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Co-occurrence of Photochemical and Microbiological TransformationProcesses in Open-Water Unit Process WetlandsCarsten Prasse,Jannis Wenk,Justin T. Jasper,Thomas A. Ternes,and David L. Sedlak†ReNUWIt Engineering Research Center and Department of Civil & Environmental Engineering, University of California at Berkeley, Berkeley, California 94720, United States ‡Department of Aquatic Chemistry, Federal Institute of Hydrology, D-56002 Koblenz, Germany§Department of Chemical Engineering and Water Innovation & Research Centre, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom ABSTRACT: The fate of anthropogenic trace organic contaminants insurface waters can be complex due to the occurrence of multiple parallel andconsecutive transformation processes. In this study, the removal of fiveantiviral drugs (abacavir, acyclovir, emtricitabine, lamivudine and zidovudine)via both bio- and phototransformation processes, was investigated inlaboratory microcosm experiments simulating an open-water unit processwetland receiving municipal wastewater effluent. Phototransformation was themain removal mechanism for abacavir, zidovudine, and emtricitabine, withhalf-lives (t1/2,photo) in wetland water of 1.6, 7.6, and 25 h, respectively. Incontrast, removal of acyclovir and lamivudine was mainly attributable toslower microbial processes (t1/2,bio = 74 and 120 h, respectively). Identificationof transformation products revealed that bio- and phototransformationreactions took place at different moieties. For abacavir and zidovudine, rapidtransformation was attributable to high reactivity of the cyclopropylamine and azido moieties, respectively. Despite substantialdifferences in kinetics of different antiviral drugs, biotransformation reactions mainly involved oxidation of hydroxyl groups to thecorresponding carboxylic acids. Phototransformation rates of parent antiviral drugs and their biotransformation products weresimilar, indicating that prior exposure to microorganisms (e.g., in a wastewater treatment plant or a vegetated wetland) would notaffect the rate of transformation of the part of the molecule susceptible to phototransformation. However, phototransformationstrongly affected the rates of biotransformation of the hydroxyl groups, which in some cases resulted in greater persistence ofphototransformation products.
leads to the formation of hydroxylated derivatives,which are Discharge of municipal wastewater effluents into surface waters more easily biodegraded than the parent compound.
can result in the presence of trace organic contaminants at Open-water unit process wetlands have been developed as a concentrations that pose potential risks to aquatic ecosystems polishing treatment step for municipal wastewater effl and drinking water resources. After their release, many trace These managed natural systems utilize sunlight to remove trace organic contaminants are attenuated by biological and photo- organic compounds and inactivate pathogens.In addition, chemical processes. Although these processes often occur microorganisms in the biomat formed at the bottom of these simultaneously or sequentially in the environment, most studies treatment basins reduce nitrate and contribute to aerobic have considered the occurrence of only one transformation biodegradation of trace organic contaminants.To assess the process at a time.−Thus, it is difficult to predict which importance of co-occurrence of biological and photochemical transformation products will be formed and whether or not transformation reactions for reaction kinetics and product transformation reactions occurring at one moiety alter the distribution, the fate of five antiviral drugs (abacavir, kinetics of subsequent transformation reactions. Furthermore, if emtricitabine, lamivudine, zidovudine, and acyclovir; see partial transformation of a compound enhances the reactivity of ) was studied under conditions comparable to those other moieties, then interaction of transformation processes encountered in open-water unit process wetlands.
could result in changes in the distribution of transformationproducts as well as their rates of removal. For example, carbamazepine, a compound that is particularly resistant to biotransformation, is slowly transformed upon exposure to Accepted: October 28, 2015 sunlight via direct photolysis and reaction with •OH.This Published: November 12, 2015 2015 American Chemical Society Environ. Sci. Technol. 2015, 49, 14136−14145 Environmental Science & Technology Laboratory Photo- and Biotransformation Experi- ments. Irradiation experiments were performed by use of acollimated beam Oriel solar simulator (Spectra Physics 91194)equipped with a 1000 W Xe lamp and either two successiveatmospheric attenuation filters (Spectra Physics 81088 and81017) or one atmospheric and one UVB filter (SpectraPhysics 81088 and 81050). Spectral irradiance was routinelymeasured with a spectroradiometer (RPS 380, internationallight) at different locations of the irradiated area to assessvariability, which was always <5%. Details on lamp irradianceenergies and the spectra of different configurations are given insection 1.1 of Irradiation experimentswere carried out in 100 mL black-painted glass beakers thatwere placed in a water bath at constant temperature(18 ± 2 °C). Initial concentrations of antivirals ofapproximately 0.5 μM were used for all kinetic experiments.
