High throughput and sensitive quantitation of tobacco-specific alkaloids and nitrosamines in wastewater.

Tobacco-specific alkaloids and nitrosamines are important biomarkers for the estimation of tobacco use and human exposure to tobacco-specific nitrosamines that can be monitored by wastewater analysis. Thus far their analysis has used solid phase extraction, which is costly and time-consuming. In this study, we developed a direct injection liquid chromatography-tandem mass spectrometry method for the quantification of two tobacco-specific alkaloids and five nitrosamines in wastewater. The method achieved excellent linearity (R2 > 0.99) for all analytes, with calibration ranging from 0.10 to 800 ng/L. Method limits of detection and quantification were 0.17 ng/L (N-nitrosonornicotine, NNN) and 1.0 ng/L (N-nitrosoanatabine (NAT) and NNN), with acceptable accuracy (100 % ± 20 %) and precision (± 15 %). Analyte loss during filtration was < 15 %, and the relative matrix effect was < 10 %. The method was applied to 43 pooled wastewater samples collected from three wastewater treatment plants in Australia between 2017 and 2021. Anabasine and anatabine were detected in all samples at concentrations of 5.0 - 33 ng/L and 12 - 41 ng/L, respectively. Three of the five tobacco-specific nitrosamines (NAT, NNN, and (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol) (NNAL)) were detected, in < 50 % of the wastewater samples, with concentrations nearly ten times lower than the tobacco alkaloids (< 1.0 - 6.2 ng/L). In-sewer stability of the nitrosamines was also assessed in this study, with four (NAT, NNAL, NNN, and N-nitrosoanabasine (NAB)) being stable (i.e. < 20 % transformation over 12 h in both control reactor (CR) and rising main reactor (RM) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) being moderately stable (< 40 % loss over 12 h in RM). This direct injection method provides a high-throughput approach in simultaneous investigation of tobacco use and assessment of public exposure to tobacco-specific nitrosamines.

Analysis of wastewater from 2013 to 2021 detected a recent increase in nicotine use in Queensland, Australia.

Previous wastewater-based epidemiology (WBE) studies have reported decreasing trends of nicotine and tobacco use in Australia before 2017, but there is concern that increasing illicit use of nicotine in vaping products and illicit tobacco could reverse this progress. This study aimed to assess temporal trends of nicotine consumption and specifically tobacco consumption via wastewater analysis in a population in Australia between 2013 and 2021. One week of daily wastewater samples were analyzed every two months from February 2013 to December 2021 in a regional city serving ∼100,000 people. A total of 340 daily samples were analyzed for anabasine (tobacco specific biomarker) and nicotine metabolites, cotinine and hydroxycotinine, using direct injection method by liquid chromatography with tandem mass spectrometry. Daily consumption estimates were calculated from daily flow data, population estimates and previously reported excretion factors. Linear spline regression was performed to identify periods when significant change of slopes occurred and to evaluate the temporal trends. Tobacco use monitored using anabasine as a biomarker, showed a decreasing trend over the whole period with a higher rate of decrease during the first two years (2013-2014, 21 % decrease) compared to the later 7 years (2015-2021, 10 % decrease). Nicotine use, monitored using cotinine and hydroxycotinine, showed a downward trend between 2013 and 2018 (2013-2014: 18 % decrease, p < 0.05; 2015-2016: 6 % increase, p = 0.48; Feb-Dec 2017: 15 % decrease, p = 0.39) followed by a significant increase from 2018 to 2021 (40 % increase, p < 0.001). This finding suggests the increasing use of non-tobacco nicotine-based products. Additionally, the tobacco use estimate by wastewater analysis was higher than the tobacco sales data, which suggests the use of illicit tobacco in the catchment.

Improving Wastewater-Based Tobacco Use Estimates Using Anabasine.

