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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:
02 April 2017 to 08 April 2017
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
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Remarks:
Total Organic Carbon (TOC) analysis
Details on sampling:
- Water samples were taken from the control and the 50 mg/L loading rate WAF test vessel at 0 and 72 hours (fresh media). Samples were also taken at 24 and 96 hours (old media).
- The samples were analysed on the day of collection.
- 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:
TEST WATER
- The test water used for the definitive test 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. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Annex 2 (attached).

EXPERIMENTAL DESIGN AND STUDY CONDUCT
- Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item. - A study to determine the general physico-chemical properties of the test item indicated the water solubility of the test item was less than 1.0 mg C/L. Given this it was considered appropriate to test up to a maximum loading rate of 50 mg/L to prevent overloading of the aqueous phase with undissolved test item.
- A 23-Hour stirring period, followed by a 1-Hour standing period was deemed sufficient to ensure equilibration between the test item and aqueous phase.

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST SYSTEM
- 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 15 March 2017.
- Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatised to test conditions from 27 March 2017 to 03 April 2017. 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 9.5 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test.
- There was zero mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 4.8 cm (sd = 0.2) and a mean weight of 0.85 g (sd = 0.19) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.3 g bodyweight/L.
- The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

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.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
Not applicable
Hardness:
Approximately 140 mg/L as CaCO3
Test temperature:
14 to 15 ºC
pH:
7.1 to 8.1 (see Table 2, attached)
Dissolved oxygen:
9.3 to 10.5 mg O2/L (see Table 2, attached)
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
- Single nominal loading rate of 50 mg/L
Details on test conditions:
PURPOSE OF THE TEST
- Rainbow trout is a freshwater fish representative of a wide variety of natural habitats, and can therefore be considered as an important non-target organism in freshwater ecosystems.
- In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

DEFINITIVE TEST
- In In accordance with the recommendations of REACH, the test was conducted according to the threshold approach recommended by ECHA. Using this approach the lowest EL50 value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as the threshold loading rate and a “Limit test” is conducted at this threshold loading rate. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LL50 is greater than the threshold loading rate.
- Therefore, as the EL50 value obtained for both the Algal Growth Inhibition study and the Acute Toxicity to Daphnia magna study were greater than 50 mg/L loading rate WAF, the test was conducted at a single loading rate of 50 mg/L loading rate WAF to ensure that toxicity was not observed at this loading rate.

EXPERIMENTAL PREPARATION
- A nominal amount of test item (1100 mg) was added to the surface of 22 L of test water to give the 50 mg/L loading rate.
- After the addition of the test item, 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 23 hours and the mixture allowed to stand for 1 hour.
- Observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 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 75-100 mL discarded) to give the 50 mg/L loading rate WAF. Microscopic observations of the WAF were performed after filtering and showed no particles or micro-dispersions of test item.
- Total Organic Carbon (TOC) analysis was performed on the fresh test solutions at 0 and 72 hours and on the old test media at 24 and 96 hours (see Annex 3, attached).

EXPOSURE CONDITIONS
- In the definitive test, 25-30 L glass exposure vessels containing 20 L 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 to 15 °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.

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.

VORTEX DEPTH MEASUREMENTS
The vortex depth was recorded at the start and end of each mixing period.

DATA VALIDATION
- The results of the test are considered valid if the following criteria are met:
(i) In the control, not more than one of the fish should die or show signs of stress during the 96 hours.
(ii) The dissolved oxygen concentration at the end of the test should be ≥60% of Air Saturation Values (ASV) in the control and test vessels.

MAJOR COMPUTERISED SYSTEMS
- Building management: Delta control system
- TOC measurement: Shimadzu TOC
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mortality
Details on results:
DEFINITIVE TEST – TOTAL ORGANIC CARBON ANALYSIS
- Total Organic Carbon (TOC) analysis of the 50 mg/L loading rate WAF fresh test preparations at 0 and 72 hours (see Annex 3, attached) showed measured carbon concentrations of less than the control. TOC analysis of the 50 mg/L old test preparations at 24 and 96 hours showed measured carbon concentrations of less than the limit of quantification (LOQ) of the method employed, which was determined to be 1.0 mg/L, and 0.36 mg/L (control corrected value) respectively.
The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

MORTALITY DATA
- There were no mortalities in 7 fish exposed to a 50 mg/L loading rate WAF for a period of 96 hours (see Table 1, attached).

SUB-LETHAL EFFECTS
- There were no sub-lethal effects of exposure observed in 7 fish exposed to a 50 mg/L loading rate WAF 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 greater than or equal to 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 2 (attached).
- Temperature was maintained at approximately 14 to 15 °C throughout the test.
- There were no treatment related differences for oxygen concentration or pH.

