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Hospital sewage constitutes an important point source for antibiotics and antibiotic-resistant bacteria due to the high antibiotic use. Antibiotic resistance can develop and cause problems in sewage systems within hospitals and municipal wastewater treatment plants, thus, interventions to treat hospital sewage on-site are important. Ozonation has proven effective in treating relatively clean wastewater, but the effect on untreated wastewater is unclear. Therefore, we piloted implementation of ozonation to treat wastewater in a tertiary hospital in Uppsala, Sweden. We measured active pharmaceutical ingredients (APIs) using liquid chromatography-mass spectrometry and antibiotic-resistant Enterobacteriaceae using selective culturing pre-and post-ozonation. Comparing low (1 m3 /h) and high (2 m3 /h) flow, we obtained a 'dose-dependent' effect of API reduction (significant reduction of 12/29 APIs using low and 2/29 APIs using high flow, and a mean reduction of antibiotics of 41% using low vs. 6% using high flow, 25% vs. 6% for all APIs). There was no significant difference in the amount of antibiotic-resistant Enterobacteiaceae pre-and post-ozonation. Our results demonstrate that ozonation of untreated wastewater can reduce API content. However, due to the moderate API decrease and numerous practical challenges in the on-site setting, this specific ozonation system is not suitable to implement at full scale in our hospital.

Neuraminidase inhibitors (NAIs) are the gold standard treatment for influenza A virus (IAV). Oseltamivir is mostly used, followed by zanamivir (ZA). NAIs are not readily degraded in conventional wastewater treatment plants and can be detected in aquatic environments. Waterfowl are natural IAV hosts and replicating IAVs could thus be exposed to NAIs in the environment and develop resistance. Avian IAVs form the genetic basis for new human IAVs, and a resistant IAV with pandemic potential poses a serious public health threat, as NAIs constitute a pandemic preparedness cornerstone. Resistance development in waterfowl IAVs exposed to NAIs in the water environment has previously been investigated in an in vivo mallard model and resistance development was demonstrated in several avian IAVs after the exposure of infected ducks to oseltamivir, and in an H1N1 IAV after exposure to ZA. The N1 and N2 types of IAVs have different characteristics and resistance mutations, and so the present study investigated the exposure of an N2-type IAV (H4N2) in infected mallards to 1, 10 and 100 µg l−1 of ZA in the water environment. Two neuraminidase substitutions emerged, H274N (ZA IC50 increased 5.5-fold) and E119G (ZA IC50 increased 110-fold) at 10 and 100 µg l−1 of ZA, respectively. Reversion towards wild-type was observed for both substitutions in experiments with removed drug pressure, indicating reduced fitness of both resistant viruses. These results corroborate previous findings that the development of resistance to ZA in the environment seems less likely to occur than the development of resistance to oseltamivir, adding information that is useful in planning for prudent drug use and pandemic preparedness.

Introduction: Negative environmental effects of active pharmaceutical ingredients (APIs) are increasingly recognized, especially concerning antibiotics, and hospitals are important point sources. "pCure" is a toilet rim block containing API-degrading enzymes; the producing company claims positive in vitro results but no implementation studies have been performed.

Materials and methods: In a university hospital setting, 16 weeks were randomized to installation or no installation of pCure in all 261 toilets connected to the same cesspit where sewage water was sampled daily. Ninety-six samples were analyzed for 102 APIs using liquid chromatography/tandem mass spectrometry.

Results and Discussion: Fifty-one APIs were detected with a large variation in levels but no significant differences in the initial statistical analysis. More statistical testing of API level ratios (pCure installed/not installed) yielded some cases of significant decrease. Differences were small and not consistent when comparing means and medians. We cannot exclude a small pCure effect but clearly pCure has no effect of biological importance. Conclusion: pCure is not useful to reduce drug residue discharge in a hospital setting. In a bigger perspective, our study exemplifies that products claiming to reduce an environmental problem need to be tested in on-site implementation studies by independent researchers before reaching the market.

