Developmental and toxicological effects of butylated hydroxytoluene metabolites on zebrafish larvae

Abstract

Butylated hydroxytoluene (BHT) is among the most widely used synthetic phenolic antioxidants. However, BHT and its metabolites have been detected in aquatic ecosystems, posing potential risks to aquatic organisms. The present study aimed to investigate the effects of BHT metabolites on embryonic development in zebrafish. To this end, embryos were exposed to BHT metabolites, including 3,5-di-tert-butyl-4 hydroxybenzaldehyde (BHT-CHO), 2,6-di-tert-butyl-4-(hydroxymethyl) phenol (BHT-OH), 3,5-di-tert-butyl-4 hydroxybenzoic acid (BHT-COOH), 2,6-di-tert-butyl-P-benzoquinone (BHT-Q), and 2,6-di-tert-butyl-4-hydroxy-4-methylcyclohexa-2,5-dien-1-one (BHT-quinol), from 1-120 h post-fertilization (hpf). BHT-CHO, -OH, -COOH, -Q, and -quinol were toxic to zebrafish larvae with 96 h LC₅₀ values of > 0.10, 15.85, 4.51, > 1.30, and 3.46 mg/L, respectively. Moreover, the acute toxicity of BHT metabolites to zebrafish larvae was indicated by morphological abnormalities, changes in heart rate, and alterations in locomotory behavior. The results indicated that exposure to BHT-COOH and BHT-OH caused intestinal developmental abnormalities, blood coagulation, tail deformities, and pericardial edema. Exposure to BHT-Q and BHT-quinol resulted in abnormal swim bladder development. Moreover, alterations in heart rate and locomotory behavior were observed in zebrafish larvae exposed to BHT-COOH, BHT-OH, and BHT-quinol. These findings demonstrate that exposure to BHT metabolites significantly affects the early growth and developmental stages of zebrafish larvae.

Keywords: Acute toxicity; Cardiotoxicity; Environmental pollution; Locomotor activity; Synthetic phenolic antioxidants.

Fig. 1

Morphological effects of BHT-COOH exposure to zebrafish larvae at 120 hpf. (A) Control; (B) fishtail deformity (FD) after exposure to 1 mg/L BHT-COOH; (C) intestinal inflammation (IF) after exposure to 1 mg/L BHT-COOH; (D) pericardial edema (PE) after exposure to 5 mg/L BHT-COOH; (E) blood coagulation (BC) and PE after exposure to 5 mg/L BHT-COOH; (F) PE after exposure to 10 mg/L BHT-COOH; (G) PE and BC after exposure to 10 mg/L BHT-COOH. At 5 mg/L, PE was confirmed in five of nine larvae sampled; at 10 mg/L, PE was confirmed in eight of nine larvae. Effects of BHT-COOH exposure on (H) body length and (I) eye size of zebrafish larvae (*p < 0.05, **p < 0.005).

Fig. 2

Zebrafish larvae exposed to control, 0.1, 1, and 2 mg/L of BHT-OH concentrations exhibit delayed swim bladder development in 0, 1, 1, and 4 zebrafish, respectively. (A) control; (B) 0.1 mg/L; (C) 1 mg/L; (D) 2 mg/L.

Fig. 3

Zebrafish larvae exposed to BHT-Q at 120 hpf exhibit delayed swim bladder development.

Fig. 4

Morphological effects of BHT-quinol exposure to zebrafish larvae at 120 hpf. (A) Control; (B) fishtail deformity (FD) and uninflated swimming bladder (USB) after exposure to 0.1 mg/L BHT-quinol; (C) USB after exposure to 0.1 mg/L BHT-quinol; (D) Pericardial edema (PE) after exposure to 1 mg/L BHT-quinol; (E) Malformed jaw and eye (JA) after exposure to 1 mg/L BHT-quinol; (F) FD after exposure to 2 mg/L BHT-quinol. (G) There was no difference in body length between the control and BHT-quinol-treated groups. (H) There was a statistically significant difference in eye size between the control and BHT-quinol-treated groups (*p < 0.05).

Fig. 5

Effects of butylated hydroxytoluene metabolites on the heartbeat of zebrafish larvae. Zebrafish embryos were exposed to each metabolite, and heartbeats were analyzed using DanioScopeTM at 120 hpf (*p < 0.05, **p < 0.005).

Fig. 6

Effects of butylated hydroxytoluene metabolites on locomotor behavior of zebrafish larvae. Zebrafish embryos were exposed to BHT-CHO, BHT-COOH, BHT-OH, BHT-Q and BHT-quinol for 120 h; their locomotor activity was analyzed using DanioVision at 120 hpf. (*p < 0.05, **p < 0.005).