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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:
Experimental start date: 06 May 2020; Experimental completion date: 20 August 2020
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:
2019
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: LESA
Batch: 8901525
Purity: 98.6%
Physical State/Appearance: Clear colorless liquid
Expiry Date: 20 March 2022
Storage Conditions: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and 30% v/v saturated solution test vessel at 0, 24, 48 and 72 hours from fresh media and at 24, 48, 72 and 96 hours from old media for quantitative analysis. The 0 and 96 hour samples were analyzed immediately whilst the other samples were either stored in the fridge or freezer prior to analysis.
Duplicate samples were taken and stored either in the fridge or frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Preliminary Media Preparation Trial:
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. A test concentration of 2.8 mg/L was obtained using a preliminary solution in dechlorinated laboratory water.
A media preparation trial was conducted in order to determine the solubility of the test item under test conditions.
Based on the result of the trial it wasevident that the initial preliminary results were incorrect and the solvent spike method of preparation was not suitable, therefore for the purpose of testing the test item was prepared using a saturated solution method of preparation at an initial loading rate of 100 mg/L, stirred via propeller stirrer for 24 hours prior to the removal of any undissolved test item by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a nominal concentration of 2.8 mg/L.

Initial Experiments:
Two initial experiments were performed at a single concentration of 100% v/v saturated solution. The first initial experiment was stopped due to the temperature of the water being outside the range required for the study. The second initial experiment was terminated due to greater than 20% mortality and sub-lethal effects of exposure in the test concentration. It was also noted that the analytical results were a lot higher compared to the initial solubility of 3.0 mg/L at 0 hours and the initial solubility indicating a level of 0.68 mg/L. Both initial experiments will not be used for reporting purposes. Following the second initial experiment further preliminary media trials were conducted and confirmed that a test concentration of 2.8 mg/L was obtained using a saturated solution method of preparation.

Definitive Test:
In accordance with the recommendations of REACh, the test was conducted according to the threshold approach recommended by ECHA. Using this approach the lowest EC50 value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as the threshold concentration and a “Limit test” is conducted at this threshold concentration. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LC50 is greater than the threshold concentration. The EC50 value obtained from the Acute Toxicity to Daphnia magna (Covance study number FD93PN) was the lowest of these two EC50 values and hence the test was conducted at a single concentration of 30% v/v saturated solution.

Experimental Preparation:
A nominal amount of test item (1100 mg) was dispersed in 11 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 Sartorius Sartopore filter (first approximate 2 liters used to pre condition the filter was discarded) to give a 100% v/v saturated solution. A further dilution was made from this saturated solution to give the test solution of 30% v/v saturated solution.
The stock solution 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:
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 22 July 2020. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 03 August 2020 to 10 August 2020. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
The water temperature was controlled at approximately 14°C with a dissolved oxygen content of greater than or equal to 9.0 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 25.5 hours prior to the start of the definitive test. There were four mortalities in the 7 days prior to the start of the test and the fish had a mean standard length of 4.8 cm (standard deviation = 0.24) and a mean body weight of 0.94 g (standard deviation = 0.16) at the end of the definitive test. Based on the mean body weight value this gave a loading rate of 0.33 g body weight/liter (static volume).
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test Water:
The test water used for both preliminary media trial, initial experiments 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 (Fleck 2750 Duplex Dechlorination unit) 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.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
140 mg/L as CaCO3
Test temperature:
Temperature was maintained at 14°C to 16°C throughout the test
pH:
pH in control and test samples ranged from 7.8 - 8.3
No treatment related differences for pH.
Dissolved oxygen:
Dissolved oxygen in control and test samples ranged from 8.9 - 9.7
No treatment related differences for oxygen concentration .
Nominal and measured concentrations:
Nominal: 30% v/v saturated solution
Geometric Mean Measured Test Concentration: 0.65 mg/L
Details on test conditions:
TEST SYSTEM
For each control and test concentration, 25 to 30 liter glass exposure vessels containing 20 liters of test media were used. 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 approximately 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.


TEST MEDIUM / WATER PARAMETERS
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.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any mortalities and sub lethal effects of exposure were recorded at 1, 3, 6, 24, 30, 48, 52.5, 72, 77 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.

Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.65 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.65 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
Definitive Test:
Analysis of the freshly prepared test media at 0, 24, 48 and 72 hours showed measured test concentrations to range from 0.78 to 0.97 mg/L. A decline in measured test concentration was observed in the old or expired test media at 24, 48, 72 and 96 hours to between 0.33 and 0.58 mg/L 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 were determined to be 0.65 mg/L.

Mortality Data:
There were no mortalities in seven fish exposed to a test concentration of 0.65 mg/L for a period of 96 hours. Inspection of the mortality data gave the following results:
LC50: >0.65 mg/L for all timepoints (1 to 96 hr).

The results of the definitive test showed the highest test concentration resulting in 0% mortality to be 0.65 mg/L. As such the No Observed Effect Concentration (NOEC) was considered to be 0.65 mg/L.

