Fatty acids, C7-9, tetraesters with pentaerythritol

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 - 16 Jan 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions (no purity)

Qualifier:

according to guideline

Guideline:

other: OECD 202 "Daphnia sp, Acute Immobilisation Test and Reproduction Test" (1984)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom (date of inspection 28 Feb 2000)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Water samples were taken from the control (replicates R1-R2 pooled) and the 1000 mg/L loading rate WAF test group (replicates R1-R2 and R3-R4 pooled) at 0 and 48 h.
- Sample storage conditions before analysis: Duplicate samples of the 1000 mg/L loading rate WAF test groups at 0 h were taken and stored frozen (approx -20°C) for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An amount of test material (10 g) was added to the surface of 10 L of reconstituted water to give a 1000 mg/L loading rate. After the addition of the test material, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. This was stirred for 23 h. After stirring the mixture was allowed to stand for 1 h. The aqueous phase was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1000 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test material to be present.
- Controls: Reconstituted water
- Evidence of undissolved material: no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: 1st instar of an in-house laboratory culture
- Age at study initiation: < 24 h
- Method of breeding: Adult Daphnia were maintained in polypropylene vessels containing approx. 2 L of reconstituted water in a temperature controlled room at 21 °C. The lighting cycle was 16 h light and 8 h dark. Each culture was fed daily with a suspension of algae (Chlorella sp.). Reproduction was by parthenogenesis.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

21.8 °C (0 h), 20.6 °C (24 h), 20.8 °C (48 h)

pH:

7.6-7.9

Dissolved oxygen:

90-98%

Nominal and measured concentrations:

Nominal: control - 1000 mg/L
Measured: < Limit of quantification (0.032 mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass jars containing approx. 200 mL of test preparation
- Type: covered to reduce evaporation
- No. of organisms per vessel: 10
- No. of vessels per concentration: 4
- No. of vessels per control: 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water

OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h dark with 20 min dawn and dusk transition periods

EFFECT PARAMETERS MEASURED: Immobilisation was recorded after 24 and 48 h test duration.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 10, 100, 1000 mg/L (WAFs)
- Results used to determine the conditions for the definitive study: No immobilisation was observed.

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(WAF)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(WAF)

Basis for effect:

mobility

Details on results:

- Mortality of control: no mortality was observed in the controls
- Effect concentrations exceeding solubility of substance in test medium: The nominal concentration of 1000 mg/L exceeded the water solubility of the test item.

No effects were observed in the range of water solubility at a nominal concentration of 1000 mg/L (WAF). Analysis of the test preparations at 0 and 48 h showed the measured test concentrations to be less than the limit of quantification of the analytical method (< 0.032 mg/L) (Table 1).

Table 1: Chemical analysis of the test loading rates

Samples	Nominal loading rate [mg/L]	Concentration found [mg/L]
0 h	control	< LOQ
	1000 R1-R2	< LOQ
	1000 R3-R4	< LOQ
48 h	control	< LOQ
	1000 R1-R2	< LOQ
	1000 R3-R4	< LOQ

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Qualifier:

according to guideline

Guideline:

other: OECD series on testing and assessment 23, 2000

Principles of method if other than guideline:

Test conducted by preparing WAFs due to low water solubility of the test substance.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: During the final test singular samples for possible analysis were taken from all test concentrations and the control at test start and test end
- Sampling method: 2 mL from the approximate centre of the test vessels
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis

At the end of the exposure period, the replicates were pooled at each concentration before sampling. Additionally, reserve samples of 2 mL were taken for possible analysis. If not used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with a loading rate of 100 mg/L applying 2 days of magnetic stirring to ensure maximum dissolution in test medium. The resulting dispersion was left to settle for 1 hour (range-finding test) or 2 hours (final test) were after the Water Accomodated Fraction (WAF) was collected by siphoning for use as highest test concentration. The lower test concentrations were prepared by subsequent dilutions in test medium.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The final test solutions were all clear and colourless.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: In-house laboratory culture with a known history. Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood
- Age at study initiation: <24 hours, from parental daphnids of more than two weeks old
- Method of breeding: Start of each batch with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel. Maximum age of the cultures is 4 weeks. Renewal of the cultures: after 7 days of cultivation half of the medium twice a week. Temperature of medium (M7, Elendt): 18 - 22 °C. Daphnids werde fed daily with a suspension of fresh water algae.
- Feeding during test: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

