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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:
The study was conducted between 24 February 2017 and 30 March 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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: LA-63P
Physical state/Appearance: Extremely pale yellow powder
Batch: 112Z5
Purity: 100%
Expiry Date: 30 December 2017
Storage Conditions: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and 100% v/v saturated solution test vessel at 0 and 72 hours from fresh media and at 24 and 96 hours from old media for quantitative 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:
The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
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.

Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000).
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in-house since 06 January 2017. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 13 March 2017 to 20 March 2017.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
140 mg/L as CaCO3
Test temperature:
14 °C
pH:
7.4 to 8.1
Dissolved oxygen:
9.3 mg O2/L
Nominal and measured concentrations:
Nominal amoumnt of test item (1100 mg) was dispersed in 22 litres of water.
Details on test conditions:
There was 1.3% mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.8 cm (sd = 0.30) and a mean weight of 1.7 g (sd = 0.22) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.60 g bodyweight/liter (static volume).

Range-finding Test
The test concentration to be used in the definitive test was determined by a preliminary range-finding test.

In the initial range-finding test fish were exposed to a series of nominal test concentrations of 1.0, 10 and 100% v/v saturated solution for a period of 96 hours.

Nominal amounts of test item (1100 and 550 mg) were dispersed in 22 and 11 liters of culture medium, respectively, with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) and both preparations pooled to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test solutions of 1.0 and 10% v/v saturated solution.
Each of the test preparations were mixed with a flat bladed stirrer for approximately 1 minute to ensure adequate mixing and homogeneity.

In the range-finding test three fish were placed in each test and control vessel and maintained in a temperature controlled room at 14 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours under static test conditions. Each 25-30 liter test and control vessel contained 20 liters of test media and was covered to reduce evaporation. After 1, 3, 6, 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish.

The control group was maintained under identical conditions but not exposed to the test item.

A sample of each test concentration was taken for chemical analysis at 0 and 24 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive Test
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration, no mortalities or sub-lethal effects of exposure were observed.

Experimental Preparation
A nominal amount of test item (1100 mg) was dispersed in 22 liters 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 item was removed by filtration through a 0.2 μm Satorius Sartopore filter (first approximate 1 liter discarded in order to pre-condition the filter) to give a 100% v/v saturated solution.

The test preparation was mixed with a flat bladed stirrer for approximately 1 minute to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations were verified by chemical analysis of the fresh media at 0 and 72 hours and of the old media at 24 and 96 hours.
A 1 liter discard was used in the definitive test, instead of the 2 liters used in the range finding test. This was considered not to have affected the study as shown by the analysis results, which are within 80% to 120% of the 0 hours measured test concentration in the range finding test where 2 litres was discarded.

Exposure Conditions
As in the range-finding test, 25-30 liter glass exposure vessels containing 20 liters of test media were used for each control and test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at 14 °C to 16 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.

The control group was maintained under identical conditions but not exposed to the test item.

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.

Assessments
Test Organism Observations
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.

Water Quality Criteria
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. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
Range-finding Test
There were no sub-lethal effects of exposure during the range-finding test.
The results showed no mortalities at the test concentrations of 1.0, 10 and 100% v/v saturated solution.
Based on this information, a single test concentration of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the highest attainable test concentration, no mortalities or sub-lethal effects of exposure were observed.
Chemical analysis of the freshly prepared 100% v/v saturated solution test preparation at 0 hours showed a measured test concentration 0.10 mg/L. Analysis of the old or expired media at 24 hours showed a measured test concentration of 0.13 mg/L, indicating that the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the freshly prepared test preparations at 0 and 72 hours showed measured test concentrations of 0.14 and 0.20 mg/L, respectively. Analysis of the old or expired test media at 24 and 96 hours showed measured test concentrations of 0.13 and 0.15 mg/L, respectively. There was a significant decline in the measured concentrations between 72 and 96 hours and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data.
The geometric mean measured test concentration was determined to be 0.15 mg/L.

Mortality Data
Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 2. There were no mortalities in 7 fish exposed to a test concentration of 0.15 mg/L for a period of 96 hours.
The test concentration of 0.15 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test item in water.

Sub-Lethal Effects
There were no sub-lethal effects of exposure observed in 7 fish exposed to a test concentration of 0.15 mg/L for a period of 96 hours.

Validation Criteria
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 ≥60% of ASV (6.1 mg O2/L) in the control and test vessels.

Water Quality Criteria
The results of the water quality measurements are given in Table 3. Temperature was maintained at 14 °C to 16 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Observations on Test Item Solubility
The test item preparations were observed to be clear colorless solutions throughout the test.

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and based on the geometric mean measured test concentration gave a 96-Hour LC50 of greater than 0.15 mg/L. The No Observed Effect Concentration was 0.15 mg/L.
Sublethal observations / clinical signs:

The test concentration of 0.15 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test item in water.

This study showed that there were no toxic effects at saturation.

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and based on the geometric mean measured test concentration gave a 96-Hour LC50 of greater than 0.15 mg/L. The No Observed Effect Concentration was 0.15 mg/L.
The test concentration of 0.15 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test item in water.

This study showed that there were no toxic effects at saturation.

Description of key information

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and based on the geometric mean measured test concentration gave a 96-Hour LC50 of greater than 0.15 mg/L. The No Observed Effect Concentration was 0.15 mg/L.

The test concentration of 0.15 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test item in water.

This study showed that there were no toxic effects at saturation.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
0.15 mg/L

Marine water fish

Marine water fish
Effect concentration:
0.15 mg/L

Additional information