Triglycerides, C16 and C18 (unsaturated)

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:

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

Adequacy of study:

key study

Study period:

14.11.1995 -17.11.1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Annex of EU Regulation 92/69/EWG of 31.07.1992

Deviations:

not specified

Principles of method if other than guideline:

The study was conducted under special consideration of the VCI concept design for (acute) aquatic testing of poorly soluble substances of 31.1.1994.

GLP compliance:

yes

Specific details on test material used for the study:

- Source and lot/batch No.of test material: Henkel KGaA, SIS no. 6239, RIS-No. 51Z1200071000
- Expiration date of the lot/batch: 10/1996
- Manufacturing date: 03/1995
- Analytical purity: 100%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Analytical monitoring:

yes

Details on sampling:

The analytical recovery of the test substance was confirmed every 24 hours during the test.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low solubility of the test substance, two different test solutions were prepared:

A) Water Accommodated Fraction (WAF): 100 mg of test substance was placed in 1L of test medium and stirred for 24 hours with a magnetic stirrer. The undissolved test substance was separated with a glass fibre filter (pre-treated with sodium hydroxide solution).
B) Direct addition of test item to test solution: the test substance was directly weighed into the vessel and shaken for 24 h in a Clim-O-Shake
To each replicate 10 mL of a 10 fold-concentrate of the mineral medium (according to DIN 38412/9) was added and subsequently filled up to 100 mL with deionised water.

- Controls: Mineral salt medium according to DIN 38412/9

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Source (laboratory, culture collection): Pflanzenphysiol. Inst. der Universität Göttingen
- Age of inoculum (at test initiation): A fresh culture was inoculated 3-4 days prior to test in test medium without test substance

ACCLIMATION
- Acclimation period: 3 - 4 days
- Culturing media and conditions (same as test or not): Algae were pre-incubated in test medium for 3 - 4 days prior to test initiation.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

23°C

pH:

Control: 8.0 at 0h; 9.8 - 10.4 at 72h
Test solution A) 7.8 at 0h; 10.4 at 72h
Test solution B) 7.8 at 0h; 8.3 at 72h
The increase in pH during the incubation time was not caused by the substance but was considered to be a result of the strong cell growth of the algal cells. It was considered that a significant influence on the test result was unlikely.

Dissolved oxygen:

Not reported.

Nominal and measured concentrations:

Nominal concentration: 100 mg/L

Measured concentrations in test solution A (prepared as WAF with separation of undissolved matter):
0.14 mg/L at 0 h
< 0.05 mg/L at 24 h
0.05 mg/L at 48 h
< 0.05 mg/L at 72 h.

The recovery of the test substance at the beginning 0 and after 48 hours was slightly above the detection limit of the analytical method, which indicate that the separation of test item from solution was not complete.

Measured concentrations in test solution B (test item directly weighed, without separation):
59.42 mg/L at 0 h
37.72 mg/L at 24 h
5.13 mg/L at 48 h
6.32 mg/L at 72 h.

Due to the increasing separation of the test suspension during the incubation period, the recovery decreased from approx. 60% at 0 hours sampling point to approximately 5% at 72h.

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flask
- Material, size, headspace, fill volume: Test vessels were filled with 100 mL of test solution
- Aeration: No
- Initial cells density: 10 E04 cells/mL
- Control end cells density: 8.6 E05 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: mineral medium according to DIN 38412/9

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: Not reported
- Photoperiod: Continuous light
- Light intensity and quality: 300 Lux (daylight spectrum)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Cell growth was determined at 0, 24, 48 and 72 hours of incubation.
- Determination of cell concentrations: Coulter Counter

Reference substance (positive control):

no

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: See 'Remark'

Remarks:

Based on growth rate of algae in solution A (prepared as Water Accomodated Fraction, with separation of undissolved test substance)

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: See 'Remark'

Remarks:

Based on growth rate of algae in solution A (prepared as Water Accomodated Fraction, with separation of undissolved test substance).

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Not reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test medium in the vessel containing solution B (prepared as direct addition without separation of undissolved test substance) was observed to be turbid at 24, 48 and 96 hours of incubation.
- Effect concentrations exceeding solubility of substance in test medium: Toxic effects were observed in the test solutions where the undissolved test substance was added directly to test solutions. However, this is attributed to the physical impact of the undissolved test item particles and not to toxicity of the test substance.

