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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 - 19 Mar 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions (no purity)
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom (date of inspection 23 Mar 1998)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Water samples were taken from the control and each replicate test vessel at 0, 24 and 48 hours.
- Sample storage conditions before analysis: Duplicate samples and samples at 24, 48 and 72 hours were taken and stored frozen (approximately -20 °C) for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Prior to addition of the test material, the dechlorinated tap water was stirred by magnetic stirrer such that a vortex depth of approximately 25% of the overall height of the water column was achieved. An amount of test material (2.0 g) was added to the vortex of 20 litres of dechlorinated tap water and stirred for 23 hours to give the 100 mg/L loading rate. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. The WSF was then removed by filtration through 0.45 µm filters.
- Controls: yes, dechlorinated tap water
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Source: Donnington Fish Farm, Upper Swell, Gloucestershire, UK (in-house since 23 Feb 1999)
- Length at the end of the definitive study (mean and SD): 4.1 cm (SD = 0.1)
- Weight at the end of the definitive study (mean and SD): 0.88 g (SD = 0.14)
- Feeding during test: None

ACCLIMATION
- Acclimation period: from 3 - 15 Mar 1999
- Acclimation conditions (same as test or not): Same as test (14 °C, lighting cycle: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods)
- Type and amount of food: commercial trout pellets
- Feeding frequency: The fish were not fed for 24 h prior to the start of the test.
- Health during acclimation (any mortality observed): No mortality in the 7 days prior to the start of the test
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
approximately 100 mg/L CaCO3
Test temperature:
14 °C
pH:
7.5 - 7.8
Dissolved oxygen:
8.6 - 9.8 mg/L
Nominal and measured concentrations:
Nominal: 0 and 100 mg/L
measured: < Limit of quantification (0.02 mg/L)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size: 20 L glass vessels
- Aeration: gentle aeration via narrow bore glass tubes
- Renewal rate of test solution (frequency): every 24 hours
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener). It was then passed through a series of computer controlled plate heat exchanges to achieve the required temperature (14 °C).
- Alkalinity: approximately 100 mg/L as CaCO3
- Intervals of water quality measurement: Daily for pH, dissolved oxygen and temperature.

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortalities and symptoms of toxicity at 3, 6, 24, 48, 72 and 96 hours. The fish from the control were weighted and measured at the end of the exposure period.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Observations on body length and weight: Control at the end of the exposure period: mean standard length of 41 mm (SD = 0.1), mean weight of 0.88 g (SD = 0.14)
- Mortality of control: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start of each mixing period the test material was observed to form pale straw coloured oily globules within the vortex and also throughout the water column. After 23 hours of stirring and 1 hour of standing the test material was observed to form an oily slick at the water surface with globules throughout the water column. After filtering and during testing the 100 mg/L loading rate WSFs were observed to be clear, colourless solutions.
Sublethal observations / clinical signs:

Table 1: Cumulative mortality data in the definitive study

Nominal loading rate
(mg/L)

Cumulative mortality (initial population)

% mortality

3 h

6 h

24 h

48 h

72 h

96 h

96 h

Control

0

0

0

0

0

0

0

100 R1

0

0

0

0

0

0

0

100 R2

0

0

0

0

0

0

0

h = hours

R = replicate

Endpoint:
short-term toxicity to fish
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 203 (Fish, Acute Toxicity Test)
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Qualifier:
according to guideline
Guideline:
ISO 7346-1 (Determination of the Acute Lethal Toxicity of Substances to a Freshwater Fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] - Part 1: Static Method)
Qualifier:
according to guideline
Guideline:
other: OECD series on testing and assessment 23, 2000
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: During the limit test singular samples for possible analysis were taken from the limit concentration and the control at t=0 h, t=24 h, and t=96 h
- 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

