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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:
07 March 2016 to 04 May 2016
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:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nominal Concentrations of 6.25, 12.5, 25.0, 50.0 and 100.0 (% saturated solution). Concentrations of test material were determined by dilution followed by liquid chromatography with tandem mass spectrometry time of flight (LC-MS/MS TOF) analysis.
- Sampling method: Samples (ca. 20 mL) were taken from the control and test concentration at 0 and 72 hours (fresh media), and 24 and 96 hours (old media) for analysis. In each case duplicate samples were taken, one for chemical analysis and one as a ‘back-up’ should further analysis be required.
Vehicle:
no
Details on test solutions:
PRELIMINARY SOLUBILITY WORK
Initial solubility work carried out indicated that a test concentration of 1 mg/L could not be achieved. Therefore a media trial was conducted using saturated solution preparation method, analytical samples were also taken. This media trial showed that the test substance would achieve higher concentrations with a saturated solution preparation method.

PREPARATION AND APPLICATION OF TEST SOLUTION
At the start of the test and at media renewals ca 1500 mg of the test substance was added to 15 L of treated mains water to give 100 mg/L top concentration. This top concentration was then stirred for ca 24 hours. After stirring the top concentration was then filtered through a 0.45 μm filter to give a 100 % saturated solution. The remaining concentrations were achieved by serially diluting from the filtered top concentration.

APPEARANCE OF TEST MEDIA
The appearance, colour and behaviour of the test substance in the freshly prepared and old test media was recorded during the test. The test preparations were observed to be colourless solutions throughout the duration of the test.

APPLICATION OF TEST SOLUTION
At the start of the test, seven Oncorhynchus mykiss, randomly selected from a holding stock, were added to each test and control vessel. The fish were not fed during the test. All fish in each test vessel were observed at ca 4 hours after addition and then at 24-hour intervals (24, 48, 72 and 96 hours) throughout the duration of the test. The number of dead fish and those fish exhibiting toxic symptoms or modified behaviour were recorded, if applicable. Any fish exhibiting toxic symptoms at or above the substantial severity limit were humanely killed and classed as mortalities for the following observational time point. At the end of the test, all surviving fish were humanely killed.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Length at study completion: A sub-sample of ten fish taken at random from all surviving fish after 96 hours gave a mean total lenght of 4.33 cm and the length ranged between 4.0 and 4.6 cm.
- Weight at study completion: Sub-sample of ten fish taken at random from all surviving fish after 96 hours gave a mean total weight of 0.703 g and the weight ranged from 0.51 to 0.90 g.
The total length and wet weight of ten randomly selected fish from the stock fish were determined periodically to ensure fish were of a suitable size and weight for testing and to ensure the correct testing volumes were used so as not to exceed the recommended loading rate.

ACCLIMATION
- Acclimation period: The fish were acclimatised to the test conditions for at least 12 days prior to testing.
- Acclimation conditions: Same as test.
- Type and amount of food during acclimation: The fish were fed with a proprietary fish food, which was added to the holding tank in quantities dictated by the size of the fish.
- Feeding frequency during acclimation: The feed was distributed to the holding tank over two feeding intervals during the day. Uneaten food and debris was siphoned or cleaned from the tanks as required.
- Health during acclimation: Fish mortalities in the holding tank were recorded as they occurred. Use of the fish for testing was considered acceptable if the cumulative mortality in the batch was <5 % during the 7-day period preceding the proposed test start date. If the mortality exceeded 5 % mortality in this same period, or disease was apparent, the batch of fish was not to be used for testing and acclimation would be increased for at least another 7 days before mortality rate re-assessment for testing. The mortality rate of the stock batch of fish was <5 % in the 7 days prior to the definitive test.

