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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:
06 January 2013 to 02 February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
see below
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
The test vessels were shielded from light for a period of 96 hours during the test. The guidelines state the need for 12-16 hours light per day; however, this is not thought to impact the validity of the test as this was required due to the nature of the test material.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentrations
- Sampling method: Samples were taken from the control and all surviving test groups at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) for quantitative analysis. Duplicate samples and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) were taken and stored for further analysis if necessary
- Sample storage conditions before analysis: The 0, 24 and 72 hour samples were stored at approximately -20 °C prior to analysis with the 96 hour samples (analysed on day pf receipt). The samples taken for further analysis were stored at approximately -20 °C.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The water solubility of the test material is 342.6 mg/L. Pre-study solubility work indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing. Based on this information, media preparation trials were conducted in order to determine the solubility of the test material under test conditions. Prolonged stirring followed by the removal of any undissolved test material by filtration was deemed an appropriate method.

An amount of test material (1100 mg) was dissolved in 11 litres of dechlorinated tap water with the aid of a propeller stirrer at a rate of approximately 1500 rpm for a period of 24 hours. The test material was weighed out under non-actinic light conditions and the stirring vessel was shielded from light as the test material was sensitive to light. After stirring, as a precautionary measure, any undissolved test material was removed with a 0.2 µm Sartorius Sartopore filter (initial approximate 2 litres discarded to pre-condition the filter) to give the 100 % v/v solution. Aliquots (200, 360, 640, 1120 and 2000 mL) of the 100 % v/v solution were each separately added to a final volume of 20 litres of dechlorinated tap water to give the 1.0, 1.8, 3.2, 5.6 and 10 % v/v test concentrations. All test preparations were shielded from light as the test material was light-sensitive.
Each prepared concentration was stirred using a flat-bladed mixer to ensure adequate mixing and homogeneity.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow Trout
- Age at study initiation (mean and range, SD):
- Length at end of definitive test (mean and SD): 4.8 cm (SD = 0.4)
- Weight at end of definitive test (mean and range, SD): 1.35 g (SD = 0.32)
- Feeding during test: No; feeding was discontinued approximately 24 hours prior to the start of the definitive test

ACCLIMATION
- Acclimation period: 13 days
- Acclimation conditions (same as test or not): No. Fish were maintained in a glass fibre tank with a "single pass" water renewal system. The lighting cycle was controlled to give a cycle of 16 hours of light and 8 hours of darkness cycle with 20 minute dawn and dusk transition periods. Fish in the main test were kept in the dark as a requirement of the nature of the test material.
The water temperature was controlled at 13 to 14 °C with a dissolved oxygen content of greater than or equal to 10.5 mg O2/L. These measured values indicate that the requirement for >80 % air saturation value (ASV) was obtained during this holding period.
- Type and amount of food: Commercial trout pellets
- Feeding frequency: Once a day
- Health during acclimation (any mortality observed): There was no mortality in the 7 days prior to the start of the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
Approximately 140 mg CaCO3/L
Test temperature:
13 to 14 °C
pH:
7.5 to 8.3
Dissolved oxygen:
Greater than or equal to 10.5 mg O2/L.
Nominal and measured concentrations:
- Nominal concentrations: 1.0, 1.8, 3.2, 5.6 and 10 % v/v
- Mean measured concentrations: 2.4, 4.5 and 7.2 mg/L for the 3.2, 5.6 and 10 % v/v nominal concentrations, respectively
Details on test conditions:
TEST SYSTEM
- Test vessel: 20 litre glass exposure vessels were used for each test concentration
- Type: closed (covered to reduce evaporation)
- Aeration: Yes; via narrow bore glass tubes
- Renewal rate of test solution (frequency/flow rate): Daily renewal of the test preparations took place to ensure that the concentrations of the test material remained near nominal and to prevent the build-up of nitrogenous waste products.
- No. of organisms per vessel: seven
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: loading rate of 0.47 g bodyweight/litre (static volume)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener). After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Total organic carbon: 0.920 mg/L
- Pesticides: 0.009 mg/L
- Chlorine: 0.270 mg/L
- Culture medium different from test medium: No. The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
- Intervals of water quality measurement: The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-hour old test preparations.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: The test vessels were shielded from light during the study as a result of the nature of the test material.

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 movement and response to physical stimulation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study. Yes
- Test concentrations: 1.0, 10 and 100 % v/v
- Results used to determine the conditions for the definitive study: Yes. The results showed no mortalities at the test concentration of 1.0 % v/v. However, mortalities were observed at 10 and 100 % v/v. After 24 hours, 100 % mortality was observed in the 100 % v/v saturated solution test concentration. After approximately 24 hours exposure, 3 out of 3 fish at 10 % v/v saturated solution were observed to exhibit prolonged sub-lethal effects. Due to animal welfare implications these fish were killed and classed as mortalities for the following observational time point.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
5.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % CL 4.6 to 6.1 mg/L
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
4.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
2.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
The results of the definitive test showed the highest test concentration based on mean-measured test concentrations resulting in 0 % mortality to be 2.4 mg/L, the lowest test concentration resulting in 100 % mortality to be 7.2 mg/L. The Lowest Observed Effect Concentration (LOEC) was considered to be 4.5 mg/L. The No Observed Effect Concentration (NOEC) was 2.4 mg/L.
Sub-lethal effects of exposure were observed at test concentrations of 7.2 mg/L. These responses were observed to be 4 out of 4 fish with swollen abdomens. Due to animal welfare implications these fish were killed and classed as mortalities for the following observational time point.
The test preparations were observed to be clear, colourless solutions for the duration of the test.