In wastewater-based epidemiology (WBE), nicotine metabolites have been used as biomarkers for monitoring tobacco use. Recently, the minor tobacco alkaloids anabasine and anatabine have been suggested as more specific biomarkers for tobacco use since nicotine use can be from both tobacco and non-tobacco sources. This study aimed to provide an in-depth evaluation of the suitability of anabasine and anatabine as WBE biomarkers of tobacco and subsequently estimate their excretion factors for WBE applications. Pooled urine (n = 64) and wastewater samples (n = 277), collected between 2009 and 2019 in Queensland, Australia, were analyzed for nicotine and its metabolites (cotinine and hydroxycotinine), as well as anabasine and anatabine. Anabasine performed as the better biomarker, showing a similar per capita load in pooled urine (2.2 ± 0.3 µg/day/person) and wastewater samples (2.3 ± 0.3 µg/day/person), while the per capita load of anatabine in wastewater was 50% higher than its load in urine. It is estimated that 0.9 µg of anabasine was excreted per cigarette smoked. Triangulation of tobacco sales data and tobacco use estimated from either anabasine or cotinine showed that anabasine-based estimates were 5% higher than sales data, while cotinine-based estimates were between 2 and 28% higher. Our results provided concrete evidence to confirm the suitability of anabasine as a specific biomarker for monitoring tobacco use by WBE.

Increased Nicotine Consumption in Australia During the First Months of the COVID-19 Pandemic.

INTRODUCTION: Mixed findings have been reported about the impact of the COVID-19 pandemic on smoking behavior in different populations. AIMS AND METHODS: In this study, we aimed to quantify changes in smoking prevalence through the proxy of nicotine consumption in the Australian population from 2017 to 2020 inclusive. Estimates of nicotine consumption between 2017 and 2020 were retrieved from a national wastewater monitoring program that covers up to 50% of the Australian population. National sales data for nicotine replacement therapy (NRT) products from 2017 to 2020 were also acquired. Linear regression and pairwise comparison were conducted to identify data trends and to test differences between time periods. RESULTS: The average consumption of nicotine in Australia decreased between 2017 and 2019 but increased in 2020. Estimated consumption in the first half of 2020 was significantly higher (~30%) than the previous period. Sales of NRT products increased gradually from 2017 to 2020 although sales in the first half of the year were consistently lower than in the second half. CONCLUSION: Total nicotine consumption increased in Australia during the early stage of the pandemic in 2020. Increased nicotine consumption may be due to people managing higher stress levels, such as from loneliness due to control measures, and also greater opportunities to smoke/vape while working from home and during lockdowns in the early stage of the pandemic. IMPLICATIONS: Tobacco and nicotine consumption have been decreasing in Australia but the COVID-19 pandemic may have temporarily disrupted this trend. In 2020, the higher impacts of lockdowns and working from home arrangements may have led to a temporary reversal of the previous downward trend in smoking during the early stage of the pandemic.

Assessing changes in nicotine consumption over two years in a population of Hanoi by wastewater analysis with benchmarking biomarkers.

Monitoring the actual change in consumption of nicotine (a proxy for smoking) in the population is essential for formulating tobacco control policies. In recent years, wastewater-based epidemiology (WBE) has been applied as an alternative method to estimate changes in consumption of tobacco and other substances in different communities around the world, with high potential to be used in resource-scarce settings. This study aimed to conduct a WBE analysis in Hanoi, Vietnam, a lower-middle-income-country setting known for high smoking prevalence. Wastewater samples were collected at two sites along a sewage canal in Hanoi during three periods: Period 1 (September 2018), Period 2 (December 2018-January 2019), and Period 3 (December 2019-January 2020). Concentrations of cotinine, 3-hydroxycotinine, and nicotine ranged from 0.73 µg/L to 3.83 µg/L, from 1.09 µg/L to 5.07 µg/L, and from 0.97 µg/L to 9.90 µg/L, respectively. The average mass load of cotinine estimated for our samples was 0.45 ± 0.09 mg/day/person, which corresponds to an estimated daily nicotine consumption of 1.28 ± 0.25 mg/day/person. No weekly trend was detected over the three monitoring periods. We found the amount of nicotine consumption in Period 1 to be significantly lower than in Period 2 and Period 3. Our WBE estimates of smoking prevalence were slightly lower than the survey data. The analysis of benchmarking biomarkers confirmed that cotinine was stable in the samples similar to acesulfame, while paracetamol degraded along the sewer canal. Further refinement of the WBE approach may be required to improve the accuracy of analyzing tobacco consumption in the poor sewage infrastructure setting of Vietnam.

