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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:
08-13 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well conducted and well described study in accordance with GLP and OECD Guideline No 203.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
hardness of the water was ≥ 250 mg/L as CaCO3
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Concentrations: single samples for analysis were taken from the control and all test concentrations.

Sampling method: samples were taken at the start (t = 0 h) and every 24 h thereafter in old and new solutions and the control.

Storage: samples were analysed directly after sampling.

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Method:
Four stock solutions were prepared with a nominal concentration of 20 mg/L for the fresh mediums at t = 0 h, t = 24 h and t = 48 h and a nominal concentration of 11 mg/L for the fresh medium at t = 72 h. For this purpose, ca. 200 mg (for the fresh mediums at t = 0 h to t = 48 h) and 112.08 mg (for the fresh medium at t = 72 h) of the test item were carefully added to 10 L of test water in a sealed glass vessel of approximately 10 L capacity containing a magnetic stirring bar. The mixing was performed during 24 h in the dark. Following mixing, the contents of the vessel were allowed to stand undisturbed for 1 h at room temperature. Then the stock solution was diluted into 5-L volumetric flasks with test water to obtain the required test concentrations. Each prepared concentration was inverted several times before filling of aquaria to ensure adequate mixing and homogeneity. After filling, the vessels were closed immediately after introduction of fish.
The test sample formed a clear colourless solution and appeared to be completely soluble when mixed with test water at the concentrations prepared.

Controls: test water without test substance but treated in the same way as the test substance solutions.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
Common name: Zebrafish
Source: Elevage de la Grande Rivière, Saint-Forgeux, France
Length at study initiation (mean, range): 2.49 cm (range: 2.23-2.70 cm)
Feeding during test: no feeding from 24 h prior to the test and during the total test period.

ACCLIMATION
Acclimation period: at least 12 days
Acclimation conditions: same as test
Type and amount of food: pelleted fish food
Feeding frequency: daily

VALIDITY OF BATCH OF FISH
In the batch of fish which was used for the test, mortality during the seven days prior to the start of the test was less than 5 %.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
no
Hardness:
≥ 250 mg/L as CaCO3
Test temperature:
23 ± 2 °C
pH:
7.16-7.94
Dissolved oxygen:
6.04-8.57 mg O2/L
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentrations: 1.0, 1.8, 3.3, 6.0, 11 and 20 mg/L
Mean measured concentrations: 0.54, 1.58, 2.72, 4.85, 8.17 and 16.01 mg/L
Details on test conditions:
TEST SYSTEM
Test vessel: glass aquaria (3.5 L) filled with minimum headspace (approximately 1 cm) and closed by placing a glass plate on the aquarium and sealed with vaseline.
Aeration: no
Renewal rate of test solution (frequency): daily
No. of organisms per vessel: 7
No. of vessels per concentration (replicates): single
No. of vessels per blank control (replicates): single
Biomass loading rate: 1.0 g/L

TEST MEDIUM / WATER PARAMETERS
Source/preparation of dilution water: reconstituted water, as prescribed by the OECD Guideline 203
Conductivity: < 10 µS/cm
Culture medium different from test medium: no
Measurements: pH, nitrate and nitrite concentration, ammonia concentration, total water hardness and dissolved oxygen were measured once a week. Temperature was recorded daily.

OTHER TEST CONDITIONS
Photoperiod: 16 h light and 8 h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): fish were inspected at least 3, 6, 24, 48, 72 and 96 h following the start of exposure.

TEST CONCENTRATIONS
Spacing factor for test concentrations: 1.82

Range finding study
Nominal test concentrations: 0.1, 1.0, 10 and 20 mg/L
Results used to determine the conditions for the definitive study: in the range-finding test, no mortality was observed at the nominal concentrations of 0.1, 1.0, 10 mg/L (even if sub-lethal effects were quickly observed at 10 mg/L) while all fish exposed to the concentration of 20 mg/L died very quickly. The chemical analyses revealed that test item concentrations were not very stable during the test period. Based on these results, test concentrations of 1.0, 1.8, 3.3, 6.0, 11 and 20 mg/L (nominal) were selected for the main test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (non-concurrent)
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
11.44 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: -
Details on results:
Mortality of control: no

After the 96 h of exposure, mortality was 0 % at 0 and 0.54, 1.58, 2.72, 4.85 and 8.17 mg/L, and 100 % at 16.01 mg/L.

