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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 April 2004 to 14 May 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
AII samples were stored frozen. On the day of analysis, the frozen samples were defrosted at room temperature.
The entire volume of each sample (5 ml) was transferred quantitatively into a 20 ml volumetric flask using methanol. The flasks were filled up to the mark with methanol.
Vehicle:
no
Details on test solutions:
Preparation of 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 5127 is a light yellow liquid. The water solubi1ity of Hatcol 5127 at 20.4 +/-0.7°C was determined to be < 0.14x10^-3 g/l in a preliminary experiment (pH was between 8.0-8.1, NOTOX Project 388035).
AII test solutions with a loading rate at or above 1.0 mg/l were prepared separately (loading rates of 1.0, 10 and 100 mg/l). These saturated solutions were stirred for three days to reach maximum solubility. After the stirring period both the 10 and the 100 mg/l loading rates were inhomogeneous emulsions with a test substance floating layer, while the 1.0 mg/l loading rate was observed to be clear and colourless. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance (see also NOTOX Project 388035). After the stirring period all three mixtures/solutions were therefore filtered twice. First through a paper filter (Schleicher and Schuell 604) to remove the larger undissolved test substance particles (ca. > 5pm) and subsequent1y through a membrane filter (Schleicher and Schuell RC55, 0.45pm). Finally a ten-fold dilution was prepared from the filtrate prepared at a loading rate of 1.0 mg/1 and indicated as 0.1 mg/1. A11 test solutions were c[ear and colourless. Part of the test so1utions was used in the simultaneously performed acute Daphnia magna test (NOTOX Project 406979).

Test concentrations
Hatcol 5127
0.45 pm filtered solutions prepared at loading rates of 1.0, 10 and 100 mg/l and a ten-fold dilution of the filtrate prepared at a loading rate of 1.0 mg/l.

Control
Test medium without test substance or other additives (blank-control).
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: 2.2 +/- 0.2 cm
Mean weight: 0.30+/- 0.10 g
Characteristics: F1 from a single parent pair bred in UV-treated water.
Reason for species selection: This system has been selected as an internationally accepted species.
Total fish used: 70
Holding
Quarantine/acclimatization
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:
CaCl22H20 293.8 mg/L
MgSO47H2o 123.3 mg/l
NaHCO3 64.8 mg/l
KCl 5.8 mg/l

Measurements: pH, nitrate and titrate concentration and ammonia concentration once a week. Temperature: Daily.
Feeding: Daily with Triyvit.
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
Hardness:
After aeration the hardness was 250 mg CaCO3 per litre
Test temperature:
20-24°C
pH:
6.0-8.5
Dissolved oxygen:
> 60% of air saturation
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
Nominal concentrations of: 0.1, 1, 10, 100 mg/L
Details on test conditions:
Test vessels: 66.5 litres, all glass, containing 4.5 litres of test medium.
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 7.8-7.9

Number of fish: 7 fish per concentration and control
Loading: 0.47 g fish/litre, i.e. 7 fish per 4.5 litres of test medium
Illumination: 16 hours photoperiod daily.
Aeration: The test media were not aerated during the test.
Feeding: No feeding from 48 hours prior to the test and during the total test period.
Introduction of fish: within 2 hours after preparation of the test media.
Euthanasia: At the end of the test surviving fish were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenyl ether in water.
Reference substance (positive control):
yes
Remarks:
This reference test was carried out to check the sensitivity of the test system as used by NOTOX. The reference substance was pentachlorophenol (PCP, SIGMA, Art. P9441, Batch 103H34BB). Concentrations: 0.10, 0.22 and 0.46 mg/l in ISO-medium.
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.009 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: No effects observed upto the limit of solubility
Key result
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: No effects observed upto the limit of solubility
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: No effects observed upto the limit of solubility
Key result
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
> 0.009 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: No effects observed upto the limit of solubility
Details on results:
Measures concentrations
The concentrations of Hatcol 5127 were determined by gas chromatography with mass spectrometric detection. 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 largest peak observed in the chromatograms of Hatcol 5127

Mortality and other effects.
No mortality or any other effects were observed.