FEEDING DURING TEST
- Feeding: The fish were not fed for at least 24 hours before the start of the definitive test and were not fed throughout the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
52 - 64 mg/L
Test temperature:
13.1 – 14.2 °C
pH:
6.88 – 7.18
Dissolved oxygen:
96 - 102 %
Conductivity:
146.5 µs/cm
Nominal and measured concentrations:
Nominal test concentrations: 6.25, 12.5, 25, 50 and 100 % saturated solutions.
Measured concentrations (Time weighted mean): 2.56, 4.02, 8.95, 15.6 and 35.4 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 20 L constructed glass aquaria.
- Type: Closed; the test vessel was fitted with a plastic lid.
- Material, size, headspace, fill volume: Test vessel contained 5 L of media.
- Renewal rate of test solution: Daily renewal of test media.
- No. of organisms per vessel: Each test vessel contained 7 fish.
- No. of vessels per concentration: One vessel per concentration.
- No. of vessels per control: One vessel per control.
- Biomass loading rate: 0.9842 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for the holding of fish stocks and for the toxicity tests was laboratory mains supply. The water was pumped to the laboratory through an activated carbon filter.
- Residual Chlorine: 0.02 – 0.04 mg/L
- Alkalinity: 22.0 mg/L HCO3
- Water Quality Determinations:
At the start of the test and at approximately 24-hour intervals during the test, the media pH, concentration of dissolved oxygen (% air saturation value and mg/L) and the individual test media temperatures were recorded in the freshly prepared and old test media.
The total hardness (mg/L as CaCO3) and residual chlorine (mg/L) of the treated mains water used to prepare the test media were measured at the start of each media renewal time point.
The protocol stated that the temperature should not vary by more than 1 °C from the initial vessel temperature. However, the maximum temperature deviation from the initial temperature (15.9 °C) was 2.7 °C. This can be put down to the unexpectedly high temperature of the treated mains water on the day of 0 hour media preparation. After reviewing the water quality data the water temp at 0 hours was ~2.5 – 3 °C warmer than the media preps at 24, 48 and 72 hours.

OTHER TEST CONDITIONS
- Adjustment of pH: Yes. The pH should be maintained within the range of 6 – 8.5. The freshly prepared top concentration was lower than this range, the media was therefore, adjusted to within this range using 2M NaOH.
- Photoperiod: 16 hour light: 8 hours dark lighting cycle plus ca 30 minute dawn/dusk phases.


EFFECT PARAMETERS MEASURED: All fish in each test vessel were observed at ca 4 hours after addition and then at 24-hour intervals (24, 48, 72 and 96 hours) throughout the duration of the test. The number of dead fish and those fish exhibiting toxic symptoms or modified behaviour were recorded, if applicable. Any fish exhibiting toxic symptoms at or above the substantial severity limit were humanely killed and classed as mortalities for the following observational time point.


TEST CONCENTRATIONS
- Range finding study: The range-finding test was conducted under static test conditions. A control group was also included. At the start of the test, ca 500 mg of the test substance was added to 5 L of treated mains water to give 100 mg/L top concentration. This top concentration was then stirred for ca 24 hours. After stirring the top concentration was then filtered through a 0.45 μm filter to give a 100 % saturated solution. The remaining concentrations were achieved by serially diluting from the filtered top concentration. Single test vessels were prepared, for the control and at each test concentration, each containing three fish.
- Test concentrations: Nominal test substance concentrations of 0.1, 1.0, 10 and 100 % saturated solutions.
- Results used to determine the conditions for the definitive study: Yes. For the duration of the range-finding test all fish in control and test vessels were observed as exhibiting no toxic symptoms.
Based on nominal concentrations, the results of the range-finding test suggested that the 96-hour LC50 value would be greater than 100 % saturated solution (nominal concentration).
However, an initial experiment was carried out as a limit test, with control and top concentration (100 % saturated solution) only. However, after 24 hours mortality was observed in the test concentration, then at 48 hours a further 2 mortalities were recorded. The test was therefore ended at 48 hours.
During the range-finder test analytical chemistry results were from repeat analysis, batches were re-run due to issues with low recoveries, below acceptance criteria of 80-120 % of nominal and poor calibrations. However, these issues were not seen during the definitive phase and therefore, are not considered to have impacted on the study integrity.
Based on the results of the range-finding test, the definitive test was conducted at nominal test concentrations of 6.25, 12.5, 25, 50 and 100 % saturated solutions.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
26.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
17.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
39.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Behavioural abnormalities: None specified
- Observations on body length and weight: The length and wet weight of a sub-sample of ten fish (from all surviving fish) were measured and recorded at the end of the test.
Mean Total Length: 4.33 cm (4.0 – 4.6 cm)
Mean Wet Weight: 0.703 g (0.51 – 0.90 g)
Fish Loading Rate: 0.9842 g/L
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test preparations were observed to be colourless solutions throughout the duration of the test.
- Effect concentrations exceeding solubility of substance in test medium: Analysis of the test preparations at 0 hours showed concentrations ranged from 1.30 to 22.3 mg/L, at 24 hours test concentrations ranged from 1.47 to 21.6 mg/L. At 72 hours concentrations ranged from 3.38 to 53.6 mg/L and at 96 hours concentrations ranged from 4.02 to 60.9 mg/L. Although analysis showed that the measured concentrations were relatively stable over the course of the 24-hour renewal periods, the measured concentrations were not within the range of 80-120 % of nominal. The results have therefore been reported in terms of the time weighted mean measured concentrations. The measured concentrations also showed that there was quite substantial variation of achieved concentration after stirring and filtering on the different media preparation days, however, this was within the variation seen for the test substance.


