Fate and Toxicity of Nitrogen Heterocyclic Azole Compounds in Biological Wastewater Treatment

Abstract

Azoles are a broad class of five-membered heterocyclic compounds containing at least one nitrogen atom and they may contain additional heteroatoms (nitrogen, sulfur, or oxygen) in the ring. Azoles such as benzotriazoles, pyrazole and 1,2,4-triazole are widely used as corrosion inhibitors in industrial processes such as chemical mechanical planarization of copper during semiconductor manufacturing, as ingredients in various commercial products and as starting materials for the synthesis of pharmaceuticals and fungicides. In the recent years, azoles have been identified as emerging contaminants due to their ubiquitous presence in wastewater effluents and receiving waters. Preliminary research suggests that azole compounds are highly toxic towards a key step of the nitrogen cycle, nitrification, which involves microbially catalyzed oxidation of ammonium to nitrite and nitrate. Moreover, evidence suggests that azoles are resistant to biodegradation during wastewater treatment. The objectives of this work are to study the nitrification inhibition caused by azole compounds and investigate potential detoxification strategies for azole-contaminated wastewaters. First, nitrification inhibition tests were carried out for selected azole compounds using return activated sludge as inoculum. The results show that azoles were severely inhibitory towards nitrification at very low concentrations. The concentrations of pyrazole, 1,2,4-triazole, benzotriazole and 5-methylbenzotriazole causing fifty percent inhibition of nitrification (IC50) were 2.7, 3.5, 2.0 and 2.2 mg L-1, respectively. A comparative study of azole inhibition in sludges from five different wastewater treatment plants was also carried out with pyrazole as the model azole compound. The IC50 values for pyrazole varied over three orders of magnitude ranging from 0.04 to 2.20 mg L-1. The results indicate that the nitrifying populations in activated sludges routinely exposed to wastewater containing high azoles concentrations showed higher tolerance towards azole toxicity. Various detoxification approaches were explored for the removal of azoles from wastewater including biodegradation, adsorption on to copper nanoparticles (NPs), and addition of excess soluble copper(II) during nitrification. Aerobic biodegradation studies with return activated sludge showed that the biodegradability of azoles was strongly dependent on the molecular structure. Slow cometabolic degradation of pyrazole was observed with glucose and NH4+ as co-substrates with a degradation rate of 0.5 mg d-1 g-1 Volatile Suspended Solids (VSS). 1,2,4-triazole was found to be highly persistent, with no significant degradation observed in 6-8 months. In contrast, the benzotriazoles were readily degraded when provided as the sole substrates as well as with glucose and NH4+ as co-substrates. The fastest rates of degradation for benzotriazole and 5-methylbenzotriazole were 8.1 and 16.5 mg d-1 g-1 VSS, respectively. Two enrichment cultures, one degrading benzotriazole and the other degrading 5-methylbenzotriazole, were developed from the activated sludge. Mass balance studies revealed complete mineralization of 5-methylbenzotriazole and partial breakdown of benzotriazole by the enrichment cultures. It was evident that some azoles such as pyrazole and 1,2,4-triazole can be persistent during biological treatment and hence other physicochemical methods such as azole removal via adsorption needed to be explored. Copper NPs were selected as the material of choice for the adsorption studies, owing to their large surface area and the known affinity of copper for azole compounds. Adsorption kinetics and isotherms were determined for three azoles, pyrazole, 1,2,4-triazole and benzotriazole. Adsorption on to copper NPs was found to be an effective method for the removal of benzotriazole and 1,2,4-triazole from wastewater. Another intervention involving addition of excess soluble copper(II) to lower azole toxicity during nitrification was tested and found to be ineffective in alleviating the toxicity. The underlying hypothesis that azoles bind soluble copper(II), resulting in reduced bioavailability of copper(II) for the ammonia monooxygenase enzyme, is likely incorrect. Efforts were also made to identify non-toxic azole alternatives with good corrosion inhibition characteristics. This included studying the effect of various chemical substituents on the toxicity of simple azole compounds with the aim of identifying patterns leading to lowering of their nitrification inhibition. The IC50 values for pyrazole and 1,2,4-triazole were very low, 0.07 and 0.11 mg L-1, respectively. In contrast, the toxicity of the dimethyl- and diethyl- substituted azoles was at least 30 to 700 times lower compared to the unsubstituted parent compounds, and in the best case, 3,5-diethyltriazole was found to be completely non-toxic. Moreover, the dialkyl- substituted azole compounds were also found to be effective corrosion inhibitors of copper in tetramethyl ammonium hydroxide at pH 10. An important implication of these results is the possibility of replacing conventional azole compounds by their non-toxic substituted counterparts in industrial applications. Lastly, biotransformation of a different azole compound, 3-nitro-1,2,4-triazol-5-one (NTO), a chemical used as an ingredient in insensitive munitions formulations was also studied. The objective of this study was to explore the ability of wastewater sludges to promote the biotransformation of NTO. Three different sludges, i.e., anaerobic granular sludge, anaerobic digested sludge, and return activated sludge, were successfully able to reduce NTO to its biotransformation product, 3-amino-1,2,4-triazol-5-one (ATO) under anaerobic conditions. This study showed that sludge from wastewater treatment plants can rapidly and readily reduce NTO to its amino- derivative. Overall, the findings laid out in this dissertation have contributed towards filling the knowledge gap related to the fate and toxicity of azole compounds. Due to the increasing stringent regulations for nitrogen removal from wastewater, efforts to detoxify azole-containing wastewaters are extremely important. A shift towards readily biodegradable azoles such as 5-methylbenzotriazole and non-toxic azoles such as 3,5-diethylpyrazole and 3,5-diethyltriazole can ensure that inhibition of nitrification process during wastewater treatment is avoided.