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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:
28 June 2018 to 28 January 2019
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0, 24, 48, 72 and 96 hours.
Water samples were taken from the control and test group, from fresh and old media throughout the 96 hour test period, for quantitative analysis. The samples were stored frozen prior to analysis. Duplicate sets of samples at 0 and 72 hours (fresh media) and 24 and 96 hours (old media) were taken and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A nominal amount of test material (2200 mg) was added to the surface of 22 L of test water to give the 100 mg/L loading rate. After the addition of the test material, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixture allowed to stand for one hour. Microscopic observations made on the WAF indicated that a significant amount of dispersed test material was present in the water column and hence it was considered justifiable to remove the dispersed material by filtering the WAFs through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid depth siphoning (the first 75 to 100 mL discarded) to give the 100 mg/L loading rate WAF.
- The vortex depth was recorded at the start and end of each mixing period.
- Evidence of undissolved material: Microscopic observations of the WAF were performed after filtering and showed microdispersions present; therefore, the WAF was filtered through filter paper. Following filtration through filter paper twice, no microdispersions were present and was observed to be a clear colourless solution throughout the test.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Age at study initiation (mean and range, SD): Juveniles
- Length at study initiation (length definition, mean, range and SD): The fish had a mean standard length of 5.1 cm (standard deviation = 0.3).
- Weight at study initiation (mean and range, SD): Mean weight of 1.1 g (standard deviation = 0.2) at the end of the definitive test.


ACCLIMATION
- Acclimation period: Fish were acclimatised to test conditions from 23 July 2018 to 30 July 2018.
- Acclimation conditions: The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Type and amount of food during acclimation: The stock fish were fed commercial trout pellets which was discontinued approximately 27 hours prior to the start of the definitive test.
- Health during acclimation: There were two mortalities in the 7 days prior to the start of the test (1.6 % of the fish batch).


FEEDING DURING TEST
- Food type: The fish received no food during exposure.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
Total hardness approximately 140 mg/L as CaCO3
Test temperature:
14 - 15 °C
pH:
7.5 - 7.9
Dissolved oxygen:
9.3 - 10.2 mg O2/L
Conductivity:
345 μS/cm at 20 °C
Nominal and measured concentrations:
100 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 25 to 30 L glass exposure vessels
- Fill volume: 20 L
- Type: Closed. The test vessels were then covered to reduce evaporation
- Material, size, headspace, fill volume: 20 L of test media
- Aeration: The test vessels were aerated via narrow bore glass tubes
- Renewal rate of test solution: daily renewal of the test preparations
- No. of organisms per vessel: Seven fish were placed in each test vessel at random, containing the test preparations
- Biomass loading rate: Loading rate of 0.4 g body weight/L

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.
- Pesticides: 0 μg/L
- Chlorine: 0.32 mg/L
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.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness and 20 minute dawn and dusk transition periods.

EFFECT PARAMETERS MEASURED: 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.

TEST CONCENTRATIONS
- Range finding study
Test concentrations: In the range finding test, fish were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L. Nominal amounts of test material (22, 220 and 2 200 mg) were each separately added to the surface of 22 L of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. After the addition of the test material, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixtures allowed to stand for 1 hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test material was present in the water column and hence it was considered justifiable to remove the dispersed material by filtering the WAFs through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid depth siphoning (the first 75 to 100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs. Microscopic observations were performed on the WAFs after filtering and the 1.0 and 10 mg/L loading rate WAFs showed no microdispersions to be present. Following filtration through a glass wool plug, the 100 mg/L loading rate was still observed to be cloudy; therefore it was filtered through filter paper. Microscopic observations after the additional filtration of the 100 mg/L loading rate showed no particles or microdispersions were present and was observed to be a clear colourless solution throughout the test.
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 to 15 °C with a photoperiod of 16 hours light and 8 hours darkness and 20 minute dawn and dusk transition periods for a period of 96 hours under static test conditions. Each 25 to 30 L test and control vessel contained 20 L 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 material.
A sample of each loading rate WAF was taken for chemical analysis at 0 and 24 hours in order to determine the stability of the test material under test conditions. All samples were stored frozen prior to analysis. Only the concentration to be used for the definitive test was analysed.
- Results used to determine the conditions for the definitive study: There were no sub lethal effects of exposure during the range finding test at 1.0 and 100 mg/L loading rate WAFs. However, increased pigmentation and swimming at the surface of the vessel were observed at the 10 mg/L loading rate WAF, but these observations were transient and unlikely to have been due to exposure to the test material.
The results showed no mortalities at 1.0, 10 and 100 mg/L loading rate WAFs.
Based on this information, a single loading rate of 100 mg/L was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no mortalities or sub lethal effects of exposure were observed.
Chemical analysis of the test preparations at 0 and 24 hours indicated that the test material concentration had increased over the 24 hour period and were therefore considered to be anomalous.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LL50
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 seven fish exposed to a 100 mg/L loading rate WAF for a period of 96 hours.
- There were no sub lethal effects of exposure observed in seven fish exposed to a 100 mg/L loading rate WAF for a period of 96 hours
- Other adverse effects control: Contamination of the control group was noted at 0 and 24 hours. Measurable levels of test mateiral were detected but the levels were considerably lower than those measured in the test group media. As no mortalities or sub-lethal effects were observed in the control group, it was considered that the presence of test material in the control media had no effect on the outcome of the study.
- Chemical analysis of the fresh test preparations at 0, 24, 48 and 72 hours showed measured test concentrations of 0.00058 to 0.10 mg/L as samarium, to range from less than the limit of quantification (LOQ; 0.00026 mg/L) to 0.016 mg/L as europium and to range from less than the LOQ (0.00026 mg/L), to 0.023 mg/L as gadolinium. Chemical analysis of the aged test preparations at 24, 48, 72 and 96 hours showed measured test concentrations to range from 0.00052 to 0.035 mg/L as samarium, less than the LOQ to 0.0057 mg/L as europium and less than the LOQ to 0.0082 mg/L as gadolinium.
The dissolved test material may have been one or several components of the test material. Given that toxicity cannot be attributed to a single component or mixture of components but to the test material as a whole, the results were based on nominal loading rates.
- Effect concentrations exceeding solubility of substance in test medium: Observations on the test media were carried out during the mixing and testing of the WAF. At the start of the mixing period the 100 mg/L loading rate preparations were observed to be clear colourless water column with particles of test material dispersed throughout the column. After 95 hours stirring and a 1 hour standing period the 100 mg/L preparations were observed to be very cloudy white dispersion with test material on the bottom of the vessel. Visual examination of the WAF showed microdispersions present and therefore it was considered justifiable to remove the dispersed material by filtering the WAF through a glass wool plug (2 to 4 cm in length) and filter paper. Microscopic examination after filtering showed the glass wool plug and filter paper had removed all undissolved test material. During the test the 100 mg/L loading rate was observed to be clear, colourless solutions.