Additionally, singular reserve samples of 2 mL were taken from all test solutions for possible analysis. If not already 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 limit test) were after the Water Accomodated Fraction (WAF) was collected by siphoning for use as highest test concentration. Note that in the final limit test the WAF was siphoned twice using a tube containing glass wool in the first siphoning step. The lower test concentrations for the range-finding test were prepared by subsequent dilutions in test medium.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The final test solutions were clear and colourless in the range-finding test and slightly hazy in the final limit test with a slight floating layer.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: carp
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands
- Age at study: F1 from a single parent-pair bred in UV-treated water
- Length at study initiation (mean): Range-finding test: 3.3 ± 0.2 cm, Limit test: 3.3 ± 0.1 cm
- Weight at study initiation (mean): Range-finding test: 1.09 ± 0.29 g, Limit test: 0.97 ± 0.06 g
- Feeding during test: No feeding from 24 hours prior to the test and during the total test period

ACCLIMATION
- Acclimation period: At least 12 days after delivery
- Type and amount of food: pelleted fish food (Cyprico Crumble Excellent (300-500 µm), Coppens International bv, Helmond, The Netherlands)
- Feeding frequency: daily
- Health during acclimation (any mortality observed): In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
180 mg/L expressed as CaCO3
Test temperature:
21.1 °C - 22.5 °C
pH:
7.3 - 8.0
Dissolved oxygen:
6.4 - 9.1 mg/L
Nominal and measured concentrations:
range finding test: 0.1, 1.0, 10 and 100% of the WAF prepared at a loading rate of 100 mg/L (nominal)
final limit test: control, 100% of the WAF prepared at a loading rate of 100 mg/L (nominal)
final limit test: control, 4.8 mg/L (measured)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, headspace, fill volume: glass vessels, 40-L, 15 L fill volume
- Aeration: no
- No. of organisms per vessel: 3 (range finding test), 7 (final limit test)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.45 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O: 211.5 mg/l
MgSO4.7H2O: 88.8 mg/l
NaHCO: 346.7 mg/l
KCl: 4.2 mg/l
- Intervals of water quality measurement: pH, nitrate, nitrite and ammonia concentration: once a week. Temperature: continuous. In addition, pH and temperature were measured before transferring the fish to the test system

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: 16 hours light, 8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Observation for any dead or severely distressed fish at 2.25, 24, 48, 72 and 96 hours following the start of exposure. In addition, every afternoon from day 0 and every morning from day 1.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10 (range-finding test)
- Justification for using less concentrations than requested by guideline: No mortality or clinical effects were observed at any of the test concentrations during the test period
- 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: No mortality or clinical effects were observed at any of the test concentrations during the test period. Based on these results samples taken from the WAF prepared at 100 mg/L were analysed. The initial concentration was 2.2 mg/L. The concentration after 24 hours was 1.1 mg/L and after 96 hours a concentration of 1.7 mg/L was measured. Hence, the concentration remained fairly stable during the test period despite the extremely low water solubility. The expected LC50 was above 2 mg/L, being three orders of magnitude above the maximum solubility in water as was determined (< 6 µg/L).
Reference substance (positive control):
yes
Remarks:
Pentachlorophenol (PCP)
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 4.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test mat. WAF
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
4.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test mat. WAF
Basis for effect:
mortality (fish)
Details on results:
Analytical results range finding test: No mortality or clinical effects were observed at any of the test concentrations during the test period. Based on these results samples taken from the WAF prepared at 100 mg/L were analysed. The initial concentration was 2.2 mg/L. The concentration after 24 hours was 1.1 mg/L and after 96 hours a concentration of 1.7 mg/L was measured. Hence, the concentration remained fairly stable during the test period despite the extremely low water solubility. The expected LC50 was above 2 mg/L, being three orders of magnitude above the maximum solubility in water as was determined (< 6 µg/L).

Analytical results final limit test: Analysis of the samples taken from the WAF prepared at 100 mg/L showed that measured concentrations decreased from 9.9 mg/L at the start to 5.1 mg/L after 24 hours and further to 3.1 mg/L after 96 hours of exposure. The decrease was expected to be related to the extremely low solubility (<6 µg/L in water) and as such it was justified to conclude that testing was performed at or above the limit of solubility. The average exposure concentration was calculated to correspond to 4.8 mg/L.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- LC50 = 0.25 mg/L (95% CL: 0.20 - 0.41 mg/L
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 Jan - 11 Jan 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study with acceptable restrictions: No monitoring of the test substance is performed. Analytical purity of the test substance not stated.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solutions were prepared by direct addition of 1000 mg of test material per litre. The test solutions were then stirred thoroughly.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Source: Upwey Trout Farm, Dorset, UK
- Feeding during test: none
- Other: The last medication given to the fish was a 1 mg/l treatment of malachite green on 30 November 1990 (5 weeks prior to the test).