Assessing alcohol consumption in a Chinese urban population and a university town using high temporal resolution wastewater-based epidemiology.

BACKGROUND: Alcohol consumption is a leading preventable risk factor for morbidity and mortality in China. Understanding drinking patterns provides important data to inform public health policies in alcohol control, especially in specific groups like university students. This study aims to assess the alcohol consumption patterns and level of use in an urban population and a university town in a Chinese city using wastewater-based epidemiology (WBE). METHODS: Daily wastewater sample was collected from an urban catchment (n = 270) and every Wednesday's sample from a university town of 10 university campus (n = 43) in 2017-2018. Concentration of alcohol consumption biomarker in wastewater, ethyl sulfate, was measured by direct injection LC-MS/MS analysis. Per capita daily alcohol consumption was then back calculated for assessment purposes. RESULTS: Per capita daily alcohol consumption was 1.4 ± 0.6 mL/ person aged 15 + /day (EPD) (range: 0.2-4.9) in the urban catchment and 1.3 ± 0.6 EPD (range: 0.3-2.6) in the university town. Trends of alcohol consumption were stable in both catchments in 2017-2018. Alcohol consumption on weekends (1.5 EPD) and weekdays (1.4 EPD) were at a similar level. Additionally, no difference was observed between holidays (1.5 EPD) and non-holidays (1.4 EPD). CONCLUSION: There is a stable weekly drinking level in the studied urban population, which is different compared to studies conducted in Western countries. Our study suggested a similar consumption level between the urban population and university students. Drinking during weekdays could negatively affect China's economic productivity and future public health policies could be adapted to reflect this pattern of drinking.

Comparison of tobacco use in a university town and a nearby urban area in China by intensive analysis of wastewater over one year period.

Understanding smoking patterns in the population is essential for formulating public health and tobacco control policies. Wastewater-based epidemiology (WBE) is a valuable complementary approach to conventional survey methods to measure tobacco use, providing non-invasive information in an objective and cost-effective manner. This study estimates tobacco use in an urban population at daily resolution and in a university town at weekly resolution in China. Wastewater samples were collected daily in an urban catchment (n = 279) and every week from a university town located within 13 km of the urban catchment (n = 43) in 2017-2018. The tobacco-related biomarkers, cotinine and hydroxycotinine, and nicotine were analyzed via direct injection liquid chromatography tandem mass spectrometry (LC-MS/MS). Per capita daily tobacco use was back-estimated based on cotinine in wastewater. Over the year of sampling, we observed an increasing trend in tobacco use in the urban catchment that corroborated with sales statistics in 2017-2018. Tobacco use in the urban area was estimated to be 1.16 cigarettes/person aged 15+/day, while it was estimated to be 0.60 cigarettes/person aged 15+/day in the university town. The level of tobacco use in the university town remained stable over the year in contrast to the urban area. The difference of tobacco use in the two catchments may be attributed to their demographic differences. Furthermore, the Tobacco-Free Campus Policy would be a possible reason for the lower level of tobacco use in the university town.

Long-term trends in tobacco use assessed by wastewater-based epidemiology and its relationship with consumption of nicotine containing products.

Measurement of population tobacco use via wastewater-based epidemiology (WBE) provides objective data to evaluate the efficacy of tobacco control strategies. However, current WBE tobacco-use estimates based on nicotine metabolites (cotinine and hydroxycotinine) can be masked by use of non-tobacco nicotine-containing products. To better understand nicotine and tobacco use, we analysed tobacco-specific biomarkers, anabasine and anatabine, as well as nicotine metabolites, cotinine and hydroxycotinine, in wastewater samples collected for 6 weeks per year over 6 years (2012-2017) from an Australian wastewater treatment plant serving approximately 100,000 people. Population-normalised mass loads were used to estimate tobacco and nicotine use trends and were compared with surveys and taxation statistics. Significant annual declines were observed for anabasine, anatabine, cotinine and hydroxycotinine of -3.0%, -2.7%, -2.4%, and -2.1%, respectively. The results corresponded with the annual declining trends reported from surveys (-5%) and taxation statistics (-4%). Significant annual decreases in the ratios of anabasine to cotinine (-1.2%) and anatabine to cotinine (-1.0%) suggested a relative increase in the use of non-tobacco nicotine products at the same time that tobacco use was declining. Monitoring tobacco use with anabasine and anatabine removed influence from nicotine-containing products, showing larger reductions in this Australian city than via nicotine biomarkers, whilst also demonstrating their suitability for monitoring long-term trends.