Pseudo-first-order phototransformation rate constants ofantivirals and photochemical probe compounds, used for Figure 1. Antiviral drugs and their most likely sites of proposed quantification of concentrations of reactive intermediates, phototransformation (outlined in orange) and biotransformation were calculated from the slopes of linear regression of the (outlined in green) reactions.
natural log of concentration versus time. No degradation ofantiviral drugs was observed in control experiments in the dark, Antiviral drugs were chosen because they are widely used for indicating that their transformation in filtered wetland water the treatment of diseases such as herpes, hepatitis, and human was attributable only to photochemical processes.
immunodeficiency virus (HIV) and have been detected at For the elucidation of biotransformation kinetics, beakers concentrations above 1 μg·L−1 in municipal wastewater were additionally supplemented with 10 mL of the biomat effluents.−No information about potential environmental taken from the bottom of a pilot-scale open-water wetland and effects resulting from the release of these compounds into the kept in the dark (see Jasper et for further details).
aquatic environment is available so far. Furthermore, little is Biodegradation of compounds followed pseudo-first-order known about the effects of these compounds on environmental degradation kinetics, indicating stable conditions throughout viruses, a group of microorganisms that play a very important the experiments. In addition, observed transformation rates role in aquatic ecosystems.
were in good agreement with results from a preliminary study By investigating transformation kinetics and mechanisms used to design the more detailed experiments.
under conditions comparable to those encountered in open- Direct and Indirect Phototransformation. Experiments to water unit process wetlands, it is possible to gain insight into assess direct phototransformation of antiviral drugs were how simultaneously occurring bio- and phototransformation conducted in buffered ultrapure water at pH values ranging reactions affect the overall fate of antiviral drugs in sunlit from 6 to 10 (pH 6−8, 5 mM phosphate buffer; pH 9−10, surface waters. These compounds also serve as models for other 5 mM borate buffer). Samples (1 mL) were collected at regular families of compounds that contain moieties susceptible to bio- time intervals and stored at 4 °C in the dark until analysis.
Electronic absorption spectra of antiviral drugs at different pHvalues (see were recorded with a UV-2600 UV−vis MATERIALS AND METHODS spectrophotometer (Shimadzu) using quartz-glass cuvettes Chemicals. Analytical reference standards of antiviral drugs (Hellma, Germany). Further details on determination of and stable isotope-labeled analogues used as internal standards quantum yields by the p-nitroanisole (PNA)/pyridine (PYR) (purity >99%) were purchased from Toronto Research methodand related calculations are provided in section 1.7 of Chemicals (Ontario, Canada). All other chemicals and solvents were obtained from Fisher Scientific (Fairlawn, NJ).
Indirect phototransformation of antiviral drugs was inves- Wetland Water Sampling Conditions. Phototransforma- tigated by the addition of specific quenchers to wetland water: tion experiments were conducted in water collected from a N,N-dimethylaniline (DMA; 10 μM) was used to scavenge pilot-scale open-water unit process wetland located in CO3 sorbic acid (2.5 mM) was used to scavenge Discovery Bay, CA. The facility treats about 10 000 gallons/ excited triplet states of the dissolved organic matter day (4.4 × 10−4 m3·s−1) of nitrified wastewater effluent from an (3DOM*histidine (20 mM) was used to scavenge singlet adjacent municipal wastewater treatment plant. Details about oxygen (1O2),and isopropyl alcohol (IPA; 26 mM) was used the open-water unit process wetland were described to scavenge •OH radicals.In addition, experiments with Water collected from the open-water wetland specific photosensitizers were conducted in ultrapure buffered typically contained 10−20 mg-N·L−1 NO − 3 , 5−10 mg-C·L−1 water to determine reaction rate constants of antiviral drugs dissolved organic carbon (DOC), and 60−80 mg-C·L−1 with individual reactive intermediates. For CO − • dissolved inorganic carbon (HCO − and CO3 ). Samples for NaNO3/NaHCO3 or duroquinone/NaHCO3 photosensititizer laboratory irradiation experiments were collected from the method was Excited triplet state photosensitizers 3- midpoint of the wetland. All samples were filtered through methoxyacetophenone (3MAP) and anthraquinone-2-sulfonate prerinsed 1 μm (nominal pore size) glass fiber filters (AQ2S) served as proxies for 3DOM*.Hydroxyl radicals (Whatman) and were stored in the dark at 4 °C until analysis, were generated by irradiation of NaNO3 solutions.For 1O2 which occurred within 5 days.