ACCLIMATION
- Acclimation period: 1 week
- Acclimation conditions: same as test, 15 ± 1°C, glass aquaria
- Type and amount of food: BP Mainstream (batch ref FF123)
- Feeding frequency: The fish was not fed for 24 h prior to the start of the test.
- Health during acclimation: No mortality was observed.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
37.3 - 38.0 mg/l as CaCO3
Test temperature:
14.4 - 15.2 °C
pH:
7.93 - 8.18
Dissolved oxygen:
8.8 - 9.8 mg/L
Nominal and measured concentrations:
Nominal: 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: vessels
- Material, size, fill volume: glass vessels, 17 L, 15 L
- Aeration: gentle aeration
- Renewal rate of test solution: every 24 hours
- No. of organisms per vessel: 10
- No. of vessels per concentration: 2
- No. of vessels per control: 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water supplied from a 100 m³ reservoir with an average retention time of 24 hours. It was
passed through activated carbon, filtered to remove particulate material and dechlorinated with sodium thiosulphate. The treated water was then held in a secondary reservoir with a capacity of 36 m³ and an average retention time of 8 hours. It was delivered to the laboratory via a temperature controlled header tank at a nominal temperature of 15°C.
- Chlorine: < 4 µg/L Cl2
- Alkalinity: 20.4 mg/L as CaCO3
- Conductivity: 138 ± 3 µS/cm
- Intervals of water quality measurement:Temperature, pH and dissolved oxygen were measured daily.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/ 8 h dark
- Light: Daylight and artificial lighting

EFFECT PARAMETERS MEASURED: Mortalities and symptoms of toxicity were recorded at at 24, 48, 72 and 96 hours test duration.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: No toxicity in the range of water solubility
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: No toxicity in the range of water solubility
Details on results:
- Other biological observations: In one of the tanks (1000(B)), a partial effect was noted, some of the fish becoming darkly discoloured. This was not significant.
- Mortality of control: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test material did not dissolve completely in the dilution water.
Sublethal observations / clinical signs:

No mortalities were observed in the dilution water control and at 1000 mg/L (nominal) during the 96 h exposure. Accordingly, an 96h-LC50 (96h) is determined to be > 1000 mg/L (nominal) and the 96h-NOEC is determined to be ≥ 1000 mg/L. Thus, no mortality within the water solubility of the substance was observed under conditions tested.

Endpoint:
short-term toxicity to fish
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
15 - 19 Mar 1999
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:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom (date of inspection 23 Mar 1998)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Water samples were taken from the control and each replicate test vessel at 0, 24 and 48 hours.
- Sample storage conditions before analysis: Duplicate samples and samples at 24, 48 and 72 hours were taken and stored frozen (approximately -20 °C) for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Prior to addition of the test material, the dechlorinated tap water was stirred by magnetic stirrer such that a vortex depth of approximately 25% of the overall height of the water column was achieved. An amount of test material (2.0 g) was added to the vortex of 20 litres of dechlorinated tap water and stirred for 23 hours to give the 100 mg/L loading rate. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. The WSF was then removed by filtration through 0.45 µm filters.
- Controls: yes, dechlorinated tap water
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Source: Donnington Fish Farm, Upper Swell, Gloucestershire, UK (in-house since 23 Feb 1999)
- Length at the end of the definitive study (mean and SD): 4.1 cm (SD = 0.1)
- Weight at the end of the definitive study (mean and SD): 0.88 g (SD = 0.14)
- Feeding during test: None