Table 1: Cell concentrations after 24, 48 and 72 hours of incubation

Nominal test substance concentration [mg/L]	Alga cell concentrations [1/mL]
	Incubation time
	24 h	48 h	72 h
Control	4.3E+04	2.4E+05	8.6E+05
100 / A*	2.0E+04	1.5E+05	1.1E+06
100 / B**	1.7E+04	5.7E+04	3.3E+05

*A = test solution prepared as Water accommodated fraction

**B = test solution prepared as direct addition without separation of undissolved test substance

 

 Table 2: Growth rates and inhibition of growth rates

Nominal test substance concentration [mg/L]	Growth rates [cells/(mL*h)]
	24 h	48 h	72 h
Control	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 [%]
	24 h	48 h	72 h
100 / A*	52.7	13.7	-4.9
100 / B**	65.2	45.2	21.2

*A = test solution prepared as Water accommodated fraction

**B = test solution prepared as direct addition without separation of undissolved test substance

Validity criteria fulfilled:

not specified

Conclusions:

No effects were observed on the growth of algae up to the limit of water solubility of glyceryl trioleate. The EL50 was determined to be >100 mg/L loading rate WAF based on nominal test concentrations.

Executive summary:

The toxicity of glyceryl trioleate to the green algae Scenedesmus subspicatus was investigated over 72 hours under static conditions in a study conducted according to EU-Method C.3 (Rieche 1996). The study was conducted as a limit test with one test concentration of 100 mg/L. The study considered the VCI concept for poorly soluble substances and therefore two different test solutions were prepared: 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 (preparation of a Water Accommodated Fraction (WAF)). Test item concentrations were verified by chemical analysis using gas chromatography.

After 72 hours no effects on the growth of algae were observed at 100 mg/L loading rate WAF. In the oversaturated solution with undissolved test substance at 100 mg/L growth inhibition of 21.2 % was observed. However, this is attributed to the physical impact of the undissolved test item particles and not to toxicity of the test substance.

The EL50 for algae after 72 hours based on growth inhibition is estimated to be above the highest loading rate tested (EL50 > 100 mg/L WAF, based on nominal concentrations).

The study is a guideline study, is GLP-compliant and it is considered to be reliable with restrictions (Klimisch 2).

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14.11.1995 -17.11.1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Annex of EU Regulation 92/69/EWG of 31.07.1992

Deviations:

not specified

Principles of method if other than guideline:

The study was conducted under special consideration of the VCI concept design for (acute) aquatic testing of poorly soluble substances of 31.1.1994.

GLP compliance:

yes

Specific details on test material used for the study:

- Source and lot/batch No.of test material: Henkel KGaA, SIS no. 6239, RIS-No. 51Z1200071000
- Expiration date of the lot/batch: 10/1996
- Manufacturing date: 03/1995
- Analytical purity: 100%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Analytical monitoring:

yes

Details on sampling:

The analytical recovery of the test substance was confirmed every 24 hours during the test.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low solubility of the test substance, two different test solutions were prepared:

A) Water Accommodated Fraction (WAF): 100 mg of test substance was placed in 1L of test medium and stirred for 24 hours with a magnetic stirrer. The undissolved test substance was separated with a glass fibre filter (pre-treated with sodium hydroxide solution).
B) Direct addition of test item to test solution: the test substance was directly weighed into the vessel and shaken for 24 h in a Clim-O-Shake
To each replicate 10 mL of a 10 fold-concentrate of the mineral medium (according to DIN 38412/9) was added and subsequently filled up to 100 mL with deionised water.

- Controls: Mineral salt medium according to DIN 38412/9

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Source (laboratory, culture collection): Pflanzenphysiol. Inst. der Universität Göttingen
- Age of inoculum (at test initiation): A fresh culture was inoculated 3-4 days prior to test in test medium without test substance

ACCLIMATION
- Acclimation period: 3 - 4 days
- Culturing media and conditions (same as test or not): Algae were pre-incubated in test medium for 3 - 4 days prior to test initiation.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

23°C

pH:

Control: 8.0 at 0h; 9.8 - 10.4 at 72h
Test solution A) 7.8 at 0h; 10.4 at 72h
Test solution B) 7.8 at 0h; 8.3 at 72h
The increase in pH during the incubation time was not caused by the substance but was considered to be a result of the strong cell growth of the algal cells. It was considered that a significant influence on the test result was unlikely.

Dissolved oxygen:

Not reported.

Nominal and measured concentrations:

Nominal concentration: 100 mg/L

Measured concentrations in test solution A (prepared as WAF with separation of undissolved matter):
0.14 mg/L at 0 h
< 0.05 mg/L at 24 h
0.05 mg/L at 48 h
< 0.05 mg/L at 72 h.

The recovery of the test substance at the beginning 0 and after 48 hours was slightly above the detection limit of the analytical method, which indicate that the separation of test item from solution was not complete.