180 mg/L expressed as CaCO3

Test temperature:

19.8 °C - 20.4 °C

pH:

7.9 - 8.0 (final test)

Dissolved oxygen:

8.7 - 8.9 mg/L (final test)

Nominal and measured concentrations:

combined limit/range finding test: control, 0.1, 1.0, 10 and 100% of the WAF prepared at a loading rate of 100 mg/L (nominal)
final test: control, 4.6, 10, 22, 46 and 100% of the WAF prepared at a loading rate of 100 mg/L (nominal)
final test: control, 0.045, 0.18, 0.47, 0.63, and 1.3 mg/L (measured)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, fill volume: glass vessels with a barrier net just below the surface to prevent Daphnids to swim to the surface in order to avoid contact to any undissolved test material, 90-mL, 70 mL fill volume
- Aeration: no
- No. of organisms per vessel: 5 (combined limit/range finding test and final test)
- No. of vessels per concentration (replicates): 4 (final test), 4 replicates at 100% WAF, 2 at 0.1, 1.0, and 10% (combined limit/range finding test)
- No. of vessels per control (replicates): 4 (combined limit/range finding test and final test)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no; M7 medium after Elendt (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33)


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light, 8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): immobility at 24 hours and 48 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10 in combined limit/range finding test, approx. 2 in final test
- Range finding study
- Test concentrations: 0.1, 1.0, 10 and 100% of the WAF
- Results used to determine the conditions for the definitive study: The two highest test groups contained a floating layer from 24 hours onwards causing entrapment of a significant number of organisms. Additional microscopic evaluation performed after 24 hours showed that bodies of the daphnids in the highest test group were all covered with test article. Hence, at least part if not all of the effects were of physical nature and caused by exposure to undissolved test material. It was concluded that the test set-up for the final study should include a barrier to prevent organisms getting stuck in the floating layer.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test mat. WAF

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.63 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

46 other: % of a WSF prepared at a loading rate of 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test mat. WAF

Basis for effect:

mobility

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: yes

Limit/range finding test: Immobility at 10 and 100% WAF were at respectively 50 and 95% at the end of the test. It should however be recognized that the two highest test groups contained a floating layer from 24 hours onwards causing entrapment of a significant number of organisms. Additional microscopic evaluation performed after 24 hours showed that bodies of the daphnids in the highest test group were all covered with test article. Hence, at least part if not all of the effects were of physical nature and caused by exposure to undissolved test material.

4.8 Samples taken from 1.0 and 100% of the WAF prepared at 100 mg/L were analysed. The initial concentration in the undiluted WAF was 2.2 mg/L. This concentration decreased to 0.22 mg/L after 48 hours. In samples taken from 1.0% WAF, the measured concentration decreased from 0.17 mg/L at the start to a concentration below the limit of detection during the test period. Concentrations in both solutions decreased significantly during the test period, which was expected to be related to the extremely low water solubility (< 6 µg/L).

Analytical results final test: Analysis of the samples taken at the start of the final test showed measured concentrations of 0.27, 0.85, 1.9, 3.6 and 7.0 mg/L in solutions representing 4.6, 10, 22, 46 and 100% of the WSF, respectively. The concentrations decreased during the test period to approximately 3 - 6% of initial. However, measured concentrations in all solutions at the end of the study still exceeded the water solubility that was determined to be less than 6 µg/L. Based on these results, the average exposure concentrations were calculated to correspond with 0.045, 0.18, 0.47, 0.63 and 1.3 mg/L.

