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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:
2015-07-31 to 2015-08-28
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)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Analytical purity: 99.9% (acid titration)
- Source and lot/batch No.of test material: A14JB3442
- Expiration date of the lot/batch: 2016-09-30 (retest date)
- Purity test date: 2015-02-18 (certificate of analysis release date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: 0.21 g/L, stability in water not indicated
Analytical monitoring:
yes
Details on sampling:
- Sampling method: singular samples (3 mL) were taken from all test concentrations and the control at t=0 h and t=96h. All samples were taken from the approximate center of the test vessels.
- Sample storage conditions before analysis: samples were stored in a freezer until analysis. Additionally, reserve samples of 3.0 mL were taken from all test solutions for possible analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A supersaturated stock solution (SS) with a loading rate of nominal 100 mg/L was prepared. The test item was mixed into the test water using magnetic stirring for 2 days at room temperature in the dark. This resulted in a clear and colourless dispersion that contained undissolved floating material and precipitate.The supersaturated stock solution was filtered through a 0.45 µm membrane filter (Whatman; RC55). The filter was preconditioned with a small volume of test solution that was discarded. The resulting SS was used as the highest test concentration.
- Eluate: no
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): final test solutions were clear and colourless.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: Carp
- Origin: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, the Netherlands
- Length at study initiation (length definition, mean, range and SD):
* range-finding test: 2.2 ± 0.1 cm
* final test: 2.2 ± 0.3 cm
- Weight at study initiation (mean and range, SD):
* range-finding test: 0.23 ±0.06 g
* final test: 0.28 ±0.10 g
- Feeding during test: no
- Food type: pelleted fish food (Essence (300-500 µm), Coppens International bv, Helmond, The Netherlands)

ACCLIMATION
- Acclimation period: At least 12 days after delivery
- Acclimation conditions (same as test or not): no data
- Type and amount of food: pelleted fish food (Essence (300-500 µm), Coppens International bv, Helmond, The Netherlands) - daily
- Health during acclimation (any mortality observed): mortality during the seven days prior to the start of the test was less than 5%.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
180 mg CaCO3/L
Test temperature:
21.6 - 22.4
pH:
7.7 ± 0.3
Dissolved oxygen:
6.0 - 8.4 mg/L
Salinity:
not applicable
Nominal and measured concentrations:
Nominal test concentrations:
Range-finding test: 10 and 100% of the filtered 100 mg/l solution
Final test: 10, 18, 32, 56 and 100% of a SS prepared at nominally 100 mg/l.

Measured test concentrations:
Range-findinf test: 0h: 0.94, 96 mg/L; 96h: 0.93, 94.6 mg/L
Analysis of samples taken during the range-finding test from the ten-fold dilution showed unexpectedly a hundred-fold lower concentration instead of a ten-fold lower concentration. However the concentrations during the test period remained stable. Overall it was concluded that all test concentrations were in agreement with nominal. This possible error did not affect the selection of a test range for the final test.

Final test:
0h: n.d., 9.88, 16.9, 31.0, 52.9, 97.4 mg/L;
96h: n.d., 9.7, 16.6 mg/L

Analysis of the samples taken at the start of the final test showed that measured concentrations were in agreement with nominal (94-99%). Concentrations remained stable during the test period at 10 and 18 mg/l, i.e. the only test groups with surviving fish. Hence, it was justified to base all further results
and the toxicity parameters on nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass aquarium
- Material, size, headspace, fill volume: 6.5 L, all-glass, containing 5 L of test solution.
- Aeration: the test media were not aerated during the test.
- No. of organisms per vessel: 7
- Biomass loading rate: 0.39 g fish/L test medium.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water purified by reverse osmosis
- Intervals of water quality measurement:
* conductivity, pH, nitrate, nitrite and ammonia concentration: once a week
* temperature: continuous

OTHER TEST CONDITIONS
- Photoperiod: 16-hour photoperiod daily

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: yes
- Test concentrations: 10 and 100 % SS
- Results used to determine the conditions for the definitive study: yes, range-finding test and results of a pre-experiment to determine the solubility of the test item in test water: yes
Reference substance (positive control):
yes
Remarks:
pentachlorophenol
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% C.L.: 18-32 mg/L
Details on results:
- Behavioural abnormalities:
* at a concentration of 18 mg/L, after 3,75 hours: swimming at bottom of tank, immobile
* at a concentration of 18 mg/L, after 24-96 hours: discoloured and immobile
- Mortality of control: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no remarkable observations, clear test medium
- Effect concentrations exceeding solubility of substance in test medium: no remarkable observation, clear test medium.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Mortality: 100% after 48 h with 0.46 mg/L
- 96-h LC50 = 0.32 mg/L
Reported statistics and error estimates:
no data
Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of T001036 was determined in a 96-hour static test according to OECD guideline 203. Due to the low water solubility, a supersaturated solution was prepared (nominal loading rate of 100 mg/L). The 96-hour LC50 was 24 mg T001036/L based on analytically confirmed nominal concentrations (95% confidence interval between 18 and 32 mg/l) and was already reached after 3¾ hours of exposure. The results of the test can be considered reliable without restriction.

Description of key information

The study of Migchielsen (2016), investigating the acute toxicity of T001036 in Carp according to OECD guideline 203, was considered as the key study for endpoint coverage.  An LC50 of 24 mg/L (based on nominal test concentrations) was found after an exposure of 96 hours in a static test in freshwater. Based on the results it can be considered that T001036 is harmful to fish.

Key value for chemical safety assessment

LC50 for freshwater fish:
24 mg/L

Additional information

One key study of Migchielsen (2016) was available on the acute toxicity of T001036 to Carp. Carp was exposed for 96 hours to T001036 under static conditions. The nominal test item concentrations were 10, 18, 32, 56 and 100% of a SS prepared at nominally 100 mg/l and in parallel the control. Analysis of the samples taken at the start of the final test showed that measured concentrations were in agreement with nominal (94-99%). Concentrations remained stable during the test period at 10 and 18 mg/l, i.e. the only test groups with surviving fish. Hence, it was justified to express the results in terms of analytically confirmed nominal concentrations. Based on the nominal test concentrations a 96 -h LC50 of 24 mg/L for T001036 (with 95% confidence limits of 18 and 32 mg/L) was obtained.

Based on the results of this study, it can be considered that T001036 is harmful to fish.