Soaps, stocks, vegetable-oil, acidulated

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:

weight of evidence

Study period:

March 2002 to April 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: ISO 14669, ISO 10253 and GESAMP protocol for preparation of poorly soluble complex mixtures

Deviations:

no

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficultly soluble test substances)
- Method: Water accommodated fraction (WAF) methodology
- Preparation: WAFs were prepared by adding test substance to the test medium (sterile marine algal media) to give loading rates of 0 and 1,000 mg/L. In order to mix the test substance, which was solid at room temperature, the sample was microwaved on full power for approximately 2 min. until it became liquid. The contents of the WAF vessels were then stirred using a magnetic stirrer, at 300 rpm, for 24 h, in a controlled environment room set at 20±2°C and then left to settle for a minimum of 2 h (range 2 – 4 h), to allow the undissolved material to separate out. The aqueous phases - the WAFs – were then drawn off via the tap for use in the tests. Control media was sterile marine algal media.

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Common name: Diatom
- Source (laboratory, culture collection): Scottish Association for Marine Science Research Services Ltd, Dunstaffnage, Oban, Scotland
- Method of cultivation: Cultures were maintained in autoclaved nutrient enriched natural seawater under constant illumination (nominal 6000 lux) at 20±2°C in a Gallenkamp orbital incubator and shaken at 100 cycles per min.

Test type:

static

Water media type:

saltwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

19.2 - 20.7

pH:

7.9 - 9.5

Dissolved oxygen:

7.5 - 6.3 mg/L

Nominal and measured concentrations:

Nominal: 0 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): Erlenmeyer flasks closed with ground glass stoppers
- Material, size, headspace, fill volume: 250 mL flasks filled with 100 mL test solution
- Initial cells density: 10000 cells/mL
- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural seawater, collected off the beach at Kinmel Bay to the west of Rhyl, Flintshire, N. Wales
- Total organic carbon for medium: 31 mg/L


OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Light intensity and quality: 6,700-8,200 lux
- Temperature: 20±2 °C


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Particle counting was performed on the contents of each flask at the start of the test and after 24, 48 and 72 h of incubation using electronic particle counter (Coulter Model ZM)
- Other: At the end of the test, estimations of the mean chain length of S. costatum for one flask from each test concentration were made by microscopic examination of at least 50 chains in a 1 mL sub sample.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The WAF prepared from palm oil initially appeared to suppress algal growth. The growth rate between 0 and 48 h was 22% lower than the controls. This resulted in reduction in biomass of 40% with respect of the controls after 48 h. However, between 48 and 72 h, good growth was achieved and at the end of the test, the chain concentration in the WAF from palm oil was almost equal to that found in controls and the average specific growth rate was only 8% lower than the controls.

Results with reference substance (positive control):

- Results with reference substance valid?: yes
- Other:The average specific growth rate was reduced by 15% compared to the control.

Validity criteria fulfilled:

no

Conclusions:

Based on the study conditions, the nominal 72 h EC50 of the constituent to Skeletonema costatum was estimated to be >1,000 mg/L.

Executive summary:

A study was conducted to evaluate the toxicity of the constituent glycerides C16 -18, C18 unsatd. (crude palm oil) to Skeletonema costatum according to the guideline ISO 14669, ISO 10253 and GESAMP under static conditions. Skeletonema costatum were exposed to the constituent at 0 and 1,000 mg/L for 0, 24, 48 and 72 h. The constituent did not have an significant effect on either average growth rate or chain length of Skeletonema costatum. 3,5 -dichlorophenol was used as a reference. After 72 h, the average specific growth rate was reduced with 15% which is less than the 20 - 80% recommended in the guidelines. Based on the study conditions, the nominal 72 h EC50 of the constituent to Skeletonema costatum was considered to be >1,000 mg/L (Worden and Sherren, 2002c).

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March 2002 to April 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: ISO 14669, ISO 10253 and GESAMP guidelines

Deviations:

no

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficultly soluble test substances)
- Method: Water accommodated fraction (WAF) methodology
- Preparation: WAFs were prepared by adding test substances to the test medium (sterile marine algal media) to give loading rates of 10, 100 and 1000 mg/L. In order to mix the test substances, which was solid at room temperature, the sample was microwaved on full power for approximately 2 min until it became liquid. The contents of the WAF vessels were then stirred using a magnetic stirrer, at 300 rpm, for 24 h, in a controlled environment room set at 20±2°C and then left to settle for a minimum of 2 h (range 2 – 4 h), to allow the undissolved material to separate out. The aqueous phases - the WAFs – were then drawn off via the tap for use in the tests. Control media was sterile marine algal media.