Contrary to the combined limit/range-finding test, no daphnids became trapped at the surface despite the fact that initial exposure concentrations in the undiluted WSF were even higher than those measured in the combined limit/range-finding test. This was the result of the use of a physical barrier in combination with placing the light source underneath the vessels. As a consequence, only very few daphnia were immobilised in comparison to the same solutions tested in the combined limit/range-finding test. This confirmed that effects in the range-finding were of physical instead of toxic nature.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50 = 0.3 mg/L (95% CL: 0.28 - 0.35 mg/L

Table: Incidence of immobility in the combined limit/range-finding test

Test group Fatty acids, C5-9, tetraesters with pentaerythritol (% WSF*)	Vessel number	Number Daphnia exposed	Response at 24 h		Response at 48 h
			number	Total %	number	Total %
Control	A B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.1	A B	5 5	0 0	0	0 0	0
1.0	A B	5 5	0 0	0	0 0	0
10	A B	5  3^1	0 (4) 0 (2)	0	2 (1) 2	50
100	A B C D	5 5 5  6^1	3 (5) 4 (5) 5 (5) 5 (5)	81	5 4 (1) 5 6	95

* = Test groups represent percentages of a WAF prepared at 100 mg/L

1 = Incorrect number of organisms exposed

( ) = number of Daphnia observed trapped at the surface of test solutions. These organisms were reimmersed into the respective solutions before recording of mobility

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

key study

Study period:

13 - 16 Jan 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions (no purity)

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to IUCLID Section 13 for the detailed justification of the Analogue Approach.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: OECD 202 "Daphnia sp, Acute Immobilisation Test and Reproduction Test" (1984)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom (date of inspection 28 Feb 2000)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Water samples were taken from the control (replicates R1-R2 pooled) and the 1000 mg/L loading rate WAF test group (replicates R1-R2 and R3-R4 pooled) at 0 and 48 h.
- Sample storage conditions before analysis: Duplicate samples of the 1000 mg/L loading rate WAF test groups at 0 h were taken and stored frozen (approx -20°C) for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An amount of test material (10 g) was added to the surface of 10 L of reconstituted water to give a 1000 mg/L loading rate. After the addition of the test material, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. This was stirred for 23 h. After stirring the mixture was allowed to stand for 1 h. The aqueous phase was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1000 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test material to be present.
- Controls: Reconstituted water
- Evidence of undissolved material: no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: 1st instar of an in-house laboratory culture
- Age at study initiation: < 24 h
- Method of breeding: Adult Daphnia were maintained in polypropylene vessels containing approx. 2 L of reconstituted water in a temperature controlled room at 21 °C. The lighting cycle was 16 h light and 8 h dark. Each culture was fed daily with a suspension of algae (Chlorella sp.). Reproduction was by parthenogenesis.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

21.8 °C (0 h), 20.6 °C (24 h), 20.8 °C (48 h)

pH:

7.6-7.9

Dissolved oxygen:

90-98%

Nominal and measured concentrations:

Nominal: control - 1000 mg/L
Measured: < Limit of quantification (0.032 mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass jars containing approx. 200 mL of test preparation
- Type: covered to reduce evaporation
- No. of organisms per vessel: 10
- No. of vessels per concentration: 4
- No. of vessels per control: 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water

OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h dark with 20 min dawn and dusk transition periods

EFFECT PARAMETERS MEASURED: Immobilisation was recorded after 24 and 48 h test duration.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 10, 100, 1000 mg/L (WAFs)
- Results used to determine the conditions for the definitive study: No immobilisation was observed.

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(WAF)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(WAF)

Basis for effect:

mobility

Details on results:

- Mortality of control: no mortality was observed in the controls
- Effect concentrations exceeding solubility of substance in test medium: The nominal concentration of 1000 mg/L exceeded the water solubility of the test item.

No effects were observed in the range of water solubility at a nominal concentration of 1000 mg/L (WAF). Analysis of the test preparations at 0 and 48 h showed the measured test concentrations to be less than the limit of quantification of the analytical method (< 0.032 mg/L) (Table 1).

Table 1: Chemical analysis of the test loading rates

Samples	Nominal loading rate [mg/L]	Concentration found [mg/L]
0 h	control	< LOQ
	1000 R1-R2	< LOQ
	1000 R3-R4	< LOQ
48 h	control	< LOQ
	1000 R1-R2	< LOQ
	1000 R3-R4	< LOQ

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (ECHA, 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to IUCLID Section 13 for the detailed justification of the Analogue Approach.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Qualifier:

according to guideline

Guideline:

other: OECD series on testing and assessment 23, 2000

Principles of method if other than guideline:

