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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:
Experimnetal phase: 14 January 2018 to 16 April 2018. Report issued: 12 July 2018.
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:
yes
Remarks:
Deviations from the study plan were not considered to have had any impact on the outcome of the study.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and all surviving test groups, from fresh and old media throughout the 96-hour test period, for quantitative analysis. Sets of duplicate 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:
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow Trout (Oncorhynchus mykiss)
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Length: fish had a mean standard length of 4.29 cm (standard deviation = 0.21) at the end of the definitive test
- Weight mean weight of 0.56 g (standard deviation = 0.10) at the end of the definitive test

ACCLIMATION
- Acclimation period: 7 days
- Acclimation conditions (same as test or not): Same as test

FEEDING DURING TEST
- Frequency: The fish were not fed during exposure to the test substance.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
none
Hardness:
140 mg/L as CaCO3
Test temperature:
14 °C
pH:
7.3 - 7.9 at 0 hours (fresh media). 8.0 - 8.2 at 96 hours (old media).
Dissolved oxygen:
9.7 mg O2/L
Conductivity:
345 μS/cm at 20 °C (typical for water source)
Nominal and measured concentrations:
Analysis of the freshly prepared test preparations at 0 and 72 hours showed that measured concentrations of between 0.41 and 8.8 mg/L were obtained, and of the old or expired media at 24 and 96 hours showed that measured concentrations of between 0.24 and 12 mg/L were obtained. 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.
Details on test conditions:
TEST SYSTEM
- Test vessel: 25 to 30 litre glass vessels
- Type (delete if not applicable): covered to prevent evaporation
- Fill volume: 20 litres.
- Aeration: No
- Temperature: approximately 13 °C to 15 °C
- Renewal rate of test solution (frequency/flow rate): 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.
- No. of organisms per vessel: 7 fish per vessel
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.20 g body weight/litre (based on mean weight values)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water declorinated through an activiated carbon filter and partly softenedusing an Elga Nimbus 1248D Duplex Water Softener.


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light, 8 hours dark.

EXAMINATIONS
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.

Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
0.89 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accommodated fraction
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
0.63 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accommodated fraction
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
LOELR
Effect conc.:
1.25 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accommodated fraction
Basis for effect:
mortality (fish)
Details on results:
There were no deaths in the control group.

Sub-lethal effects of exposure were observed at the 1.25, 5.0 and 10 mg/L loading rate WAF. These responses were swimming on the bottom, loss of equilibrium and a moribund state.

After approximately 24 hours exposure seven out of seven fish at 5.0 mg/L loading rate WAF were observed to be moribund. Due to animal welfare implications these fish were killed and classed as mortalities.

After approximately 96 hours exposure seven out of seven fish at 1.25 mg/L loading rate WAF was observed to be moribund. Due to animal welfare implications these fish were killed and classed as mortalities.

Reported statistics and error estimates:
The LL50 values at 1, 3 and 6 hours were determined by inspection of the data. The LL50 values and associated confidence limits 24, 48, 72 and 96 hours and the slope of the response curve and its standard error were calculated by the Trimmed Spearman-Karber Procedure. The LOEL and the NOEL at 0, 3, 6, 24 and 48 hours were calculated using the Fisher’s Exact Binomial Test with Bonferroni correction, whilst at 72 and 96 hours were calculated by the Step-down Cochran-Armitage Test Procedure. All results were calculated using the ToxRat Professional computer software package (TOXRAT).
Sublethal observations / clinical signs:

Inspection of the mortality data at 1, 3 and 6 hours, and analysis of the mortality data by Trimmed Spearman-Karber Procedure at 24, 48, 72 and 96 hours based on the nominal loading rates gave the following results:

 Time

(Hours)

 LL50

(mg/L Loading Rate WAF)

95% Confidence Limits

(mg/L Loading Rate WAF) 

 1  >10  -
 3  >10  -
 6  >10  -
 24  3.5  Not determined due to the nature of the data
 48  1.8   Not determined due to the nature of the data
 72  1.5  1.1 - 1.8
 96  0.89   Not determined due to the nature of the data
Validity criteria fulfilled:
yes
Conclusions:
Based on the water accommodated fraction (WAF) the test substance 96-hour LL50 (mg/L loading rate WAF) was 0.89 mg/L. The No Observed Effect Level (NOEL) was 0.63 mg/L and the Lowest Observed Effect Level (LOEL) was 1.25 mg/L.
Executive summary:

Introduction

The study was undertaken 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.

 

Method

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

 

Following a preliminary range-finding test, fish were exposed, in groups of seven, to WAFs of the test item over a range of nominal loading rates of 0.63, 1.25, 2.5, 5.0 and 10 mg/L for a period of 96 hours at a temperature of approximately 13 °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.

 

Results

Analysis of the freshly prepared test preparations at 0 and 72 hours showed that measured concentrations of between 0.41 and 8.8 mg/L were obtained, and of the old or expired media at 24 and 96 hours showed that measured concentrations of between 0.24 and 12 mg/L were obtained.

 

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.

 

Exposure of rainbow trout to the test item gave the following results:

 Time Point

(Hours)

LL50

(mg/L Loading Rate WAF) 

 95% Confidence Limits

(mg/L Loading Rate WAF)

 NOEL

(mg/L)

 LOEL

(mg/L)

 96  0.89  Not determined due to the nature of the data  0.63  1.25

 

Conclusion

Based on the water accommodated fraction (WAF) the test substance 96 -hour LL50 (mg/L loading rate WAF) was 0.89 mg/L. The No Obderved Effect Level (NOEL) was 0.63 mg/L and the Lowest Observed Effect Level (LOEL) was 1.25 mg/L.

Description of key information

Fish acute toxicity result for the test substance.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
0.89 mg/L

Additional information

Based on the water accommodated fraction (WAF) the test substance 96-hour LL50 (mg/L loading rate WAF) was 0.89 mg/L.  The No Observed Effect Level (NOEL) was 0.63 mg/L and the Lowest Observed Effect Level (LOEL) was 1.25 mg/L.