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Common name: Diatom
- Source (laboratory, culture collection): Scottish Association for Marine Science Research Services Ltd, Dunstaffnage, Oban, Scotland
- Method of cultivation: Cultures were maintained in autoclaved nutrient enriched natural seawater under constant illumination (nominal 6,000 lux) at 20±2°C in a Gallenkamp orbital incubator and shaken at 100 cycles min-1

Test type:

static

Water media type:

saltwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

19.2-21.3°C

pH:

8.2-9.6

Dissolved oxygen:

7.4-6.2 mg/L O2

Nominal and measured concentrations:

0 and 1,000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): Erlenmeyer flasks closed with ground glass stoppers
- Material, size, headspace, fill volume: 250 mL flasks filled with 100 mL test solution
- Initial cells density: 10,000 cells/mL
- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural seawater, collected off the beach at Kinmel Bay to the west of Rhyl, Flintshire, N. Wales
- Total organic carbon for medium: 31 mg/L


OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Light intensity and quality: 6,700-8,200 lux
- Temperature: 20±2°C


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Particle counting was performed on the contents of each flask at the start of the test and after 24, 48 and 72 h of incubation using electronic particle counter (Coulter Model ZM)
- Other: At the end of the test, estimations of the mean chain length of S. costatum for one flask from each test concentration were made by microscopic examination of at least 50 chains in a 1 mL sub sample.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

78 h

Dose descriptor:

EC0

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Other: Average chain density in the control vessels increased by a factor of 50 in 72 h and indicates that a good growth took place in control conditions. The mean chain-length in the starter culture was 6.6 cells per chain. In the test, the mean chain lengths after 72 h were 4.7 and 4.1 cells for S. costatum taken from the control and crude soybean oil respectively.

Results with reference substance (positive control):

- Results with reference substance valid?: yes
- Other:The average specific growth rate was reduced by 15% compare to the control.

Validity criteria fulfilled:

no

Conclusions:

Based on the study conditions, the nominal 72 h EC0 of the constituent to Skeletonema costatum was estimated to be >1,000 mg/L.

Executive summary:

A study was conducted to evaluate the toxicity of water accommodated fractions (WAF) of the constituent glycerides, C16 -18 and C18 unsatd. (crude soybean oil) to Skeletonema costatum according to the guidelines ISO 14669, ISO 10253 and GESAMP under static conditions. Skeletonema costatum were exposed to crude soybean oil at 0 and 1,000 mg/L for 0, 24, 48 and 72 h. The result of the test showed that the constituent did not have an significant effect on the chain length of Skeletonema costatum. Based on the study conditions, the nominal 72 h EC0 of the constituent to Skeletonema costatum was estimated to be >1,000 mg/L (Worden and Sherren, 2002d).

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January 12 2004 to January 17 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Deviations:

no

GLP compliance:

not specified

Analytical monitoring:

not specified

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficultly soluble test substances)
- Method: Water accommodated fraction (WAF) methodology
- Preparation: WAFs were prepared by adding test substance to the test medium (sterile marine algal media) to give loading rates of 10, 100 and 1,000 mg/L. In order to mix the test substance, which was solid at room temperature, the sample was microwaved on full power for approximately 2 min until it became liquid. The contents of the WAF vessels were then stirred using a magnetic stirrer, at 300 rpm, for 24 h, in a controlled environment room set at 20±2°C and then left to settle for a minimum of 2 h (range 2 – 4 h), to allow the undissolved material to separate out. The aqueous phases - the WAFs – were then drawn off via the tap for use in the tests. Control media was sterile marine algal media.

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Common name: Diatom
- Source (laboratory, culture collection): Scottish Association for Marine Science Research Services Ltd, Dunstaffnage, Oban, Scotland
- Method of cultivation: Cultures were maintained in autoclaved nutrient enriched natural seawater under constant illumination (nominal 6000 lux) at 20±2°C in a Gallenkamp orbital incubator and shaken at 100 cycles min-1

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

20±2°C

pH:

Control: 8.5 at the start to 9.2 after 72 h
Test: 8.5 at the start to 8.3 - 9.3 after 72 h

Nominal and measured concentrations:

Nominal concentration: 10, 100 and 1,000 mg/L
1,000 mg/L was equivalent to 4 mg total carbon/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): Erlenmeyer flasks closed with ground glass stoppers
- Material, size, headspace, fill volume: 250 mL flasks filled with 100 mL test solution
- Initial cells density: 10,000 cells/mL
- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural seawater, collected off the beach at Kinmel Bay to the west of Rhyl, Flintshire, N. Wales
- Total organic carbon for medium: 31 mg/L


OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Light intensity and quality: 6,700-8,200 lux
- Temperature: 20±2°C


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Particle counting was performed on the contents of each flask at the start of the test and after 24, 48 and 72 h of incubation using electronic particle counter (Coulter Model ZM)
- Other: At the end of the test, estimations of the mean chain length of S. costatum for one flask from each test concentration were made by microscopic examination of at least 50 chains in a 1 mL sub sample.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol; 1.5 mg/L

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

>= 10 - < 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

>= 100 - < 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes

Results with reference substance (positive control):

- Results with reference substance: Valid
- Average specific growth rate was not reduced over 72 h compared to the control, but that a reduction of 26% was seen in Skeletonema costatum biomass (as measured by area under the growth curve).

