Understanding Toxicity and Formation of Chlorinated Products of 1,3-Diphenylguanidine (DPG) in Water

Importance of the Topic

1,3-Diphenylguanidine (DPG) is extensively used in tire and rubber production and is increasingly detected in surface water, drinking water systems and leachates from HDPE pipes. Its transformation during disinfection may generate chlorinated by-products with unknown health impacts. Understanding the toxicity and formation pathways of DPG chlorination products is critical for assessing environmental risks and informing water treatment practices.

Goals and Study Overview

This study aimed to (1) evaluate the cytotoxicity and effects on cellular bioenergetics of DPG and five chlorinated derivatives, and (2) simulate disinfection conditions to characterize the formation and yield of these chlorinated products under typical water treatment and distribution scenarios.

Methodology and Instrumentation

• Cell viability assays: End-point alamarBlue HS and real-time impedance monitored by Agilent xCELLigence RTCA eSight using A549 lung epithelial cells exposed to DPG and chlorinated analogues.
• Bioenergetics analysis: Agilent Seahorse XF96 extracellular flux analyzer measured oxygen consumption rate (OCR) under mitochondrial stress (oligomycin, FCCP, rotenone/antimycin A) to assess basal respiration, ATP production and proton leak.
• Simulated disinfection: DPG (5 μM) reacted with free chlorine (7–80 μM) or monochloramine (35–40 μM) under controlled pH and temperature. Reaction quenching with ascorbic acid.
• Quantitation and separation: Agilent 1290 Infinity II HPLC coupled to 6495C Triple Quadrupole LC/MS in MRM mode using a Poroshell 120 EC-C18 column. External calibration (2.5–50,000 ng/L) and optimized source and collision parameters via MassHunter Software.

Main Results and Discussion

• Cytotoxicity (alamarBlue HS): CC15 was least toxic (EC50 ≈152 μM); only CC11 exhibited higher toxicity than DPG (EC50 lower than DPG).
• Real-time cytotoxicity (RTCA): Initial 24-h exposure confirmed alamarBlue trends; after 168 h, all chlorinated products exceeded DPG toxicity (rank: CC11 > CC04 > VD03 > CC05 > CC15 > DPG).
• Bioenergetics (Seahorse): Except CC05, all compounds reduced basal respiration and ATP production; DPG, CC04 and CC11 induced significant proton leak, indicating mitochondrial membrane damage.
• Chlorination by-product formation: Low chlorine doses (≤40 μM) favored di-chlorinated products in the order CC15 > CC05 > CC04. Tri- and tetra-chlorinated species (CC11, VD03) were not detected. At higher chlorine doses, target products accounted for ≤10% of DPG, suggesting additional transformation routes; no target chlorinated products formed during chloramination.

Benefits and Practical Applications

The integrated workflow combining real-time cytotoxicity, bioenergetics profiling and triple-quadrupole LC/MS offers a sensitive, high-throughput platform for early environmental toxicology screening. It enables rapid hazard identification of parent compounds and disinfection by-products, supporting safer product development and optimized water treatment strategies.

Future Trends and Potential Applications

• Extension to other emerging micropollutants and their transformation products under disinfection conditions.
• High-throughput and automated optimization of MRM methods for broader environmental monitoring.
• Integration with predictive models of disinfection by-product formation in distribution networks.
• Application in risk assessment frameworks to inform regulatory guidelines for drinking water safety.

Conclusion

This study demonstrates that chlorination of DPG produces di-chlorinated derivatives with varying cytotoxic and mitochondrial effects. CC11 and CC04 exhibited the most significant toxicity and mitochondrial impairment. Simulated chlorination conditions yield chiefly di-halogenated products, with potential environmental risks in treated waters. The combined in vitro assays and triple-quadrupole LC/MS workflow provide a robust approach for early screening of hazardous transformation products in water treatment contexts.

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