VORTEX DEPTH MEASUREMENTS
- 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.

OBSERVATIONS ON TEST ITEM SOLUBILITY
- Observations on the test media were carried out during the mixing and testing of the WAF.
- At the start of the mixing period the 50 mg/L loading rate was observed to be a clear colourless water column with particles of test item floating at the surface. After 23 hours stirring and a 1-Hour standing period the 50 mg/L loading rate was observed to remain as at the start of the mixing period, with the additional observation of fine particles of test item dispersed throughout the water column.
- Observation of the WAF showed fine particles dispersed throughout the water column and therefore it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length). Microscopic examination after filtering showed the glass wool plug had removed all particles of test item. During the test the control and 50 mg/L loading rate were observed to be clear, colourless solutions.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
STATISTICAL ANALYSIS
- An estimate of the LL50 values was given by inspection of the mortality data.
Validity criteria fulfilled:
yes
Conclusions:
Exposure of rainbow trout to the test item gave LL50 values of greater than 50 mg/L loading rate WAF. The No Observed Effect Loading Rate was 50 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 50 mg/L.
Executive summary:

GUIDELINE

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.

 

METHODS

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF). A study to determine the general physico-chemical properties of the test item indicated the water solubility of the test item was less than 1.0 mg C/L. Given this it was considered appropriate to test up to a maximum loading rate of 50 mg/L to prevent overloading of the aqueous phase with undissolved test item. In accordance with the recommendations of REACH, the test was conducted according to the threshold approach recommended by ECHA. Using this approach the lowest EL50 value from either the algal growth inhibition study or acute toxicity to Daphnia magna study is set as the threshold loading rate and a “Limit test” is conducted at this threshold loading rate. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LL50 is greater than the threshold loading rate. Therefore, as the EL50 value obtained for both the Algal Growth Inhibition study and the Acute Toxicity to Daphnia magna study were greater than 50 mg/L loading rate WAF, the test was conducted at a single loading rate of 50 mg/L loading rate WAF to ensure that toxicity was not observed at this loading rate. Seven fish were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 50 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 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

 

RESULTS

Total Organic Carbon (TOC) analysis of the 50 mg/L loading rate WAF fresh test preparations at 0 and 72 hours showed measured carbon concentrations of less than the control. TOC analysis of the 50 mg/L old test preparations at 24 and 96 hours showed measured carbon concentrations of less than the limit of quantification (LOQ) of the method employed, which was determined to be 1.0 mg/L, and 0.36 mg/L (control corrected value) respectively. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

 

CONCLUSION

Exposure of rainbow trout to the test item gave LL50 values of greater than 50 mg/L loading rate WAF. The No Observed Effect Loading Rate was 50 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 50 mg/L.

Description of key information

Exposure of rainbow trout to the test item gave LL50 values of greater than 50 mg/L loading rate WAF. The No Observed Effect Loading Rate was 50 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 50 mg/L (OECD 203 and EU Method C.1).

Key value for chemical safety assessment

Additional information

GUIDELINE

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.

 

METHODS

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF). A study to determine the general physico-chemical properties of the test item indicated the water solubility of the test item was less than 1.0 mg C/L. Given this it was considered appropriate to test up to a maximum loading rate of 50 mg/L to prevent overloading of the aqueous phase with undissolved test item. In accordance with the recommendations of REACH, the test was conducted according to the threshold approach recommended by ECHA. Using this approach the lowest EL50 value from either the algal growth inhibition study or acute toxicity to Daphnia magna study is set as the threshold loading rate and a “Limit test” is conducted at this threshold loading rate. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LL50 is greater than the threshold loading rate. Therefore, as the EL50 value obtained for both the Algal Growth Inhibition study and the Acute Toxicity to Daphnia magna study were greater than 50 mg/L loading rate WAF, the test was conducted at a single loading rate of 50 mg/L loading rate WAF to ensure that toxicity was not observed at this loading rate. Seven fish were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 50 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 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

 

RESULTS

Total Organic Carbon (TOC) analysis of the 50 mg/L loading rate WAF fresh test preparations at 0 and 72 hours showed measured carbon concentrations of less than the control. TOC analysis of the 50 mg/L old test preparations at 24 and 96 hours showed measured carbon concentrations of less than the limit of quantification (LOQ) of the method employed, which was determined to be 1.0 mg/L, and 0.36 mg/L (control corrected value) respectively. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

 

CONCLUSION

Exposure of rainbow trout to the test item gave LL50 values of greater than 50 mg/L loading rate WAF. The No Observed Effect Loading Rate was 50 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 50 mg/L.