Non-ignorable dipeptides as nitrogenous precursors in chlor(am)ination: Tyrosyl-L-phenylalanine generates higher toxicity to mammalian cells than aromatic monomeric amino acids

Abstract

Amino acids and peptides are key precursors for disinfection byproducts (DBPs) in wastewater. Aromatic amino acids generate high levels of DBPs during chlorination and chloramination, yet the toxicity of dipeptides containing these amino acids remains understudied. This study detected six amino acids and five dipeptides in secondary effluents at μg/L concentrations. Among them, the dipeptide Tyrosyl-L-phenylalanine (Tyr-Phe) showed the highest cytotoxicity and genotoxicity following one-hour disinfection with 5 mg-Cl₂/L. During chlorination, its cytotoxicity increased from 14.2 to 86.1 mg-Phenol/L, and genotoxicity rose from 1.6 to 19.6 μg-4-NQO/L. Tyr-Phe was 1–2 times more toxic than its monomers phenylalanine (Phe) and tyrosine (Tyr) at the same concentration. Similar results were observed during chloramination. Total organic chlorine (TOCl) levels showed that most dipeptides produced more TOCl than monomers under both disinfection methods, aligning with the toxicity trends. Ultra-performance liquid chromatography–high-resolution mass spectrometry (UPLC-HRMS) analysis revealed that Tyr-Phe generated more DBPs with higher molecular weight, aromaticity, and nitrogen content than those generated from Phe and Tyr. Seven DBPs were identified, including novel compounds such as 4-chloro-d-phenylalanine and 3,5-dichloro-4-hydroxybenzonitrile, which were unique to Tyr-Phe. These DBPs showed cytotoxicity and genotoxicity comparable to or greater than known DBPs like dichloroacetonitrile. These DBPs were detected at μg/L levels in secondary effluents and contributed 0.1%–1.1% to overall cytotoxicity. This study highlights dipeptides as significant DBP precursors that increase toxicity in disinfected wastewater, emphasizing the need for targeted monitoring and risk mitigation strategies.

Graphical abstract

Keywords

Dipeptide / Aromatic amino acid / Tyrosyl-L-phenylalanine / Disinfection byproducts / Toxicity

Highlight

	● Tyr-Phe generates higher toxicity than monomers upon chlorination/chloramination.
	● Tyr-Phe produces more DBPs and higher molecular weights than its monomers.
	● Four novel, highly toxic nitrogen-containing DBPs were identified.
	● Novel Tyr-Phe DBPs were detected at μg/L levels in secondary effluents.

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