Fatty acids, C6-12, mixed tetraesters with heptanoic acid, pentaerythritol, 3,5,5-trimethylhexanoic acid and valeric acid

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:

23 Apr -07 Aug 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance Reaction product of pentaerythritol and trimethylolpropane with n-pentanoic acid, 2-methylbutyric acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid. 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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

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: 100 mg/L
- Sampling method: Water samples were taken from the control and each replicate test vessel at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 h (old media) for quantitative analysis. Duplicate samples were taken.
A volume of sample was extracted with three portions (3 x 50 mL) of dichloromethane and the extracts filtered through anhydrous sodium sulphate. The combined extracts were evaporated to dryness and the residue re-dissolved in hexane.
- Sample storage conditions before analysis: stored frozen (approximately -20 °C) for further analysis, if necessary and at ambient temperature in light and dark conditions for approximately 24 h before analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For range-finding test, amounts of test material (210 and 2100 mg) were each separately placed on the surface of 2 L of dechlorinated tap water to give 10 and 100 mg/L loading rates which were then stirred by magnetic stirrer, to achieve a vortex depth of approximately 25 % of the distance to the bottom of the vessel, for 24 hours. The stirring was stopped after 24 h and the mixtures allowed to stand for 1 h prior to removing the aqueous phase or Water Accommodated Fraction by siphon. Based on the results of the range-finding study a "Limit test" was conducted for the definitive study at a 100 mg/L loading rate WAF to confirm that at the maximum test concentration given in the test guidelines, no mortalities or sublethal effects of exposure were observed.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Brow Well Fisheries Ltd, Phoenix Cottage, Hebden, Nr Skipton, UK
- Feeding during test: no

ACCLIMATION
- Acclimation period: from 22 Jul 1998 to 3 Aug 1998
- Acclimation conditions (same as test or not): temperature: 14 °C, DO: 9.3 mgO2/L, light:darkness=16 h:8 h
- Type and amount of food: commercial trout pellets
- Health during acclimation (any mortality observed): 1% mortality in the 7 days prior to the start of the test. The fish had a mean standard length of 4.6 cm (sd = 0.4 cm) and a mean weight of 1.36 g (sd = 0.29 g) at the end of the definitive test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

109 mg/L as CaCO3

Test temperature:

14 °C

pH:

7.6 (mean)

Dissolved oxygen:

9.3 mgO2/L

Nominal and measured concentrations:

control and 100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Type: closed
- Material, size: 20 L glass vessel
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water
- Total organic carbon: 2.3 mgC/L
- Particulate matter: 0.10 mg/L
- Pesticides: <0.03 μg/L
- Chlorine: 0.11 mg/L
- Alkalinity: 102 mg/L as CaCO3
- Conductivity: 468 μs/cm
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light and 8 h darkness cycle with 20 min dawn and dusk transition periods

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Any mortailites and sub-lethal effects of exposure were recorded at 3, 6, 24, 28, 72 and 96 h after the start of exposure.

TEST CONCENTRATIONS
- Range finding study:
- Test concentrations: control, 10, 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: The results showed no mortalities at the test concentrations of 10 and 100 mg/L. Based on this information, a single test concentration of 100 mg/L was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the highest attainable test concentration of 100 mg/L, no mortalities or sub-lethal effects of exposure were observed.

Reference substance (positive control):

no

Key result

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:

no mortalities occurred during the study

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

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

Remarks:

3 substances available for read across

Adequacy of study:

weight of evidence

Justification for type of information:

see the attached justification in section 13 for the full details.

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.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other:

Remarks:

PE/TMP tetra/tri C5, i-C5, C7, C8, i-C9, C10 ester

Key result

Duration:

95 h

Dose descriptor:

LC50

Effect conc.:

> 0.22 - < 0.27 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

mortality (fish)

Remarks on result:

other: Hatcol 3331

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:

Hatcol 5236

Validity criteria fulfilled:

not applicable

Remarks:

read across

Conclusions:

The read across for substance, CAS: 156558-98-4; EC: 451-190-0; is based upon the analogous substances to which basic form, degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of short-term toxicity to fish. The EC50 for the substance based on the mean of the information available is deemed to be 33.47 mg/L, to which no adverse effects were noted upto the limit of solubility.

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 April 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:

Preparation of the test solution started with a stock solution of 100 mg/l applying 2 days of magnetic stirring to ensure maximum solubility in test medium. The resulting solution was colourless, but contained a floating layer and test substance particles. 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.

Details on test solutions:

Nominal test concentration: A 5 µm filtrate prepared at a loading rate of 100 mg/l, the regulatory limit concentration.
Blank –control: Test medium without test substance or other additives (0 mg/l).

