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EC number: 202-216-2
CAS number: 93-08-3
Range finding test
95% confidence interval- Lower Fiducial limit
95% confidence interval - Upper Fiducial limit
Median Lethal Concentration
Body weight (g)
This study was conducted as per OECD
203 (2019) to assess the acute toxicity effects of test chemical on
zebrafish (Danio rerio) following exposure up to 96 h under
static condition. Juvenile fish of same age and normal in appearance
were used in this (originate from same source and population). The
average length and weight (10 fish) were observed 1.4 cm and 0.012g,
respectively. Fish were fed (commercial fish food) daily during
acclimatization. Photoperiod and light intensity were maintained such
as16 h light- 8 h dark, 732-804 Lux during experiment. Hardness of water
was measured once during acclimatization and found to be 165 mg CaCO3/L,
temperature, pH and dissolve oxygen were maintained between 22.1-22.7
°C,6.4-7.1,6.8-8.5 mg/L, respectively, throughout the test. Fish were
acclimatized for 7 days prior dosing. Natural water was used as dilution
control and acetone in natural water (500µL/5000 mL conc) was used as
vehicle control and the same concentration was used for formulation of
test chemical. The study was initiated with a range finding test by
using following concentrations of 0 (control), 0 (vehicle control),
2.35, 4.23, 7.61, 13.71, 24.67 mg/L. During range finding test, no
mortality or abnormality was found in control groups and 2.35, 4.23,
7.61 mg/L conc., but 100% mortality were found in 13.71, 24.67 mg/L
concentrations. Hence, a main study was conducted using 0 (control), 0
(vehicle control), 6.61, 7.93, 9.52, 11.42, 13.71 mg/L concentrations.
No mortality or abnormality was found in control groups and 6.61, 7.93
mg/L concentration but abnormal behavioural responses like head shaking,
loss of buoyancy of control, corkscrew swimming were observed in 9.52,
11.42, 13.71 mg/L conc. 7 fish were used/concentration during range
finding and main study. HPLC method was used for method validation and
active ingredient analysis along with stability of the test item in the
test medium. Test item was found to be stable in the test medium. The
active ingredient content results were considered acceptable (80-120%)
as during main study 0 h and 96 h avg. recovery for 6.61, 9.52, 13.71
mg/L conc were 96.80%, 95.72%, 95.72% & 96.92%, 95.83%, 95.58%,
respectively, were found in acceptable range. The test is valid as all
the validity criteria are fulfilled: No mortality in control or vehicle
control were found throughout the 96-h test duration; Dissolve oxygen
conc. was maintained above 60% in all test vessels throughout the test;
The recovery active ingredient content was found between 80-120% up to
96h. The 96 h LC50 of test chemical to zebrafish (Danio rerio)
was determined to be 10.44 mg/L with 95% confidence upper and lower
limit were found to be 10.79 and 10.09 mg/L, respectively. 96h LC50
value with 95% confidence limits (upper limit, lower limit) were
calculated by probit analysis using NCSS Software 2019, version 19.0.3.
Hence, as per CLP classification category, the test chemical can be
categorized as aquatic chronic category 3.
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