Cytogenotoxic potential and toxicity in adult Danio rerio (zebrafish) exposed to chloramine T

Abstract

Background: Chloramine-T (CL-T) is a synthetic sodium salt used as a disinfectant in fish farms to combat bacterial infections in fish gills and skin. While its efficacy in pathogen control is well-established, its reactivity with various functional groups has raised concerns. However, limited research exists on the toxicity of disinfection by-products to aquatic organisms. Therefore, this study aims to assess the sublethal effects of CL-T on adult zebrafish by examining biomarkers of nucleus cytotoxicity and genotoxicity, acetylcholinesterase (AChE) inhibition, and histopathological changes.

Methods: Male and female adult zebrafish (wildtype AB lineage) specimens were exposed to 70, 140, and 200 mg/L of CL-T and evaluated after 96 h. Cytotoxic and genotoxic effects were evaluated by estimating the frequencies of nuclear abnormalities (NA), micronuclei (MN), and integrated optical density (IOD) of nuclear erythrocytes. Histopathological changes in the gills and liver were assessed using the degree of tissue changes (DTC). AChE activity was measured in brain samples.

Results and conclusions: At a concentration of 200 mg/L, NA increased, indicating the cytogenotoxic potential of CL-T in adult zebrafish. Morphological alterations in the nuclei were observed at both 70 and 200 mg/L concentrations. Distinct IOD profiles were identified across the three concentrations. There were no changes in AChE activity in adult zebrafish. The DTC scores were high in all concentrations, and histological alterations suggested low to moderate toxicity of CL-T for adult zebrafish.

Keywords: Acetylcholinesterase activity; Gill and liver histopathology; Integrated optical density (IOD); Micronucleus test; Nuclear abnormalities.

Figure 1. Nuclear abnormalities frequency verified in zebrafish adults exposed to 70, 140 and 200 mg/L of Chloramine-T for 96h.

Values are expressed as medians, 25 and 75th quartiles. Medians with different letters are significantly different. Distinct letters represent a significative difference p < 0.05; +, mean.

Figure 2. Histograms representing the integrated optical density (IOD) from erythrocytes nuclei of zebrafish adults exposed to 70, 140 and 200 mg/L of Chloramine-T for 96 h.

Figure 3. Acetylcholinesterase (AChE) activity observed in zebrafish adults exposed to 70, 140 and 200 mg/L of Chloramine-T for 96 h.

Values are expressed as medians, 25 and 75th quartiles for AChE activity in micromoles per minute per milligram of protein (µmols/min − mg protein). The same letters represent that there was no significant difference (p < 0.05) between the control and concentration groups (mg/L).

Figure 4. Degree of Tissues Changes (DTC) and frequency of w1, w2, and w3 reversibility degree of damage for the gills and livers in zebrafish adults exposed to 70, 140 and 200 mg/L of Chloramine-T for 96 h.

(A) and (B), box plot for the median, 25th, 75th quartiles and mean (+) degree of tissues changes (DTC). (C) and (D), radar graphic visualization for the w1, w2, and w3 frequency distribution for gill and liver, respectively. Distinct letters among boxes represent difference (P < 0.05).

Figure 5. Histological sections of gills stained in HE from zebrafish adults control and exposed to 70, 140 and 200 mg/L of Chloramine-T for 96 h.

(A) Control specimen on the sagittal section; primary lamella (pl) and secondary lamella (sl) appear intact morphology; er (erythrocytes); sinus venous (black star); bar scale = 50 µm; in highlighted, squamous epithelial cells (ep) form a thin lining layer along the primary lamella; chlorite cells (white arrow) and pillar cells (p) are usually observed; bar scale = 10 µm. (B) A transversal section of a specimen exposed to 70 mg/L; melanomacrophages (Mm) are readily aggregated peripherally to the arterioles; bar scale = 100 µm; in highlighted, an initial aneurism formation (arrow); bar scale = 10 µm. (C) A gill segment in a sagittal section of specimen exposed to 140 mg/L showing secondary lamellae fusioned (arrowhead) and focal epithelial cells necrosis; bar scale = 100 µm. (D), (E) and (F) Sagittal sections from specimens exposed to 200 mg/L showing distinct lamellar fusion morphology: bar scale = 50 µm. In (D), an initial lamellar fusion due to epithelial cells proliferation (white arrow); in (E), a thin layer of epithelial cells lines the fully fused lamellae (arrows); basophilic nuclei niches are diffusely distributed diffusely distributed according to the extensive cellular proliferation; and (F), layers of the same cells thicken the gill lining, suggesting a metaplasia adaptation (white arrow); arrowhead shows epithelial cells desquamation.

Figure 6. Histological sections of liver parenchyma in zebrafish adults exposed to 70, 140 and 200 mg/L of Chloramine-T for 96 h.

(A)–(C) Control samples. (A) Low magnification showing a typical and homogeneous architecture; portal vessels (bv) are randomly distributed and partially filled by blood cells; sinusoids displayed narrow unfilled spaces which usually connect to portal vessels; bar scale = 100 µm. (B) Detail of intact sinusoidal web; some hepatocyte displayed normal cytoplasmatic vacuoles suggestive of lipids or glycogen stocks; a cordonal hepatocytes arrangement are commonly noted; bar scale = 50 µm. (C) Sinusoid aspect in high magnification; intra sinusoidal macrophage (M) are adhered to the endothelial wall; eosinophilic granular pigments are typical in the cytoplasm of hepatocytes which present nuclei basophilic with evident nucleoli; bar scale = 10 µm. (D) and (E) Sample from a specimen exposed to 70 mg/L. Sinusoidal web and portal vessels (pv) are mildly distended and filled by leucocyte (L) cells; hepatocytes cytoplasm showing enlarged aspect; bar scale = 50 µm; in (E), sinusoidal architecture is disrupted with lumen content serous exudate (E); hepatocytes are notably modified displayed increased cytoplasmic content suggestive of hydropic degeneration; bar scale = 50 µm. (F) Sample from a specimen exposed to 140 mg/L; general aspect showing hepatocytes clearly vacuolated, typical of fatty degeneration; portal vessel is distended and hyperemic; the sinusoidal web is not apparent due the hepatocellular compression; bar scale = 100 µm. (G) Sample from a specimen exposed to 200 mg/L; necrosis areas are diffusely distributed into the parenchyma, although some region the tissue architecture is preserved; bar scale = 150 µm. (H) and (I) high magnification of the 200 mg/L sample; cell necrosis (n) area surrounded by hepatocytes in degeneration process; nuclei are pyknotic (pk), in karyorrhexis (kh) or karyolisis (ka) stages; bar scale = 10 µm. HE stain.