Table 1: Growth of Skeletonema costatum exposed to WAFs of coconut oil and 1.5 mg/L solution of reference compound 3,5-DCP

Treatment	Mean reduction in area under the growth curve (A) relative to unfiltered controls (%)			Mean reduction in average specific growth rate (µ) relative to unfiltered controls (%)			Mean number cells per chain
	0-24 h	0-48 h	0-72 h	0-24 h	0-48 h	0-72 h	72 h
Control	-	-	-	-	-	-	3.4
Control (filtered)	17	8.3	-1.6	10	1.4	-6	2.4
Coconut 10 mg/L	-31	-16	-9.9	-21	-6.4	-3.2	3.2
Coconut 100 mg/L	96	84	57	92	48	-2	4.1
Coconut 1000 mg/L	100	100	100	100	>100	>100	0
3,5-DCP (1.5 mg/L)	32	38	26	13	13	-5.6	3.4

Validity criteria fulfilled:

not specified

Conclusions:

Based on the study conditions, the 72 h EbL50 and ErL50 of the constituent were determined to be between 10 - 100 and 100 - 1,000 mg/L, respectively.

Executive summary:

A study was conducted to evaluate the acute toxicity of the constituent glycerides C8 -18 and C18 unsatd. (coconut oil) to Skeletonema costatum according to the guideline ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum) under static conditions. Skeletonema costatum were exposed to water accommodated fractions prepared from the constituent at concentrations ranging from 10 -1,000 mg/L for 72 h. The range of loading rates within which a 50% reduction in algal growth occurred after 72 h was determined on the basis of both the area under the growth curve (EbL50) and specific growth rate (ErL50). Based on the study conditions, the 72 h EbL50 and ErL50 of the constituent were determined to be between 10 - 100 and 100 - 1,000 mg/L, respectively (Worden and Sherren, 2004e).

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Deviations:

no

GLP compliance:

not specified

Analytical monitoring:

not specified

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficultly soluble test substances)
- Method: Water accommodated fraction (WAF) methodology
- Preparation: WAFs were prepared by adding test substance to the test medium (sterile marine algal media) to give loading rates of 10, 100 and 1000 mg/L. In order to mix the test substance, which was solid at room temperature, the sample was microwaved on full power for approximately 2 min until it became liquid. The contents of the WAF vessels were then stirred using a magnetic stirrer, at 300 rpm, for 24 h, in a controlled environment room set at 20 ± 2°C and then left to settle for a minimum of 2 h (range 2 – 4 h), to allow the undissolved material to separate out. The aqueous phases - the WAFs – were then drawn off via the tap for use in the tests. Control media was sterile marine algal media.

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Source (laboratory, culture collection): Scottish Association for Marine Science Research Services Ltd, Dunstaffnage, Oban, Scotland
- Method of cultivation: An appropriate amount of culture was used to inoculate starter culture flasks containing sterile media. The flasks were incubated at 20 ± 2°C in a Gallenkamp orbital incubator under full constant illumination (9,000 lux) for 4 d. This starter culture was used for inoculation of test cultures.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

20 ± 2°C

pH:

Control: 8.4 at the start to 9.5 after 72 h
Test: 8.2 - 8.4 at the start to 8.0 - 9.6 after 72 h

Nominal and measured concentrations:

Nominal concentration: 10, 100 & 1000 mg/L
- 1000 mg/L was equivalent to 4.2 mg total carbon/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): Erlenmeyer flasks closed with ground glass stoppers
- Material, size, headspace, fill volume: 250 mL flasks filled with 100 mL test solution
- Initial cells density: 10,000 cells/mL
- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural seawater, collected off the beach at Kinmel Bay to the west of Rhyl, Flintshire, N. Wales
- Total organic carbon of medium: 31.0 mg/L


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Light intensity and quality: 7,400-8,550 lux
- Temperature: 20 ± 2°C
- Other: Flasks incubated in a cooled, orbital incubator (100 cycles/min)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Particle counting was performed on the contents of each flask at the start of the test and after 24, 48 and 72 h of incubation using electronic particle counter (Coulter Model ZM)
- Other: At the end of the test, estimations of the mean chain length of S. costatum for one flask from each test concentration were made by microscopic examination of at least 50 chains in a 1 mL sub sample.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol; 1.5 mg/L

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

>= 10 - < 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

>= 100 - < 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Test material was completely toxic to Skeletoname costatum at a loading rate of 1,000 mg/L and showed some toxicity at 100 mg/L, where algal growth was initially inhibited but recovered after 48 h (see Table 1 for details).