production, rose bengal was used as a photosensitizer.To Environ. Sci. Technol. 2015, 49, 14136−14145 Environmental Science & Technology further verify the role of 1O2, some experiments wereperformed in D2O. Reaction rate constants were determinedeither by competition kinetics or by comparing reaction rates ofantiviral drugs with those of established photochemical probecompounds (experimental details and calculations are providedin sections 1.5 and 1.6). For all indirectphototransformation experiments, the concentration changes ofphotochemical probe compounds and antiviral drugs duringirradiation were determined by HPLC-UV. Experimental andanalytical details, including comprehensive results, are providedin section 1.2.
Given the structural similarities of antivirals with DNA bases, additional irradiation experiments were performed withadenine, 2-aminoadenosine, cytosine, cytidine, guanine, thymi-dine, and thymine section 2.1.1) toobtain further information about photoreactive moieties in themolecules to aid in identification of transformation products.
Figure 2. Phototransformation kinetics of antiviral drugs in experi- Identification of Photo- and Biotransformation Products.
ments with wetland water at different pH values and contribution of High-resolution mass spectrometry (HRMS; LTQ Orbitrap direct and indirect photolysis processes by comparison with results Velos, Thermo Scientific, Bremen, Germany) was used to obtained in ultrapure water. Data for wetland water are corrected for conduct accurate MS and MS/MS analysis of transformation light absorption. Error bars show 95% confidence intervals.
products of antiviral drugs. To this end, experiments at elevatedconcentrations (40 μM) were used. The LTQ Orbitrap Velos degradation of antiviral drugs in wetland water occurred in was coupled to a Thermo Scientific Accela liquid chromatog- the dark, indicating that their removal was solely attributable to raphy system (Accela pump and autosampler). HRMS was photochemical processes. Photosynthetic activity leads to conducted in the positive electrospray ionization (ESI) mode.
significant diurnal fluctuations of pH in open-surface wet- To obtain information on the chemical structure of trans- lands.Therefore, phototransformation kinetics of antiviral formation products, MSn fragmentation experiments were drugs in wetland water were also determined at pH 6.5 and 10 conducted with data-dependent acquisition. Further informa- tion on the applied setup and data-dependent acquisition Phototransformation of abacavir in wetland water increased parameters can be found in (section when the pH value was adjusted to 6.5 or 10. This can be 1.3). Product formation of antiviral drugs in laboratory attributed to a higher contribution of direct photolysis due to experiments was determined by liquid chromatography/tandem higher quantum yields at lower pH values (i.e., Φapp is 4.2−11.4 mass spectrometry (LC/MS/MS). Details are provided in times higher between pH 6 and 8 compared to pH 9 and 10; (section 1.4).
) and faster indirect photolysis at higher pH values.
Combined Bio- and Photodegradation Experiments. The Comparison of transformation kinetics with results obtained in fate of antiviral drugs in the presence of sunlight and ultrapure water revealed the dominance of indirect photo- microorganisms was investigated over a 72 h period in the degradation processes at pH 8.9 and 10, whereas direct laboratory. Black-painted glass beakers (250 mL) were filled photolysis was more important at pH 6.5. The addition of with 180 mL of wetland water and 20 mL of freshly collected sorbic acid and histidine significantly reduced phototransfor- biomat material from the bottom of the Discovery Bay open- mation rates of abacavir in wetland water Although water unit process wetland. The experimental setup was the interpretation of results from experiments with scavengers same as described above for photochemical experiments but requires these results suggest the involvement of with three day/night cycles to simulate field conditions (8 h of 3DOM* and 1O2 in the photochemical fate of this compound.
daily irradiation followed by 16 h of darkness; 72 h total).
