Abstract
The release of antifungal (AF) in the environment is a growing concern due to the potential risks posed to humans, animals, and microbial communities. The most immediate concern regarding the release of AFs in the aquatic environment is related to the spread of antifungal resistance. AFs are widely detected in wastewater treatment plants, sludge, and river surface waters at up to μg L-1 levels. Furthermore, the stereochemistry of antifungal agents has been largely overlooked due to lack of analytical methods enabling studies at the enantiomeric level. This work introduces a new analytical methodology for combined separation of achiral and chiral antifungal agents and their metabolites with the utilization of chiral chromatography coupled with triple quadrupole tandem mass spectrometry to enable comprehensive profiling of wide-ranging antifungal agents and their metabolites in environmental matrices. The method showed very good linearity and range (r2 > 0.997), method accuracy (61-143%) and precision (3-31%) as well as low (ng L-1). MQLs for most analytes. The new multi-residue analytical method was applied to determine the concentration of antifungal drugs and plant fungicides in river water, influent and effluent samples collected in South-West England.AFs were also identified and quantified in environmental aqueous samples using post-acquisition data mining and retrospective quantification and confirmation using bbCID method in the positive mode. The molecular mass plus a hydrogen ion was selected as a quantifier ion and analyte retention time, fragments m/z, precursor and fragments isotopic pattern and also precursor-to-fragment signals intensity ratios were used as the criteria for identification and quantification of AFs according to the EU guidelines. The following parameters were measured to test instrument performance: linearity, the instrumental limit of detection (IDL) and the instrumental limit of quantification (IQL). The linearity of calibration curves was generally very good with r2 on average ≥ 0.997 for the concentration range 0.008-1000 ng mL-1. Intra and interday accuracy was typically within the range 83-117% and the method was characterized by high precision below 23% RSD for most chemicals. The SPE methodology utilized a hydrophilic lipophilic balanced (HLB) copolymer as the extraction phase. The SPE recoveries of most antifungal agents were between 80-119%. However, some compounds provided low or high recoveries due to matrix effects. MQLs for liquid matrices ranged from 58 ng L-1 for terbinafine metabolite in surface water, to 7,262 ng L-1 for clotrimazole in effluent. The method was applied to monitor over 7 days in five WWTPs serving five cities in the UK and the Eerste River Catchment in South Africa.
For South-West England samples, the fungicide tebuconazole was found at the following concentrations: 252.4 ±70.2, 927.5 ±2.4 and 115.1 ±37.6 ng L-1 in river water, effluent and influent respectively. Its metabolite, hydroxy-tebuconazole was quantified only in the river water at 228.9 ±54.8 ng L-1 confirming its usage and environmental transformation. Terbinafine (used in both human and animal treatment) was also determined in river water (50.2 ±6.5 ng L-1) at higher concentrations than in wastewater influent (30.5 ±2.4 ng L-1). Its metabolite N-desmethyl-carboxyterbinafine was identified only in river water at < MDL indicating other than communal sources of this contaminant. Fluconazole was present at < MQL and 101.0 ±35.6 ng L-1 in river water and effluent respectively. Epoxiconazole enantiomers (with primary usage of crops) were quantified only in river water with significant predominance of E1 enantiomer: 67.3 ±26.5 ng L-1 and 13.2 ±4.4 ng L-1 for E1 and E2 respectively. Propiconazole enantiomers (with primary usage of crops) were also quantified only in river water at concentrations denoting 32.2 ±2.0 ng L-1 and 41.3 ±0.9 ng L-1 for E1 and E2 enantiomers respectively. However, only one enantiomer of deacetyl-ketoconazole was determined in effluent wastewater at the concentration of 218.21 ±38.62 ng L-1.
Five WWTPs in five cities were monitored over 7 days. Interestingly, all sites showed high presence of fluconazole: The Eerste catchment monitoring programme showed widespread presence of AFs with prothioconazole being recorded at the highest concentrations. Terbinafine metabolite, terbinafine, fluconazole, tebuconazole, tebuconazole metabolite, propiconazole epoxiconazole and prothioconazole metabolite followed. Clotrimazole and prochloraz were not quantified at any time. Prothioconazole, a wide spectrum synthetic fungicide used on crops, was recorded with the highest average concentrations >20 μg L-1 in the river water affected by anthropogenic discharges (both WWTP treated effluent and informal settlement runoff). It was followed by tebuconazole and terbinafine and their metabolites at >100 ng L-1. Fluconazole, propiconazole and epoxiconazole were also frequently recorded at >50 ng L-1. Interestingly, combined wastewater from WWTP effluent and informal settlement runoff showed the highest concentrations for most fungicides. Furthermore, WBE-based chemical mining pipelines were utilised to calculate AF intake in studied communities, followed by data triangulation with NHS per postcode AF prescription database. The environmental risk assessment of the target antifungal agent was assessed using their concentration in river water and the wastewater through the risk quotient (RQ) method. RQ values for fluconazole were low risk ranged from 0.00 to 0.03 for all river water and wastewater discharge. Following by tebuconazole, RQs were low to high risk in the range of 0.00-6.92. Propiconazole and terbinafine show low risk for general scenario and high risk in the worst scenario (0.00-45.87 and 0.00-101.23, respectively), whereas RQs were high risk for ketoconazole in all seasons for influent wastewater. Due to no information of PNEC available for many compounds no risk assessment for these compounds were possible. Although antifungal drugs and fungicides are determined at relatively low levels in the environment, there are effects on the aquatic environment, humans, animals, especially antifungal resistance, that require immediate attention.
| Date of Award | 26 Jul 2023 |
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| Original language | English |
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| Supervisor | Barbara Kasprzyk-Hordern (Supervisor) & Mirella Di Lorenzo (Supervisor) |