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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:
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)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
not specified
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

6.21

7.8

8.6

7.8

8.7

100

7.5

8.5

7.4

6.9

7.3

6.61

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.

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)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
not specified
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.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Mar - 01 Apr 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions (no control)
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 2: Semi-static method)
Qualifier:
equivalent or similar 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 test substance was weighed directly into the test vessels, filled up with standard dilution water to a final volume of 5L and than stirred for 3 minutes by ultra-turrax.- Controls: no data
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM- Common name: Zebra fish- Feeding during test: none- Other: < 0.1% mortality prior to test
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
about 250 mg CaCO3/L
Test temperature:
22.5 - 23.5 °C
pH:
7.6 - 7.9
Dissolved oxygen:
58 - 99%
Nominal and measured concentrations:
Nominal: 3000 and 10000 mg/L
Details on test conditions:
TEST SYSTEM- Test vessel (size, headspace, fill volume): 10 L fish basins containing 5 L test water- Renewal rate of test solution (frequency/flow rate): daily- No. of organisms per vessel: 10- No. of vessels per concentration (replicates): 1TEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: Standard dilution waterOTHER TEST CONDITIONS- Photoperiod: 16 h illumination per dayEFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortalities are recorded at 0, 6, 24, 48, 72 and 96 h.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
>= 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Behavioural abnormalities: None
Sublethal observations / clinical signs:

Table 1: Mortality [%] of Brachydaniorerio

Test concentration [mg product/L]

Incubation time [h]

0

6

24

48

72

96

3000

0

0

0

0

n.d.*

0

10000

0

0

0

0

n.d.*

0

 * n.d. = not determined

Validity criteria fulfilled:
yes
Conclusions:
The 96h LC50 was determined to be > 10000 mg/L. No mortality was observed in the study.
Executive summary:

The 96h LC50 was determined to be > 10000 mg/L. No mortality was observed in the study.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
07 - 11 Jan 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions (no purity)
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 (especially for difficult test substances)- Method: The test solutions were prepared by direct addition of 1000 mg of test material per litre. The test solutions were then stirred thoroughly.- Controls: Yes, freshwater control
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 (27 Nov 1990)- 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 or not): Same as test (15 ± 1 °C, glass aquaria under daylight and artificial lighting)- 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 (any mortality observed): No mortality
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:
7.92 - 8.09
Dissolved oxygen:
9.0 - 9.8 mg/L
Nominal and measured concentrations:
Nominal: 0 and 1000 mg/L
Details on test conditions:
TEST SYSTEM- Test vessel: - Material, size, headspace, fill volume: 17 L glass vessels with a fill volume of 15 litres - Aeration: gentle aeration- 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): 1TEST 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.- Alkalinity: 20.4 mg/L as CaCO3- Conductivity: 136 - 142 µS/cm- Intervals of water quality measurement: Daily for pH, dissolved oxygen and temperature.OTHER TEST CONDITIONS- Photoperiod: 16 h light/ 8 h darkEFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortalities and symptoms of toxicity at 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):
not specified
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)
Details on results:
- Observations on body length and weight: Control at the end of the exposure period: length 37 - 47 mm (mean 41 mm), weight 0.47 - 1.31 g (mean 0.81 g)- 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:

Table 1: Percentage mortalities during the test period

Nominal concentration of test substance (mg/L)

Percentage mortality observed

24 h

48 h

72 h

96 h

1000 (A)

0

0

0

0

1000 (B)

0

0

0

0

Dilution control water

0

0

0

0

 

Validity criteria fulfilled:
yes
Conclusions:
The 96h LC50 was determined to be > 1000 mg/L. No mortality was observed in the study.
Executive summary:

The 96h LC50 was determined to be > 1000 mg/L. No mortality was observed in the study.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
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: noACCLIMATION- 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: 7TEST 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.
Sublethal observations / clinical signs:

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.

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.
Executive summary:

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.

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
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: No toxicity in the range of water solubility
Key result
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

Key value determind using OECD guideline 203 and EU test standard C1.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
0.22 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.

CAS 11138 -60 -6

In thestudy with CAS 11138-60-6 (conducted in accordance with OECD Guideline 203),no mortality was observed and therefore the LL50 was determined to be > 10000 mg/l WAF. Due to the low water solubility (< 0.3 mg/l), no mortality within the water solubility of the test substance was observed and therewith the LC50 (96h) is > water solubility of the test substance.

HATCOL 2937

In the study with CAS 91050-89-4 (conducted in accordance with ISO 7346 -1), the 96h LC50 was determined to be > 10000 mg/L. No mortality was observed in the study.

CAS 78 -16 -0

In the study with CAS 78 -16 -0 (conducted in accordance with OECD Guideline 203), the 96h LC50 was determined to be > 1000 mg/L. No mortality was observed in the study.

HATCOL 1765

One study, investigating the short-term toxicity to freshwater fish, was available for pentaerythritol tetraesters of n-decanoic, n-heptanoic, n-octanoic and n-valeric acids (CAS 68424-31-7). This semi-static study (Combert and Caunter, 1991) with Salmo gairdneri equivalent or similar to OECD 203 determined LC50 (96h) > 1000 mg/L (nominal). No mortality was observed within the tested concentration (1000 mg/L nominal) during 96h exposure. As the test material did not dissolved completely in the dilution water, the tested concentration demonstrated a worst case. Due to the low water solubility of the test substance (< 1 mg/l), no toxicity was observed in the range of water solubility of the test substance. Therefore, the substance is not harmful to fish.