Results with reference substance (positive control):

- Results with reference substance: Valid
- The area under the growth curve was reduced by 87% and the average specific growth rate was reduced by 37% compared to controls

Table 1: Growth of Skeletonema costatum exposed to WAFs of palm kernel oil and 1.5 mg/L solution of reference compound 3,5-DCP

Treatment	Mean reduction in area under the growth curve (A) relative to unfiltered controls (%)			Mean reduction in average specific growth rate (µ) relative to unfiltered controls (%)			Mean number cells per chain
	0-24 h	0-48 h	0-72 h	0-24 h	0-48 h	0-72 h	72 h
Control	-	-	-	-	-	-	4.3
Palm kernel oil 10 mg/L	18	15	8.4	12	5.8	1.4	4.4
Palm kernel oil 100 mg/L	81	93	83	65	81	28	4.6
Palm kernel oil 1000 mg/L	80	97	100	64	>100	>100	-
3,5 -DCP (1.5 mg/L)	79	94	87	66	90	3.7	4.1

Validity criteria fulfilled:

not specified

Conclusions:

Based on the study conditions, the nominal 72 h EbL50 and ErL50 of the constituent were determined to be between 10 - 100 and 100 - 1,000 mg/L, respectively.

Executive summary:

A study was conducted to evaluate the acute toxicity of the constituent glycerides C8 -18 and C18 unsatd. (palm kernel oil) to Skeletonema costatum according to the guideline ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum) under static conditions. Skeletonema costatum were exposed to water accommodated fractions prepared with the constituent at concentrations ranging between 10 - 1,000 mg/L for 72 h. The range of loading rates within which a 50% reduction in algal growth occurred after 72 h was determined on the basis of both the area under the growth curve (EbL50) and specific growth rate (ErL50). Based on the study conditions, the nominal 72 h EbL50 and ErL50 of the constituent were determined to be between 10 - 100 and 100 - 1,000 mg/L, respectively (Worden and Sherren, 2004h).

 

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Deviations:

no

GLP compliance:

not specified

Analytical monitoring:

not specified

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficultly soluble test substances)
- Method: Water accommodated fraction (WAF) methodology
- Preparation: WAFs were prepared by adding test substance to the test medium (sterile marine algal media) to give loading rates of 10, 100 and 1,000 mg/L. In order to mix the test substance, which was solid at room temperature, the sample was microwaved on full power for approximately 2 min until it became liquid. The contents of the WAF vessels were then stirred using a magnetic stirrer, at 300 rpm, for 24 h, in a controlled environment room set at 20 ± 2°C and then left to settle for a minimum of 2 h (range 2 – 4 h), to allow the undissolved material to separate out. The aqueous phases - the WAFs – were then drawn off via the tap for use in the tests. Control media was sterile marine algal media.

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Source (laboratory, culture collection): Scottish Association for Marine Science Research Services Ltd, Dunstaffnage, Oban, Scotland
- Method of cultivation: An appropriate amount of culture was used to inoculate starter culture flasks containing sterile media. The flasks were incubated at 20 ± 2°C in a Gallenkamp orbital incubator under full constant illumination (9,000 lux) for 4 d. This starter culture was used for inoculation of test cultures.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

20 ± 2°C

pH:

Control: 8.4 at the start to 9.5 after 72 h
Test: 8.2 - 8.4 at the start to 8.0 - 9.6 after 72 h

Nominal and measured concentrations:

Nominal concentration: 10, 100 and 1,000 mg/L
1,000 mg/L was equivalent to 5.1 mg total carbon/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): Erlenmeyer flasks closed with ground glass stoppers
- Material, size, headspace, fill volume: 250 mL flasks filled with 100 mL test solution
- Initial cells density: 10,000 cells/mL
- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural seawater, collected off the beach at Kinmel Bay to the west of Rhyl, Flintshire, N. Wales
- Total organic carbon of medium: 31.0 mg/L


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Light intensity and quality: 7,400-8,550 lux
- Temperature: 20 ± 2°C
- Other: Flasks incubated in a cooled, orbital incubator (100 cycles/min)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Particle counting was performed on the contents of each flask at the start of the test and after 24, 48 and 72 h of incubation using electronic particle counter (Coulter Model ZM)
- Other: At the end of the test, estimations of the mean chain length of S. costatum for one flask from each test concentration were made by microscopic examination of at least 50 chains in a 1 mL sub sample.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol; 1.5 mg/L

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

>= 10 - < 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

>= 100 - < 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Test material was completely toxic to S. costatum at a loading rate of 1,000 mg/L and showed some toxicity at 100 mg/L, where algal growth was initially inhibited but recovered after 48 hours (see Table 1 for details).