Reported statistics and error estimates:
Statistical analysis was performed using the CETIS program v 1.8.6.8. The LC50 values were estimated using linear interpolation. The NOEC and LOEC values were established by observation of the data.
Sublethal observations / clinical signs:

Toxicity of the test material to Oncorhynchus mykiss

The LC50 values and corresponding NOEC and LOEC values are presented in the table below. The results are presented in terms of geometric mean measured concentration.

Summary of results for acute toxicity to Oncorhynchus Mykiss

Parameter

Toxicity Value (mg/L)

24 hours

48 hours

72 hours

96 hours

LC50

> 39.6

> 39.6

28.1

26.3

NOEC

39.6

39.6

17.3

17.3

LOEC

 > 39.6

 > 39.6

39.6

 39.6

Validity Criteria

The validity criteria for solvent control mortality (less than 10 %) and dissolved oxygen (>60 % ASV) were both satisfied. The test was therefore considered to be valid.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 96-hour LC50 value was estimated to be 26.3 mg/L and the corresponding NOEC and LOEC values were 17.3 mg/L and 39.6 mg/L, respectively.
Executive summary:

The acute toxicity of the test material to Oncorhynchus mykiss (Rainbow Trout), was assessed in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 203.

A study was conducted to determine the 96-hour acute toxicity of the test substance to Oncorhynchus mykiss. The study was conducted using a semi-static test design with daily renewal of test media during the test.

Based on the results of a range-finding test, the definitive test was conducted at nominal test concentration of 6.25, 12.5, 25, 50 and 100 % saturated solutions. A control group was also included.

Single 20 L constructed glass aquaria, containing 5 L of media, were used for the control and test concentration each containing 7 fish.

Analysis of the test media samples was conducted on fresh media at 0, and 72 hours and on corresponding old media at 24 and 96 hours.

Chemistry analysis showed that the measured concentrations were relatively stable over the course of the 24-hour renewal period.

As measured concentrations were not within the range of 80-120 % of nominal, and the test was run semi-static the results were reported in terms of the time weighted mean measured concentrations.

Time weighted mean measured concentrations were calculated to be 2.54, 4.55, 10.6, 17.3 and 39.6 mg/L.

The validity criteria for control mortality (≤10 %) and dissolved oxygen (≥60 %) were both satisfied. The test is therefore considered valid.

Based on time weighted mean measured concentrations, the 96-hour LC50 value was estimated to be 26.3 mg/L. The corresponding NOEC and LOEC values were 17.3 mg/L and 39.6 mg/L, respectively.

The validity criteria for control mortality (≤10 %) and dissolved oxygen (≥60 %) were both satisfied. The test is therefore considered valid.

Description of key information

Under the conditions of the study, the 96-hour LC50 value was estimated to be 26.3 mg/L and the corresponding NOEC and LOEC values were 17.3 mg/L and 39.6 mg/L, respectively.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
26.3 mg/L

Additional information

The acute toxicity of the test material to Oncorhynchus mykiss (Rainbow Trout), was assessed in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 203. 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 study was conducted to determine the 96-hour acute toxicity of the test substance to Oncorhynchus mykiss. The study was conducted using a semi-static test design with daily renewal of test media during the test.

Based on the results of a range-finding test, the definitive test was conducted at nominal test concentration of 6.25, 12.5, 25, 50 and 100 % saturated solutions. A control group was also included.

Single 20 L constructed glass aquaria, containing 5 L of media, were used for the control and test concentration each containing 7 fish.

Analysis of the test media samples was conducted on fresh media at 0, and 72 hours and on corresponding old media at 24 and 96 hours.

Chemistry analysis showed that the measured concentrations were relatively stable over the course of the 24-hour renewal period.

As measured concentrations were not within the range of 80-120 % of nominal, and the test was run semi-static the results were reported in terms of the time weighted mean measured concentrations.

Time weighted mean measured concentrations were calculated to be 2.54, 4.55, 10.6, 17.3 and 39.6 mg/L.

The validity criteria for control mortality (≤10 %) and dissolved oxygen (≥60 %) were both satisfied. The test is therefore considered valid.

Based on time weighted mean measured concentrations, the 96-hour LC50 value was estimated to be 26.3 mg/L.The corresponding NOEC and LOEC values were 17.3 mg/L and 39.6 mg/L, respectively.