This was also supported by experiments with specific singlet Antiviral drugs were added individually at concentrations of oxygen and excited triplet state sensitizers (see below).
approximately 0.5 μM to ensure detection of both parent Negligible removal of the structural analogues adenine and 2- antiviral compounds and their transformation products.
aminoadenosine further indicated that the photolability of Samples were collected at regular time intervals and stored at abacavir can be attributed to the cyclopropyl moiety (see 4 °C in the dark prior to LC/MS/MS analysis, which occurred section 2.1.1).
within 24 h. Further details about the analytical method can be Rates of phototransformation of zidovudine were not affected by changes in pH. Comparison with reaction rates inboth ultrapure water and wetland water in the presence of RESULTS AND DISCUSSION scavengers revealed the dominance of direct photolysis Phototransformation in Wetland Water. Phototransfor- Similar to abacavir, comparison with the depletion of mation of the five investigated antiviral drugs in wetland water structural analogues thymine and thymidine indicated that the followed first-order kinetics (r2 ≥ 0.98; ). In azide moiety was responsible for the observed photoreactivity native wetland water (pH 8.9), the fastest phototransformations of zidovudine, as both analogues showed no removal when were observed for abacavir (kobs = 0.52 ± 0.06 h−1), zidovudine exposed to light (see section 2.1.1).
(kobs = 0.09 ± 0.002 h−1) and emtricitabine (kobs = Phototransformation rates of acyclovir in wetland water 0.03 ± 0.002 h−1) whereas the transformations of acyclovir increased with increasing pH. Comparison with results from and lamivudine were significantly slower (kobs = 0.012 ± 0.001 ultrapure water revealed that removal at pH 8.9 was solely due and 0.011 ± 0.001 h−1, respectively) (). No to indirect photolysis, whereas at pH 10 direct photolysis was Environ. Sci. Technol. 2015, 49, 14136−14145 Environmental Science & Technology Table 1. Quantum Yields (pH 9) and Apparent Second-Order Reaction Rate Constants of Indirect Phototransformation ofAntiviral apparent second-order reaction rate constants (M−1·s−1) Φapp(300−400nm) at 1.2 × 109 (±18%) 1.1 × 1011 (±3%) 1.2 × 109 (±4%) 1.3 × 1010 (±2%) 2.4 × 106 (±5%) 1.3 × 106 (±4%) 1.2 × 107 (±25%) 5.0 × 109 (±2%) 1.2 × 108 (±2%) 6.3 × 107 (±4%) 9.3 × 109 (±2%) 3.0 × 106 (±4%) 4.3 × 106 (±12%) 9.2 × 109 (±1%) 1.2 × 106 (±3%) 1.7 × 106 (±3%) aIndirect phototransformation occurred via reaction with 1O 2, •OH, •CO3 , and excited triplet states (values are given relative to degradation of the 3Sens* probe compound TMP). Quantum yields of antiviral drugs at pH 6−8 and pH 10 can be found in . bNot applicable due to reactionof abacavir with DQ in the dark. cND, not detected above the level of uncertainty.
also important. Significantly reduced rates of acyclovir photo- with measured second-order reaction rate constants of antivirals transformation in the presence of histidine and sorbic acid 2, •OH, and •CO3 ) were in good agreement, indicating indicated the importance of 1O2 and 3DOM* to indirect reasonable results.
photolysis (In contrast to abacavir and zidovudine, Comparison of Photo- versus Biotransformation phototransformation kinetics were similar to those observed for Rates. Dark experiments conducted with wetland water in the structural analogue guanine Thus, photo- the presence of biomat material indicated that biotransforma- transformation of acyclovir can be attributed primarily to the tion rates varied considerably among antiviral drugs. Bio- guanine moiety.
transformation half-lives (t1/2,bio) ranged from 74 h for acyclovir For lamivudine and emtricitabine, phototransformation to 500 h (21 days) for emtricitabine kinetics in wetland water decreased with increasing pH. Noremoval of lamivudine was observed in ultrapure water,indicating that its removal was entirely attributable to indirectphotolysis. Higher phototransformation rates of emtricitabinerelative to lamivudine further indicated the strong influence ofthe fluorine atom for emtricitabine's photolability. Thepresence of the fluorine substituent led to greater lightabsorption at 300−320 nm (). Even though theabsorption spectrum of emtricitabine did not change with pH,the quantum yield steadily decreased with increasing pH Phototransformation of lamivudine in wetland water wasfully inhibited by sorbic acid, histidine, and IPA but wasunaltered in the presence of DMA (This indicatesthe importance of 3DOM*, 1O2, and OH radicals for its indirectphototransformation. For emtricitabine, phototransformationrates in wetland water were affected only by IPA and sorbic acid(), suggesting that reactions with 1O Figure 3. Photo- and biotransformation rate constants k (per hour) and associated half-life t important for this compound. The high photostability of its 1/2 (days) of antiviral drugs in laboratory experiments. Small bars within phototransformation columns indicate associated DNA base cytosine and nucleotide cytidine revealed half-lives based on daily sunshine hours (9−15 h). For determination the importance of structural modifications [thiol group (both of biodegradation half-lives, experiments were conducted in the compounds) and fluorine (emtricitabine)] to the observed presence of the biomat in the dark. Error bars represent 95% confidence intervals obtained from linear regressions.