Measured concentrations in test solution B (test item directly weighed, without separation):
59.42 mg/L at 0 h
37.72 mg/L at 24 h
5.13 mg/L at 48 h
6.32 mg/L at 72 h.

Due to the increasing separation of the test suspension during the incubation period, the recovery decreased from approx. 60% at 0 hours sampling point to approximately 5% at 72h.

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flask
- Material, size, headspace, fill volume: Test vessels were filled with 100 mL of test solution
- Aeration: No
- Initial cells density: 10 E04 cells/mL
- Control end cells density: 8.6 E05 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: mineral medium according to DIN 38412/9

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: Not reported
- Photoperiod: Continuous light
- Light intensity and quality: 300 Lux (daylight spectrum)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Cell growth was determined at 0, 24, 48 and 72 hours of incubation.
- Determination of cell concentrations: Coulter Counter

Reference substance (positive control):

no

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: See 'Remark'

Remarks:

Based on growth rate of algae in solution A (prepared as Water Accomodated Fraction, with separation of undissolved test substance)

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: See 'Remark'

Remarks:

Based on growth rate of algae in solution A (prepared as Water Accomodated Fraction, with separation of undissolved test substance).

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Not reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test medium in the vessel containing solution B (prepared as direct addition without separation of undissolved test substance) was observed to be turbid at 24, 48 and 96 hours of incubation.
- Effect concentrations exceeding solubility of substance in test medium: Toxic effects were observed in the test solutions where the undissolved test substance was added directly to test solutions. However, this is attributed to the physical impact of the undissolved test item particles and not to toxicity of the test substance.

Table 1: Cell concentrations after 24, 48 and 72 hours of incubation

Nominal test substance concentration [mg/L]	Alga cell concentrations [1/mL]
	Incubation time
	24 h	48 h	72 h
Control	4.3E+04	2.4E+05	8.6E+05
100 / A*	2.0E+04	1.5E+05	1.1E+06
100 / B**	1.7E+04	5.7E+04	3.3E+05

*A = test solution prepared as Water accommodated fraction

**B = test solution prepared as direct addition without separation of undissolved test substance

 

 Table 2: Growth rates and inhibition of growth rates

Nominal test substance concentration [mg/L]	Growth rates [cells/(mL*h)]
	24 h	48 h	72 h
Control	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 [%]
	24 h	48 h	72 h
100 / A*	52.7	13.7	-4.9
100 / B**	65.2	45.2	21.2

*A = test solution prepared as Water accommodated fraction

**B = test solution prepared as direct addition without separation of undissolved test substance

Validity criteria fulfilled:

not specified

Conclusions:

No effects were observed on the growth of algae up to the limit of water solubility of glyceryl trioleate. The EL50 was determined to be >100 mg/L loading rate WAF based on nominal test concentrations.

Executive summary:

The toxicity of glyceryl trioleate to the green algae Scenedesmus subspicatus was investigated over 72 hours under static conditions in a study conducted according to EU-Method C.3 (Rieche 1996). The study was conducted as a limit test with one test concentration of 100 mg/L. The study considered the VCI concept for poorly soluble substances and therefore two different test solutions were prepared: 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 (preparation of a Water Accommodated Fraction (WAF)). Test item concentrations were verified by chemical analysis using gas chromatography.

After 72 hours no effects on the growth of algae were observed at 100 mg/L loading rate WAF. In the oversaturated solution with undissolved test substance at 100 mg/L growth inhibition of 21.2 % was observed. However, this is attributed to the physical impact of the undissolved test item particles and not to toxicity of the test substance.

The EL50 for algae after 72 hours based on growth inhibition is estimated to be above the highest loading rate tested (EL50 > 100 mg/L WAF, based on nominal concentrations).

The study is a guideline study, is GLP-compliant and it is considered to be reliable with restrictions (Klimisch 2).

Description of key information

The EL50 based on growth inhibition of algae was determined to be >100 mg/L loading rate WAF based on nominal test concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

Additional information

No data on toxicity Tallicin K2 to algae is available. Therefore, a study with glyceryl trioleate is used to complete this endpoint. The study was conducted according to a standard guideline, is GLP-compliant and is considered to be reliable with restrictions (Klimisch 2).

Toxicity of glyceryl trioleate to the green algae Scenedesmus subspicatus was investigated in a limit test conducted according to EU method C.3. The study was conducted with additional consideration of the VCI concept for poorly soluble substances. Therefore, two different test solutions were prepared: 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 (preparation of a Water Accommodated Fraction (WAF)).

After 72 hours no effects on the growth of algae were observed at 100 mg/L loading rate WAF and 21.1 % of growth inhibition was observed in the oversaturated solution with undissolved test substance at 100 mg/L.