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 Netheriands.
Mean length 2.4 ± 0.2cm
Mean weight 0.32 ± 0.1g
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 MiIli-Ro water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
CaCl22H2O 293.8 mg/l
MgSO47H2O 123.3 mg/l
NaHCO3 64.8 mg/l
KCL 5.8 mg/l
Hardness is 250 mg CaCO3/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 required for this study.

Hardness:

Not specified in the study report.

Test temperature:

20-24°C

pH:

pH: 6.0-8.5, not varying more than 1 unit

Dissolved oxygen:

oxygen: >60% of air saturation;

Salinity:

Not applicable - freshwater study

Nominal and measured concentrations:

Nominal concentration of 100 mg/l

Details on test conditions:

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 CaCO3 per litre and the pH was 8.0.
Number of fish: 7 fish per concentration and controls.
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.
Feeding: No feeding from 48 hours prior to the test and during the total test period.
Introduction of fish: Within 35 minutes after preparation of the test media.
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.

SAMPLING FOR ANALYSIS OF TEST CONCENTRATIONS
During the test duplicate samples for analysis were taken from the test solution and the blank control.
Sampling:
Frequency: at t=O 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.

MEASUREMENTS AND RECORDINGS
Mortality and other effects: At 3.5, 24, 48, 72 and 96 hours following the start of exposure. Dead fish were removed when observed.
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 of the medium: Daily in all vessels, beginning at the start of the test (day 0).

Reference substance (positive control):

yes

Remarks:

Historical study

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.22 - 0.27 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

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 three largest peaks observed in the chromatograms of HATCOL 3331.

The analytical results showed that the filtered solution prepared at a loading rate of 100 mg/I contained an initial concentration of 0.50 mg/l or 0.48 mg/l. After 24 hours of exposure the test concentration had decreased to 0.30 mg/l or 0.44 mg/l. At the end of the test period the test concentration had decreased below the limit of quantification (i.e. below 0.2 mg/I).
The average exposure concentration, was 0.22, 0.22 or 0.27 mg/l. Hence, average concentrations remained above or approximated the solubility limit of HATCOL 3331 (i.e. < 0.2 mg/I). The observed decrease was probably a consequence of this extremely low solubility.

Mortality and other effects
No fish died during the test. No undissolved test substance was observed during the test.

Experimental conditions
The test conditions all met the requirements described in the protocol (pH: 6.0-8.5, not varying more than 1 unit; oxygen: > 60% of air saturation; temperature: 20-24°C, constant within 2°C).

Results with reference substance (positive control):

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/I (95 % confidence interval between 0.19 and 0.27 mg/l). The 24h-LC50 was 0.24 mg/I (95% confidence interval between 0.21 and 0.30 mg/I), and remained unchanged until 72h. The range of the 96h-LC50 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:

Not specified in the report

Sublethal observations / clinical signs:

Table 1: Incidence of mortality and total mortality during the test

Loading rate HATCOL 3331 (mg/l)	Initial number of fish	Cumulative mortality					Total Mortality (%)
		3.5h	24h	48h	72h	96h
Blank-control	7	0	0	0	0	0	0
100	7	0	0	0	0	0	0

 

Table 2: pH-values and dissolved oxygen concentrations (mg/l) during the test

Loading rate HATCOL 3331 (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	6.2^1	7.8	8.6	7.8	8.7
100	7.5	8.5	7.4	6.9	7.3	6.6^1	7.9	8.7	7.8	8.7

¹After this measurement, aeration was introduced to prevent further decrease of the oxygen concentration.

 

Table 3: Temperatures (°C) measured during the final test

Loading rate HATCOL 3331 (mg/l)	Day 0	Day 1	Day 2	Day 3	Day 4
Blank-control	22.1	21.2	21.3	21.1	21.0
5.6	22.5	21.2	21.3	21.0	20.9

 

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
0.06	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:

Under the conditions of the present test HATCOL 3331 induced no visible effects in carp at the concentration obtained in a filtered solution prepared at a loading rate of 100 mg/l.
In conclusion: Due to the very low solubility of HATCOL 3331 in water, concentration levels that might be toxic for carp could not be reached. Therefore, the 96h-LC50 exceeded the maximum solubility of HATCOL 3331 in test medium.

Executive summary:

96-Hour Acute Toxicity Study in Carp with HATCOL 3331.

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.L. "Acute toxicity for fish"; and the OECD guideline No. 203: "Fish Acute Toxicity Test", Adopted 17 July, 1992.

