Decanoic acid, mixed esters with heptanoic acid, octanoic acid and pentaerythritol

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 - 09 April 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study with acceptable restrictions

Qualifier:

according 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 (especially for difficult test substances)
- Method: Water accommodated fraction (WAF) were prepared by stirring the test substance in the dilution water for 24 hours to reach equilibrium
concentration. A 20 L complete glass basin was appointed with a glass tube, by what the aqueous phase could be take off witout contamination of the unsolved film of the hydrocarbon. Before the take off of the aqueous phase the preparation was left for 1 hour for complete phase separation.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Source: MPI für Entwicklungsbiologie, Tübingen, Germany
- Length at study initiation (length definition, mean, range and SD): 3.1 - 3.5 cm
- Feeding during test: no

ACCLIMATION
- Acclimation period: At least 12 d
- Health during acclimation (any mortality observed): Mortality ≤ 5%

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test temperature:

23 ± 2 °C

pH:

8.2 - 8.4

Dissolved oxygen:

> 91%

Nominal and measured concentrations:

Nominal: 10000 mg/L (WAF)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 10 L/ basin
- Aeration: yes, constant with Pasteur pipettes
- Renewal rate of test solution (frequency): every 24 h
- No. of organisms per concentration: 7

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: By aeration (1-2 d) dechlorinated tap water
- Intervals of water quality measurement: Oxygen content, pH-value and temperature were registered after 24 h, 48 h, 72 h and 96 h.

OTHER TEST CONDITIONS
- Photoperiod: 14 light, 10 h dark (Gro-Lux)

EFFECT PARAMETERS MEASURED: Schooling behaviour, activity (mobility), surfacing, floatability
(equilibrium behaviour), dead animals, breathing frequency

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: WAF loading rate

Details on results:

- Behavioural abnormalities: 2 of the 7 test fish showed abnormal behaviour during the 96 h test period. This 2 fish were seperated from the school
and showed lower activity. After 72 h the schooling behaviour of the rest of the fish was also disturbed. The activity and the equilibrium behaviour
was equal to the control fish.
- Other biological observations: The feed ingestion after test end was only reduced at the 2 more reacted fish. All other fish showed the same
behaviour as the control fish.
- Mortality of control: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: WAFs showed light turbidity, but
contained no visible emulsion drops.

Validity criteria fulfilled:

yes

Conclusions:

No mortality of the fish could be observed in the control and the test concentration (10000 mg/l WAF) after 96h under conditions tested. 2 of the 7 test fish showed abnormal behaviour during the 96 h test period. This 2 fish were seperated from the swarm and showed lower activity. But after 72 h the swarm behaviour of the rest of the fish was also disturbed. However, the activity and the equilibrium behaviour was equal to the control fish. The feed ingestion after test end was only reduced at the 2 more reacted fish. All other fish showed the same behaviour as the control fish. As WAFs showed light turbidity and the water solubility of the test substance is < 0.3 mg/l, the abnormal behaviour could cause in unsolved test substance. Overall no mortality could be observed and therefore the LL50 is > 10000 mg/l. Due to the low water solubility (< 0.3 mg/l), no mortality within the water solubility of the test substance were observed and therewith the LC50 (96h) is > water solubility of the test substance.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The product was added directly in the aquaria after weighting to obtain the desired final concentration.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Oily patches on the surface of the water

Test organisms (species):

Salmo trutta

Details on test organisms:

TEST ORGANISM
- Source: Nordmarka, fish was obtained as fry from a nearby hatchery (OFA) 3 weeks prior to testing.
- Age at study initiation (mean and range, SD): Fry (0+)
- Length at study initiation: 5.5 cm
- Weight at study initiation: 1.64 g

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

11.5 ± 1.0°C

pH:

Start: 6.4 - 6.5
24 hours range (mean): 6.6 - 6.8 (6.8)

Dissolved oxygen:

Start: 100%
24 hours range (mean): 88 - 94% (93%)

Nominal and measured concentrations:

Nominal: 100, 320 and 1000 mg/L
Weighted in (mean): 107, 319 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: Glass aquarium with 14 L water
- Aeration: Water was moderately aerated through a glass diffuser.
- Renewal rate of test solution (frequency/flow rate): Daily
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.8 g fish/l

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural lake water
- Intervals of water quality measurement: Dissolved oxygen (% saturation) and pH was measured each 24 hours.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The fishes were observed each day and behaviour and condition was
recorded, dead fishes were counted and removed.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: Due to expected low toxicity the definitive test was therefore carried out at
only three concentrations.
- Range finding study
- Test concentrations: 100 and 1000 mg/L with 1 fish at each concentration
- Results used to determine the conditions for the definitive study: No mortality was observed after 4 days.

