In recent years, the emerging persistent organic matter attracted great attention in the environmental protection industry [1
]. Owing to their high consumption and refractory chemical properties [4
], antibiotics are considered as a new persistent organic substances [5
]. Antibiotics enter the aquatic environment through industrial and medical wastewater from the pharmaceutical industry [8
], as well as human and animal feces [10
]. Trimethoprim (TMP) is a commonly used antibacterial synergetic agent which can be combined with sulfonamides to greatly enhance their antibacterial activity [11
]. It is widely used in the treatment of human diseases and animal bacterial infections [13
]. Only 25% of TMP can be metabolized by human body after ingestion [15
], and the remaining TMP and its metabolites are discharged out of the body through the digestive system and subsequently enter domestic wastewater [16
]. TMP has been detected in wastewater from sewage treatment plants, surface water environment, drinking water, and other water bodies [19
]. Even in large-scale wastewater treatment plants with advanced technologies such as chlorination and ultraviolet disinfection radiation, the concentration of TMP in the effluent ranges from the ng/L to the μg/L level [24
]. TMP in water cannot be effectively removed through the existing traditional wastewater treatment processes [26
]. Therefore, exploring an efficient and environmentally friendly technology to degrade TMP in water is of great significance. At present, some advanced technologies and materials have been studied to treat refractory wastewater. For instance, it was reported that MBR technology can effectively degraded TMP from hospital wastewater [29
]. Sharma et al. [30
] discovered nanofiber membrane for wastewater treatment, which greatly reduced the cost of membrane technology. In addition, advanced oxidation processes (AOPs) have been studied to eliminate TMP, including photolysis [33
], photo-Fenton [34
], photocatalysis [35
], ozonation, and so on.
was often used in water treatment [36
], and was demonstrated to oxidize antibiotics [38
is one of the strongest oxidizing substances in nature, and its oxidation potential can reach 2.07 V [39
]. There are two modes of reaction between O3
and organic pollutants in aqueous solution. The first mode is the direct reaction of O3
molecules with organic pollutants. The second mode is the decomposition of O3
to produce hydroxyl radicals (·OH), which then reacts with organic pollutants [41
]. The reduction product of O3
is oxygen, which will not bring secondary pollution. Most antibiotics (e.g., amoxicillin, doxycycline, ciprofloxacin, and sulphadiazine) can readily react with O3
]. TMP is an organic compound with a high O3
reaction rate constant (k
= 2.7 × 105
at pH 7) [43
]. Therefore, TMP can be directly removed by O3
oxidation. Moreover, TMP can be removed by ·OH produced by O3
= 6.9 × 109
at pH 7) [46
]. Ling et al. [47
] found that norfloxacin and levofloxacin had high reactivity to O3
. Kuang et al. discovered that TMP can be effectively removed when the molar ratio of O3
dose to TMP reached 3 [48
]. In addition, four degradation pathways of TMP were proposed in this reference: hydroxylation, carbonylation, deamination, and demethylation. As a common free radical quencher, methanol was used to distinguish between direct and indirect oxidation [49
]. Ozonation has been shown to have a high potential for the oxidation of pharmaceuticals in drinking water [37
] and wastewater [50
]. Although in practice, the amount of ozone used in water treatment can only lead to partial oxidation; however, partial oxidation was sufficient to significantly reduce pharmacological activity and toxicity. The ozone-derived oxidation products formed from parent pharmaceutical compounds may be more susceptible to biological degradation [37
Natural organic matters (NOM), such as humic acids (HA), are present in water [51
]. The presence of these NOM which may react directly with O3
or indirectly with free radicals will affect the ozonation of TMP. Therefore, the presence of HA will reduce the amount of O3
and ·OH, and lead to the decrease of the oxidation rate of micro pollutants [53
]. At present, many researches focus on the effect of NOM on the photocatalytic degradation of antibiotics as a photosensitizer [54
]. However, study on the effect of HA on the ozonation of TMP and its pathway is limited.
To summarize, most of the studies only focused on the effect of HA on the degradation rate of TMP by ozonation, as for the degradation pathway, most of the studies just stayed in pure solution. This study not only discusses the effect of HA on the degradation effect, but also compares the change of intermediate products in the degradation process. In addition, the effect of methanol on the experiment was also studied. The normalized abundances of the intermediate products of TMP in water in the presence and absence of HA during ozonation process were detected by using HPLC-MS/MS to explore the degradation mechanisms of TMP. Methanol, which can inhibit the oxidation of TMP by ·OH, was added to the solution to judge the main method of reaction. The results reflect that ·OH played an important role in the reaction. In addition, the 3D-EEM analysis of the solution with HA addition was carried out to discuss the change of the fluorescent substance during the experiment. This study could provide theoretical basis for the control of TMP and its ozonation products during water treatment.
2. Materials and Methods
The solutions were prepared using corresponding chemical reagents with high purity. TMP (>99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol (HPLC-grade, CH3
OH), acetonitrile (HPLC–grade, CH3
CN), and formic acid (HCOOH, 98%), which were used for chromatographic analysis, were supplied by Sigma-Aldrich. The HA used in this study was Suwannee River Humic Acid, (SRHA, 2S101H), which was obtained from the International Humic Substances Society (IHSS, US) (Table S1
). Sodium thiosulfate (Na2
, chemically pure) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All of these chemicals were used without any pre-treatment.
Ultrapure water used for sample preparation was obtained from the Milli-Q water purification system (Millipore Synergy 185, US). The pharmaceutical sample solutions were stored in the dark at 4 °C. All the experiments were performed at pH 6.0 ± 0.1.