Additional experiments with individual reactive species revealed second-order reaction rates with •OH at or above Under typical wetland treatment conditions (i.e., hydraulic (abacavir, zidovudine) diffusion-controlled rates ranging from 5 retention times of 2−3 days), significant biological attenuation × 109 to 1.1 × 1011 M−1·s−1 (Antiviral compounds of acyclovir and abacavir is expected, whereas removal of the were reactive with CO − • at rates between 1.2 × 106 and 1.2 × other antiviral drugs via microbial processes is unlikely to be 109 M−1·s−1, while only abacavir (1.2 × 109 M−1·s−1) and important. Comparison of transformation rates of antiviral acyclovir (1.2 × 107 M−1·s−1) reacted with 1O2. With the drugs in the dark to those observed in irradiated wetland water exception of abacavir, no depletion of antiviral compounds was indicated that phototransformation processes were dominant observed in the presence of the model triplet photosensitizer for abacavir, zidovudine, and emtricitabine, while for acyclovir 3MAP. However, depletion of all compounds was observed in and lamivudine, biotransformation was similar or more the presence of AQ2S at rates similar to or higher than the important than photolysis during typical summertime con- reference probe compound trimethylphenol (TMP), indicating selective reactivity with excited triplet states. Comparison of Transformation of Abacavir. HRMS analysis indicated measured and predicted rate constant for antivirals under that four primary transformation products (TP318, TP288, wetland conditions (obtained by multiplication of steady-state TP284, and TP246) were formed during photolysis of abacavir concentrations of reactive species measured in wetland water in wetland water section 2.2; Environ. Sci. Technol. 2015, 49, 14136−14145 Environmental Science & Technology Figure 4. Transformation of abacavir (left, top) and resulting formation of photo-TPs (left, middle) and bio/bio-photo-TPs (left, bottom), as well asproposed transformation pathway (right), in combined 3-day experiments in the presence of biomat with 8 h of daily irradiation. In thetransformation pathway, photo- and biotransformation reactions and structural changes in the molecules are indicated in orange and green,respectively.
In agreement with results obtained for the structural tigated in buffered water (direct photolysis only), wetland water analogues 2-aminoadenosine and adenine, fragmentation (direct and indirect photolysis), and wetland water in the patterns of TP318, TP288, and TP246 revealed that the presence of different reactive intermediate scavengers. The cyclopropylamine moiety was the main site of reaction, leaving results revealed that both direct and indirect photolysis of the 2-aminoadenine (fragments m/z 151.073, 134.046, and abacavir produced the same suite of TPs at similar relative 109.051) and the 2-cyclopenten-1-methanyl moieties (frag- concentrations, despite the fact that disappearance of the parent ments m/z 95.353 and 79.054) unaltered.
compound was significantly accelerated in the presence of Exact mass calculations of TP318 showed addition of two DOM and individual reactive intermediates ( oxygen atoms to the cyclopropyl moiety (Δm + 31.9898 Da).
Similar results have been reported for irgarol, an algaecide Results from MS2 experiments were consistent with scission of that is structurally similar to abacavir, suggesting that the the cyclopropyl ring and the presence of a terminal hydroxyl cycloproylamine moiety is the main site of reaction under all group, as indicated by the cleavage of H2O and CH2O.
conditions.Photodegradation experiments in buffered ultra- For TP288, MS data suggested modification of the pure water with different optical filters indicated that cyclopropyl moiety via loss of one carbon atom and addition wavelengths below 320 nm preferentially led to cleavage of of one oxygen atom, leading to the formation of an acetamide, the cyclopropyl moiety (TP246), whereas wavelengths above whereas TP246 was formed via cleavage of the cyclopropyl ring.
