The increasing prevalence of cancer in Thailand over the past decade has resulted in a substantial rise in the use of anticancer drugs, which are eventually discharged into municipal wastewater through hospital and domestic effluents. The inability of conventional wastewater treatment systems to completely remove these pharmaceuticals has been widely reported. The continuous release of these emerging anticancer agents into aquatic environments reduces water quality and threatens biodiversity. Even at trace levels, these compounds may act as persistent pollutants capable of impairing ecosystem. This study investigated the occurrence and concentration levels of three widely used chemotherapeutic agents including cyclophosphamide (COP), doxorubicin (DOX), and vincristine (VIN) in Bangkok’s municipal wastewater to evaluate their potential environmental risks. Thirty-two influent and effluent wastewater samples were collected from eight large-scale wastewater treatment plants (WWTPs) from October 2024 to January 2025. Samples were processed using solid-phase extraction (SPE) and analyzed by liquid chromatography–triple quadrupole mass spectrometry (LC–MS/MS). The analytical method demonstrated high precision and reproducibility, with relative standard deviations (%RSD) below the 20% acceptance limit for all compounds. Method accuracy ranged from 81.84% to 107.21%. Results showed the presence of only COP in almost influent and effluent at levels ranging from 0.26 to 2.06 µg/L. In contrast, DOX and VIN levels remained consistently below the limits of quantitation (LOQ) in all WWTP samples. This study establishes the first baseline for COP, DOX, and VIN contamination in Bangkok’s municipal wastewater. Notably, the residue of COP in wastewater suggests that current wastewater treatment facilities in Thailand are insufficient for its removal, posing a potential long-term risk to local aquatic ecosystems.

Currently, emerging pharmaceutical contaminants (EPCs) pose a significant risk to aquatic biodiversity on both a global and regional scale. Their accumulation in the environment presents several challenges to both human health and ecological integrity. Among EPCs, cyclophosphamide (COP), a widely used alkylating cytotoxic and immunosuppressive drug in human and veterinary oncology, has become one of the most frequently detected environmental contaminants. It has been reported to impair the immune system, induce oxidative stress, and exhibit genotoxic and cytotoxic effects in various fish species. However, limited research has evaluated its toxicity in the Siamese fighting fish (Betta splendens), an endemic significant aquatic species in Thailand. This study evaluated the acute toxicity of COP in Betta splendens by assessing systemic oxidative stress responses, alterations in gene expression, and localized histopathological changes within both hepatic and renal tissues. The 96 h of median lethal concentration (LC₅₀) value for COP in Betta splendens was determined to be 793.4 mg/L. High-concentration exposure (800 mg/L) induced severe systemic oxidative stress, evidenced by a significant reduction in superoxide dismutase (SOD) activity and a marked elevation in malondialdehyde (MDA) levels across both liver and kidney. At the transcriptomic level, acute COP exposure significantly upregulated pro-inflammatory (interleukin-1β, IL-1β and tumor necrosis factor, TNF-α), cellular stress (heat shock protein 70, HSP70), and apoptotic (caspase-3, Casp3) genes. Semi-quantitative histopathological evaluation revealed severe concentration-dependent structural damage. Hepatic lesions peaked at 800 mg/L characterized by diffuse vacuolation, vascular congestion, and extensive necrosis. Similarly, severe renal damage occurred at 800 mg/L, featuring marked vascular congestion, melanomacrophage aggregation, and widespread tubular necrosis. Overall, these findings demonstrate that acute waterborne COP exposure induces severe hepatotoxicity and nephrotoxicity in Betta splendens driven by oxidative stress, pro-inflammatory signaling, and apoptotic pathways. This study provides essential baseline toxicity thresholds and highlights the physiological risks COP spills pose to tropical freshwater labyrinth fish.