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)

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: There was no observable toxic sign related to behaviour or pigmentation.
- Any observations that might cause a difference between measured and nominal values: After 24 h a small amount of "emulsion"
was observed at the two highest concentrations, also some increase in turbidity was noted.

Validity criteria fulfilled:

yes

Conclusions:

No mortality of the fish could be observed in the control and the test concentrations (100, 320 and 1000 mg/L nominal) during the 96 hours exposure.
After 24 h a small amount of "emulsion" was observed at the two highest concentrations, also some increase in turbidity was noted. Hence, the LC50 (96h) for Salmo trutta exposed to the test substance was > 1000 mg/L (nominal). Due to the low water solubility (< 0.3 mg/L), no effect within the water solubility of the test substance were observed.

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

The substance is a member of a group of pentaerythritol and a mixture of alkyl carboxylic acids which share similar characteristics across the group. Please see attached justification for read across.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: WAF Loading rate

Remarks:

Brachydanio rerio (CAS 11138-60-6)

Key result

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:

Remarks:

Salmo trutta (CAS 11138-60-6)

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other:

Remarks:

Salmo trutta (CAS 11138-60-6)

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 0.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other:

Remarks:

96h-LC50 for carp exceeded the maximum solubility (CAS 70693-33-3)

Conclusions:

The substance, CAS 68441-67-8, is analogous to the substances to be read across to, in terms of basic form, and the degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of aquatic toxicity to fish. The substance is considered to be not toxic to fish for the defined endpoints on the basis of read across. This will be confirmed by appropriate study data as soon as this is available.

Executive summary:

The substance, CAS 68441-67-8, is analogous to the substances to be read across to, in terms of basic form, and the degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of aquatic toxicity to fish. The substance is considered to be not toxic to fish for the defined endpoints on the basis of read across. This will be confirmed by appropriate study data as soon as this is available.

Reference 4

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

06 May 2003 to 18 August 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

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

Analytical monitoring:

yes

Details on sampling:

During the limit test duplicate samples were taken from the blank-control and the filtered solution prepared at a loading rate of 100 mg/l for analysis. The method of analysis is described in the appended Analytical Report.
Sampling:
Frequency: at t= 0 h, t= 24 hand t= 96 h
Volume: 5 ml, from the approximate centre of the test vessels.
Storage: Not applicable, samples were analysed on the day of sampling.

Vehicle:

no

Details on test solutions:

The standard test procedures required generation of test solutions that contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions.
The testing of concentrations that disturbed the test system were prevented (e.g. film of the test substance on the water surface).
HATCOL 5236 is a clear pale yellow liquid with a purity of 96.7%. The water solubility of HATCOL 5236 at 20.0 ± 0.8°C was determined to be< 2.0x10e-4 g/l using the flask method (NOTOX Project 365041 ).
A stock solution was prepared at a loading rate of 100 mg/l. This supersaturated solution was stirred for two days to reach maximum solubility. After the stirring period the mixture was colourless but contained a test substance floating layer and a deposit of test substance.
Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance. After the stirring period the mixture was therefore filtered through a paper filter (Schleicher and Schuell604) to remove the larger undissolved test substance particles (ca.> 5 pm). The filtrate was clear and colourless. Note that the blank-control received the same treatment.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

Species: Carp (Cyprinus carpio, Teleostei, Cyprinidae) (Linnaeus, 1758)
Source: Zodiac, proefacc, “De Haar Vissen", L.U, Wageningen, the Netherlands.
Mean length: 2.4±0.2cm
Mean weight: 0.32 ± 0.1 g
Characteristics: F1 from a single parent pair bred in UV treated water
Reason for selection: This system has been selected as an internationally accepted species.
Total fish used: 14