Results with reference substance (positive control):

- Results with reference substance: Valid
- The area under the growth curve was reduced by 87% and the average specific growth rate was reduced by 37% compared to controls

Reported statistics and error estimates:

None

Table 1: Growth of Skeletonema costatum exposed to WAFs of palm kernel olein and 1.5 mg/L solution of reference compound 3,5-DCP

Treatment	Mean reduction in area under the growth curve (A) relative to unfiltered controls (%)			Mean reduction in average specific growth rate (µ) relative to unfiltered controls (%)			Mean number cells per chain
	0-24 h	0-48 h	0-72 h	0-24 h	0-48 h	0-72 h	72 h
Control	-	-	-	-	-	-	4.3
Palm kernel olein 10 mg/L	26	27	17	20	13	4.2	4.4
Palm kernel olein 100 mg/L	87	97	93	76	>100	49	4.7
Palm kernel olein 1000 mg/L	87	98	100	76	>100	>100	-
3,5 -DCP (1.5 mg/L)	79	94	87	66	90	3.7	4.1

Validity criteria fulfilled:

not specified

Conclusions:

Based on the study conditions, the nominal 72 h EbL50 and ErL50 of the constituent were determined to be between 10 - 100 and 100 - 1,000 mg/L, respectively.

Executive summary:

A study was conducted to evaluate the acute toxicity of the constituent glycerides C8 -18 and C18 unsatd. (palm kernel olein) to Skeletonema costatum according to the guideline ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum) under static conditions. Skeletonema costatum were exposed to water accommodated fractions of the constituent at concentrations ranging from 10 - 1,000 mg/L for 72 h. The range of loading rates within which a 50% reduction in algal growth occurred after 72 h was determined on the basis of both the area under the growth curve (EbL50) and specific growth rate (ErL50). Based on the study conditions, the nominal 72 h EbL50 and ErL50 of the constituent were determined to be between 10 - 100 and 100 - 1,000 mg/L, respectively (Worden and Sherren, 2004f).

 

Reference 6

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Deviations:

no

GLP compliance:

not specified

Analytical monitoring:

not specified

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficultly soluble test substances)
- Method: Water accommodated fraction (WAF) methodology
- Preparation: WAFs were prepared by adding test substance to the test medium (sterile marine algal media) to give loading rates of 10, 100 and 1,000 mg/L. In order to mix the test substance, which was solid at room temperature, the sample was microwaved on full power for approximately 2 min until it became liquid. The contents of the WAF vessels were then stirred using a magnetic stirrer, at 300 rpm, for 24 h, in a controlled environment room set at 20 ± 2°C and then left to settle for a minimum of 2 h (range 2 – 4 h), to allow the undissolved material to separate out. The aqueous phases - the WAFs – were then drawn off via the tap for use in the tests. Control media was sterile marine algal media.

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Source (laboratory, culture collection): Scottish Association for Marine Science Research Services Ltd, Dunstaffnage, Oban, Scotland
- Method of cultivation: An appropriate amount of culture was used to inoculate starter culture flasks containing sterile media. The flasks were incubated at 20 ± 2°C in a Gallenkamp orbital incubator under full constant illumination (9,000 lux) for 4 d. This starter culture was used for inoculation of test cultures.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

20 ± 2°C

pH:

Control: 8.4 at the start to 9.5 after 72 h
Test: 8.2 - 8.4 at the start to 8.0 - 9.6 after 72 h

Nominal and measured concentrations:

Nominal concentration: 10, 100 and 1,000 mg/L
1,000 mg/L was equivalent to 1.3 mg total carbon/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): Erlenmeyer flasks closed with ground glass stoppers
- Material, size, headspace, fill volume: 250 mL flasks filled with 100 mL test solution
- Initial cells density: 10,000 cells/mL
- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural seawater, collected off the beach at Kinmel Bay to the west of Rhyl, Flintshire, N. Wales
- Total organic carbon of medium: 31.0 mg/L


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Light intensity and quality: 7,400-8,550 lux
- Temperature: 20 ± 2°C
- Other: Flasks incubated in a cooled, orbital incubator (100 cycles/min)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Particle counting was performed on the contents of each flask at the start of the test and after 24, 48 and 72 h of incubation using electronic particle counter (Coulter Model ZM)
- Other: At the end of the test, estimations of the mean chain length of S. costatum for one flask from each test concentration were made by microscopic examination of at least 50 chains in a 1 mL sub sample.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol; 1.5 mg/L

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

>= 100 - < 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Test material was initially toxic to S. costatum at 1,000 mg/L, but algal growth recovered after 48 hours exposure. WAFs prepared from 100 and 10 mg/L were not toxic (see Table 1 for details).

