media was placed into a beaker (2L nominal volume) 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 with a positive displacement pipette on the water surface outside of the glass tubes. The beakers were covered with Parafilm and shaken gently (approx. 50 RPM, to avoid emulsion formation) for about one day. After shaking, the required volume of aqueous fraction was drawn off for testing via the glass sampling tube. The exposure was started after separation of the undissolved material by adding inoculum culture to the WAF at a ratio of 1:100.
Undissolved test substance was visible outside the glass sampling tube at the water surface in all test treatments. Inside of the glass sampling tube no undissolved test substance was observed. All stock test solutions appeared colorless and clear.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: SAG 86.81
- Source (laboratory, culture collection): The algae were obtained at regular intervals from SAG (Collection of algal cultures in Göttingen) and were kept in liquid culture in the Laboratory of Ecotoxicology of the Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, 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).
- Inoculum culture: An inoculum culture in exponential growth phase was prepared with an aliquot of the stock algal culture added to sterile test media to provide an initial cell density of 0.5 x 10E04 cells/mL. The inoculum culture is incubated under test conditions for 4 days prior to test initiation. The increase in biomass is verified to ensure that growth is within the normal range and algal cells are examined microscopically for normal morphology prior to use for test inoculation. Inoculum culture cell density (4 days growth): 184x 10E04 cells/mL (368 fold increase).
Inoculum culture morphology: normal and healthy

ACCLIMATION
- Culturing media and conditions: Same as in the final test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.9 - 23.2 °C

pH:

7.5 - 8.0

Nominal and measured concentrations:

Nominal: control, 3.2, 10, 32, 100, and 320 mg/L (loading rate, WAF)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Erlenmeyer flasks (nominal volume 250 mL) plugged with gas permeable silicone sponge caps and filled with 100 mL test solution
- Test chamber: Vötsch Industrietechnik GmbH Bioline (VB1014) controlled climate cabinet
- Initial cells density: 0.5 x 10E04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes. The test medium was prepared according to OECD Guideline 201

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Standard medium according to OECD 201

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: permanent
- Light intensity and quality: Artificial light, type universal white (OSRAM L 25); Average 7115 lux (within ± 15% variability) at a wave length of 400 - 700 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: 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
- Chlorophyll measurement: Algal growth measured as in vivo chlorophyll-a fluorescence (pulsed excitation with light flashes having a wave length of 430 nm)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- 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): EC50 >100 mg/L for both fluorescence and growth rate. Pretest results show slight growth inhibition at 100 and 320 mg/L. Therefore this test was performed using a broad concentration range up to 320 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: not determined

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: not determined

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

22.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 9.41 - 54.1

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

54.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 31.0 - 96.4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

296 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 176 - 500

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

172 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 93.8 - 314

Details on results:

- Inhibition of growth rate was observed at nominal loading rates of 100 and 320 mg/L.
- All 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):

EC50 (72 h, growth) = 0.90 mg/L

Table: Growth inhibition test

Test Group (loading rate) [mg/L]	Mean Cell Density (cells/mL) x 10E04 at 72 hours	Mean yield at 72 hours	Mean specific growth rate at 72 hours
0 (control)	42.4	42.1	1.63
3.2	42.0	41.6	1.61
10	39.1	38.8	1.58
32	40.9	40.6	1.60
100	26.2	25.9	1.47
320	22.1	21.8	1.43

Validity criteria fulfilled:

yes

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Justification for type of information:

Toxicity data from a structurally related source substance (Glycerides, C14-C18 and C 16-C18 unsatd., mono-, di-, and tri- (even numbered), CAS 91052-28-7) are used as read-across in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. Both are UVCB substances, formed from the esterification of fatty acids and glycerol, having a high content of C18 fatty acids and to a certain extent C14 and C16 fatty acids. Both are composed of mono- to tri esters (Target substance: monoester: 61.9%, diester<: 29.7%, triester: 3.2%; Source substance: monoester: 20-95%, diester: 5-60%, triester: 0.5-15%). Furthermore, their water solubilities are comparable (< 1 mg/L). Therefore, these two substances are expected to show similar environmental behaviour and comparable degrees of toxicity to aquatic organisms. Based on the above information, the use of data from Glycerides, C14-C18 and C 16-C18 unsatd., mono-, di-, and tri- (even numbered)as read-across for Glycerides, C14-18 mono- and di- is considered justified.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: not determined

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Description of key information

ErL50 (72 h) > 320 mg/L (nominal, loading rate) for Desmodesmus subspicatus (OECD 201)

NOELR (72 h) = 32 mg/L (nominal, loading rate) for Desmodesmus subspicatus (OECD 201)

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

32 mg/L

Additional information

No study is available investigating the toxicity of Glyceride, rape oil mono-, di- and tri-, hydrogenated (CAS 91744-94-4) to freshwater algae. Therefore, toxicity data from a structurally related source substance (Glycerides, C14-C18 and C 16-C18 unsatd., mono-, di-, and tri- (even numbered), CAS 91052-28-7) are used as read-across in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. Both are UVCB substances, formed from the esterification of fatty acids and glycerol, having a high content of C18 fatty acids and to a certain extent C14 and C16 fatty acids. Both are composed of mono- to tri esters (Target substance: monoester: 61.9%, diester<: 29.7%, triester: 3.2%; Source substance: monoester: 20-95%, diester: 5-60%, triester: 0.5-15%). Furthermore, their water solubilities are comparable (< 1 mg/L). Therefore, these two substances are expected to show similar environmental behaviour and comparable degrees of toxicity to aquatic organisms. Based on the above information, the use of data from Glycerides, C14-C18 and C 16-C18 unsatd., mono-, di-, and tri- (even numbered)as read-across for Glycerides, C14-18 mono- and di- is considered justified.

One study evaluating the toxicity of the source substance Glycerides,C14-18 and C16-18 unsaturated, mono-, di- and tri- (CAS No. 91052-28-7) to algae is available (Salinas, 2013). This test was conducted according to OECD Guideline 201, under GLP conditions. Desmodesmus subspicatus was exposed for 72 hours to the test substance at test concentrations of 3.2, 10, 32, 100 and 320 mg/L (nominal, loading rate). Chlorophyll-a fluorescence (pulsed excitation with light flashes having a wavelength of 430 nm) was the measured test parameter. Nevertheless, cell density was also determined in order to derive the linear correlation between both parameters. After 72 hours of exposure, effects on algal growth rate were observed at concentrations of 100 mg/L and 320 mg/L, leading to an ErL10 of 172 mg/L and a NOELR of 32 mg/L (nominal, loading rate). The ErL50 was determined to be > 320 mg/L (nominal, loading rate). The reported effects are well above the water solubility of the substance (water solubility < 0.05 mg/L) and therefore, they could be caused by physical interference or adsorption between algae and the test substance. Nevertheless, stock solutions were reported to be colourless and clear, and therefore, effects due to toxicity cannot be discarded. Based on the results obtained for the structurally related analogue (in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5), Glyceride, rape oil mono-, di- and tri-, hydrogenated could exhibit low toxicity to algae at chronic exposure.