Sub-Lethal Effects:
There were no sub lethal effects of exposure observed in seven fish exposed to a test concentration of 0.64 mg/L for a period of 96 hours.

Water Quality Criteria:
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 of Test Item Solublity:
The control and test item preparations were observed to be clear colorless solutions throughout the test.

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 (9.2 mg O2/L) in the control and test vessels.
Sublethal observations / clinical signs:

Cumulative Mortality Data in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)

Cumulative Mortality (Initial Population = 7)
(Hours)

Mortality (%)

1
Hour

3
Hours

6
Hours

24
Hours

30
Hours

48
Hours

52.5
Hours

72
Hours

77
Hours

96
Hours

 

Control

0

0

0

0

0

0

0

0

0

0

0

0.65

0

0

0

0

0

0

0

0

0

0

0

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 concentrations gave a 96 hour LC50 of greater than 0.65 mg/L. The NOEC was 0.65 mg/L.
Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2019) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Methods

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 2.8 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

In accordance with the recommendations of REACh, the test was conducted according to the threshold approach recommended by ECHA. Using this approach the lowest EC50 value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as the threshold concentration and a “Limit test” is conducted at this threshold concentration. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LC50is greater than the threshold concentration. The EC50value obtained from the Acute Toxicity to Daphnia magna was the lowest of these two EC50 values (0.60 mg/L) and hence the test was conducted at a single concentration of 30% v/v saturation solution.

Seven fish were exposed to an aqueous solution of the test item, at a single concentration of 30% v/v saturated solution for a period of 96 hours at a temperature of 14 ºC to 16 °C under semi‑static test conditions. The test item solution was prepared by stirring an excess (100 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µmSartorius Sartopore filter, first approximate 2 litersused to pre‑condition the filter was discarded) to produce a saturated solution of the test item with a nominal concentration of 100% v /v saturation solution. This saturated solution was the further diluted as necessary to provide the required test group. The number of mortalities and any sub‑lethal effects of exposure in each test and control vessel were determined 1, 3, 6, 24, 30, 48, 52.5, 72, 77 and 96 hoursafter the start of exposure.

Results

Analysis of the freshly prepared test media at 0, 24, 48 and 72 hours showed measured test concentrations to range from 0.78 to 0.97 mg/L. A decline in measured test concentration was observed in the old or expired test media at 24, 48, 72 and 96 hours to between 0.33 and 0.58 mg/L and hence it was considered appropriate to calculate the results based on 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 0.65 mg/L.

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LC50values based on the geometric mean measured test concentrations of greater than 0.65 mg/L. The No Observed Effect Concentration (NOEC) was 0.65 mg/L.

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

Description of key information

Key study (Covance 2020):

Introduction

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2019) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Methods

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 2.8 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

In accordance with the recommendations of REACh, the test was conducted according to the threshold approach recommended by ECHA. Using this approach the lowest EC50value from either the Algal Growth Inhibition study or Acute Toxicity toDaphnia magnastudy is set as the threshold concentration and a “Limit test” is conducted at this threshold concentration. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LC50is greater than the threshold concentration. The EC50value obtained from the Acute Toxicity to Daphnia magna was the lowest of these two EC50values (0.60 mg/L) and hence the test was conducted at a single concentration of 30% v/v saturation solution.

Seven fish were exposed to an aqueous solution of the test item, at a single concentration of 30% v/v saturated solution for a period of 96 hours at a temperature of 14 ºC to 16 °C under semi‑static test conditions. The test item solution was prepared by stirring an excess (100 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters used to pre‑condition the filter was discarded) to produce a saturated solution of the test item with a nominal concentration of 100% v /v saturation solution. This saturated solution was the further diluted as necessary to provide the required test group. The number of mortalities and any sub‑lethal effects of exposure in each test and control vessel were determined1, 3, 6, 24, 30, 48, 52.5, 72, 77 and 96 hoursafter the start of exposure.

Results

Analysis of the freshly prepared test media at 0, 24, 48 and 72 hours showed measured test concentrations to range from 0.78 to 0.97 mg/L. A decline in measured test concentration was observed in the old or expired test media at 24, 48, 72 and 96 hours to between 0.33 and 0.58 mg/L and hence it was considered appropriate to calculate the results based on 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 0.65 mg/L.

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LC50values based on the geometric mean measured test concentrations of greater than 0.65 mg/L. The No Observed Effect Concentration (NOEC) was 0.65 mg/L.

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:
0.64 mg/L

Additional information

The acute toxicity of (Z)-3 -hexenyl salicylate was assessed in orange killifish in the supporting study. The study was non-GLP and performed to sound scientific principles but the study duration was 48 hours and therefore less suitable to assess the acute toxicity of the substance for classification and labelling and risk assessment purposes. The substance is readily biodegradable and so a semi-static or flow-through test system should have been used and no analytical measurements of the test material concentration were made. The results, however, concur with the results and conclusions of the key study i.e. lower toxicity observed in fish than Daphnia/algae.