320 nm (UVA and visible light) led to scission of the The chemical structure of TP246 was confirmed by comparison cyclopropyl ring followed by partial oxidation (TP318) with a commercially available reference standard. The exact mass and fragmentation pattern of TP284 was consistent with These findings suggest that phototransformation of abacavir loss of two protons from either the cyclopropylamine or the 2- is initiated by a one-electron oxidation of the cyclopropylamine aminoadenosine moiety (fragments m/z 149.069 and 189.088 moiety, leading to formation of a cyclopropylaminium radical instead of m/z 151.073 and 191.104 compared to abacavir and cation,,followed by subsequent reactions resulting in the other TPs). When the high photolability of the cyclopropyl formation of various products. Interestingly, this phenomenon moiety is considered, these structural changes were most likely has also been utilized for the investigation of electron-hopping due to formation of a cyclopropylimine.
in DNA by modifying guanine and adenine with cyclopropyl To assess the relative importance of direct and different Due to the instability of the initially formed indirect photolysis processes in formation of the observed closed ring radical cation, the modification results in rapid abacavir transformation products, their formation was inves- cyclopropyl ring opening as well as 1,2-hydrogen migration, Environ. Sci. Technol. 2015, 49, 14136−14145 Environmental Science & Technology Figure 5. Transformation of acyclovir in the presence of biomat in the dark (a) and in combined photo- and biotransformation experiments (b), aswell as formation of TP257 via reaction of acyclovir with 1O2 in D2O and H2O by use of rose bengal as photosensitizer (c) and its proposedphototransformation pathway (d). Occurrence of acyclovir carboxylate at t0 in panels a and b is due to its emission by the wastewater treatment plantthat feeds the wetland.
leading to formation of an ionized Scission of slower. When the light was turned back on, nearly all remaining the ring is followed either by complete cleavage of the abacavir disappeared. As expected, the light-induced trans- cyclopropyl moiety (TP246) or by reaction of the ring-opened formation of abacavir gave rise to the four photo-TPs described radical cation with H2O/O2.In the latter case, electron above (see middle panel of ). The concentrations of release from the carbon-centered radical followed by hydrolysis these photo-TPs decreased by approximately 25% over the next leads to formation of a 3-hydroxypropanaminium cation,and 2.5 days, indicating that further transformation took place, via subsequent addition of water results in formation of a 3- either photolytic or microbial processes.
hydroxypropanamide (TP318). In our system, TP288 is formed Additional biodegradation experiments with the four photo- by photolytic cleavage of the hydroxymethyl group, which leads TPs of abacavir revealed that biotransformation occurs at the to the formation of the acetamide TP284 was same moiety as observed for the parent compound, leading to most likely formed via H-atom abstraction, resulting in the corresponding TP246, TP284, TP288, and TP318 formation of a neutral cyclopropyl radical, followed by an carboxylates (Exact mass data and fragmentation electron-transfer reaction and/or hydrolysis and elimination of patterns of biophoto-TPs determined by HRMS analysis are water, even though this reaction has only been shown to be included in section 2.2. Consequently, catalyzed by enzymes so far.
the observed decrease in concentration of photo-TPs shown in Experiments with biomat material in the dark to determine the middle panel of was mainly attributable to the relative importance of biotransformation reactions indicated biotransformation, leading to a steady formation of carboxylate that microbial transformation of abacavir mainly occurred via photo-TPs (bottom panel of ). Faster transformation oxidation of the primary alcohol group of the 2-cyclopenten-1- rates of abacavir photo-TPs observed during irradiation periods hydroxymethanyl side chain to produce the corresponding may have been attributable to enhanced biotransformation due carboxylic acid (abacavir carboxylate, ). This was to elevated oxygen concentrations or elevated pH values that consistent with previous experiments conducted with mixed occurred when photosynthetic microbes in the biomat were liquor-suspended solids from an activated sludge treatment active. Differences in biotransformation rates of TP246, TP284, TP288, and TP318, compared to abacavir When abacavir was exposed simultaneously to light and indicate that alteration of chemical structure influences microorganisms (a rapid loss of the compound was biotransformation kinetics, for example, by affecting enzyme observed during the first 8-h light period (i.e., initial binding affinities or steric properties. Light exposure of abacavir concentration decreased by approximately 90%). For the next carboxylate formed in the dark led to its phototransformation, 16 h (i.e., the dark period), abacavir removal was significantly ultimately yielding the same photo-TPs as abacavir (bottom Environ. Sci. Technol. 2015, 49, 14136−14145 Environmental Science & Technology panel of ). When it is considered that abacavir is during dark periods, whereas its concentration decreased upon already transformed extensively to abacavir carboxylate in exposure to sunlight. This indicates that the compound was activated sludge treatment,rapid elimination of both transformed further by photolytic processes, most likely via the compounds can be expected in open-water unit process same mechanisms as acyclovir. This was confirmed by wetlands. In contrast to biotransformation reactions, similar additional irradiation experiments with acyclovir carboxylate phototransformation kinetics were observed for abacavir and in wetland water (results not shown).