2.2. Ozonation Experiments Setup
The ozonation experimental system for the study was consisted of an oxygen cylinder, an O3 gas generator (Model 3S-X, KIRIS, China), and a reaction reactor device. The O3 gas was generated by passing pure O2 feed gas (99.9%) through the O3 generator equipped with a gaseous flow meter. Then continuously sparged O3 gas into the bottom of the reactor using a fine bubble diffuser at a constant flow rate of 1 L/min. Excess O3 was passed into two gas absorption bottles containing 2% KI solution.
2.3. TMP Ozonation Study
2.3.1. Removal of TMP in O3 Solution
Four experimental conditions were prepared, including TMP (10 mg/L) in ultrapure water, in methanol solution (0.25 mol/L), in HA solution (15 mg/L), and in mixed solution of HA (15 mg/L) and methanol (0.25 mol/L). The solutions were designed to study the effect of the additional NOM and free radical quencher (methanol for ·OH) on the ozonation of TMP.
First, O3 gas was introduced into ultrapure water at 0 °C (in ice bath). The concentration of O3 in the water increased gradually, and the corresponding concentration of O3 solution was obtained by controlling the aeration time. Afterward, 50 mL of the water matrix (the concentration was twice that of the above four conditions) was added into a conical flask, and 50 mL of O3 solution from the reactor was added to the conical flask. In this way, the concentration of the corresponding substance in the mixed solution meet the set experimental conditions. The solution was mixed before determining its concentration to ensure that the reaction was complete (in 1 min), and sodium thiosulfate solution was added to the sample to stop the reaction. At the same time, 5 mL of O3 solution was obtained to determine the concentration of O3. The initial concentration of dissolved O3 was calculated by dividing the concentration in O3 solution by 2. To ensure accuracy, all experiments were conducted in triplicate.
The effects of HA and OH on TMP degradation were studied by comparing the degradation of TMP in four experimental conditions.
2.3.2. Ozonation of TMP in O3 Contactor
The experimental condition in this study was similar to that in Section 2.3.1
except that the reactor was stirred continuously using a magnetic stirrer at 20 °C, and O3
gas was directly introduced into four water substrates to react with the pollutants. Samples were obtained at corresponding times, and sodium thiosulfate solution was added to the sample to stop the reaction. To ensure that the amount of the products was above the detection limit of the HPLC-MS/MS, the initial TMP concentration was set at 10 mg/L. The experiments were carried out in a continuously aerated reactor containing TMP solution with an initial pH of 6.0 ± 0.1, and the amounts of the residual parent compounds and the formation of intermediate products were detected by HPLC-MS/MS at regular time intervals. Samples for EEM detection were also taken before the beginning of the experiment and at the 5th, 10th, and 30th min of the experiment.
2.4. Analytical Methods
The concentration of O3
was determined according to the “Standard Methods for the Examination of Water and Wastewater (21st Ed)-APHA (2005)-Method 4500-O3
-Indigo Colorimetric Method”. More detailed information and procedures about indigo method can be found in the literature [55
TMP concentration and the abundance of intermediate products during the TMP degradation process was determined according to the HPLC-MS/MS system (Agilent 1290-6460, USA) with an Agilent C18 column (3.5 μm, 2.1 × 150 mm). The column temperature was maintained at 40 °C. Mobile phase A was ultrapure water containing 0.1% formic acid, and mobile phase B was methanol (99%). The following gradient program was used for the analysis: 90% A (initial), 90–80% A (1.0–1.5 min), 80–60% A (1.5–3.5 min), 60% A (3.5–6.0 min), 60–40% A (6.0–6.5 min), 40% A (6.5–7.0 min), 40–0% A (7.0–7.5 min), 0% A (7.5–10.5 min), and 0–90% A (10.5–11.0 min). The injection volume was 10 μL. The column temperature was maintained at 40 °C. The flow rate of the mobile phase was 0.3 mL/min. For the elucidation of the intermediates, positive ionization mode was used to acquire the intermediates with a scan range of m/z 50 to m/z 600.
Fluorescence spectrometer (FS5, Edinburgh Instruments, Livingston, UK) was used to measure the changes of the fluorescent substances in the reaction process. The parameters were set as follows: scanning speed of 12,000 nm/min, data interval of 5.0 nm, excitation light bandwidth of 3.0 nm, and emission light bandwidth of 3.0 nm. The excitation wavelength (Ex) was set to 230–450 nm and the emission wavelength (Em) was 260–550 nm.
The toxicity of TMP and its degradation products were predicted using the Toxicity Estimation Software Tool (V4.2.1, USEPA, 2016).
Excessive O3 can degrade TMP rapidly, and the addition of HA and methanol had little effect on the reaction, indicating that TMP could be directly oxidized by O3 molecules. When O3 was not excessive, the removal efficiency of TMP increased with the increase of O3 dosage. The addition of HA and methanol inhibited the degradation of TMP. Therefore, indirect oxidation also played an important role in the removal of TMP.
The reaction pathways proposed were hydroxylation, demethylation, cleavage, and carbonylation. In particular, hydroxylation was the dominant reaction. The existence of HA in water may affect the generation of cleavage and carbonylation products of TMP significantly, and the formation of hydroxylated products and demethylation products also decreased slightly. According to the free radical quenching test, the cleavage and demethylation products were mainly produced by indirect oxidation. With the extension of ozonation time, the fluorescence value in the solution decreased and the fluorescence peak blue shifted, indicating that the structure of HA changed in the reaction and was competitively degraded with TMP.
Ozone oxidation technology not only has a fast degradation rate, but also can remove odor without producing secondary pollution. Although ozone oxidation only leads to partial oxidation of drugs, due to the high removal rate and the small quantities of products, partial oxidation is enough to reduce pharmacological activity and toxicity. Ozonation has a high potential for the oxidation of pharmaceuticals in drinking water and wastewater. This study could provide theoretical basis for the control of TMP and its ozonation products during water treatment.