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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:
6 June 2016 to 29 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control samples and test sample at nominal concentration of 100 % saturated solution. In each case, duplicate samples were taken, one for chemical analysis and one as a ‘back-up’ should further analysis be required.
- Sampling method: Concentrations of test material in Treated Mains Water (TMW) were determined by dilution followed by liquid chromatography with tandem mass spectrometry time of flight (LC-MS/MS TOF) analysis.
- Sample storage conditions before analysis: Samples (ca 20 mL) were taken from the control and test concentrations at 0 hours (fresh media) and 96 hours (old media) for analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: At the start of the range-finding 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 0.45µm filter to give a 100 % saturated solution. The remaining concentrations were achieved by serially diluting from the filtered top concentration.
Based on the results of the range-finding test, the definitive limit test was conducted at a nominal test concentration of 100 % saturated solution. At the start of the test, ca 1000 mg of the test substance was added to 10 L of treated mains water to give 100 mg/L concentration. This concentration was then stirred for ca 24 hours, prior to filtration through a 0.45µm filter to give a 100 % saturated solution.
- Controls: The control was prepared by adding treated mains water only to the control vessel.
- Chemical name of vehicle: Not applicable, the test material was added to water.
- Evidence of undissolved material: The control media was observed to be colourless solutions throughout the duration of the test. The 100 % saturated solution test vessel was observed as 100 % colourless solution at 0 hours for the remainder of the test it was observed as a non-homogenous liquid dispersion with white particulates at the surface and in suspension.
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 length of 4.82 cm and the length ranged of between 4.1 and 5.2 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 1.02 g and the weight ranged from 0.727 to 1.27 g.

ACCLIMATION
- Acclimation period: The fish were acclimatised to the test conditions for at least 12 days prior to testing.
- Acclimation conditions (same as test or not): 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: On a weekly basis during the holding period, a sub sample of 10 fish was weighed and a mean wet weight calculated. The wet weight was used to determine the quantity of food required for the number of fish remaining in the holding tank. The amount of food required per holding tank was weighed and stored in labelled feed containers place in the fish holding room. 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:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
63 – 73 mg/L
Test temperature:
13.3 – 16.4 °C
pH:
7.19 - 7.34
Dissolved oxygen:
85 – 87 %
Conductivity:
146.5 µs/cm
Nominal and measured concentrations:
Definitive test:
- Nominal concentrations of 100 % saturated solution
- Geometric mean measured concentrations: 68.8 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 15 L constructed glass aquaria
- Type: closed. Each glass aquaria was fitted with a plastic lid
- Material, size, headspace, fill volume: 10 L of test media.
- Renewal rate of test solution: There was no renewal of test solution. The test was conducted using static test conditions with media preparation at 0 hours only.
- No. of organisms per vessel: Seven fish per vessel.
- No. of vessels per concentration: One vessel per concentration.
- No. of vessels per control: One vessel per control.
- Biomass loading rate: 0.72 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 a particulate filter, an activated carbon filter and a UV steriliser.
- Colour: <0.6 mg/L Pt/Co
- Turbidity: 0.10 NTU
- Alkalinity: 22.0 mg/L HCO3
- Aluminium: <6.65 µg/L Al
- pH: 7.26
- BOD (Biological Oxygen Demand): 1.9 mg/L
- Cadmium: <0.040 µg/L Cd
- Ammoniacal Nitrogen: 0.01 mg/L NH4
- TON: 19.970
- Copper: <5.07 µg/L Cu
- Nitrite: <0.0130 mg/L NO2
- Calcium Hardness: 27.9501 mg/L CaCO3
- Iron: <9.5 µg/L Fe
- Lead: <0.39 µg/L Pb
- Manganese: <3.6 µg/L Mn
- Residual Chlorine: 0.06 – 0.06 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: No pH adjustment
- Photoperiod: The test was conducted in a temperature-controlled laboratory (15 ± 2 ºC) with a 16-hour light: 8-hours dark lighting cycle with ca 30-minute dawn/dusk phases.

EFFECT PARAMETERS MEASURED: All fish in each vessel were observed at ca 3.5 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 severe severity limit were humanely killed, in accordance with Schedule 1 of the UK Home Office Scientific Procedures Act (ASPA), 1986, and classed as mortalities for the following observational time point.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: Nominal concentrations of 0.1, 1.0, 10 and 100 % saturated solution.
- Results used to determine the conditions for the definitive study: No mortality was observed for the range-finding test, all fish in control and test vessels were observed as exhibiting no toxic symptoms.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: The result is based on the loading rate of the test material
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: The result is based on the loading rate of the test material
Details on results:
For the duration of the test, fish from the control and test concentration vessels showed no signs of toxic symptoms. Based on the loading rate the 96-hour LC50 value was estimated to be >100 mg/L. The corresponding NOEC value was 100 mg/L. The LOEC value was not calculated as there were no effects observed during the 96-hour test period.