ACCLIMATION
- Acclimation period: from 3 - 15 Mar 1999
- Acclimation conditions (same as test or not): Same as test (14 °C, lighting cycle: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods)
- Type and amount of food: commercial trout pellets
- Feeding frequency: The fish were not fed for 24 h prior to the start of the test.
- Health during acclimation (any mortality observed): No mortality in the 7 days prior to the start of the test
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
approximately 100 mg/L CaCO3
Test temperature:
14 °C
pH:
7.5 - 7.8
Dissolved oxygen:
8.6 - 9.8 mg/L
Nominal and measured concentrations:
Nominal: 0 and 100 mg/L
measured: < Limit of quantification (0.02 mg/L)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size: 20 L glass vessels
- Aeration: gentle aeration via narrow bore glass tubes
- Renewal rate of test solution (frequency): every 24 hours
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener). It was then passed through a series of computer controlled plate heat exchanges to achieve the required temperature (14 °C).
- Alkalinity: approximately 100 mg/L as CaCO3
- Intervals of water quality measurement: Daily for pH, dissolved oxygen and temperature.

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortalities and symptoms of toxicity at 3, 6, 24, 48, 72 and 96 hours. The fish from the control were weighted and measured at the end of the exposure period.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Observations on body length and weight: Control at the end of the exposure period: mean standard length of 41 mm (SD = 0.1), mean weight of 0.88 g (SD = 0.14)
- Mortality of control: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start of each mixing period the test material was observed to form pale straw coloured oily globules within the vortex and also throughout the water column. After 23 hours of stirring and 1 hour of standing the test material was observed to form an oily slick at the water surface with globules throughout the water column. After filtering and during testing the 100 mg/L loading rate WSFs were observed to be clear, colourless solutions.
Sublethal observations / clinical signs:

Table 1: Cumulative mortality data in the definitive study

Nominal loading rate
(mg/L)

Cumulative mortality (initial population)

% mortality

3 h

6 h

24 h

48 h

72 h

96 h

96 h

Control

0

0

0

0

0

0

0

100 R1

0

0

0

0

0

0

0

100 R2

0

0

0

0

0

0

0

h = hours

R = replicate

Endpoint:
short-term toxicity to fish
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 203 (Fish, Acute Toxicity Test)
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Qualifier:
according to guideline
Guideline:
ISO 7346-1 (Determination of the Acute Lethal Toxicity of Substances to a Freshwater Fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] - Part 1: Static Method)
Qualifier:
according to guideline
Guideline:
other: OECD series on testing and assessment 23, 2000
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: During the limit test singular samples for possible analysis were taken from the limit concentration and the control at t=0 h, t=24 h, and t=96 h
- 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

Additionally, singular reserve samples of 2 mL were taken from all test solutions for possible analysis. If not already 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 limit test) were after the Water Accomodated Fraction (WAF) was collected by siphoning for use as highest test concentration. Note that in the final limit test the WAF was siphoned twice using a tube containing glass wool in the first siphoning step. The lower test concentrations for the range-finding test were prepared by subsequent dilutions in test medium.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The final test solutions were clear and colourless in the range-finding test and slightly hazy in the final limit test with a slight floating layer.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: carp
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands
- Age at study: F1 from a single parent-pair bred in UV-treated water
- Length at study initiation (mean): Range-finding test: 3.3 ± 0.2 cm, Limit test: 3.3 ± 0.1 cm
- Weight at study initiation (mean): Range-finding test: 1.09 ± 0.29 g, Limit test: 0.97 ± 0.06 g
- Feeding during test: No feeding from 24 hours prior to the test and during the total test period

ACCLIMATION
- Acclimation period: At least 12 days after delivery
- Type and amount of food: pelleted fish food (Cyprico Crumble Excellent (300-500 µm), Coppens International bv, Helmond, The Netherlands)
- Feeding frequency: daily
- Health during acclimation (any mortality observed): In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
180 mg/L expressed as CaCO3
Test temperature:
21.1 °C - 22.5 °C
pH:
7.3 - 8.0
Dissolved oxygen:
6.4 - 9.1 mg/L
Nominal and measured concentrations:
range finding test: 0.1, 1.0, 10 and 100% of the WAF prepared at a loading rate of 100 mg/L (nominal)
final limit test: control, 100% of the WAF prepared at a loading rate of 100 mg/L (nominal)
final limit test: control, 4.8 mg/L (measured)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, headspace, fill volume: glass vessels, 40-L, 15 L fill volume
- Aeration: no
- No. of organisms per vessel: 3 (range finding test), 7 (final limit test)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.45 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O: 211.5 mg/l
MgSO4.7H2O: 88.8 mg/l
NaHCO: 346.7 mg/l
KCl: 4.2 mg/l
- Intervals of water quality measurement: pH, nitrate, nitrite and ammonia concentration: once a week. Temperature: continuous. In addition, pH and temperature were measured before transferring the fish to the test system

