Glycerides, C18-36

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:

14 Nov-17 Nov 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study with acceptable restrictions (precipitation occurred in those test vessels where the substance was directly added, probably creating the observed effects by physical interference)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

. Additionally consideration of the "VCI-Konzept-Entwurfes für die (akute) aquatische Prüfung von schwerlöslichen Stoffen vom 31.1. 1994"

Deviations:

yes

Remarks:

only 300 lux light intensity

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Both test runs (a) separated from undissolved test substance and b) directly weighed in
- Sampling method: Samples were taken at 0, 24, 48, 72 hours

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Due to the low solubility of the test substance two different test solutions were prepared according to the VCI-concept for poorly soluble substances
a) Test solution with 100 mg/L and subsequently separation of unsolved test substance: 100 mg test substance were added to 1 L test medium and stirred for 24h. Afterwards the undissolved test substance was separated by filtration with a fibre glass filter (pretreated with 1 molar NaOH). For each replicate 100 mL aliquot of the filtrate were used, with algae inoculated and incubated
b) 100 mg/L directly weighed in solution
The test substance was directly weighed in and shaked for 24h in a Clim-O-Shake.
To each replicate 10 mL of a 10times-concentrate of the mineral medium (according to DIN 38412/9) was added and subsequently filled up to 100 mL with deionised water. Afterwards the algae suspension was added and the incubation started

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Source (laboratory, culture collection): Pflanzenphysiol. Inst. of University Göttingen


ACCLIMATION
- Acclimation period: 3 to 4 days of preincubation in test medium without test substance

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

23°C

pH:

Control: 8.0 (0h); 9.8 - 10.4 (72h)
Test solution a) 7.8 (0h); 10.4 (72h)
Test solution b) 7.8 (0h); 8.3 (72h)
The increase of the pH during the incubation time was not caused by the substance but results from the strong cell growth of the algal cells. A significant influence onto the test result is unlikely

Nominal and measured concentrations:

Nominal concentration: 100 mg/L
Measured concentrations:
Test solution a) separation of undissolved matter: 0h - 0.14 mg/L; 24h - < 0.05 mg/L; 48 h - 0.05 mg/L; 72h < 0.05 mg/L. The recovery of the test substance at the beginning and after 48 hours was slightly above the detection limit, which means that the separation was not complete.
Test solution b) directly weighed in without separation: 0h - 59.42 mg/L; 24h - 37.72 mg/L; 48 h-5.13 mg/L; 72h- 6.32 mg/L. Due to the increasing separation of the test suspension during the incubation period, the recovery decreased from approx. 60% to 5% from 0h to 72h.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 300 mL Erlenmeyer flasks, filled with 100 mL
- Initial cells density: 1x10E4 cells/mL
- Control end cells density: 8.6x10E5 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3


GROWTH MEDIUM
- Standard medium used: yes, according to DIN 38412/9


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water
- Culture medium different from test medium: preadaption to test medium for 3-4 days
- Intervals of water quality measurement: after 72h


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: not mentioned
- Photoperiod: continuous
- Light intensity and quality: 300 Lux (daylight spectrum)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : growth rate and biomass after 24, 48 and 72h
- Determination of cell concentrations: Coulter counter


TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: test solution a) with separation of undissolved test substance; no effect up to the limit of the water solubility of test substance was observed.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: test solution a) with separation of undissolved test substance; no effect up to the limit of the water solubility of test substance was observed.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: test solution a) with separation of undissolved test substance; no effect up to the limit of the water solubility of test substance was observed.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

< 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: test solution b) without separation of undissolved test substance

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: yes, in test solution b) where the undissolved matter was not filtered out
- Effect concentrations exceeding solubility of substance in test medium: In test solution b) an effect was observed which can be attributed to the undissolved matter (see Remarks on results including tables and figures)

The test with test solution a), a test solution where the undissolved test substance was filtered out (preparation of a Water Accommodated Fraction (WAF)), showed no effects on the growth rate of Scenedesmus subspicatus. Therewith, it can be stated that no effect up to the limit of the water solubility of the test substance occurred under conditions tested.

Thus, the slight effect on algae growth observed in test solution b) where the undissolved test substance was not filtered out, can be attributed to physical impacts of the undissolved test substance and not to a systemic toxicity of the test substance.