Results with reference substance (positive control):

- Results with reference substance: Valid
- The area under the growth curve was reduced by 87% and the average specific growth rate was reduced by 37% compared to controls

Reported statistics and error estimates:

None

Table 1: Growth of Skeletonema costatum exposed to WAFs of palm kernel stearin and 1.5 mg/L solution of reference compound 3,5-DCP

Treatment	Mean reduction in area under the growth curve (A) relative to unfiltered controls (%)			Mean reduction in average specific growth rate (µ) relative to unfiltered controls (%)			Mean number cells per chain
	0-24 h	0-48 h	0-72 h	0-24 h	0-48 h	0-72 h	72 h
Control	-	-	-	-	-	-	4.3
Palm kernel stearin 10 mg/L	4.9	10	9.7	9.5	8.1	6.3	4.3
Palm kernel stearin 100 mg/L	19	16	10	12	6	1.5	4
Palm kernel stearin 1000 mg/L	80	94	84	64	92	28	4.6
3,5 -DCP (1.5 mg/L)	79	94	87	66	90	3.7	4.1

Validity criteria fulfilled:

not specified

Conclusions:

Based on the study conditions, the nominal 72 h EbL50 and ErL50 of the constituent were determined to be between 100 - 1,000 and greater than 1,000 mg/L, respectively.

Executive summary:

A study was conducted to evaluate the acute toxicity of the constituent glycerides C8 -18 and C18 unsatd. (palm kernel stearin) to Skeletonema costatum according to the guideline ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum) under static conditions. S. costatum were exposed to water accommodated fractions of the constituent at concentrations ranging from 10 - 1,000 mg/L for 72 h. The range of loading rates within which a 50% reduction in algal growth occurred after 72 h was determined on the basis of both the area under the growth curve (EbL50) and specific growth rate (ErL50). Based on the study conditions, the nominal 72 h EbL50 and ErL50 of the constituent were determined to be between 100 - 1,000 and greater than 1,000 mg/L, respectively (Worden and Sherren, 2004g).

 

Reference 7

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

13 April 2010 to 17 April 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Refer to section 13 for details on the read across justification. The algae study with the read across substance is used as a supporting study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Appropriate amounts of the stock solution were diluted with pure water to get TC standard solutions in the range from 1 to 50 mg Carbon/L and IC standard solutions in the range of 1 to 25 mg Carbon/L.
- Sample storage conditions before analysis: The samples from the end of the test were filtered (0.45 µm cellulose acetate) before analysis

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficultly soluble test substances)
- Method: Inoculation of a biomass of 5,000 algal cells per mL test medium
- Controls: Yes (six replicates)
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): A supersaturated stock solution of 100 mg test substance/L.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Freshwater green algae
- Strain: Strain No. 61.81 SAG
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen", 37073 Göttingen, Germany
- Method of cultivation: The algae were cultivated in the laboratories under standardised conditions according to the test guidelines

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (= 24 mg/L) as CaCO3.

Test temperature:

23 -24°C

pH:

8.0 at the start of the test
8.7 - 8.8 at the end of the test

Nominal and measured concentrations:

Nominal: 0 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): Closed
- Material, size, headspace, fill volume: 50 mL volume with 50 mL of test medium
- No. of vessels per concentration (replicates): Six
- No. of vessels per control (replicates): Six


GROWTH MEDIUM
- Standard medium used: Yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (OECD Medium)

OTHER TEST CONDITIONS
- Adjustment of pH: Yes
- Light intensity and quality: 6120 - 6440 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Spectrophotometer

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water accommodated fraction

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water accommodated fraction

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

water accommodated fraction

Basis for effect:

other:

Remarks:

growth rate and yield

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape: No
- Colour differences: No remarkable observations
- Adherence to test vessels: No

Results with reference substance (positive control):

- Results with reference substance valid: Yes

Analytical Results:

Determination of the test substance	Based on the results of TOC measurements the concentration of the test substance was determined using a calibration curve.
Calibration Range	1 to 50 mg Carbon/L in pure water in the case of TC-calibration and 1 to 25 mg Carbon/L in pure water in the case of IC-calibration
Linearity of Response	Correlation of peak area of different standard solutions with their corresponding concentrations, using a linear regression
Regression Coefficient	At least 0.9972 in the case of TC at least 0.9926 in the case of IC
Typical Calibration Curve	y = 3.9115 * x + 1.4425 in the case of TC y = 3.5092 * x – 0.9996 in the case of IC
Limit of Detection	LODTC: 3.2470 mg Carbon/L LODIC: 3.0318 mg Carbon/L
Limit of Quantification	5 mg Carbon/L 113 % (n = 4, RSD 12 %)
Mean Recovery in the Fortified Samples	109 % (n = 8, RSD 10 %)
Mean recovery in the test samples	Since the test substance is not well water soluble, all results were below LOQ

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the read across study, 72 h ErC50, EyC50 and LOEC of the test substance, Soaps, stocks, vegetable oil, acidulated, is considered to be >100 mg/L WAF (nominal).

