TITLE: Acute toxicity of injected drugs and substances in fish 
AUTHORS: Bruna Patrícia D.c., Sabrina Alana Gomes Pinto, Layana Aquino Moura, Diógenes Silva, Kelly Christina Ferreira Castro, Caio Maximino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol, modified from OECD 203 (Acute toxicity test, adult fish), tests toxic (lethal and nonlethal) effects of substances that have been injected intraperitoneally in adult fish. While OECD 203 is appropriate for testing the effects of waterborne substances (e.g., sewage effluents, pesticides, and other toxicants that can reach water bodies), the modified version can be added in a drug screening pipeline</div></div>

[STEPS]
?. [Anesthesia and injection procedures]
Anesthesize fish in ice-cold water (12-14 ºC). Remove animal from water as soon as movements stop.
on ice
?. [Anesthesia and injection procedures]
Transfer animal to surgical bed (water-soaked sponge with a cut to fixate the fish), kept in a vessel with ice-cold water.
on ice
?. [Anesthesia and injection procedures]
Inject the chemical, using the protocol by Kinkel et al. (2010).
Recommended injection volumes are of 1 μL/0.1g body weight. Using a 10 µL microsyringe is useful in the case of small fish, such as zebrafish.
?. [Anesthesia and injection procedures]
Gently remove animal from the surgical bed and return to observation tank, or leave to rest until behavioral experiment.
?. [Behavioral experiment]
Optionally, running a behavioral experiment 30 min after injection can decrease the number of animals used in a drug screening pipeline. We commonly screen chemicals for anxiolytic-like effects using the light/dark preference test and/or the novel tank test.
?. [Observe lethal and sublethal endpoints]
Register lethal and sublethal endpoints at the following time points after injection:2.5 h5.5 h24 h30 h48 h54 h72 h78 h96 h
?. Mortality: Definition of "mortality" is derived from OECD 203: "Fish are considered dead if there is no visible movement (e.g. gill movements) and if touching of the caudal peduncle produces no reaction. Mortalities are recorded, and dead fish are removed as soon as they are observed".
?. Register sublethal endpoints, as follows:  AB1DomainClinical sign2DistributionLoss or densing of schooling / shoaling behavior3Vertical distribution - Surfacing or bottom-dwelling4Equilibrium and buoyancyAbnormal horizontal orientation5Abnormal vertical orientation6Loss of buoyancy control7Observed behaviorsHypoactivity or hyperactivity8Spiral swimming9Hyperventilation or hypoventilation10Irregular ventilation11Increased ventilation depth12Convulsions13Coughing, gulping, or gasping14Surface escape / avoidance behaviors15Bottom escape / avoidance behaviors16Irritated skin behaviors17Aggression and/or cannibalism18AppearanceTetany19Skin color - Darkening, lightening, or mottled skin20Oedema21Hemorrhagic areas or petechiae22Exophthalmia23Mucus secretion24Faecal casts25Provoked behaviorVisual and tank knocking stimulus - over reactivity or under reactivity26Tactile stimulus - over reactivity or under reactivity
AB1DomainClinical sign2DistributionLoss or densing of schooling / shoaling behavior3Vertical distribution - Surfacing or bottom-dwelling4Equilibrium and buoyancyAbnormal horizontal orientation5Abnormal vertical orientation6Loss of buoyancy control7Observed behaviorsHypoactivity or hyperactivity8Spiral swimming9Hyperventilation or hypoventilation10Irregular ventilation11Increased ventilation depth12Convulsions13Coughing, gulping, or gasping14Surface escape / avoidance behaviors15Bottom escape / avoidance behaviors16Irritated skin behaviors17Aggression and/or cannibalism18AppearanceTetany19Skin color - Darkening, lightening, or mottled skin20Oedema21Hemorrhagic areas or petechiae22Exophthalmia23Mucus secretion24Faecal casts25Provoked behaviorVisual and tank knocking stimulus - over reactivity or under reactivity26Tactile stimulus - over reactivity or under reactivity
?. [Anesthesia and injection procedures]
Repeat injection procedures in 2 animals, for a total of 3 animals per dose.
?. Humanely sacrifice animals after the last observation (i.e., 96 h after injection).
?. After sacrifice, animals can be dissected, and organs be harvested for pathology.
?. [Stepwise procedure]
After running the control (vehicle) group, proceed to inject animals with the lowest dose and follow the stepwise procedure:
?. [Stepwise procedure]
Inject 3 animals with the lowest dose (e.g., 5 mg/kg) and repeat steps 7-9, above.
?. [Stepwise procedure]
If, at the end of the last time interval (96 h), 0-1 animals are dead, replicate the experiment with 3 more animals to confirm non-lethality. If 2-3 animals are dead, proceed to determine the dose as a lethal dose.
?. [Stepwise procedure]
If non-lethality is confirmed, inject 3 animals with the next dose (e.g., 50 mg/kg), and repeat steps 7-9. . At the end of the last time interval for this dose, determine mortality ( )
?. [Stepwise procedure]
Stop experiment when the lethal dose is reached. No more animals must be exposed to the test chemical once the dose is established.
Results should be reported as:Mortality in the control(s)The LD value at 96 hIncidence and description of visible abnormalities, as describd in step 8