Neuraminidase inhibitors are a cornerstone of influenza pandemic preparedness before vaccines can be mass-produced and thus a neuraminidase inhibitor-resistant pandemic is a serious threat to public health. Earlier work has demonstrated the potential for development and persistence of oseltamivir resistance in influenza A viruses exposed to environmentally relevant water concentrations of the drug when infecting mallards, the natural influenza reservoir that serves as the genetic base for human pandemics. As zanamivir is the major second-line neuraminidase inhibitor treatment, this study aimed to assess the potential for development and persistence of zanamivir resistance in an in vivo mallard model; especially important as zanamivir will probably be increasingly used. Our results indicate less potential for development and persistence of resistance due to zanamivir than oseltamivir in an environmental setting. This conclusion is based on: (1) the lower increase in zanamivir IC50 conferred by the mutations caused by zanamivir exposure (2-17-fold); (2) the higher zanamivir water concentration needed to induce resistance (at least 10 µg l^-1); (3) the lack of zanamivir resistance persistence without drug pressure; and (4) the multiple resistance-related substitutions seen during zanamivir exposure (V116A, A138V, R152K, T157I and D199G) suggesting lack of one straight-forward evolutionary path to resistance. Our study also adds further evidence regarding the stability of the oseltamivir-induced substitution H275Y without drug pressure, and demonstrates the ability of a H275Y-carrying virus to acquire secondary mutations, further boosting oseltamivir resistance when exposed to zanamivir. Similar studies using influenza A viruses of the N2-phylogenetic group of neuraminidases are recommended.

This study investigated the i) kinetics, and ii) proportion of photolysis of 30 relatively stable active pharmaceutical ingredients (APIs) during artificial UV irradiation for 28 d in ammonium acetate buffer, filtered and unfiltered river water. Buffer was included to control removal kinetics under stable pH conditions and without particulate matter. Dark controls were used to determine removal due to other processes than photolysis and calculate the proportion of photolysis of the total removal. The removal of each API in each matrix was determined using online solid phase extraction/liquid chromatography tandem mass spectrometry (online SPE/LC-MS/MS). Most APIs transformed during the 28 d of UV irradiation and the dark controls showed that photolysis was the major removal process for the majority of the APIs studied. The half-lives ranged from 6 h (amitriptyline) in unfiltered river water to 884 h (37 d, carbamazepine) in buffer. In unfiltered river water, the proportion of APIs with short half-lives (<48 h) was much higher (29%) than in the other matrices (4%), probably due to additional organic carbon, which could have promoted indirect photolysis. Furthermore, two APIs, memantine and fluconazole, were stable in all three matrices, while alprazolam was stable in buffer and unfiltered river water and four additional APIs were stable in buffer. Considering the relatively long-term UV-exposure, this study enabled the investigation of environmentally relevant half-lives in natural waters. Many APIs showed high persistence, which is environmentally concerning and emphasizes the importance of further studies on their environmental fate and effects.

Anti-influenza drugs constitute a key component of pandemic preparedness plans against influenza. However, the occurrence of such drugs in water environments, the potential of resistance development in the natural hosts, and the risk for transmission of antiviral resistance to humans call for measures to increase removal in wastewater treatment plants (WWTPs). In this study, removal of three anti-influenza drugs; amantadine (AM), oseltamivir carboxylate (OC) and zanamivir (ZA), and formation/removal of their transformation products during ozonation of wastewater effluents from two Swedish WWTPs in Uppsala and Stockholm were studied. The removal profile of target antivirals and formation/removal of their transformation products were studied by liquid chromatography/high resolution mass spectrometry. 3.5 h of ozone exposure (total dose of ozone 5.95 g) led to complete removal of the three anti-influenza drugs with a degradation in the following order ZA > OC > AM. Two, five and one transformation products were identified and semi-quantified for AM, OC and ZA, respectively. Increasing and later decreasing transformation products concentration followed the decrease in concentration of target compounds. All transformation products detected, except one of AM in wastewater from Stockholm WWTP, were removed at the end of the experiment. The removal efficiency was higher for all studied compounds in wastewater from Uppsala WWTP, which had lower TOC and COD values, less phosphorus, and also higher pH in the water. Ozonation thus offers multiple benefits through its potential to degrade influenza antivirals, hence decrease the risk of environmental resistance development, in addition to degrading other pharmaceuticals and resistant microorganisms.

