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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 - 19 Jan 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions
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)
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Accommodated Fraction (WAF): This WAF was prepared by moderately stirring 100 mg test substance per liter of medium overnight
followed by filtration (MILLIPORE AP15 glass fiber filter).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain: Straus
- Length at study initiation (length definition, mean, range and SD): < 2mm
- Method of breeding: Parental and young Daphnia were held in 40L glass aquaria.

BREEDING
- Breeding conditions (same as test or not): Reconstituted water (ISO 6341) was the same as test, prepared with distilled water. The medium in the
breeding system was continuously aerated in comparison to the test.
- Type and amount of food: 15 g sera micropan (sera GmbH, Heinsberg) homogenized in 1L deionized water
- Feeding frequency: Once a day, except weekends

ACCLIMATION
- Acclimation period: Daphnia, which had been selected in size, were acclimatized to the reconstituted water for 4 hours before introduction into
the test media.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Test temperature:
20.0 ± 0.5 °C
pH:
control: 7.3 at 0h, 7.7 at 48h
100 mg/l: 7.5 at 0h, 7.7 at 48h
Dissolved oxygen:
control: 6.2 mg/l at 0h, 6.0 mg/l at 48h
100 mg/l: 6.0 mg/l at 0h, 5.9 mg/l at 48h
Nominal and measured concentrations:
nominal: 100 mg/L (WAF)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 100 mL flasks, all-glass, with 50 mL of test medium
- Aeration: no
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO 6341), prepared with distilled water. The ratio of Ca:Mg ions is 4:1 and of Na:K ions is 10:1.
- Alkalinity: 0.8 mmol/L
- Ca/mg ratio: 4:1
- Culture medium different from test medium: No, but test medium was not aerated as the culture medium.
- Intervals of water quality measurement: Temperature determined in a control flask at the start and at the end of the test. Oxygen and pH were
determined in all vessels prior to the addition of Daphnia to the test medium and at the end of the test.

OTHER TEST CONDITIONS
- Photoperiod: 16 h a day, supplied by overhead white fluorescent tubes.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations of immobile Daphnia were made after 24 and 48 h of
exposure
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EL0
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility

The test substance show no acute toxicity for daphnids under test conditions.

Table 1: Immobilisation of Daphnia magna after 24 and 48 hours

Nominal concentration (mg/l)

Code

Number of individuals per test vessel

Immobile Daphnia (No./vessel)

24h

Immobile Daphnia (No./vessel)

48h

% immobile Daphnia after 24h of exposure

% immobile Daphnia after 48h of exposure

Control
0

A

B

C

20
20
20

0
1
0

0
1
0

0
5
0

0
5
0

100

A

B

20
20

0
1

1
1

0
5

5
5

 

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:
other: GLP 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

 31

0 (4)

0 (2)

0

2 (1)

2

50

   100

A

B

C

D

5

5

5

 61

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

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:
17 - 19 Jan 2007
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)
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Accommodated Fraction (WAF): This WAF was prepared by moderately stirring 100 mg test substance per liter of medium overnight
followed by filtration (MILLIPORE AP15 glass fiber filter).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain: Straus
- Length at study initiation (length definition, mean, range and SD): < 2mm
- Method of breeding: Parental and young Daphnia were held in 40L glass aquaria.

BREEDING
- Breeding conditions (same as test or not): Reconstituted water (ISO 6341) was the same as test, prepared with distilled water. The medium in the
breeding system was continuously aerated in comparison to the test.
- Type and amount of food: 15 g sera micropan (sera GmbH, Heinsberg) homogenized in 1L deionized water
- Feeding frequency: Once a day, except weekends

ACCLIMATION
- Acclimation period: Daphnia, which had been selected in size, were acclimatized to the reconstituted water for 4 hours before introduction into
the test media.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Test temperature:
20.0 ± 0.5 °C
pH:
control: 7.3 at 0h, 7.7 at 48h
100 mg/l: 7.5 at 0h, 7.7 at 48h
Dissolved oxygen:
control: 6.2 mg/l at 0h, 6.0 mg/l at 48h
100 mg/l: 6.0 mg/l at 0h, 5.9 mg/l at 48h
Nominal and measured concentrations:
nominal: 100 mg/L (WAF)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 100 mL flasks, all-glass, with 50 mL of test medium
- Aeration: no
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO 6341), prepared with distilled water. The ratio of Ca:Mg ions is 4:1 and of Na:K ions is 10:1.
- Alkalinity: 0.8 mmol/L
- Ca/mg ratio: 4:1
- Culture medium different from test medium: No, but test medium was not aerated as the culture medium.
- Intervals of water quality measurement: Temperature determined in a control flask at the start and at the end of the test. Oxygen and pH were
determined in all vessels prior to the addition of Daphnia to the test medium and at the end of the test.

OTHER TEST CONDITIONS
- Photoperiod: 16 h a day, supplied by overhead white fluorescent tubes.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations of immobile Daphnia were made after 24 and 48 h of
exposure
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EL0
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility

The test substance show no acute toxicity for daphnids under test conditions.

Table 1: Immobilisation of Daphnia magna after 24 and 48 hours

Nominal concentration (mg/l)

Code

Number of individuals per test vessel

Immobile Daphnia (No./vessel)

24h

Immobile Daphnia (No./vessel)

48h

% immobile Daphnia after 24h of exposure

% immobile Daphnia after 48h of exposure

Control
0

A

B

C

20
20
20

0
1
0

0
1
0

0
5
0

0
5
0

100

A

B

20
20

0
1

1
1

0
5

5
5

 

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
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

 31

0 (4)

0 (2)

0

2 (1)

2

50

   100

A

B

C

D

5

5

5

 61

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

Key value for chemical safety assessment

Additional information

Since no studies investigating the short-term toxicity of Pentaerythritol, mixed esters with linear and branched fatty acids to aquatic invertebrates are available, in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5 a read-across to the structurally related source substances and Fatty acids, C5-9 tetraesters with pentaerythritol (CAS 67762-53-2) and Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (CAS 11138-60-6) was conducted. The target substance is characterized as a tetraester of pentaerythritol and linear and branched fatty acids. The source substances are structurally very similar to the target substance. Fatty acids, C5-9 tetraesters with pentaerythritol (CAS 67762-53-2) is characterized as a tetraester of pentaerythritol with fatty acids of C5-C9. The source substance Fatty acids, C8-10 (even numbered), di-and triesters with propylidynetrimethanol (CAS 11138-60-6) is a triester of trimethylolpropane and C8-C10 fatty acids (even numbered).

This read-across is justified in detail in the overall summary (IUCLID Section 6.1) and within the analogue justification in IUCLID Section 13.

The first study investigated the short-term toxicity of the source substance 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (CAS 11138-60-6) in a limit test according to OECD Guideline 202 (Häner, 2007). Daphnia magna was exposed to a WAF loading rate of 100 mg/L. No effects on mobility of D. magna were observed resulting in an EL50 (48 h) > 100 mg/L.

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 source 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 Pentaerythritol, mixed esters with linear and branched fatty acids will not exhibit short-term effects to aquatic invertebrates up to the limit of water solubility.