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: 16 hours light, 8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Observation for any dead or severely distressed fish at 2.25, 24, 48, 72 and 96 hours following the start of exposure. In addition, every afternoon from day 0 and every morning from day 1.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10 (range-finding test)
- Justification for using less concentrations than requested by guideline: No mortality or clinical effects were observed at any of the test concentrations during the test period
- 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: No mortality or clinical effects were observed at any of the test concentrations during the test period. Based on these results samples taken from the WAF prepared at 100 mg/L were analysed. The initial concentration was 2.2 mg/L. The concentration after 24 hours was 1.1 mg/L and after 96 hours a concentration of 1.7 mg/L was measured. Hence, the concentration remained fairly stable during the test period despite the extremely low water solubility. The expected LC50 was above 2 mg/L, being three orders of magnitude above the maximum solubility in water as was determined (< 6 µg/L).
Reference substance (positive control):
yes
Remarks:
Pentachlorophenol (PCP)
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 4.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test mat. WAF
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
4.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test mat. WAF
Basis for effect:
mortality (fish)
Details on results:
Analytical results range finding test: No mortality or clinical effects were observed at any of the test concentrations during the test period. Based on these results samples taken from the WAF prepared at 100 mg/L were analysed. The initial concentration was 2.2 mg/L. The concentration after 24 hours was 1.1 mg/L and after 96 hours a concentration of 1.7 mg/L was measured. Hence, the concentration remained fairly stable during the test period despite the extremely low water solubility. The expected LC50 was above 2 mg/L, being three orders of magnitude above the maximum solubility in water as was determined (< 6 µg/L).

Analytical results final limit test: Analysis of the samples taken from the WAF prepared at 100 mg/L showed that measured concentrations decreased from 9.9 mg/L at the start to 5.1 mg/L after 24 hours and further to 3.1 mg/L after 96 hours of exposure. The decrease was expected to be related to the extremely low solubility (<6 µg/L in water) and as such it was justified to conclude that testing was performed at or above the limit of solubility. The average exposure concentration was calculated to correspond to 4.8 mg/L.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- LC50 = 0.25 mg/L (95% CL: 0.20 - 0.41 mg/L
Endpoint:
short-term toxicity to fish
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
07 Jan - 11 Jan 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study with acceptable restrictions: No monitoring of the test substance is performed. Analytical purity of the test substance not stated.
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:
equivalent or similar to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solutions were prepared by direct addition of 1000 mg of test material per litre. The test solutions were then stirred thoroughly.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Source: Upwey Trout Farm, Dorset, UK
- Feeding during test: none
- Other: The last medication given to the fish was a 1 mg/l treatment of malachite green on 30 November 1990 (5 weeks prior to the test).

ACCLIMATION
- Acclimation period: 1 week
- Acclimation conditions: same as test, 15 ± 1°C, glass aquaria
- Type and amount of food: BP Mainstream (batch ref FF123)
- Feeding frequency: The fish was not fed for 24 h prior to the start of the test.
- Health during acclimation: No mortality was observed.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
37.3 - 38.0 mg/l as CaCO3
Test temperature:
14.4 - 15.2 °C
pH:
7.93 - 8.18
Dissolved oxygen:
8.8 - 9.8 mg/L
Nominal and measured concentrations:
Nominal: 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: vessels
- Material, size, fill volume: glass vessels, 17 L, 15 L
- Aeration: gentle aeration
- Renewal rate of test solution: every 24 hours
- No. of organisms per vessel: 10
- No. of vessels per concentration: 2
- No. of vessels per control: 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water supplied from a 100 m³ reservoir with an average retention time of 24 hours. It was
passed through activated carbon, filtered to remove particulate material and dechlorinated with sodium thiosulphate. The treated water was then held in a secondary reservoir with a capacity of 36 m³ and an average retention time of 8 hours. It was delivered to the laboratory via a temperature controlled header tank at a nominal temperature of 15°C.
- Chlorine: < 4 µg/L Cl2
- Alkalinity: 20.4 mg/L as CaCO3
- Conductivity: 138 ± 3 µS/cm
- Intervals of water quality measurement:Temperature, pH and dissolved oxygen were measured daily.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/ 8 h dark
- Light: Daylight and artificial lighting