Environmental conditions
AII test conditions remained within the ranges prescribed by the protocol (pH: 6.0-8.5, constant within 1 unit; temperature 20-24°C, constant within 2°C; oxygen > 60% of air saturation).
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 PCP induced on lethal effects in carp at or below 0.22 mg/l. The 96h-LC50 for carp exposed to PCP was 0.32 mg/l (95 % confidence interval between 0.22 and 0.46 mg/l) and already reached within 24 hours of exposure. 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 ln carp originating from the present batch falls within this range.
Sublethal observations / clinical signs:

Incidence of mortality and total mortality during the final test

Loading rate Hatcol 5127 mg/L

Initial number of fish

Cumulative mortality

Total mortality %

4 h

24 h

48 h

72 h

96 h

Blank control

7

0

0

0

0

0

0

0.1

7

0

0

0

0

0

0

1.0

7

0

0

0

0

0

0

10

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 final test

Loading rate Hatcol 5127 mg/L

Day 0

Day 1

Day 2

Day 3

Day 4

 

pH

O2*

pH

O2

pH

O2

pH

O2

pH

O2

O2

Blank control

7.9

9.1/8.1

7.4

7.4

7.2

7.1

7.5

7.0

7.6

7.2

 

0.1

7.9

8.9/8.0

7.4

7.2

7.2

6.8

7.5

6.9

7.6

7.3

 

1.0

7.8

7.0/6.8

7.4

7.1

7.2

6.8

7.5

6.9

7.3

6.7

 

10

7.9

7.1/6.9

7.4

7.0

7.2

6.8

7.4

6.7

7.3

6.9

 

100

7.9

7.1/6.6

7.4

7.1

7.2

6.7

7.4

6.8

7.3

7.0

 

* oxygen concentration measured in the morning/afternoon

 

Temperatures (°C} measured during the final test

Loading rate Hatcol 5127 mg/L

Day 0

Day 1

Day 2

Day 3

Day 4

Blank control

21.3

21.7

21.7

21.5

21.6

0.1

21.4

21.7

21.6

21.5

21.4

1.0

21.3

21.8

21.7

21.6

21.6

10

21.3

21.7

21.7

21.6

21.6

100

21.5

21.4

21.7

21.5

21.6

 

Incidence of mortality observed in the reference study

Loading rate PCP (mg/L) nominal

Initial number of fish

Cumulative mortality

Total mortality %

2.5 h

24 h

48 h

72 h

96 h

0.1

5

0

0

0

0

0

0

0.22

5

0

0

0

0

0

0

0.46

5

0

5

5

5

5

100

 


 

Procedural recovery samples

Date of preparation

[DD/MM/YY]2

Date of pre-treatment

[DD/MM/YY]1

Date of analysis

[DD/MM/YY]3

Concentration nominal (mg/L)

Concentration analysed (mg/L)

Recovery (%)

Mean Recovery (%)

10-05-04

24-05-04

27-05-04

0.00503

0.00503

0.00532

0.00518

106

103

104

10-05-04

24-05-04

27-05-04

0.0503

0.0503

0.0679

0.0687

135

137

136

10-05-04

24-05-04

27-05-04

10.1

10.1

9.21

10.4

92

104

98

1: Samples were stored frozen until pre-treatment on 24-05-04

2: Combined with NOTOX Project 406979

3: Due to problems with the HPLC equipment, the samples and calibration solutions were stored at ambient temperature and analysed on 27-05-04

 

Concentrations of Hatcol 5127 in test medium (final test)

Time of sampling (hours)

Date of sampling

[DD/MM/YY]

Date of pre-treatment

[DD/MM/YY]2

Date of analysis

[DD/MM/YY]4

Concentration

Loading rate (mg/L)1

Analysed (mg/L)

Relative to initial (%)

0

10-05-043

24-05-04

26-05-04

0

0

100

100

<0.001

<0.001

0.0338

0.0275

 

24

11-05-04

24-05-04

26-05-04

0

0

100

100

<0.001

<0.001

0.0215

0.0261

n.a.

n.a.

70

85

96

14-05-04

24-05-04

26-05-04

0

0

100

100

<0.001

<0.001

<0.001

<0.001

n.a.

n.a.

n.a.

n.a.

 

1: 0.45µm filtered solution

2: Samples were stored frozen until pre-treatment on 24-05-04

3: Combined with NOTOX project 406979

4: Due to problems with the HPLC equipment, the samples and calibration solutions were stored at ambient temperature and analysed on 27-05-04

n.a.: Not applicable.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study Hatcol 5127 induced no visible effects in carp exposed to a filtered solution prepared at a loading rate of 100 mg/l, containing an average exposure concentration of 0.0093 mg/l (NOEC).

Due to the extremely low water solubility of Hatcol 5127, concentration levels that might be toxic to carp could not be reached.
Executive summary:

96-Hour Acute Toxicity Study in Carp with Hatcol 5127.

The study procedure described in this report was based on the ISO lnternational Standard 7346-1: Static method, 1996. ln addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C.1, 1992, the OECD guideline No. 203, 1992 and the OECD series on testing and assessment number 23, December 14, 2000.

Hatcol 5127 is a light yellow liquid. The water solubility of Hatcol 5127 at 20.4 +/- 0.7°C was determined to be < 0.14x10^-3 g/l in a preliminary experiment (pH was between 8.0-8.1, NOTOX Project 388035).

