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Ecotoxicological information

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:
19 September 2014 to 22 May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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
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 Appendix 2 (attached).

VALIDATION OF 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).

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 the 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. - Since the EL50 value obtained for the algal growth inhibition study was > 160 mg/L and the EL50 value obtained for the acute toxicity to Daphnia magna study was > 100 mg/L, the test was conducted at a single loading rate of 100 mg/L WAF to ensure that toxicity was not observed at this loading rate.

EXPERIMENTAL PREPARATION
- A nominal amount of test item (2200 mg) was added to a glass side and suspended in the water column of 22 L of test water to give the 100 mg/L loading rate.
- After 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 was 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.
- Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item was present.
- 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.
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 17 February 2015.
- Fish were maintained in a glass fibre tank with a 'single pass' water renewal system.
- Fish were acclimatised to test conditions from 28 February 2015 to 02 March 2015.
- 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 4.3 cm (sd = 0.1) and a mean weight of 0.67 g (sd 0.12) at the end of the definitive test. Based on the mean weight value, the loading rate was 0.23 g bodyweight/L
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 °C to 15 °C
pH:
7.6 to 8.2
Dissolved oxygen:
8.9 to 10.4 mg O2/L
Salinity:
Not applicable
Nominal and measured concentrations:
100 mg/L (nominal loading rate)
Details on test conditions:
- The lighting cycle was controlled to give 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Water temperature was controlled at approximately 14 °C with a dissolved oxygen content of ≥ 9.9 mg O2/L. These parameters were recorded daily.
- Diet and diluent water were not considered to contain any contaminant that would affect the integrity and outcome of the study.
- 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.
- Test vessels were covered to reduce evaporation and 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 involving daily renewal of the test preparations to prevent build up of nitrogenous waste products.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
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 test preparations at 0 and 72 hours (fresh media) and 24 and 96 hours (old media) (see Appendix 4, attached) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.010 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.
- 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
- Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 1 (below).
- 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.
- The No Observed Effect Loading Rate (NOELR) was considered to be 100 mg/L loading rate WAF.
- It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

SUB-LETHAL EFFECTS
- There were no sub-lethal effects of exposure observed in 7 fish exposed to a 100 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 ≥ 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 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 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 test item ashered to a glass slide, suspended just below the surface of the water.
- After 23 hours stirring and a 1 hour standing period the 100 mg/L loading rate was observed to remain as at the start of stirring.
- Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
- After siphoning and for the duration of the test, the 100 mg/L loading rate was observed to be a clear, colourless, solution with the exception of the fresh media at 0 hours and the corresponding old media at 24 hours, which was observed to be a slightly opaque solution.
- While it is unclear as to why the media became an opaque solution once it was in the test vessel at 0 hours, as no toxicity was observed, this was considered not to have had an impact on the outcome of the test.

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity to fish of the test item was assessed in accordance with OECD Guideline 203. 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. It was considered unnecessary and unrealistic to test at loading rates in excess of 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 the 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. Since the EL50 value obtained for the algal growth inhibition study was > 160 mg/L and the EL50 value obtained for the acute toxicity to Daphnia magna study was > 100 mg/L, the test was conducted at a single loading rate of 100 mg/L 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 approximately 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 at 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 test preparations at 0 and 72 hours (fresh media) and 24 and 96 hours (old media) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method, which was determined to be 0.010 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.

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

Description of key information

Exposure of rainbow trout to the test item using a method compatible with OECD 203 and EU Method C.1 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. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF.

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 the 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. Since the EL50 value obtained for the algal growth inhibition study was > 160 mg/L and the EL50 value obtained for the acute toxicity to Daphnia magna study was > 100 mg/L, the test was conducted at a single loading rate of 100 mg/L 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 approximately 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 at 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 test preparations at 0 and 72 hours (fresh media) and 24 and 96 hours (old media) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method, which was determined to be 0.010 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.

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