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Ecotoxicological information

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:
Definitive study: 4th October 1999 to 8th October 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: March 23rd 1998 Date of signature: July 21st 1998
Specific details on test material used for the study:
No additional information
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1.0, 1.8, 3.2, 5.6 and 10 mg/l (nominal concentrations)

- Sampling method: Replicates were verified by chemical analysis at 0, 24 and 96 hours. A C18 (EC) solid phase extraction cartridge was pre-conditioned with methanol (10 ml) and water (10 ml). A volume of test sample was drawn through a preconditioned cartridge under vacuum and and the cartridge dried. The test material was eluted from the dried cartridge with acetone to give a final theoretical test material concentration of approximately 100 mg/l.

- Sample storage conditions before analysis: Duplicate samples were taken at each occasion and stored frozen (approximately -20°C) for further analysis if necessary.
Vehicle:
no
Details on test solutions:
For the purpose of the definitive study the test material was prepared by direct solution in water.

An amount of test material (500 mg) was dissolved in dechlorinated tap water with the aid of ultrasonication and shaking for approximately 30 minutes prior to adjusting the volume to 1 litre to give the 500 mg/l stock solution. Aliquots (44, 79.2, 140.8,264.4 and 440 mil of the stock solution were separately dispersed in dechlorinated tap water while stirring with a flat bladed mixer for approximately 1 minute prior to adjusting the volume to 22 Iitres to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/l test concentrations respectively.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Strain: no data
- Source: Brow Well Fisheries, Hebden, Yorkshire, UK.
- Age at study initiation (mean and range, SD): Juvenile
- Length at study initiation (length definition, mean, range and SD): not stated in study report
- Weight at study initiation (mean and range, SD): not stated in study report
- Method of breeding: asexual
- Feeding during test: not applicable, as feeding discontinued 24 hours prior to the start of the defnitive study

ACCLIMATION
- Acclimation period: 22 September 1999 to 4 October 1999
- Acclimation conditions (same as test or not): Yes, apart from no feeding during test
- Type and amount of food: Commercial trout pellets
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): no mortalities

QUARANTINE (wild caught)
- not applicable
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
Not applicable
Hardness:
Approximately 100 mg/l as CaCO3.
Test temperature:
Recorded daily throughtout the study. Maintained at 14 +/- 1°C.
pH:
Recorded daily thoughout the study. No treatment related effects observed.
Range of 7.2 to 7.9
Dissolved oxygen:
Recorded daily thoughout the study. Greater than or equal to 9.4 mg O2/l.
Salinity:
Not applicable, as freshwater study
Nominal and measured concentrations:
Range finding study: 1.0, 10 and 100 mg/l (nominal)

Definitive study: 1.0, 1.8, 3.2, 5.6 and 10 mg/l

Measured test concentrations to be in the range 90% to 122% of the nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass exposure vessels
- Type (delete if not applicable): closed - covered with lids and sealed to reduce evaporation
- Material, size, headspace, fill volume: 22 litres of test material with test vessels completely filled to reduce headspace
- Aeration: test vessels received no auxillary aeration
- Type of flow-through (e.g. peristaltic or proportional diluter): semi-static
- Renewal rate of test solution (frequency/flow rate): daily renewal of test solution
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: 0.62 g bodyweight /litre

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water dechlorinated by passage through an activated carbon filter and aprtly softened.
- Total organic carbon:
Mean: 1 mg/l Minimum: 1 mg/l Maximum; 1 mg/l
- Metals: no total values for metals provided
- Pesticides:
Mean: <0.02 µg/l Minimum: <0.01 µg/l Maximum: <0.03 µg/
- Chlorine:
Free chlorine: Mean: <0.06 mg/l Minimum: 0.01 mg/l Maximum: 0.24 mg/l
Total chlorine: Mean: 0.12 mg/l Minimum: 0.03 mg/l Maximum: 0.32 mg/l
- Conductivity: Mean: 434 µS/cm Minimum: 269 µS/cm Maximum: 570 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: Maximum of 46 samples obtained during period of 01/01/1998 to 30/09/1998

OTHER TEST CONDITIONS
- Adjustment of pH: not required
- Photoperiod: 16 hours light and 8 hours darkness with a 20 minute dawn and dusk transition periods
- Light intensity: not stated in report

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. the criteria of death were taken to be the absence of both respiratory movements and response to physical stimulation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.0, 1.8, 3.2, 5.6 and 10 mg/l
- Justification for using less concentrations than requested by guideline: not applicable

- Range finding study
- Test concentrations: nominal concentrations of 1.0, 10 and 100 mg/l.
- Results used to determine the conditions for the definitive study: The results showed no mortalities at the test concentration of 1.0 mg/l. after approximately 30 minutes exposure 3/3 fish were observed to be moribund with arched backs in the 100 mg/l test group. Therefore, due to animal welfare implications these fish were killed in extremis and were classed as mortalities. After approximately 24 hours exposure 1/3 fish was observed to be swimming at the bottom of the test vessel with a swollen abdomen and 2/3 fish were observed to be morbund with swollen abdomens in the 10 mg/l test group. These fish were also killed in extremis due to animal welfare implications and classed as mortalities.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
7.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
other: The highest test concentration causing 0% mortality
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
other: The highest test concentration causing no sub-lethal effects
Effect conc.:
1.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: sub-lethal effects
Details on results:
Range finding Results:

The results showed no mortalities at the test concentration of 1.0 mg/l. After approximately 30 minutes exposure 3/3 fish were observed to be moribund with arched backs in the 100 mg/l test group. Therefore, due to animal welfare implications these fish were killed in extremis and were classed as mortalities. After approximately 24 hours exposure 1/3 fish was observed to be swimming at the bottom of the test vessel with a swollen abdomen and 2/3 fish were observed to be moribund with swollen abdomens in the 10 mg/I test group. These fished were also killed in extremis due to animal welfare implications and classed as mortalities.

Based on this information test concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg/I were selected for the definitive study.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
None.

Definitive study:

The results of the definitive study showed the highest test concentration resulting in 0% mortality to be 3.2 mg/L, the lowest test concentration resulting in 100% mortality to be 10 mg/L and the highest test concentration resulting no sub-lethal effects to be 1.8 mg/L.

Cumulative mortality in the definitive study

Nominal concentration (mg/l)

Cumulative mortality (initial population = 10)

% mortality

96 hours

3 hours

6 hours

24 hours

48 hours

72 hours

96 hours

Control

0

0

0

0

0

0

0

1.0

0

0

0

0

0

0

0

1.8

0

0

0

0

0

0

0

3.2

0

0

0

0

0

0

0

5.6

0

0

0

0

1

1

10

10

0

0

0

10

10

10

100

Sub-lethal effects

Sub-lethal effects of exposure were observed at test concentrations of 3.2 mg/l and above. These responses were swimming at the surface, swimming at the bottom of test vessels and increased pigmentation.

Sub-lethal effects of exposure in the definitive study

Nominal concentration (mg/l)

Sub-lethal effects

Time (hours)

3

6

24

48

72

96

Control

No abnormalities detected

 

 

 

 

 

 

1.0

No abnormalities detected

 

 

 

 

 

 

1.8

No abnormalities detected

 

 

 

 

 

 

3.2

Increased pigmentation

 

 

 

1/10

3/10

3/10

5.6

Increased pigmentation

Swimming at bottom with increased pigmentation

 

 

6/10

10/10

9/9

8/9

1/9

10

Swimming at bottom

Swimming at surface

Swimming at bottom with increased pigmentation

Swimming at surface with increased pigmentation

Increased pigmentation

1/10

2/10

1/10

2/10

 

 

1/10

 

2/10

 

7/10

 

 

A/D

 

 

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test material to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 value of 7.1 mg/I with 95% confidence limits of 6.3 - 7.9 mg/l. The highest test concentration causing no mortality was 3.2 mg/L, the lowest test concentration causing 100% mortality was 10 mg/L and the highest test concentration causing no sub-lethal effects was 1.8 mg/L.
Executive summary:

Methods: A study was performed to assess the acute toxicity of the test material to rainbow trout (Oncorynchus mykiss). The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No. 203, "Fish, Acute Toxicity Test" referenced as Method C1 of commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Procedures: Following a preliminary range-finding study, fish were exposed, in groups of 10, to an aqueous solution of the test material over a range of concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg/l for a period of 96 -hours under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughtout the study until termination after 96 hours.

Results: The 96 -hour LC50 based on nominal test concentrations was 7.1 mg/l with 95% confidence limits of 6.3 to 7.9 mg/l. The highest test concentration causing no mortality was 3.2 mg/L, the lowest test concentration causing 100% mortality was 10 mg/L and the highest test concentration causing no sub-lethal effects was 1.8 mg/L.

Analysis of the test solutions at 0, 24 and 96 -hours showed the measured test concentrations to be in the range 90% to 122% of nominal; therefore, the results are based on nominal test concentrations.

Description of key information

OECD Guideline 203, EU Method C1, GLP, key study, validity 1:

96h-LC50 (Oncorhynchus mykiss) = 7.1 mg/L (95% CL: 6.3-7.9 mg/L) based on analytically confirmed nominal test concentrations.

Key value for chemical safety assessment

LC50 for freshwater fish:
7.1 mg/L

Additional information

Based on the results of one key study (SPL, 1999), performed according to OECD Guideline 203 and EU Method C1 with GLP statement, the 96 -hour LC50 (Oncorhynchus mykiss) based on nominal test concentrations was 7.1 mg/L with 95% confidence limits of 6.3 to 7.9 mg/L. The highest test concentration causing no mortality was 3.2 mg/L and the highest test concentration causing no sub-lethal effects was 1.8 mg/L.

Analysis of the test solutions at 0, 24 and 96 -hours showed the measured test concentrations to be in the range 90% to 122% of nominal; therefore, the results are based on nominal test concentrations.