Executive summary:

A study was conducted to determine the inhibitory effect of read across substance, 'oils, vegetable, deodorizer distillates' on the growth of the freshwater green algae Pseudokirchneriella subcapitata according to OECD Guideline 201 and EU Method C.3. Six replicates of exponentially growing algal cultures were exposed to water accommodated fractions (WAFs) of the read across substance at only nominal test concentration of 100 mg/L for a period of 72 h under static conditions. The inhibition of growth rate and yield was determined at 24, 48 and 72 h time intervals and compared to data for the control and a reference substance potassium dichromate. The quantification of the read across substance was performed using the TOC-method at 0 and 72 hrs. Since the read across substance is not well water soluble, all analytical results were below the LOQ (limit of quantification). The microscopic examination of the shape of the algal cells after 72 h of test duration did not show any difference between the test and the control groups. Therefore, the NOEC was determined to be at least 100 mg/L (nominal) and the 72 h LOEC, ErC50 and EyC50 were clearly higher than the only test concentration of nominal 100 mg/L. These values could not be quantified due to the absence of toxicity of the read across substance up to the only test concentration of nominal 100 mg/L, the concentration representing the limit of solubility (Kley and Deierling, 2010e). Based on the results of the read across study, 72 h ErC50, EyC50 and LOEC of the test substance, Soaps, stocks, vegetable oil, acidulated, is considered to be >100 mg/L WAF (nominal).

Reference 8

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

12 April 2010 to 17 April 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 for details on the read across justification. The algae study with the read across substance is used as a supporting study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Appropriate amounts of the stock solution were diluted with pure water to get TC standard solutions in the range from 1 to 50 mg Carbon/L and IC standard solutions in the range of 1 to 25 mg Carbon/L.
- Sample storage conditions before analysis: The samples from the end of the test were filtered (0.45 µm cellulose acetate) before analysis

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Inoculation of a biomass of 5000 algal cells per mL test medium
- Controls: Yes (Six replicates)
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): A supersaturated stock solution of 100 mg test item/L.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Freshwater green algae
- Strain: Strain No. 61.81 SAG
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen", 37073 Göttingen, Germany
- Method of cultivation: The algae were cultivated in the laboratories under standardised conditions according to the test guidelines

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

23°C

pH:

8.0 - 9.1

Nominal and measured concentrations:

Nominal: 0 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): Closed
- Material, size, headspace, fill volume: 50 mL volume with 50 mL of test medium
- No. of vessels per concentration (replicates): Six
- No. of vessels per control (replicates): Six


GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (OECD Medium)

OTHER TEST CONDITIONS
- Adjustment of pH: Yes
- Light intensity and quality: 6120 - 6440 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Spectrophotometer

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water accommodated fraction

Basis for effect:

other: growth rate and yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other:

Basis for effect:

growth rate

Remarks:

water accommodated fraction

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water accommodated fraction

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape: No
- Colour differences: No remarkable observations
- Adherence to test vessels: No

Results with reference substance (positive control):

- Results with reference substance valid: Yes

The concentration of test substance recorded by total organic carbon analysis was below the limit of quantification in the control and test vessels. The LOQ was equivalent to 3 mg/L. Test concentrations were therefore based on nominal values.

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the read across study, 72 h ErC50, EyC50 and LOEC of the test substance, Soaps, stocks, vegetable oil, acidulated, is considered to be >100 mg/L WAF (nominal).

Executive summary:

A study was conducted to determine the inhibitory effect of the read-across substance 'soybean oil, deodorizer distillate' on the growth of the freshwater green algae Pseudokirchneriella subcapitata according to OECD Guideline 201 and EU method C.3 under static conditions. Exponentially growing cultures were exposed to water accommodated fraction of the only tested concentration 100 mg/L (nominal) under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 h, and over several algal generations. A limit test was performed in compliance with the test guidelines to demonstrate that the read-across substance has no toxic effect on Pseudokirchneriella subcapitata at this concentration. Analytical verification of the test concentrations were conducted on samples of the freshly prepared and aged (72 h) test medium via the TOC-method. Under the test conditions, the nominal 72 h NOEC was determined to be 100 mg/L. The nominal 72 h ErC50 and EyC50 were clearly higher than the only tested concentration of 100 mg/L. These values could not be quantified due to the absence of toxicity to the read-across substance at the concentration representing the limit of solubility (Kley and Deierling, 2010f). Based on the results of the read across study, 72 h ErC50, EyC50 and NOEC of the test substance, Soaps, stocks, vegetable oil, acidulated, is considered to be >100 mg/L (nominal). Based on the results of the read across study, 72 h ErC50, EyC50 and LOEC of the test substance, Soaps, stocks, vegetable oil, acidulated, is considered to be >100 mg/L WAF (nominal).