Reported statistics and error estimates:
An estimate of the LL50 values was given by inspection of the mortality data.
Sublethal observations / clinical signs:

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

Temperature was maintained at 14 °C to 15 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Vortex Depth Measurement

The vortex depth was recorded at the start and end of each mixing period and was observed to be a dimple at the water surface on each occasion.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, the LL50 value was >100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was100 mg/L loading rate WAF.
Executive summary:

The acute toxicity of the test material to rainbow trout (Oncorhynchus mykiss) was assessed according to OECD Guideline 203 and EU Method C.1, and in compliance with GLP.

Due to the low aqueous solubility and complex nature of the test material, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, seven fish were exposed to a WAF of the test material, at a single nominal loading rate of 100 mg/L for a period of 96 hours at a temperature of 14°C to 15 ºC under semi-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.

Samples of the control and 100 mg/L loading rate WAF were taken at 0, 24, 48 and 72 hours (fresh media) and at 24, 48, 72 and 96 hours (old media) for chemical analysis.

Chemical analysis of the fresh test preparations at 0, 24, 48 and 72 hours showed measured test concentrations of 0.00058 to 0.10 mg/L as samarium, to range from less than the limit of quantification (LOQ; 0.00026 mg/L) and 0.016 mg/L as europium and to range from less than the LOQ (0.00026 mg/L) and 0.023 mg/L as gadolinium. Chemical analysis of the aged test preparations at 24, 48, 72 and 96 hours showed measured test concentrations to range from 0.00052 to 0.035 mg/L as samarium, less than the LOQ to 0.0019 mg/L as europium and less than the LOQ to 0.0082 mg/L as gadolinium.

The dissolved test material may have been one of several components of the test material. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test material as a whole, the results were based on nominal loading rates only.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L loading rate WAF.

Under the conditions of the test, the LL50 value was >100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was 100 mg/L loading rate WAF.

Description of key information

Under the conditions of the test, the LL50 value was >100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was100 mg/L loading rate WAF.

Key value for chemical safety assessment

Additional information

The acute toxicity of the test material to rainbow trout (Oncorhynchus mykiss) was assessed according to OECD Guideline 203 and EU Method C.1, and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Due to the low aqueous solubility and complex nature of the test material, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, seven fish were exposed to a WAF of the test material, at a single nominal loading rate of 100 mg/L for a period of 96 hours at a temperature of 14°C to 15 ºC under semi-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.

Samples of the control and 100 mg/L loading rate WAF were taken at 0, 24, 48 and 72 hours (fresh media) and at 24, 48, 72 and 96 hours (old media) for chemical analysis.

Chemical analysis of the fresh test preparations at 0, 24, 48 and 72 hours showed measured test concentrations of 0.00058 to 0.10 mg/L as samarium, to range from less than the limit of quantification (LOQ; 0.00026 mg/L) and 0.016 mg/L as europium and to range from less than the LOQ (0.00026 mg/L) and 0.023 mg/L as gadolinium. Chemical analysis of the aged test preparations at 24, 48, 72 and 96 hours showed measured test concentrations to range from 0.00052 to 0.035 mg/L as samarium, less than the LOQ to 0.0019 mg/L as europium and less than the LOQ to 0.0082 mg/L as gadolinium.

The dissolved test material may have been one of several components of the test material. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test material as a whole, the results were based on nominal loading rates only.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L loading rate WAF.

Under the conditions of the test, the LL50 value was > 100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was 100 mg/L loading rate WAF.