Quarantine/Acclimatisation: At least 12 days after delivery.
Medium: ISO-medium, formulated using Milli-Ro water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
CaCl2•2H2O 293.8 mg/l
MgSO4•7H2O 123.3 mg/l
NaHCO3 64.8 mg/l
KCI 5.8 mg/l
Measurements: pH, nitrate and nitrite concentration and ammonia concentration: once a week. Temperature: every day.
Feeding: Daily with Trouvit.
Validity of batch: 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

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

After aeration the hardness was 250 mg CaC03 per litre

Test temperature:

The temperature measured ranged between 21.0 and 22.3 °C during the 96-hour test period

pH:

pH: 6.0-8.5

Dissolved oxygen:

oxygen: >5 mg/l at 22°C throughout the test).

Salinity:

Not applicable

Nominal and measured concentrations:

Measured concentrations
The test substance consisted of a mixture of molecules with different molecular weights, which differed in water solubility, resulting in a number of peaks in the chromatogram of the test substance solutions. It was not possible to determine which molecule was responsible for the toxicological response, if any. Furthermore, since not all components were quantifiable in the calibration chromatograms, it was not possible to determine the concentration of the total test substance. Therefore, the toxicological evaluation was based on the water soluble fraction at the loading rate. In addition, the actual concentration was estimated from the two largest peaks (peaks 1 and 2) observed in the chromatograms of HATCOL 5236.

Details on test conditions:

TEST CONCENTRATIONS
HATCOL 5236: A 5 µm filtrate of a supersaturated solution prepared at a loading rate of 100 mg/l.
Control: Test medium without test substance or other additives (Blank-control).

TEST PROCEDURE AND CONDITIONS
Test duration: 96 hours
Test type: Static
Test vessels: 6.5 litres, all-glass.
Test medium: ISO-medium, aerated until the dissolved oxygen concentration had reached saturation and the pH had stabilised. After aeration the hardness was 250 mg CaC03 per litre and the pH was 8.0.
Number of fish: 7 fish per test concentration and control.
Loading: 0.45 g fish/litre, i.e. 7 fish per 5 litres of test medium.
Illumination: 16 hours photoperiod daily
Aeration: Aeration was introduced after 48 hours of exposure and was stopped at the end of the test period.
Feeding: No feeding from 48 hours prior to the test and during the total test period.
Introduction of fish: Within 1 hour after preparation of the test solutions.
Euthanasia: At the end of the test the surviving fish were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

MEASUREMENTS AND RECORDINGS
Mortality and other effects: At 3½, 24, 48, 72 and 96 hours following the start of exposure.
Fish length and weight: Ten fish of the batch used for the test, were weighed and measured prior to the start of the test.
Dissolved oxygen content pH and temperature: Daily in all vessels, beginning at the start of the test (day 0).

Reference substance (positive control):

yes

Remarks:

pentachlorophenol

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 0.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Measured concentrations
The test substance consisted of a mixture of molecules with different molecular weights, which differed in water solubility, resulting in a number of peaks in the chromatogram of the test substance solutions. It was not possible to determine which molecule was responsible for the toxicological response, if any. Furthermore, since not all components were quantifiable in the calibration chromatograms, it was not possible to determine the concentration of the total test substance. Therefore, the toxicological evaluation was based on the water soluble fraction at the loading rate. In addition, the actual concentration was estimated from the two largest peaks (peaks 1 and 2) observed in the chromatograms of HATCOL 5236.
The analytical results showed that the filtered solution prepared at a loading rate of 100 mg/l had an initial concentration of 0.84 mg/l (when based on peak 1) or 0.64 mg/l (when based on peak 2). After 24 hours of exposure the test concentration had decreased to 0.38 mg/l (when based on peak 1) or 0.23 mg/l (when based on peak 2). At the end of the test period the test concentration had decreased below the limit of quantification (i.e. below 0.1 mg/l).
The average exposure concentration, based on peak 1, was 0.25 mg/l, while the average exposure concentration, based on peak 2, was 0.18 mg/l. Hence, average concentrations remained above or approximated the solubility limit of HATCOL 5236 (i.e.< 0.2 mg/l). The observed decrease was probably a consequence of this extremely low solubility.