Description of key information

Based on the studies with major constituents as well as by read-across to the similar deodorizer distillates, the 72 h ErC50 and EyC50 >100 mg/L (nominal) has been considered further for the hazard assessment of ‘soaps, stocks, vegetable oil, acidulated’.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

Additional information

In absence of studies with ‘soaps, stocks, vegetable oil, acidulated’, its acute aquatic toxicity potential to algae has been evaluated on the basis of available studies with major constituents as well as by read-across to the similar deodorizer distillates.

A study was conducted to determine the inhibitory effect of read across substance, 'oils, vegetable, deodorizer distillates' on the growth of the freshwater green algae Pseudokirchneriella subcapitata according to OECD Guideline 201 and EU Method C.3. Six replicates of exponentially growing algal cultures were exposed to water accommodated fractions (WAFs) of the read across substance at only nominal test concentration of 100 mg/L for a period of 72 h under static conditions. The inhibition of growth rate and yield was determined at 24, 48 and 72 h time intervals and compared to data for the control and a reference substance potassium dichromate. The quantification of the read across substance was performed using the TOC-method at 0 and 72 hrs. Since the read across substance is not well water soluble, all analytical results were below the LOQ (limit of quantification). The microscopic examination of the shape of the algal cells after 72 h of test duration did not show any difference between the test and the control groups. Therefore, the NOEC was determined to be at least 100 mg/L (nominal) and the 72 h LOEC, ErC50 and EyC50 were clearly higher than the only test concentration of nominal 100 mg/L. These values could not be quantified due to the absence of toxicity of the read across substance up to the only test concentration of nominal 100 mg/L, the concentration representing the limit of solubility (Kley and Deierling, 2010e). Based on the results of the read across study, 72 h ErC50, EyC50 and LOEC of the test substance, Soaps, stocks, vegetable oil, acidulated, is considered to be >100 mg/L WAF (nominal).

 

A study was conducted to determine the inhibitory effect of the read-across substance 'soybean oil, deodorizer distillate' on the growth of the freshwater green algae Pseudokirchneriella subcapitata according to OECD Guideline 201 and EU method C.3 under static conditions. Exponentially growing cultures were exposed to water accommodated fraction of the only tested concentration 100 mg/L (nominal) under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 h, and over several algal generations. A limit test was performed in compliance with the test guidelines to demonstrate that the read-across substance has no toxic effect on Pseudokirchneriella subcapitata at this concentration. Analytical verification of the test concentrations were conducted on samples of the freshly prepared and aged (72 h) test medium via the TOC-method. Under the test conditions, the nominal 72 h NOEC was determined to be 100 mg/L. The nominal 72 h ErC50 and EyC50 were clearly higher than the only tested concentration of 100 mg/L. These values could not be quantified due to the absence of toxicity to the read-across substance at the concentration representing the limit of solubility (Kley and Deierling, 2010f). Based on the results of the read across study, 72 h ErC50, EyC50 and NOEC of the test substance, Soaps, stocks, vegetable oil, acidulated, is considered to be >100 mg/L (nominal). Based on the results of the read across study, 72 h ErC50, EyC50 and NOEC of the test substance, Soaps, stocks, vegetable oil, acidulated, is considered to be >100 mg/L WAF (nominal).

Further, several acute toxicity studies were conducted on the saltwater algae species Skeletonema costatum using water accommodated fractions of glyceride constituent representative substances i.e., ‘glycerides, C8-18 and C18-unsatd.’ in the form of palm kernel stearin, palm kernel olein and palm kernel oil (Worden and Sherren, 2004 a and b). The lowest loading dose which caused 50% reduction in growth rate of the exposed test species (72 h ErL50) was > 100 mg/L, suggesting a low acute toxicity. This is in line with results obtained with ‘glycerides, C16-18 and C18-unsatd.’ (Worden and Sherren, 2002).

 

Overall based on the above studies, ‘soaps, stocks, vegetable oil, acidulated’ is considered to have low acute toxicity potential in algae and the 72 h ErC50 and EyC50 >100 mg/L (nominal) has been considered further for the hazard assessment.