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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:
20 September 2015 to 07 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP 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)
Analytical monitoring:
yes
Details on sampling:
- Water samples were taken from the control and 100 mg/L loading rate WAF test vessel at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) for quantitative analysis.
- Samples were stored frozen prior to analysis.
- Duplicate samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) were 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.

VALIDATION OF THE MIXING PERIOD
- Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF (see Appendix 3, attached).
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 from 04 August 2015.
- Fish were maintained in a glass fibre tank with a 'single pass' water renewal system.
- Fish were acclimatised to test conditions from 14 September 2015 to 21 September 2015.
- Lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle.
- Water temperature was controlled at 14 °C with a dissolved oxygen content of ≥ 8.1 mg O2/L. These parameters were recorded daily.
- The stock fish were fed commercial trout pellets, which were discontinued approximately 24 hours prior to the start of the definitive test.
- There were 3 mortalities in the 7 days prior to the start of the test and the fish had a mean standard length of 4.7 cm (sd = 0.2) and a mean weight of 1.4 g (sd = 0.17) at the end of the definitive test. Based on mean weight value this gave a loading rate of 0.49 g bw/L.
- The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.
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:
14-15 °C (see Table 3, attached)
pH:
7.7 to 8.1 ( see Table 3, attached)
Dissolved oxygen:
9.2 to 10.0 mg O2/L ( see Table 3 attached)
Salinity:
Not applicable
Nominal and measured concentrations:
100 mg/L nominal loading rate
Details on test conditions:
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 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.
- The EL50 value obtained from both the algal growth inhibition study and the acute toxicity to Daphnia magna study were greater than 100 mg/L and hence the test was conducted at a single loading rate of 100 mg/L loading rate WAF.

EXPERIMENTAL PREPARATION
- A nominal amount of test item (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 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.
- Stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour.
- 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.
- The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF.
- Microscopic observations of the WAF showed no micro dispersions or undissolved test item present.
- Glass exposure vessels (25 to 30 L) containing test media (20 L) were used for each control and test concentration.
- At the start of the test, seven fish were placed in the test preparations in each test vessel at random.
- 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.
- Test vessels were aerated via narrow bore glass tubes.
- 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 which daily renewal of the test preparations took place to prevent build-up of nitrogenous waste products.
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)
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
VALIDATION OF MIXING PERIOD
- Preliminary investigational work (see Appendix 3) indicated that there was no significant increase in the amount of dissolved test item when the preparation period was extended for longer than 24 hours.
- Therefore, for the purpose of testing, the WAF was prepared using a stirring period of 23 hours followed by a 1-hour settlement period.

CHEMICAL ANALYSIS OF TEST LOADING RATES
- Chemical analysis of the freshly prepared 100 mg/L loading rate test samples at 0 and of the expired media at 24 and 96 hours showed measured concentrations of less than limit of quantification (LOQ) of analytical method which was determined to be 0.51 mg/L.
- Does not infer that no test item was in solution, just that any dissolved test item was at concentration of less than the LOQ.
- Chemical analysis of the freshly prepared test sample at 72 hours showed a measured test concentration of 1.08 mg/L.
- The duplicate sample for the 72 hour sample was also analysed and showed a measured test concentration of 2.00 mg/L.
- This slightly higher than expected measured concentration was considered to be due to the presence of small amount of undissolved test item and was considered to have no adverse effect on the outcome of the test given that no effects to exposure were observed.
- The dissolved test item may have been one or several components of the test item.
- 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.

MORTALITY DATA
- Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 2 (attached).
- There were no mortalities in 7 fish exposed to a 100 mg/L loading rate WAF for a period of 96 hours.
- Inspection of mortality data gave LL50 values > 100 mg/L loading rate WAF at 3, 6, 24, 48, 72 and 96 hours.

SUB-LETHAL EFFECTS
- Sub-lethal effects of exposure were observed at the 100 mg/L loading rate WAF after 3 hours of exposure.
- Response was an increased pigmentation in 3 out of 7 fish, which were not observed at 6 hours (see Table 2, attached).

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 (9.27mg O2/L) in the control and test vessels.

WATER QUALITY CRITERIA
- The results of the water quality measurements are given in Table 3 (attached).
- Temperature was maintained at 14-15 °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 100 mg/L loading rate was observed to be a clear colourless water column with a lump of test item floating at the surface.
- After 23 hours stirring and a 1-hour standing period the 100 mg/L loading rate was observed to be a clear colourless water column with a lump of test item floating at the surface.
- Microscopic observation of the WAF showed no micro-dispersions or undissolved test item present.
- After siphoning and during the test, the 100 mg/L loading rate was observed to be a clear, colorless solution.
Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated using the threshold approach and gave a 96-hour LL50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.
Executive summary:

GUIDELINE

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method 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, the test medium was prepared as a Water Accommodated Fraction (WAF).

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 loading threshold 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 100 mg/L loading rate WAF, the test was conducted at a single loading rate of 100 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 100 mg/L for a period of 96 hours at a temperature of 14°C to 15°C 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

Chemical analysis of the freshly prepared test samples at 0 hours and of the expired media at 24 and 96 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method were obtained which was determined to be 0.51 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ. Chemical analysis of the freshly prepared test sample at 72 hours showed a measured test concentration of 1.08 mg/L to be obtained. The duplicate sample was also analysed and showed a measured test concentration of 2.00 mg/L. This slightly higher than expected measured concentration was considered to be due to the presence of a small amount of undissolved test item. This was considered to have had no adverse effect on the outcome of the test given that no effects to exposure were observed. 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 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Description of key information

Exposure of rainbow trout to the test item gave LL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF (OECD 203 and EU Method C.1).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
100 mg/L

Additional information

GUIDELINE

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method 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, the test medium was prepared as a Water Accommodated Fraction (WAF).

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 loading threshold 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 100 mg/L loading rate WAF, the test was conducted at a single loading rate of 100 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 100 mg/L for a period of 96 hours at a temperature of 14 °C to 15 °C 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

Chemical analysis of the freshly prepared test samples at 0 hours and of the expired media at 24 and 96 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method were obtained which was determined to be 0.51 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ. Chemical analysis of the freshly prepared test sample at 72 hours showed a measured test concentration of 1.08 mg/L to be obtained. The duplicate sample was also analysed and showed a measured test concentration of 2.00 mg/L. This slightly higher than expected measured concentration was considered to be due to the presence of a small amount of undissolved test item. This was considered to have had no adverse effect on the outcome of the test given that no effects to exposure were observed.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 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.