Test conducted by preparing WAFs due to low water solubility of the test substance.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: During the final test singular samples for possible analysis were taken from all test concentrations and the control at test start and test end
- Sampling method: 2 mL from the approximate centre of the test vessels
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis

At the end of the exposure period, the replicates were pooled at each concentration before sampling. Additionally, reserve samples of 2 mL were taken for possible analysis. If not used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with a loading rate of 100 mg/L applying 2 days of magnetic stirring to ensure maximum dissolution in test medium. The resulting dispersion was left to settle for 1 hour (range-finding test) or 2 hours (final test) were after the Water Accomodated Fraction (WAF) was collected by siphoning for use as highest test concentration. The lower test concentrations were prepared by subsequent dilutions in test medium.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The final test solutions were all clear and colourless.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: In-house laboratory culture with a known history. Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood
- Age at study initiation: <24 hours, from parental daphnids of more than two weeks old
- Method of breeding: Start of each batch with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel. Maximum age of the cultures is 4 weeks. Renewal of the cultures: after 7 days of cultivation half of the medium twice a week. Temperature of medium (M7, Elendt): 18 - 22 °C. Daphnids werde fed daily with a suspension of fresh water algae.
- Feeding during test: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

180 mg/L expressed as CaCO3

Test temperature:

19.8 °C - 20.4 °C

pH:

7.9 - 8.0 (final test)

Dissolved oxygen:

8.7 - 8.9 mg/L (final test)

Nominal and measured concentrations:

combined limit/range finding test: control, 0.1, 1.0, 10 and 100% of the WAF prepared at a loading rate of 100 mg/L (nominal)
final test: control, 4.6, 10, 22, 46 and 100% of the WAF prepared at a loading rate of 100 mg/L (nominal)
final test: control, 0.045, 0.18, 0.47, 0.63, and 1.3 mg/L (measured)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, fill volume: glass vessels with a barrier net just below the surface to prevent Daphnids to swim to the surface in order to avoid contact to any undissolved test material, 90-mL, 70 mL fill volume
- Aeration: no
- No. of organisms per vessel: 5 (combined limit/range finding test and final test)
- No. of vessels per concentration (replicates): 4 (final test), 4 replicates at 100% WAF, 2 at 0.1, 1.0, and 10% (combined limit/range finding test)
- No. of vessels per control (replicates): 4 (combined limit/range finding test and final test)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no; M7 medium after Elendt (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33)


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light, 8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): immobility at 24 hours and 48 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10 in combined limit/range finding test, approx. 2 in final test
- Range finding study
- Test concentrations: 0.1, 1.0, 10 and 100% of the WAF
- Results used to determine the conditions for the definitive study: The two highest test groups contained a floating layer from 24 hours onwards causing entrapment of a significant number of organisms. Additional microscopic evaluation performed after 24 hours showed that bodies of the daphnids in the highest test group were all covered with test article. Hence, at least part if not all of the effects were of physical nature and caused by exposure to undissolved test material. It was concluded that the test set-up for the final study should include a barrier to prevent organisms getting stuck in the floating layer.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test mat. WAF

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.63 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

46 other: % of a WSF prepared at a loading rate of 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test mat. WAF

Basis for effect:

mobility

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: yes

Limit/range finding test: Immobility at 10 and 100% WAF were at respectively 50 and 95% at the end of the test. It should however be recognized that the two highest test groups contained a floating layer from 24 hours onwards causing entrapment of a significant number of organisms. Additional microscopic evaluation performed after 24 hours showed that bodies of the daphnids in the highest test group were all covered with test article. Hence, at least part if not all of the effects were of physical nature and caused by exposure to undissolved test material.

4.8 Samples taken from 1.0 and 100% of the WAF prepared at 100 mg/L were analysed. The initial concentration in the undiluted WAF was 2.2 mg/L. This concentration decreased to 0.22 mg/L after 48 hours. In samples taken from 1.0% WAF, the measured concentration decreased from 0.17 mg/L at the start to a concentration below the limit of detection during the test period. Concentrations in both solutions decreased significantly during the test period, which was expected to be related to the extremely low water solubility (< 6 µg/L).