 

The batch of HATCOL 3331 tested was a clear colourless liquid with a purity of >97.3% and the substance was not completely soluble in test medium at the concentration tested. The water solubilityof Hatcol 3331 at 20.3 ± 0.8°C was determined to be < 2.0 x 10^-4g/l, according to the flask method (NOTOX Project 365052). The partition coefficient (n-octanol/water), Pow, was determined to be ≥ 5.1*10 ^-6 (log Pow ≥ 6.7) at 20.3 ± 0.8°C (NOTOX Project 365085).

Preparation of the test solution started with a stock solution of 100 mg/l applying 2 days of magnetic stirring to ensure maximum solubility in test medium. The resulting solution was colourless, but contained a floating layer and test substance particles. 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 to a filtered (ca. 5 µm) HATCOL 3331 solution prepared at a loading rate of 100 mg/l and to a blank-control. The total test period was 96 hours. Samples for determination of actual exposure concentrations were taken at the start, after 24 hours of exposure and at the end of the test period.

Analysis of the samples showed that average exposure concentrations were at or above the solubility limit of HATCOL 3331 (i.e. < 0.2 mg/l).

The study met the acceptability criteria prescribed by the protocol and was considered valid. HATCOL 3331 induced no visible effects in carp at the concentration obtained in a filtered solution prepared at a loading rate of 100 mg/l, corresponding to an average exposure concentration approaching the solubility limit, i.e. 0.22 -0.27 mg/l.

 

In conclusion: Due to the very low solubility of HATCOL 3331 in water, concentration levels that might be toxic for carp could not be reached. Therefore, the 96h-LC50 exceeded the maximum solubility of HATCOL 3331 in test medium.

Reference 4

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key 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

The read across for substance, CAS: 156558-98-4; EC: 451-190-0; is based upon the analogous substances to which basic form, degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of short-term toxicity to fish The EC50  for the substance based on the mean of the information available is deemed to be 33.47 mg/L, to which no adverse effects were noted upto the limit of solubility.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

33.47 mg/L

Additional information

HATCOL 3331

96-Hour Acute Toxicity Study in Carp with HATCOL 3331.

The batch of HATCOL 3331 tested was a clear colourless liquid with a purity of >97.3% and the substance was not completely soluble in test medium at the concentration tested. The water solubility of Hatcol 3331 at 20.3 ± 0.8°C was determined to be < 2.0 x 10^-4g/l, according to the flask method (NOTOX Project 365052). The partition coefficient (n-octanol/water), Pow, was determined to be ≥ 5.1*10^-6(log Pow ≥ 6.7) at 20.3 ± 0.8°C (NOTOX Project 365085).

Preparation of the test solution started with a stock solution of 100 mg/l applying 2 days of magnetic stirring to ensure maximum solubility in test medium. The resulting solution was colourless, but contained a floating layer and test substance particles. 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 to a filtered (ca. 5 µm) HATCOL 3331 solution prepared at a loading rate of 100 mg/l and to a blank-control. The total test period was 96 hours. Samples for determination of actual exposure concentrations were taken at the start, after 24 hours of exposure and at the end of the test period.

Analysis of the samples showed that average exposure concentrations were at or above the solubility limit of HATCOL 3331 (i.e. < 0.2 mg/l).

The study met the acceptability criteria prescribed by the protocol and was considered valid. HATCOL 3331 induced no visible effects in carp at the concentration obtained in a filtered solution prepared at a loading rate of 100 mg/l, corresponding to an average exposure concentration approaching the solubility limit, i.e. 0.22 -0.27 mg/l.

In conclusion: Due to the very low solubility of HATCOL 3331 in water, concentration levels that might be toxic for carp could not be reached. Therefore, the 96h-LC50 exceeded the maximum solubility of HATCOL 3331 in test medium.

 

HATCOL 5236

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 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.

One GLP Guideline study according to OECD 203 is available investigating the short-term toxicity of the source substance PE/TMP tetra/tri C5, i-C5, C7, C8, i-C9, C10 ester to freshwater fish (Wetton, and Mullee, 1998). The rainbow trout (Oncorhynchus mykiss) was exposed for 96 h to a limit Water Accommodated Fraction (WAF) of nominal 100 mg/L. GC analysis resulted in a measured initial concentration of 0.53 mg/L. No mortality was observed resulting in a LL50 (96 h) of > 0.53 mg/L (measured initial concentration) and > 100 mg/L (nominal), respectively.

Based on the results from a structurally similar source substance (in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5) it can be concluded that Pentaerythritol tetraesters of n-C5, n-C7, n-C8, i-C9 and n-C10 fatty acids will not show effects up to the limit of water solubility.