EFFECT PARAMETERS MEASURED: Mortalities and symptoms of toxicity were recorded at at 24, 48, 72 and 96 hours test duration.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: No toxicity in the range of water solubility
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: No toxicity in the range of water solubility
Details on results:
- Other biological observations: In one of the tanks (1000(B)), a partial effect was noted, some of the fish becoming darkly discoloured. This was not significant.
- Mortality of control: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test material did not dissolve completely in the dilution water.
Sublethal observations / clinical signs:

No mortalities were observed in the dilution water control and at 1000 mg/L (nominal) during the 96 h exposure. Accordingly, an 96h-LC50 (96h) is determined to be > 1000 mg/L (nominal) and the 96h-NOEC is determined to be ≥ 1000 mg/L. Thus, no mortality within the water solubility of the substance was observed under conditions tested.

Description of key information

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

Key value for chemical safety assessment

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 fish are available, in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5 a read-across to the structurally related source substances Pentaerythritol tetraesters of n-decanoic, n-heptanoic, n-octanoic and n-valeric acids (CAS 68424-31-7), 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 source substances are representative to evaluate the short-term toxicity of the target substance to fish. 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. Tetra-esterification products of C5, C7, C8, C10 fatty acids with pentaerythritol (CAS 68424-31-7) is a UVCB substance consisting of C5-C10 fatty acids tetra-esterified with pentaerythritol. 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 investigated the short-term effects of the source substance Pentaerythritol tetraesters of n-decanoic, n-heptanoic, n-octanoic and n-valeric acids (CAS 68424-31-7) to freshwater fish (Comber and Caunter, 1991). The study was performed according to OECD 203 (GLP) using a semi-static test design. Salmo gairdneri was exposed to a limit concentration of 1000 mg/L (nominal). After an exposure of 96 h no mortality was observed resulting in a LC50 (96 h) > 1000 mg/L (nominal). As the test material did not dissolved completely in the dilution water, the tested concentration demonstrated a worst case. The soluble fraction as well as the insoluble particles did not cause any toxic effects.

The second study with 3,5,5-trimethylhexanoic acid mixed tetraesters with PE and valeric acid (CAS 131459-39-7) was performed according to OECD 203 (GLP) using Oncorhynchus mykiss as test organism (Mullee & Wetton, 1999). Due to the low water solubility of the substance in water a limit test with a saturated solution was tested. This solution was prepared by 23 h of stirring followed by filtration. After an exposure of 96 h no mortality was recorded resulting in a LL50 (96 h) > 100 mg/L (nominal). The measured concentrations were below the quantification limit reflecting the very low water solubility of the source substance. 

The third study investigated the short-term toxicity of the source substance fatty acids, C5-9 tetraesters with pentaerythritol (CAS 67762-53-2) to freshwater fish according to OECD 203 (GLP) under static conditions using Cyprinus carpio as test organism (Migchielsen, 2012). A nominal loading rate of 100 mg/L (prepared as WAF) was applied. Test substance monitoring measured a concentration of 4.8 mg/L. The final test solution was slightly hazy with a floating layer; however, this is considered negligible, because no mortality was observed in the treatment throughout the test period of 96 h. Hence, the LL50 (96 h) is determined to be > 100 mg/L (nominal) and > 4.8 mg/L (measured) and thus above the limit of water solubility (< 0.006 mg/L, purified water).

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 Tetraesters of pentaerythritol with heptanoic acid and 3,5,5-trimethylhexanoic acid (List number 805-289-4) will not exhibit short-term effects to fish up to the limit of water solubility.