abacavir carboxylate ). TP246 carboxylate was Transformation of Zidovudine, Lamivudine, and identified as the main product that accumulates over time Emtricitabine. Mass spectra of the phototransformation because it is not susceptible to further reactions.
products of emtricitabine, lamivudine, and zidovudine indicated Transformation of Acyclovir. In contrast to abacavir, the structural changes at different positions on the molecules transformation of acyclovir was dominated by microbial (For lamivudine and emtricitabine, HRMS processes ), with biotransformation resulting in the analysis revealed oxidation of the riboside moiety (lamivudine formation of acyclovir carboxylate, which was not susceptible to TP245 and emtricitabine TP263), most likely via S-oxidation.
further microbial transformation. These results are consistent This was confirmed by comparison with commercially available with previous biotransformation experiments conducted with reference standards. Addition of H2O to the 5-fluorocytosine acyclovir in sewage sludge.
moiety was observed for emtricitabine (emtricitabine TP265).
In the absence of biomat material, exposure of wetland water Experiments conducted with the fluorine-free analogue to simulated sunlight resulted in formation of two main photo- lamivudine illustrate the importance of fluorine substitution: TPs (TP257 and TP223). HRMS analysis indicated that TP257 the F-moiety increases light absorbance at wavelengths >300 contains two additional oxygen atoms on the guanine moiety, nm () for emtricitabine and leads to faster as evidenced by detection of fragment m/z 184 instead of m/z photodegradation Emtricitabine TP265 152 (Photosensitized degradation of was formed via hydration of the double bond of the 5- guanine and guanosine occurs by reaction with excited triplet fluorocytosine moiety, yielding a hydroxyl group at position C6.
2, •OH, or •CO3 .,The main product of reaction For zidovudine, observed phototransformations were mainly of guanine with 1O2 has been identified as spiroiminodihy- attributable to the photolability of the azido moiety. Formation dantoiTo assess the role of 1O2 in the photo- of zidovudine TP239 can be explained by cleavage of N2, transformation of acyclovir in wetland water, experiments yielding a nitrene intermediate, which reacts further via were conducted in both H2O and D2O in the presence of the intramolecular C−H insertion to an Subsequent 1O2 sensitizer rose bengal (Lifetimes of 1O2 in D2O nucleophilic attack of the aziridine by water leads to are more than an order of magnitude higher than in H2O,and hydroxylation of the C atom in β-position or formation of a faster transformation of acyclovir in D2O confirmed the role of hydroxylamine (zidovudine TP257).Results from HRMS 1O2 in the indirect photolysis of acyclovir. In addition, the yield analysis of zidovudine TP221 were inconclusive but indicated of TP257 increased in D Due to its photochemical 2 and H2O from the furanosyl moiety.
properties, acyclovir is likely to undergo self-sensitization via In addition, photolytic cleavage of the nitrogen−carbon bond photoexcitation and subsequent formation of 1O between DNA base moieties and riboside analogue side chains guanine and −For the second acyclovir photo- was observed for all three compounds, resulting in formation of TP (TP223), HRMS analysis indicated the loss of two protons, 5-fluorocytosine (emtricitabine TP129), cytosine (lamivudine most likely from the side chain, as evidenced by the detection TP111), and thymine (zidovudine TP126). None of these TPs of fragments m/z 152, 135, and 110, suggesting that the was detected in sunlight experiments in the presence of biomat guanine moiety remained unchanged Additional (), indicating that they were rapidly information obtained from fragmentation of the side chain was transformed, most likely via microbial processes. For inconclusive but indicated oxidation of the terminal alcohol to zidovudine, this was confirmed by additional biodegradation the corresponding aldehyde via reaction with •OH.