Table 1: Cell concentrations and pH-values

Nominal test substance concentration [mg/L]	Alga cell concentrations [1/mL]			pH values
	Incubation time [h]			Incubation time [h]
	24	48	72	0	72
Control	4.3E+04	2.4E+05	8.6E+05	8.0	9.8-10.4
100 a)	2.0E+04	1.5E+05	1.1E+06	7.8	10.4
100 b)	1.7E+04	5.7E+04	3.3E+05	7.8	8.3

 

 

Table 2: Growth rates and inhibition of growth rates

Concentration [mg/L]	Growth rates after …h incubation
	24 [cells/(mL*h)]	48 [cells/(mL*h)]	72 [cells/(mL*h)]
0	0.0611	0.0659	0.0618
100 a)	0.0289	0.0569	0.0649
100 b)	0.0213	0.0361	0.0487
	Inhibition of growth rates after … h incubation
	24 [%]	48 [%]	72 [%]
100 a)	52.7	13.7	-4.9
100 b)	65.2	45.2	21.2

Reference 2

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:

other: GLP 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

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

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:

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:

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

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Study period:

14 Nov-17 Nov 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study with acceptable restrictions (precipitation occurred in those test vessels where the substance was directly added, probably creating the observed effects by physical interference)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

. Additionally consideration of the "VCI-Konzept-Entwurfes für die (akute) aquatische Prüfung von schwerlöslichen Stoffen vom 31.1. 1994"

Deviations:

yes

Remarks:

only 300 lux light intensity

GLP compliance:

yes

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: test solution a) with separation of undissolved test substance; no effect up to the limit of the water solubility of test substance was observed.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: test solution a) with separation of undissolved test substance; no effect up to the limit of the water solubility of test substance was observed.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: test solution a) with separation of undissolved test substance; no effect up to the limit of the water solubility of test substance was observed.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

< 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: test solution b) without separation of undissolved test substance

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Study period:

20 Aug - 24 Aug 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP 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

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

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:

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:

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

Description of key information

No effects up to the limit of water solubility; read-across.

Additional information

Since no studies investigating the toxicity of Dub TGI 24 to aquatic algae are available, in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5 a read-across to the structurally related source substances, Glycerides, C16-18 and C18-unsatd. (CAS No. 67701-30-8) and Glycerides,C14-18 and C16-18 unsaturated, mono-, di- and tri- (CAS No. 91052-28-7) was conducted. The source substances are representative to evaluate the toxicity of the target substance to aquatic algae. The target substance is characterized as a di and triesters with glycerol and 2-decyltetradecanoic acid. The source substances are structurally very similar to the target substance. Glycerides, C16-18 and C18-unsatd. (CAS No. 67701-30-8) is an UVCB substance consisting of C16 – C18 (even) and C18 unsaturated fatty acids with glycerol mainly as triglycerides. Glycerides,C14-18 and C16-18 unsaturated, mono-, di- and tri- (CAS No. 91052-28-7) is characterized as mono, di and triester of glycerol with fatty acids of C14-18 and C16-18 unsaturated. 

This read-across is justified in detail in the overall summary (IUCLID Section 6.1) and within the analogue justification in IUCLID Section 13. In this case of read-across, the best suited (highest degree of structural similarity, nearest physico-chemical properties) read-across substance was used for the assessment.

The first study with the read-across substance Glycerides, C16-18 and C18-unsatd. (CAS No. 67701-30-8) was conducted as limit test according to EU-Method C.3, under GLP conditions. The test was performed with additional consideration of the VCI concept for poorly soluble substances. Thus, two different test solutions were prepared with 100 mg/L: One solution where the test substance was directly weighed into the test vessel and undissolved substance was not removed and a second solution with separation of undissolved substance through filtration. No effect on the growth rate of Scenedesmus subspicatus was observed in the filtrated solutions (EC50 (72h) > 10 µg/L, meas. TWA; > 100 mg/L, nominal) and thus, it can be stated that no effect up to the limit of the water solubility of the test substances occurred under conditions tested. The effects observed in the oversaturated solutions with undissolved test substance (EC50 < 100 mg/L, nominal) can therefore be attributed to physical impacts of the undissolved test substances and not to a systemic toxicity of the tested substances.

The second study with the read-across substance Glycerides,C14-18 and C16-18 unsaturated, mono-, di- and tri- (CAS No. 91052-28-7) 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 available results from structurally related read-across substances (in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5) which are characterized by a similar ecotoxicological profile and comparable structure, it can be concluded that Dub TGI 24 will not expected effects on aquatic algae up to the limit of water solubility.