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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:
24 March 2017 to 10 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The control and all surviving test groups
- Sampling method: Water samples were taken at 0 and 72 hours from fresh media and at 24 and 96 hours from old media for quantitative analysis.
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis. Duplicate samples and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) were taken and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Preliminary solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. Based on this information the test material was categorised as being a ‘difficult substance’ as defined by OECD. Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.
Following the trial, the maximum dissolved test material concentration was obtaining following a 24-hour preparation period. There were no significant differences between the pre-treatment types (centrifugation under different conditions and filtration). As near nominal concentrations were obtained, the initial loading rate of the saturated solution was increased to 100 mg/L to ensure the maximum dissolved test material concentration was obtained.
Based on this information the test material was prepared using a saturated solution method of preparation at an initial loading rate of 100 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test material by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded) to give a 100 % v/v saturated solution.
In the definitive test, a nominal amount of test material (2200 mg) was dispersed in 22 litres of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to give a 100 % v/v saturated solution. An aliquot (2.5 litres) of this 100 % v/v saturated stock solution was taken and the volume adjusted to 5 litres to give the 50 % v/v saturated solution. A series of dilutions was made from the 50 % v/v saturated solution to give test concentrations of 0.16, 0.40, 1.0, 2.5 and 6.25 % v/v saturated solution. Each stock solution was inverted several times and each test concentration was mixed with a flat bladed stirrer for approximately 1 minute 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: juvenile
- Length at study end: 4.4 cm (sd = 0.41)
- Weight at study end: 1.1 g (sd = 0.30)
- Method of breeding: Not specified; obtained from commercial suppler

ACCLIMATION
- Acclimation period: 03 April 2017 to 10 April 2017
- Acclimation conditions: Fish were maintained in a glass fibre tank with a "single pass" water renewal system. The water temperature was controlled at 14 to 15 °C with a dissolved oxygen content of greater than or equal to 8.9 mg O2/L. The lighting cycle was controlled to give a cycle with 16 hours of light and 8 hours of darkness with 20 minute dawn and dusk transition periods. The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
- Type and amount of food during acclimation: The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test.
- Health during acclimation: 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:
140 mg/L as CaCO3
Test temperature:
14 to 15 °C
pH:
7.6 to 8.2
Dissolved oxygen:
8.9 to 11.0 mg O2/L
Nominal and measured concentrations:
- Nominal: 0.16, 0.40, 1.0, 2.5 and 6.25 % v/v saturated solution
- Measured: 0.030, 0.18, 0.41, 1.2 and 3.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 25-30 litre glass exposure vessels
- Type: Covered to reduce evaporation
- Material, size, headspace, fill volume: 20 litres
- Aeration: The test vessels were aerated via narrow bore glass tubes
- Renewal rate of test solution: A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build-up of nitrogenous waste products.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.40 g bodyweight/litre

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) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Metals: Aluminium 7.46 mg/L, iron 19.9 µg/L, manganese 2.9 µg/L, sodium 22.35 mg/L, copper 0.02 mg/L, lead 0.51 µg/L and nickel 3.1 µg/L.
- Chlorine: 0.32 mg/L (prior to dechlorination)
- Culture medium different from test medium: No
- 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
- Photoperiod: 16 hours of light and 8 hours of darkness with 20 minute dawn and dusk transition periods

EFFECT PARAMETERS MEASURED:
Any mortalities and sub-lethal effects of exposure were recorded at 1, 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: 2.5
- Range finding study: Yes
- Test concentrations: 1.0, 10 and 100 % v/v saturated solution
- Results used to determine the conditions for the definitive study: Yes. The results showed 100 % mortality at 10 and 100 % v/v saturated solution and 33 % mortality at 1.0 % v/v saturated solution at 96 hours. After approximately 1 hour of exposure 1 fish at 10 % v/v saturated solution was observed to have a loss of equilibrium and to be swollen. Due to animal welfare implications this fish was killed and classed as a mortality for this observational time point. Based on this information test concentrations were selected for the definitive test.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.9 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: 95 % CL 1.2 to 3.0 mg/L
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
DEFINITIVE TEST
Cumulative mortality data from the exposure of rainbow trout to the test material during the definitive test is given in Table 1. Inspection of the mortality data at 1, 3 and 6 hours and analysis of the mortality data using Binomial distribution at 24, 48, 72 and 96 hours based on the average fresh measured test concentrations gave the following results: at 1, 3 and 6 hours the LC50 was >3.0 mg/L; at 24, 48, 72 and 96 h the LC50 was 1.9 mg/L (95 % CL 1.2 to 3.0 mg/L, concentrations resulting in 0 and 100 % mortality, respectively).
The Lowest Observed Effect Concentration (LOEC) was considered to be 3.0 mg/L and the No Observed Effect Concentration (NOEC) was 1.2 mg/L.
Sub-lethal effects of exposure were observed at test concentrations of 1.2 and 3.0 mg/L and above. These responses were swimming at the surface and pale in colour.