AII test solutions with a loading rate at or above 1.0 mg/l were prepared separately (loading rates of 1.0, 10 and 100 mg/l). These saturated solutions were stirred for three days to reach maximum solubility. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance (see also NOTOX Project 388035). After the stirring period all three mixtures/solutions were therefore filtered twice. First through a paper filter to remove the larger undissolved test substance particles (ca. > 5pm) and subsequently through a membrane filter (0.45µm). Finally a ten fold dilution was prepared from the filtrate prepared at a loading rate of 1.0 mg/l. AII test solutions were clear and colourless.

A final test was performed exposing seven fish per concentration to a blank control, 0.45µm filtered solutions prepared at loading rates of 1.0, 10 and 100mg/l and in addition to a ten fold dilution of the filtrate prepared at a loading rate 1.0 mg/l for a period of 96 hours. Samples for analytical confirmation of actual exposure concentrations were taken from the blank-control and the highest test concentration at the start, after 24 hours of exposure and at the end of the test.

Since not all components of Hatcol 5127 were quantifiable in the calibration chromatograms,  analytical results were based on the largest peak. Based on this peak the filtered solution prepared at a loading rate of 100 mg/l contained a mean initial concentration of 0.031 mg/l. After 24 hours the concentration was relatively stable at 0.024 mg/l (77% of initial) but after 72 hours of exposure this test concentration had decreased to below the lowest calibration solution (<0.001 mg/l). The average exposure concentration at a loading rate of 100 mg/l was calculated to be 0.0093 mg/l. The observed decrease after 24 hours of exposure was probably a consequence of the extremely low solubility.

The study met the acceptability criteria prescribed by the protocol and was considered valid. Hatcol 5127 induced no visible effects in carp exposed to a filtered solution prepared at a loading rate of 100 mg/l, containing an average exposure concentration of 0.0093 mg/l {NOEC).

Due to the extremely low water solubility of Hatcol 5127, concentration levels that might be toxic to carp could not be reached. 

Description of key information

Under the conditions of the study Hatcol 5127 induced no visible effects in carp exposed to a filtered solution prepared at a loading rate of 100 mg/l, containing an average exposure concentration of 0.0093 mg/l (NOEC).

 

Due to the extremely low water solubility of Hatcol 5127, concentration levels that might be toxic to carp could not be reached.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
100 mg/L

Additional information

96-Hour Acute Toxicity Study in Carp with Hatcol 5127.

The study procedure described in this report was based on the ISO lnternational Standard 7346-1: Static method, 1996. ln addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C.1, 1992, the OECD guideline No. 203, 1992 and the OECD series on testing and assessment number 23, December 14, 2000.

Hatcol 5127 is a light yellow liquid. The water solubility of Hatcol 5127 at 20.4 +/- 0.7°C was determined to be < 0.14x10^-3 g/l in a preliminary experiment (pH was between 8.0-8.1, NOTOX Project 388035).

AII test solutions with a loading rate at or above 1.0 mg/l were prepared separately (loading rates of 1.0, 10 and 100 mg/l). These saturated solutions were stirred for three days to reach maximum solubility. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance (see also NOTOX Project 388035). After the stirring period all three mixtures/solutions were therefore filtered twice. First through a paper filter to remove the larger undissolved test substance particles (ca. > 5pm) and subsequently through a membrane filter (0.45µm). Finally a ten fold dilution was prepared from the filtrate prepared at a loading rate of 1.0 mg/l. AII test solutions were clear and colourless.

A final test was performed exposing seven fish per concentration to a blank control, 0.45µm filtered solutions prepared at loading rates of 1.0, 10 and 100mg/l and in addition to a ten fold dilution of the filtrate prepared at a loading rate 1.0 mg/l for a period of 96 hours. Samples for analytical confirmation of actual exposure concentrations were taken from the blank-control and the highest test concentration at the start, after 24 hours of exposure and at the end of the test.

Since not all components of Hatcol 5127 were quantifiable in the calibration chromatograms,  analytical results were based on the largest peak. Based on this peak the filtered solution prepared at a loading rate of 100 mg/l contained a mean initial concentration of 0.031 mg/l. After 24 hours the concentration was relatively stable at 0.024 mg/l (77% of initial) but after 72 hours of exposure this test concentration had decreased to below the lowest calibration solution (<0.001 mg/l). The average exposure concentration at a loading rate of 100 mg/l was calculated to be 0.0093 mg/l. The observed decrease after 24 hours of exposure was probably a consequence of the extremely low solubility.

The study met the acceptability criteria prescribed by the protocol and was considered valid. Hatcol 5127 induced no visible effects in carp exposed to a filtered solution prepared at a loading rate of 100 mg/l, containing an average exposure concentration of 0.0093 mg/l {NOEC).

Due to the extremely low water solubility of Hatcol 5127, concentration levels that might be toxic to carp could not be reached.