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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:
This study was conducted between 29 August 2017 and 04 November 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:
EC No. 440/2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Identification: Jeffcat LE 30
- Chemical Name: 2-(2-(dimethylamino)ethoxy)-N-(2-(2-(dimethylamino)ethoxy)ethyl)-N-methylethanamine
- CAS-No.: 65286-55-7
- Batch: DR74271215
- Purity: 98.2%
- Physical state/Appearance: clear colorless liquid
- Expiry Date: 30 December 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
Storage Conditions: room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Range-Finding Test
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, however, analysis of the range-finding samples was not carried out.

Definitive Test
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 72 and 96 hours.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Range-finding Test
The test concentration to be used in the definitive test was determined by a preliminary range finding test.
In the range finding test fish were exposed to a series of nominal test concentrations of 1.0, 10 and 100 mg/L. The test item was dissolved directly in test water.
An amount of test item (4000 mg) was dissolved in test water and the volume adjusted to 1 liter to give the 4000 mg/L test concentration from which a series of dilutions was made to give the remaining test concentrations of 1.0, 10 and 100 mg/L.
The stock solution was inverted several times to ensure adequate mixing and homogeneity.
The prepared concentrations were stirred for approximately 1 minute with a flat bladed stirrer to ensure homogeneity.
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, however, analysis of the range-finding samples was not carried out.

Definitive Test
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no mortalities or sub lethal effects of exposure were observed.
An amount of test item (2000 mg) was dissolved in test water and the volume adjusted to 500 mL to give the 4000 mg/L test concentration from which a dilution was made to give the test concentration of 100 mg/L.
The stock solution was inverted several times to ensure adequate mixing and homogeneity.
The prepared concentration was stirred for approximately 1 minute with a flat bladed stirrer to ensure homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 72 and 96 hours
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Strain: Oncorhynchus mykiss
- Source: Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since 12 September 2017
- Age at study initiation (mean and range, SD): juvenile
- Length at study initiation (length definition, mean, range and SD): mean standard length of 5.3 cm (standard deviation = 0.5)
- Weight at study initiation (mean and range, SD): mean body weight of 1.16 g (standard deviation = 0.32) at the end of the definitive test.


ACCLIMATION
- Acclimation period: Fish were acclimatized to test conditions from 23 October 2017 to 30 October 2017.
- Acclimation conditions (same as test or not): Fish were maintained in a glass fiber tank with a "single pass" water renewal system.
- Type and amount of food during acclimation: The stock fish were fed commercial trout pellets which was discontinued approximately 21 hours prior to the start of the definitive test. The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study. The fish were not individually identified and received no food during exposure.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
approximately 140 mg/L as CaCO3
Test temperature:
Temperature was maintained at approximately 14”C to 15”C throughout the test.
pH:
concentration dependent differences in pH were observed throughout the test.
Dissolved oxygen:
6.1 mg O2/L at 15 °C in the control and test vessels.
Nominal and measured concentrations:
Chemical analysis of the fresh test preparations at 0 and 72 hours showed measured test concentrations of 102 and 100 mg/L. Analysis of the corresponding expired test media at 24 and 96 hours showed measured test concentrations of 102 and 103 mg/L indicating that the concentration of test item in the test solutions had been satisfactorily maintained and so the results are based on nominal test concentrations only.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass vessels
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 25 to 30 liter glass exposure vessels containing 20 liters of test media were used for each control and test concentration.
- Aeration: The test vessels were aerated via narrow bore glass tubes.
- Renewal rate of test solution (frequency/flow rate): semi- static regime
- No. of organisms per vessel: At the start of the test 7 fish were placed in each test vessel at random, in the test preparations.
- Biomass loading rate: Based on the mean body weight value this gave a loading rate of 0.41 g body weight/liter (static volume).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 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.
- 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 the 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. Water samples were taken from the control and test groups 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 samples were stored frozen prior to analysis.
Duplicate samples at 0 and 72 hours (fresh media), 24 and 96 hours (old media) were taken and stored frozen for further analysis if necessary.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods for a period of 96 hours.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : An estimate of the LC50 values was given by inspection of the mortality data.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study
- Test concentrations: 1.0, 10 mg/L, 100 mg/L
- Results used to determine the conditions for the definitive study: yes (limit test)
Range-finding Test
The results showed no mortalities or sub-lethal effects at 1.0 and 10 mg/L, however, a single mortality was observed at 100 mg/L. Prior to the mortality occurring, the affected fish was observed with loss of equilibrium, however, this observation was believed to be due to the aggressive behavior of the other fish in the test vessel and not necessarily as a result of exposure to the test item. Due to a technical error, analysis of the range-finding test samples was not carried out. Data from the algal and daphnia tests indicated that near nominal concentrations were achieved in the freshly prepared test solutions and that these concentrations were maintained for the duration of the exposure periods. Based on this information, a single test concentration of 100 mg/L was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/EC Test Guidelines, no mortalities or sub lethal effects of exposure were observed.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- There were no mortalities in 7 fish exposed to a test concentration of 100 mg/L for a period of 96 hours.
- The results of the definitive test showed the highest test concentration resulting in 0% mortality to be 100 mg/L. The NOEC was 100 mg/L.
- There were no sub lethal effects of exposure observed in the test.


Sublethal observations / clinical signs:

Cumulative Mortality Data in the Definitive Test 

Nominal 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

100

0

0

0

0

0

0

0

0

 

Validity criteria fulfilled:
yes
Remarks:
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.
Conclusions:
Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LC50 values based on the nominal test concentrations of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) was 100 mg/L.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Description of key information

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 (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008. Seven fish were exposed to an aqueous solution of the test item, at a single concentration of 100 mg/L for a period of 96 hours at a temperature of approximately 15 °C under static test conditions. 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. Chemical analysis of the fresh test preparations at 0 and 72 hours showed measured test concentrations of 100 and 102 mg/L. Analysis of the corresponding expired test media at 24 and 96 hours showed measured test concentrations of 102 and 103 mg/L indicating that the concentration of test item in the test solutions had been satisfactorily maintained and so the results are based on nominal test concentrations only. Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LC50 values based on the nominal test concentrations of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) was 100 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Key value for chemical safety assessment

Additional information