Analytical results final test: Analysis of the samples taken at the start of the final test showed measured concentrations of 0.27, 0.85, 1.9, 3.6 and 7.0 mg/L in solutions representing 4.6, 10, 22, 46 and 100% of the WSF, respectively. The concentrations decreased during the test period to approximately 3 - 6% of initial. However, measured concentrations in all solutions at the end of the study still exceeded the water solubility that was determined to be less than 6 µg/L. Based on these results, the average exposure concentrations were calculated to correspond with 0.045, 0.18, 0.47, 0.63 and 1.3 mg/L.

Contrary to the combined limit/range-finding test, no daphnids became trapped at the surface despite the fact that initial exposure concentrations in the undiluted WSF were even higher than those measured in the combined limit/range-finding test. This was the result of the use of a physical barrier in combination with placing the light source underneath the vessels. As a consequence, only very few daphnia were immobilised in comparison to the same solutions tested in the combined limit/range-finding test. This confirmed that effects in the range-finding were of physical instead of toxic nature.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50 = 0.3 mg/L (95% CL: 0.28 - 0.35 mg/L

Table: Incidence of immobility in the combined limit/range-finding test

Test group Fatty acids, C5-9, tetraesters with pentaerythritol (% WSF*)	Vessel number	Number Daphnia exposed	Response at 24 h		Response at 48 h
			number	Total %	number	Total %
Control	A B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.1	A B	5 5	0 0	0	0 0	0
1.0	A B	5 5	0 0	0	0 0	0
10	A B	5  3^1	0 (4) 0 (2)	0	2 (1) 2	50
100	A B C D	5 5 5  6^1	3 (5) 4 (5) 5 (5) 5 (5)	81	5 4 (1) 5 6	95

* = Test groups represent percentages of a WAF prepared at 100 mg/L

1 = Incorrect number of organisms exposed

( ) = number of Daphnia observed trapped at the surface of test solutions. These organisms were reimmersed into the respective solutions before recording of mobility

Description of key information

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

Additional information

Since no studies investigating the short-term toxicity of Tetraesters of pentaerythritol with heptanoic acid and 3,5,5-trimethylhexanoic acid (List number 805-289-4) to aquatic invertebtrates are available, in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5 a read-across to the structurally related source substances 3,5,5-trimethylhexanoic acid mixed tetraesters with PE and valeric acid (CAS 131459-39-7) and Fatty acids, C5-9 tetraesters with pentaerythritol (CAS 67762-53-2) was conducted. The target substance is characterized as a tetraester of pentaerythritol and heptanoic acid and 3,5,5-trimethylhexanoic acid. The source substances are structurally very similar to the target substance. 3,5,5-trimethylhexanoic acid mixed tetraesters with PE and valeric acid (CAS 131459-39-7) is mainly a tetraester of 3,5,5-methylhexanoic acid and pentaerythritol. Fatty acids, C5-9 tetraesters with pentaerythritol (CAS 67762-53-2) is characterized as a tetraester of pentaerythritol with fatty acids of C5-C9. 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 3,5,5-trimethylhexanoic acid mixed tetraesters with PE and valeric acid (CAS 131459-39-7) was performed according to OECD 202 (1984; GLP) under static conditions with the water flea Daphnia magna (Sewell & McKenzie, 2003). A Water Accommodated Fraction of nominal 1000 mg/L was tested in this study. Measured concentrations were below the limit of quantification (GC/FID analysis). After 48 h no effects on the test organisms were observed resulting in an EL50 (48 h) > 1000 mg/L (nominal).

The second study with the read-across substance fatty acids, C5-9 tetraesters with pentaerythritol (CAS 67762-53-2) was performed according to OECD 202 (GLP) using Daphnia magna as test organism under static conditions (Migchielsen, 2012). The WAFs were prepared by adding the appropriate amount of test substance with subsequent stirring and sampling of the aqueous portions. A nominal loading rate of 100 mg/L, corresponding to a measured concentration of 1.3 mg/L was tested. No immobilisation was observed in the treatment throughout the test period of 48 h. Hence, the EL50 (48 h) is determined to be > 100 mg/L based on the nominal loading rate and > 1.3 mg/L based on the measured concentration.
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 Tetraesters of pentaerythritol with heptanoic acid and 3,5,5-trimethylhexanoic acid (List number 805-289-4) will not exhibit short-term effects to aquatic invertebrates up to the limit of water solubility.