Mortality and other effects
No lethal or clinical effects were observed during the 96-hour test period. No undissolved test substance particles were observed during the test.
Experimental conditions
These test conditions remained within the limits prescribed by the protocol (pH: 6.0-8.5, not varying by more than 1 unit; oxygen: >5 mg/l at 22°C throughout the test).
The temperature measured ranged between 21.0 and 22.3 °C during the 96-hour test period, which was within the range prescribed by the protocol (20-24 °C, constant within 2 °C).

Results with reference substance (positive control):

During the test the pH, oxygen concentration and the temperature of the medium were within the optimal ranges for fish.
Under the conditions of the present test PENTACHLOROPHENOL induced no lethal effects in carp at or below 0.15 mg/l. The 96h-LC50 for carp exposed to PCP was 0.21 mg/l (95% confidence interval between 0.19 and 0.27 mg/l). The 24h-LC50 was 0.24 mg/l (95% confidence interval between 0.21 and 0.30 mg/l), and remained unchanged until 72h. The range of the 96hLC50 for carp is generally between 0.10 and 0.46 mg/l based on historical data of reference tests performed approximately every 3 months from April 1988 until the end 2000, and annually since then. The response observed in carp originating from the present batch falls within this range.

Reported statistics and error estimates:

ACCEPTABILITY OF THE TEST
1. No mortality was observed in the control group.
2. The oxygen concentration was maintained at least 60% of the air saturation value throughout the test (> 5 mg/l at 22 °C). Other test conditions (pH and temperature) were maintained within the limits prescribed by the guidelines.
3. Owing to the extremely low solubility of HATCOL 5236 in water, actual concentrations in the filtered solution could not be maintained at more than 80% of the initial concentration.
However, during the test period concentrations were above or approximated the water solubility level.
4. The 96h-LC50 of the reference chemical, for the stock of fish was in reasonable agreement with results obtained previously in our laboratory.

Sublethal observations / clinical signs:

Incidence of mortality and total mortality during the limit test

Loading rate HATCOL 5236 (mg/l)	Initial number of fish	Cumulative mortality					Total Mortality (%)
		3½h	24h	28h	72h	96h
Blank-control	7	0	0	0	0	0	0
100	7	0	0	0	0	0	0

 

pH-values and dissolved oxygen concentrations (mg/l) during the limit test

Loading rate HATCOL 5236 (mg/l)	Day 0		Day 1		Day 2*		Day 3		Day 4
	pH	O2	pH	O2	pH	O2	pH	O2	pH	O2
Blank-control	8.0	8.6	7.5	7.1	7.3	6.2	7.8	8.6	7.8	8.7
100	7.9	8.5	7.4	7.0	7.2	5.3	7.9	8.7	7.8	8.8

*Aeration was introduced after 48 hours of exposure and was stopped at the end of the test period.

 

Temperatures (°C) measured during the limit test

Loading rate HATCOL 5236 (mg/l)	Day 0	Day 1	Day 2	Day 3	Day 4
Blank-control	22.2	21.2	21.3	21.1	21.0
100	22.3	21.2	21.3	21.1	21.0

 

Incidence of mortality observed in the reference study

Concentration PCP (mg/l) Nominal	Initial Number of Fish	Cumulative number of dead fish recorded at various time points after start of exposure					Total Mortality (%)
		2.5h	24h	48h	72h	96h
Control	5	0	0	0	0	0	0
0.05	5	0	0	0	0	0	0
0.10	5	0	0	0	0	0	0
0.15	5	0	0	0	0	0	0
0.22	5	0	1	1	1	3	60
0.32	5	0	5	5	5	5	100

 

Validity criteria fulfilled:

yes

Conclusions:

Owing to the extremely low solubility of HATCOL 5236 in water, concentration levels toxic for fish could not be reached. Therefore, the 96h-LC50 for carp exceeded the maximum solubility of HATCOL 5236 in water.

Executive summary:

96-Hour Acute Toxicity Study in Carp with HATCOL 5236.

The study procedures described in this report were based on the ISO International Standard 7346-1:"Water quality-Determination of the acute lethal toxicity of substances to a freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei,Cyprinidae)]"-Part 1: Static method, Second edition, 1996-06-15. In addition, the guidelines met the EEC directive 92/69, Part C.1. "Acute toxicity for fish"; and the OECD guideline No. 203: "Fish Acute Toxicity Test, Adopted 17 July, 1992.