- Behavioural abnormalities: An aggressive fish was observed in the control vessel at ca 21.5-hours, the fish was observed occasionally charging at the other fish, it was also noted that the fish was patrolling the centre of the tank. An aggressive fish was also observed at ca 53 hours, the fish was observed charging at the other fish and patrolling main body of water. Therefore, these fish were removed from the main test vessels and added to a separate 2 L glass beakers filled to ca 1.4 L to minimise the impact of the aggression to the other fish.No other signs of aggression were observed during the test.

- Observations on body length and weight:
The mean total length for all surviving fish was 4.82 cm; with minimum and maximum of 4.1 - 5.2 cm.
The mean wet weight for all surviving fish was 1.02 g, with minimum and maximum of 0.727 - 1.27 g.

- Any observations that might cause a difference between measured and nominal values: The limit of quantitation of the test material in treated mains water using this method was 0.1 mg/L. Analysis of the test preparations at 0 hours showed concentration of 70.5 mg/L and at 96 hours showed concentration of 67.1 mg/L. Although analysis showed that the measured concentration was relatively stable over the course of the 96-hour test period, the measured concentration was not within the range of 80-120 % of nominal. The geometric mean measured concentration was calculated to be 68.8 mg/L.

- Concentration of test material solutions
The measured value of the solutions represents just the soluble fraction. Having consulted the guidance documents OECD 23 Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures and OECD 2000 Use of the Harmonized system for the Classification of Chemicals which are Hazardous for the Aquatic Environment, both confirm that use of the loading rate is advocated for expressing exposure/reporting data concerning WAFs. It is therefore considered to be scientifically sound to report the concentration of the limit dose solution as 100 mg/L based on the loading rate.
Reported statistics and error estimates:
Statistical analysis was not carried out as no toxic effects were observed during the test. Therefore, results were derived empirically.

Test Media Descriptions

The control media was observed to be colourless solutions throughout the duration of the test. The 100 % saturated solution test vessel was observed as a colourless solution at 0 hours for the remainder of the test it was observed as a non-homogenous liquid dispersion with white particulates at the surface and in suspension.

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 >100 mg/L and the corresponding NOEC value was 100 mg/L based on the loading rate of the test material.
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 static test design. Based on the results of a range-finding test, the definitive limit test was conducted at a nominal test concentration of 100 % saturated solution. A control group was also included. Single 15 L constructed glass aquaria, containing 10 L of media, were used for the control and test concentration each containing 7 fish per vessel.

Analysis of the test media samples were conducted on fresh media at 0 hours and on corresponding old media at 96 hours. Chemistry analysis showed that the measured concentrations were relatively stable over the course of the 96-hour test period. The measured value of the solutions represents just the soluble fraction. Having consulted the guidance documents OECD 23 Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures and OECD 2000 Use of the Harmonized system for the Classification of Chemicals which are Hazardous for the Aquatic Environment, both confirm that use of the loading rate is advocated for expressing exposure/reporting data concerning WAFs. It is therefore considered to be scientifically sound to report the concentration of the limit dose solution as 100 mg/L based on the loading rate.

For the duration of the test, fish from the control and test concentration vessels showed no signs of toxic symptoms. The validity criteria for control mortality (=10 %) and dissolved oxygen (=60 %) were both satisfied. The test is therefore considered valid.

Under the conditions of the study, the 96-hour LC50 value was estimated to be >100 mg/L and the corresponding NOEC value was 100 mg/L based on the loading rate of the test material.

Description of key information

Under the conditions of the study, the 96-hour LC50 value was estimated to be >100 mg/L and the corresponding NOEC value was 100 mg/L based on the loading rate of the test material.

Key value for chemical safety assessment

LC50 for freshwater fish:
100 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 awarded a reliability score of 1 in accordance with the criteria 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 static test design. Based on the results of a range-finding test, the definitive limit test was conducted at a nominal test concentration of 100 % saturated solution. A control group was also included. Single 15 L constructed glass aquaria, containing 10 L of media, were used for the control and test concentration each containing 7 fish per vessel.

Analysis of the test media samples were conducted on fresh media at 0 hours and on corresponding old media at 96 hours. Chemistry analysis showed that the measured concentrations were relatively stable over the course of the 96-hour test period. The measured value of the solutions represents just the soluble fraction. Having consulted the guidance documents OECD 23 Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures and OECD 2000 Use of the Harmonized system for the Classification of Chemicals which are Hazardous for the Aquatic Environment, both confirm that use of the loading rate is advocated for expressing exposure/reporting data concerning WAFs. It is therefore considered to be scientifically sound to report the concentration of the limit dose solution as 100 mg/L based on the loading rate.

For the duration of the test, fish from the control and test concentration vessels showed no signs of toxic symptoms. The validity criteria for control mortality (=10 %) and dissolved oxygen (=60 %) were both satisfied. The test is therefore considered valid.

Under the conditions of the study, the 96-hour LC50 value was estimated to be >100 mg/L and the corresponding NOEC value was 100 mg/L based on the loading rate of the test material.