2.3 Fish acute toxicity test
The water used for marine fish toxicity assessmentwere obtained from the same location that the fish were
collected from. For freshwater fish, the lake water was used instead of tap water. This is to avoid chlorine
contamination that exists in the tap water.The fish were kept under normal laboratory illumination with a daily
photoperiod of 12-16 hours. The temperature of the water was between 23 ± 2°C. The dissolved oxygen and pH of
the water were 5-7 ppm and 7 respectively. The fish were observed carefully every day for signs of disease, stress,
physical damage, and mortality. Dead and abnormal specimens were removed immediately upon observation. After
the adaptation period was complete; a group of 10 healthy fish were selected randomly and placed into 6.5 litter
plastic tanks provided with electric air pumps. Healthy fish can be recognized by their normal swimming style. Fish
swimming abnormally should not be selected as they may have a weak resistance to the chemicals used in testing.
Each tank was equipped with 5 litters of water. No food was provided for the fish during the test. The weight of each
fish is approximately 0.5 g. The acute fish toxicity test was performed according to the OECD standard methods
[20]. Each IL was tested for at least four concentrations. Different concentrations were added to each testing tank.
Behaviours of the fish were monitored closely and dead fish were removed immediately. The number of dead fish
for each concentration was recorded after 24, 48, 72, and 96 hours. Then the median lethal concentration (LC50)
value (that is the concentration required to kill 50% of test fish in 96 hours) was calculated. The results from the
experiment were compared with acute toxicity rating scale provided by U.S. Fish and Wildlife Service (USFWS)
(Table 1).