HATCOL 5236 is a clear pale yellow liquid with a purity of 97.6%. The water solubility of HATCOL 5236 at 20.0 ±0.8°C was determined to be <2.0x10 ^-4g/l using the flask method (NOTOX Project 365041).

A stock solution was prepared at a loading rate of 100 mg/l. This supersaturated solution was stirred for two days to reach maximum solubility. After the stirring period the mixture was colourless but contained a test substance floating layer and a deposit of test substance.

Collection of the water phasebysiphoning or centrifugation was not an option considering the specific gravity of the test substance. After the stirring period the mixture was therefore filtered through a paper filter (ca.> 5 µm). The filtrate was clear and colourless.

A limit test was performed exposing seven carp per concentration to a 5 µm filtered solution prepared at a loading rate of 100 mg/l and a blank-control for a maximum of 96 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 hours of exposure and at the end of the test.

Analysis of the samples showed that the average exposure concentration was at or above the solubility limit of HATCOL 5236 (i.e.< 0.2 mg/l).

The study met the acceptability criteria prescribed by the protocol and was considered valid.

HATCOL 5236 induced no visible/lethal effects in carp in a filtered solution prepared at a loading rate of 100 mg/l, corresponding to an average exposure concentration at or above the water solubility.

In conclusion: Owing to the extremely low solubility of HATCOL 5236 in water, concentration levels toxic for fish could not be reached. Therefore, the 96h-LC50 for carp exceeded the maximum solubility of HATCOL 5236 in water.

Description of key information

Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (CAS-No. 11138-60-6) is not harmful to fish.

96h-LC50 for carp exceeded the maximum solubility of HATCOL 5236 in water.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (CAS-No. 11138-60-6)

Two semi-static studies, investigating the short-term toxicity to freshwater fish of Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (CAS-No. 11138-60-6), were available. The key study (Kuttler, 1998) with Brachydanio rerio (Danio rerio) according to OECD 203 determined LL50 (96 h) > 10000 mg/L (WAF loading rate). In the supporting study (Källqvist and Tobiesen, 1995) with Salmo trutta according to OECD 203 the EC50 (96 h) is > 1000 mg/L and the LOEC > 1000 mg/L. As at the two highest concentrations (320 and 1000 mg/L) a small amount of emulsion and some increase in turbidity was observed, the highest concentration (1000 mg/L) demonstrated a worst case. In both studies no mortality was observed within the water solubility of the test substance under conditions tested. Therefore, it can be concluded that no toxicological effects on fish are expected up to the water

solubility for Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (CAS-No. 11138-60-6).

96-Hour Acute Toxicity Study in Carp with HATCOL 5236.

HATCOL 5236 is a clear pale yellow liquid with a purity of 97.6%. The water solubility of HATCOL 5236 at 20.0 ±0.8°C was determined to be <2.0x10-4g/l using the flask method (NOTOX Project 365041).

A stock solution was prepared at a loading rate of 100 mg/l. This supersaturated solution was stirred for two days to reach maximum solubility. After the stirring period the mixture was colourless but contained a test substance floating layer and a deposit of test substance.

Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance. After the stirring period the mixture was therefore filtered through a paper filter (ca.> 5 μm). The filtrate was clear and colourless.

A limit test was performed exposing seven carp per concentration to a 5 μm filtered solution prepared at a loading rate of 100 mg/l and a blank-control for a maximum of 96 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 hours of exposure and at the end of the test.

Analysis of the samples showed that the average exposure concentration was at or above the solubility limit of HATCOL 5236 (i.e.< 0.2 mg/l).

The study met the acceptability criteria prescribed by the protocol and was considered valid.

HATCOL 5236 induced no visible/lethal effects in carp in a filtered solution prepared at a loading rateof 100 mg/l, corresponding to an average exposure concentration at or above the water solubility. In conclusion: Owing to the extremely low solubility of HATCOL 5236 in water, concentration levels toxic for fish could not be reached. Therefore, the 96h-LC50 for carp exceeded the maximum solubility of

HATCOL 5236 in water.