Environmental Chemistry covers a range of topics within the discipline of chemistry, from toxicology to legislation, which warrants interdisciplinary study. Consequently, problem-based learning (PBL), a style of student-centered learning which facilitates the integration of multiple subjects, was investigated to determine if it would be a more appropriate instructional method for teaching Environmental Chemistry than the traditional teacher-centered education model. This article describes the practical aspects of course development and implementation of PBL in a master’s level course in Environmental Chemistry. Overall, the results, which were collected from the initial two years of the course, indicated that the students were pleased and found PBL to be an efficient methodology for not only learning, but also acquiring an in-depth understanding of Environmental Chemistry. This is intended as a case-study with the target audience consisting primarily of high school and undergraduate chemistry teachers, but may also be useful for teachers in other subject areas with an interest in student-centered education.

Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and new human IAVs often contain gene segments originating from avian IAVs. Treatment options for severe human influenza are principally restricted to neuraminidase inhibitors (NAIs), among which oseltamivir is stockpiled in preparedness for influenza pandemics. There is evolutionary pressure in the environment for resistance development to oseltamivir in avian IAVs, as the active metabolite oseltamivir carboxylate (OC) passes largely undegraded through sewage treatment to river water where waterfowl reside. In an in vivo mallard (Anas platyrhynchos) model, we tested if low-pathogenic avian influenza A(H7N9) virus might become resistant if the host was exposed to low levels of OC. Ducks were experimentally infected, and OC was added to their water, after which infection and transmission were maintained by successive introductions of uninfected birds. Daily fecal samples were tested for IAV excretion, genotype, and phenotype. Following mallard exposure to 2.5 μg/liter OC, the resistance-related neuraminidase (NA) I222T substitution, was detected within 2 days during the first passage and was found in all viruses sequenced from subsequently introduced ducks. The substitution generated 8-fold and 2.4-fold increases in the 50% inhibitory concentration (IC50) for OC (P < 0.001) and zanamivir (P = 0.016), respectively. We conclude that OC exposure of IAV hosts, in the same concentration magnitude as found in the environment, may result in amino acid substitutions, leading to changed antiviral sensitivity in an IAV subtype that can be highly pathogenic to humans. Prudent use of oseltamivir and resistance surveillance of IAVs in wild birds are warranted.

Pandemics are unique public health emergencies that can result in a large sudden increase in the use of a restricted set of pharmaceuticals within a short time period. In the case of an influenza pandemic, antiviral use will greatly exceed inter-pandemic use in most countries by several orders of magnitude, as few countries maintain significant inter-pandemic usage-Japan being a notable exception [1]. Depending on the severity of the pandemic, antibiotics have the potential to significantly exceed inter-pandemic usage for the treatment of secondary bacterial respiratory infections [2]. Decongestant usage is also predicted to increase with an increase in upper-and lower-respiratory tract infections [3].

Abstract Zanamivir (Za) is a highly polar and hydrophilic antiviral drug used for the treatment of influenza A viruses. Za has been detected in rivers of Japan and it's environmental occurrence has the risk of inducing antiviral resistant avian influenza viruses. In this study, a rapid automated online solid phase extraction liquid chromatography method using bonded zwitterionic stationary phases and tandem mass spectrometry (SPE/LC–MS/MS) for trace analysis of Za was developed. Furthermore, an internal standard (IS) calibration method capable of quantifying Za in Milli-Q, surface water, sewage effluent and sewage influent was evaluated. Optimum pre-extraction sample composition was found to be 95/5 v/v acetonitrile/water sample and 1% formic acid. The developed method showed acceptable linearities (r2≥0.994), filtration recovery (≥91%), and intra-day precisions (RSD≤16%), and acceptable and environmentally relevant LOQs (≤20 ng L−1). Storage tests showed no significant losses of Za during 20 days and +4/−20 °C (≤12%) with the exception of influent samples, which should be kept at −20 °C to avoid significant Za losses. The applicability of the method was demonstrated in a study on phototransformation of Za in unfiltered and filtered surface water during 28 days of artificial UV irradiation exposure. No significant (≤12%) phototransformation was found in surface water after 28 days suggesting a relatively high photostability of Za and that Za should be of environmental concern.