VERIFICATION OF TEST CONCENTRATIONS
Analysis of the 3.2, 5.6 and 10 % v/v test preparations at 0, 24, 72 and 96 hours showed measured test concentrations to range from 1.8 to 7.9 mg/L. Given the slight variability in measured concentrations obtained with each media measured it was considered appropriate to calculate the results based on the mean measured test concentrations.
Reported statistics and error estimates:
The LC50 values were determined by analysis of the mortality data by the geometric mean method at 24 hours and by the trimmed Spearman-Karber method (Hamilton et al, 1977) at 48, 72 and 96 hours based on the mean-measured test concentrations.

Table 1: Cumulative Mortality Data in the Definitive Test

Nominal Concentration (%v/v)

Mean Measured Concentration (mg/L)

Cumulative Mortality (Initial Population 7)

% 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

Not analysed

0

0

0

0

0

0

0

1.8

Not analysed

0

0

0

0

0

0

0

3.2

2.4

0

0

0

0

0

0

0

5.6

4.5

0

0

0

1

1

1

14

10

7.2

0

0

7*

7

7

7

100

*3 fish were found dead and the remaining 4 had to be euthanised for humane reasons due to significant sub-lethal effects approaching the substantial severity limit at 21 hours after exposure (swollen abdomen).

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, a 96 h LC50 value of 5.3 mg/L was obtained with 95 % confidence limits of 4.6 - 6.1 mg/L. The Lowest Observed Effect Concentration and No Observed Effect Concentration based on mean-measured test concentrations were considered to be 4.5 and 2.4 mg/L, respectively.
Executive summary:

The toxicity of the test material to Rainbow Trout (Oncorhynchus mykiss) was investigated in accordance with the standardised guidelines OECD 203 and EU Method C.1 under GLP conditions.

A pre-study media preparation trial indicated that the use of a prolonged stirring method of preparation followed by the removal of any undissolved test material by filtration was appropriate for this test material.

Following a preliminary range-finding test, fish were exposed, in groups of seven, to an aqueous solution of the test material over a range of nominal concentrations of 1.0, 1.8, 3.2, 5.6 and 10 % v/v for a period of 96 hours at a temperature of 13 to 14 °C under semi-static test conditions. The test material solution was prepared by dissolving 100 mg/L of test material in test medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period, as a precautionary measure, any undissolved test material was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a 100 % v/v solution of the test material from which dilutions were prepared to give the required test concentrations. 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 throughout the test until termination after 96 hours.

Water samples were taken from the control and all surviving test groups at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) for quantitative analysis. Duplicate samples and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) were taken and stored at approximately -20 °C for further analysis if necessary.

Analysis of the 3.2, 5.6 and 10 % v/v test preparations at 0, 24, 72 and 96 hours showed measured test concentrations to range from 1.8 to 7.9 mg/L. Given the slight variability in measured concentrations obtained with each media renewal it was considered appropriate to calculate the results based on the mean measured test concentrations.

Three mortalities occurred in the 7.2 mg/L dose group within 24 hours. One mortality was observed in the 4.5 mg/L dose group after 48 hours. Sub-lethal effects of exposure were observed at test concentrations of 7.2 mg/L at 24 hours. These responses were observed to be 4 out of 4 fish with swollen abdomens. Due to animal welfare implications these fish were killed and classed as mortalities for the following observational time point.

Under the conditions of the study, a 96 h LC50 value of 5.3 mg/L was obtained with 95 % confidence limits of 4.6 - 6.1 mg/L. The Lowest Observed Effect Concentration and No Observed Effect Concentration based on mean-measured test concentrations were considered to be 4.5 and 2.4 mg/L, respectively.

Description of key information

A 96 h LC50 value of 5.3 mg/L was obtained with 95 % confidence limits of 4.6 - 6.1 mg/L.

Key value for chemical safety assessment

LC50 for freshwater fish:
5.3 mg/L

Additional information

The toxicity of the test material to Rainbow Trout (Oncorhynchus mykiss) was investigated in accordance with the standardised guidelines OECD 203 and EU Method C.1 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

A pre-study media preparation trial indicated that the use of a prolonged stirring method of preparation followed by the removal of any undissolved test material by filtration was appropriate for this test material.

Following a preliminary range-finding test, fish were exposed, in groups of seven, to an aqueous solution of the test material over a range of nominal concentrations of 1.0, 1.8, 3.2, 5.6 and 10 % v/v for a period of 96 hours at a temperature of 13 to 14 °C under semi-static test conditions. The test material solution was prepared by dissolving 100 mg/L of test material in test medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period, as a precautionary measure, any undissolved test material was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a 100 % v/v solution of the test material from which dilutions were prepared to give the required test concentrations. 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 throughout the test until termination after 96 hours.

Water samples were taken from the control and all surviving test groups at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) for quantitative analysis. Duplicate samples and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) were taken and stored at approximately -20 °C for further analysis if necessary.

Analysis of the 3.2, 5.6 and 10 % v/v test preparations at 0, 24, 72 and 96 hours showed measured test concentrations to range from 1.8 to 7.9 mg/L. Given the slight variability in measured concentrations obtained with each media renewal it was considered appropriate to calculate the results based on the mean measured test concentrations.

Three mortalities occurred in the 7.2 mg/L dose group within 24 hours. One mortality was observed in the 4.5 mg/L dose group after 48 hours. Sub-lethal effects of exposure were observed at test concentrations of 7.2 mg/L at 24 hours. These responses were observed to be 4 out of 4 fish with swollen abdomens. Due to animal welfare implications these fish were killed and classed as mortalities for the following observational time point.

Under the conditions of the study, a 96 h LC50 value of 5.3 mg/L was obtained with 95 % confidence limits of 4.6 - 6.1 mg/L. The Lowest Observed Effect Concentration and No Observed Effect Concentration based on mean-measured test concentrations were considered to be 4.5 and 2.4 mg/L, respectively.