Severity Classification of Clinical Signs and Defining the Moribund State as an Experimental Endpoint for Acute Toxicity Testing Using Japanese Medaka (Oryzias latipes)

Abstract

Use of the moribund state as an endpoint for acute toxicity testing in fish is complicated by the lack of consensus about the sublethal signs that define the state or that can predict imminent death. Defining the moribund state in fish requires determination of the association between each observable sublethal sign and transition to imminent death. To establish a criterion for defining the moribund state in Japanese medaka (Oryzias latipes), we determined death/clinical‐sign ratios, defined as the proportion of fish with each clinical sign that transitioned to imminent death, for 13 clinical signs observed in individually housed fish exposed to one of two typical toxicants, three pharmaceuticals, two pesticides, and one metal. Also, the time from onset of each clinical sign to transition to imminent death (referred to as the survival time) was determined by continuous monitoring. Three of the observed clinical signs—immobility, lethargy, and immobility at the surface—were found to be indicators of the moribund state, with each of these signs having a death/clinical‐sign ratio of 1.0. Evaluation of the survival time after onset of the other 10 signs enabled determination of whether timely euthanasia would be appropriate, thereby providing a means of reducing the suffering of laboratory fish in the period before death. Environ Toxicol Chem 2022;41:1089–1095. © 2022 SETAC

Copper sulfate induces clinico-hematological, oxidative stress, serum biochemical and histopathological changes in freshwater fish rohu (Labeo rohita)

“…On days 12th, 24th, and 36th post-treatment, ~2.0 mL of blood from the caudal vein of six randomly selected fish was collected using a 26-gauge hypodermic sterile needle after the fish was anesthetized with clove oil (4 mg.L −1 ) ( 27 ). A blood sample was placed in anticoagulant-coated glass test tubes on each collection day.…”

“…Various hematological parameters, including hemoglobin (Hb) quantity, hematocrit (Hct), red blood cells (RBCs) counts, total and differential white blood cells (WBCs) counts, lymphocytes, and neutrophils were determined as described previously ( 28 ). Total proteins (Cat # 997180) were assessed according to the earlier method ( 27 ). After centrifugation, serum was separated and stored at −20°C.…”

“…Similarly, the liver, kidneys, brain, and gills were preserved in 10% paraformaldehyde for microscopic studies. After fixation, all the visceral tissues were processed for histopathological changes using eosin and hematoxylin staining procedures ( 27 ). Tissue slides were examined for the presence of lesions by two pathologists, and if discrepancy come across, then a third pathologist was asked for judgment.…”

“…Various hematological parameters, including hemoglobin (Hb) quantity, hematocrit (Hct), red blood cells (RBCs) counts, total and differential white blood cells (WBCs) counts, lymphocytes, and neutrophils were determined as described previously (28). Total proteins (Cat # 997180) were assessed according to the earlier method (27). After centrifugation, serum was separated and stored at −20 • C. Serum biochemical parameters like aspartate aminotransferase (AST; Cat # 30243), alanine aminotransferase (ALT; Cat # 30254), creatinine (Cat # 99108), cholesterol (Cat # 30183), alkaline phosphatase (ALP; Cat # 30134) and triglycerides (Cat # 30364) were measured using commercial kits (Canovelles; Barcelona, Spain).…”

“…Due to anthropogenic activities, several toxicants, including CuSO 4 , enter the aquatic ecosystem and cause detrimental effects on the exposed aquatic organisms (13). Fishes are considered an important component of the aquatic food chain among different aquatic organisms and reflect almost all the adverse effects upon exposure to toxicants (25)(26)(27)(28). High pollution due to various heavy metals in water has become a significant problem because of the threat to aquatic life and public health concerns related to fish consumption (23, 29, 30).…”