The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was greater than or equal to 60 % of ASV (6.1 mg O2/L) in the control and test vessels.
The test material preparations were observed to be clear colourless solutions throughout the test.
Reported statistics and error estimates:
An estimate of the LC50 values at 1, 3 and 6 hours was given by inspection of the mortality data. The LC50 values and associated confidence limits at 24, 48, 72 and 96 hours and the slope of the response curve and its standard error were calculated by Binomial distribution. The LOEC and NOEC values at 96 hours were calculated using the Fisher’s Exact Binomial Test with Bonferroni correction. All results were calculated using the ToxRat Professional computer software package (TOXRAT).
Sublethal observations / clinical signs:

Table 1: Cumulative Mortality Data in the Definitive Test

Average Fresh Measured Test
Concentration
(mg/L)

Cumulative Mortality (Initial Population = 7)

%

Mortality

1
Hour

3
Hours

6
Hours

24 Hours

48 Hours

72 Hours

96 Hours

96
Hours

Control

0

0

0

0

0

0

0

0

0.030

0

0

0

0

0

0

0

0

0.18

0

0

0

0

0

0

0

0

0.41

0

0

0

0

0

0

0

0

1.2

0

0

0

0

0

0

0

0

3.0

0

0

0

7

7

7

7

100

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study the 96 hour LC50 was 1.9 mg/L with 95 % CL of 1.2 to 3.0 mg/L and the No Observed Effect Concentration (NOEC) was 1.2 mg/L.
Executive summary:

The acute toxicity of the test material to fish was determined in accordance with the standardised guidelines OECD 203 and EU Method C.1 under GLP conditions using Oncorhynchus mykiss.

Preliminary solubility work conducted 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. A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 46 mg/L was obtained from a saturated solution method of preparation, with an initial loading rate of 50 mg/L, indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test, fish were exposed, in groups of seven, to an aqueous solution of the test material at nominal concentrations of 0.16, 0.40, 1.0, 2.5 and 6.25 % v/v saturated solution for a period of 96 hours at a temperature of 14 to 15 °C under semi-static test conditions. The test material solution was prepared by stirring an excess (100 mg/L) of test material in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test material was removed by filtration to produce a 100 % v/v saturated solution of the test material. This saturated solution was then further diluted as necessary, to provide the remaining test groups. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 1, 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Analysis of the freshly prepared test preparations at 0 and 72 hours showed measured test concentrations to range from less than the limit of quantification (0.059 mg/L) to 3.0 mg/L. There was no significant change in the measured concentrations at 24 and 96 hours and so the results are based on average fresh measured test concentrations only. Where values of less than the LOQ were obtained the average concentration was calculated substituting that value with half of the LOQ (0.030 mg/L). The average fresh measured test concentrations were determined to be 0.030, 0.18, 0.41, 1.2 and 3.0 mg/L.

After 96 hours, 100 % mortality was observed in the highest test material concentration. Sub-lethal effects of exposure were observed at test concentrations of 1.2 and 3.0 mg/L and above. These responses were swimming at the surface and pale in colour.

The validity criteria of the test were fulfilled.

Under the conditions of this study the 96 hour LC50 was 1.9 mg/L with 95 % CL of 1.2 to 3.0 mg/L and the No Observed Effect Concentration (NOEC) was 1.2 mg/L. 

Description of key information

The acute toxicity of the test material to fish was determined in accordance with the standardised guidelines OECD 203 and EU Method C.1 under GLP conditions using Oncorhynchus mykiss. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).


 


Preliminary solubility work conducted 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. A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 46 mg/L was obtained from a saturated solution method of preparation, with an initial loading rate of 50 mg/L, indicating this to be the limit of water solubility of this material under test conditions.


 


Following a preliminary range-finding test, fish were exposed, in groups of seven, to an aqueous solution of the test material at nominal concentrations of 0.16, 0.40, 1.0, 2.5 and 6.25 % v/v saturated solution for a period of 96 hours at a temperature of 14 to 15 °C under semi-static test conditions. The test material solution was prepared by stirring an excess (100 mg/L) of test material in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test material was removed by filtration to produce a 100 % v/v saturated solution of the test material. This saturated solution was then further diluted as necessary, to provide the remaining test groups. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 1, 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.


 



Analysis of the freshly prepared test preparations at 0 and 72 hours showed measured test concentrations to range from less than the limit of quantification (0.059 mg/L) to 3.0 mg/L. There was no significant change in the measured concentrations at 24 and 96 hours and so the results are based on average fresh measured test concentrations only. Where values of less than the LOQ were obtained the average concentration was calculated substituting that value with half of the LOQ (0.030 mg/L). The average fresh measured test concentrations were determined to be 0.030, 0.18, 0.41, 1.2 and 3.0 mg/L.



After 96 hours, 100 % mortality was observed in the highest test material concentration. Sub-lethal effects of exposure were observed at test concentrations of 1.2 and 3.0 mg/L and above. These responses were swimming at the surface and pale in colour.



The validity criteria of the test were fulfilled.



Under the conditions of this study the 96 hour LC50 was 1.9 mg/L with 95 % CL of 1.2 to 3.0 mg/L and the No Observed Effect Concentration (NOEC) was 1.2 mg/L. 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
LC50
Effect concentration:
1.9 mg/L

Additional information