experiments with photo-TPs (thymine, TP239, and TP257), Results from the 72-h simulated sunlight experiments showing the rapid elimination of thymine (When conducted in the presence of the biomat revealed a steady the importance of both thymine and cytosine as DNA building decrease of acyclovir during light and dark periods, indicating blocks is considered, it is likely that they were incorporated into the dominance of biotransformation processes the microbial biomass. The fate of 5-fluorocytosine remains However, biotransformation of acyclovir was significantly faster in the sunlight experiments compared to dark controls Similar to abacavir and acyclovir, biotransformation of suggesting that the higher oxygen concentrations and emtricitabine, lamivudine, and zidovudine was shown to result elevated pH values that occurred when microorganisms in the in the formation of carboxylated TPs via oxidation of the biomat were undergoing photosynthesis played a role in terminal alcohol as observed previously for abacavir and biotransformation In the presence of simulated acyclovir (As carboxylated TPs are expected to sunlight, production of two phototransformation products (i.e., follow the same phototransformation mechanisms as the parent TP257 and TP224) was observed. No significant removal of compounds, the interaction of photo- and biotransformation TP257 was detected during dark periods, suggesting limited reactions is likely to result in their complete elimination via biotransformation via oxidation of the terminal hydroxyl group mineralization and/or microbial uptake of the side chain. Although the exact reason for this is Environmental Implications. Differences between ki- unknown, a plausible explanation is that structural modifica- netics and transformation product formation in the presence tions of the guanine core moiety prevented enzymatic oxidation and absence of the biomat highlight the complexity of of TP257. In contrast, concentrations of TP223 decreased in transformation reactions that lead to the removal of trace the dark. For the biotransformation product (i.e., acyclovir organic contaminants in open-water unit process wetlands and carboxylate), increasing concentrations were observed only other sunlit waters. Attempts to predict the environmental fate Environ. Sci. Technol. 2015, 49, 14136−14145 Environmental Science & Technology was found to occur at the same location as in the parentcompound. As a result, mechanisms and kinetics were similar tothose observed for parent antiviral compounds. This isimportant because carboxylate biodegradation products aretypically present in much higher concentrations in biologicaltreated wastewater compared to parent compounds.Incontrast, biodegradation kinetics of phototransformationproducts of antiviral drugs differed substantially from thatobserved for the parent compound even though the site ofenzymatic oxidation did not change. This can be explained bydifferences in enzyme affinities and steric hindrance. Forexample, phototransformation of acyclovir created a trans-formation product (TP257) that was not susceptible tobiotransformation by microorganisms that could oxidize theparent compound in the dark.
Combining kinetic studies with investigations of trans- formation product formation provides a better understandingof mechanisms relevant for the removal of trace organiccontaminants in sunlit waters. By conducting biotransformationstudies in the presence and absence of light, it is possible toassess interactions between transformation processes and thelikelihood that complete mineralization of trace organiccontaminants will occur. These data also suggest that relativeratios of antiviral compounds and their transformation productsmight be useful as in situ probes to assess the relativeimportance of microbial and photochemical transformationpathways. This study highlights the need to consider theformation of different transformation products in sunlit andlight-shaded systems and the possibility of using knowledge ofthe reactivity of specific moieties in chemical fate assessment.
When the variety of formed transformation products isconsidered, there is a need for appropriate risk assessmenttools to assess potential adverse effects of transformationproducts with unknown toxicities on aquatic ecosystems.
Additional field studies may further confirm these laboratorymicrocosm results and help to assess the suitability ofapproaches for determining the relative importance ofindividual transformation processes.
■ ASSOCIATEDCONTENT *S Supporting InformationThe Supporting Information is available free of charge on theat DOI: Additional text, 22 figures, and 16 tables withinformation on sample analysis, UV spectra of antiviraldrugs, phototransformation kinetics plots, determinationof indirect photolysis reaction rate constants, quantumyields, steady-state concentrations of reactive intermedi-ates in wetland water, experiments with DNA modelcompounds, MSn fragments of transformation products,formation and fate of abacavir photo-TPs by different Figure 6. Proposed photo- and biodegradation pathway of lamivudine reactive intermediates, and results of combined bio- and (top), emtricitabine (middle), and zidovudine (bottom) in open-water phototransformation experiments with emtricitabine, wetland cells. Orange and green arrows indicate photo- and lamivudine and zidovudine ) biotransformation reactions, respectively.
of organic contaminants in these systems require an under- ■ AUTHORINFORMATION standing of both processes as well as their potential Corresponding Author Identification of TPs showed that bio- and phototransforma- tion reactions took place at different positions of the antiviral molecules. Phototransformation of biodegradation products The authors declare no competing financial interest.
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