Sub-lethal effects were quickly observed (t ≤ 3 h) for fish exposed to the concentration of 8.17 mg/L (but no mortality at the end of the test) and fish exposed to the concentration of 20 mg/L died very quickly (t ≤ 3 h). Hence, the highest concentration resulting in no mortality at 96 h was 8.17 mg/L and the lowest concentration resulting in 100 % mortality at 96 h was 16.01 mg/L.
Results with reference substance (positive control):
Results with reference substance valid: yes

Historical data (dated: July 2012): LC50 (24 h) = 179.39 mg/L
Reported statistics and error estimates:
no data
Sublethal observations / clinical signs:

none

Validity criteria fulfilled:
yes
Remarks:
mortality in control was 0 %; semi-static test conditions; oxygen content was ≥ 60 % ASV in the control and test vessels throughout the test; results were based on measured concentrations
Conclusions:
Under the test conditions, the 96h-LC50 of nopyl acetate for zebrafish (Danio rerio) was estimated to be 11.44 mg/L, based on mean measured concentrations.
Executive summary:

An acute aquatic toxicity study was performed on zebrafish (Danio rerio) in accordance with GLP and OECD Guideline No 203. Following a preliminary range-finding test, groups of seven fish were exposed to an aqueous solution of nopyl acetate, at nominal test concentrations of 1.0, 1.8, 3.3, 6.0, 11 and 20 mg/L, in aquaria closed over the study period with a minimum of headspace. The total test period was 96 h and test solutions were renewed on a daily basis. The mortality of the fish was determined in a semi-static 96-h test by visual observation after 24, 48, 72 and 96 h.

Exposure concentrations were measured at the start of the test and every 24 h thereafter in old and new solutions. Concentrations of the test item were not satisfactorily maintained within ± 20 % of the initial concentrations throughout the test. Thus, the evaluation of the effects on Danio rerio was based on the geometric mean of the measured concentrations: 0.54, 1.58, 2.72, 4.85, 8.17 and 16.01 mg/L.

After 96 h of exposure, mortality was 0 % at 0 and 0.54, 1.58, 2.72, 4.85 and 8.17 mg/L, and 100 % at 16.01 mg/L. Sub-lethal effects (without mortality at the end of the test) were quickly observed (t ≤ 3 h) for fish exposed to a concentration of 8.17 mg/L and fish exposed to a concentration of 20 mg/L died very quickly (t ≤ 3 h). Hence, the highest concentration resulting in no mortality at 96 h was 8.17 mg/L and the lowest concentration resulting in 100 % mortality at 96 h was 16.01 mg/L. Based on measured concentrations, the LC50 determined for each experimental time point was 11.44 mg/L.

Therefore, the 96h-LC50 of nopyl acetate for zebrafish (Danio rerio) was estimated to be 11.44 mg/L, based on mean measured concentrations.

Description of key information

An acute aquatic toxicity study was performed on zebrafish (Danio rerio) in accordance with OECD Guideline No 203.
The 96h-LC50 of nopyl acetate for freshwater fish is 11.44 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
11.44 mg/L

Additional information

Key study conducted according to OECD 203 Guideline and GLP without deviation.

Fish (Danio rerio) were exposed to nopyl acetate at nominal concentrations of 1.0, 1.8, 3.3, 6.0, 11 and 20 mg/L over a test period of 96 hours (semi-static test, daily renewal). Concentrations were measured at the start of the study and daily until the end of the test. The concentrations of the test item were not satisfactorily maintained within ± 20% of the initial concentrations throughout the test, thus the LC50 were based on the geometric mean of the measured concentrations. The 96h-LC50 determined in this study can be used as key value for chemical safety assessment.