Determination of anabasine, anatabine, and nicotine biomarkers in wastewater by enhanced direct injection LC-MS/MS and evaluation of their in-sewer stability.

Wastewater-based epidemiology (WBE) has been used to estimate tobacco use in the population. However, the increased use of nicotine replacement therapies and e-cigarettes contributes to the load of nicotine metabolites in wastewater, causing over-estimation of tobacco use if nicotine metabolites were used in WBE back-estimation. This study aims to develop a rapid method for determining the tobacco-specific biomarkers, anabasine and anatabine, in wastewater and to evaluate their in-sewer stability for better estimation of tobacco use by WBE. An enhanced direct injection LC-MS/MS was developed to quantify anabasine and anatabine as well as nicotine biomarkers (nicotine, cotinine and hydroxycotinine). The method was optimal when wastewater was filtered through 0.2 µm RC syringe filters and a pre-conditioned SPE cartridge (Oasis HLB 1 cc, 30 mg) before 50 µL was injected into the LC-MS/MS system. Limits of quantification varied between 2.7 and 54.9 ng/L with recoveries from 76% to 103% for all five compounds. In sewer reactors, anabasine and anatabine were less stable than cotinine and hydroxycotinine. They were more stable in the gravity sewer reactor with <20% loss in 12 h than in the rising main sewer reactor with ~30% loss in the same period. We then applied the new method to 42 daily wastewater influent samples collected from an Australian wastewater treatment plant. The five biomarkers were detected in all samples with concentrations ranging from 9.2 to 7430 ng/L. All five compounds were positively correlated with one another. Our results suggested a high throughput analytical method for feasible application in anabasine and anatabine as biomarkers of tobacco use in routine wastewater monitoring.

Using wastewater-based epidemiology to estimate consumption of alcohol and nicotine in major cities of China in 2014 and 2016.

Monitoring the use of alcohol and tobacco in the population is important for public health planning and evaluating the efficacy of intervention strategies. The aim of this study was to use wastewater-based epidemiology (WBE) to estimate alcohol and tobacco consumption in a number of major cities across China and compare WBE estimates with other data sources. Daily composite influent wastewater samples were collected from wastewater treatment plants (WWTPs) across China in 2014 (n = 53) and 2016 (n = 45). The population-normalized daily consumption estimated by WBE were compared with other data sources where available. The average consumption of alcohol was 8.1 ± 7.0 mL ethanol/person aged 15+/day (EPD) in the investigated cities of 2016 while those involved in 2014 had an average consumption of 4.7 ± 3.0 EPD. The average tobacco consumption was estimated to be 3.7 ± 2.2 cigarettes/person aged 15+/day (CPD) in 2016 and 3.1 ± 1.9 CPD in 2014. The changes in the average consumption in those cities from 2014 to 2016 were supported by the results from a limited number of WWTPs where samples were collected in both years. Consumption of alcohol and tobacco in urban China is at a medium level compared with other countries on a per capita basis. WBE estimates of tobacco consumption were relatively comparable with results of traditional surveys and sales statistics. WBE estimates of alcohol consumption were lower than WHO survey results, probably due to EtS degradation and uncertainty in the EtS excretion factor.

Estimating nicotine consumption in eight cities using sewage epidemiology based on ammonia nitrogen equivalent population.

Sewage epidemiology is a real-time tool used to monitor tobacco consumption. In this study, we investigated the tobacco consumption in eight cities in Jilin province using sewage epidemiology. We collected influent wastewater samples from ten wastewater treatment plants (WWTPs) that serve nearly four million people. Mean nicotine (NIC) loads ranged from 1.42 to 14.2mg/d/capita, whereas mean cotinine (COT) loads showed lower levels with 0.33 to 2.15mg/d/capita. Population size was estimated to provide an accurate and real-time population based on ammonia nitrogen (NH4-N) concentration in influent. To verify the NH4-N equivalent population, we compared these results with the corresponding population estimated based on the expert knowledge of the local WWTPs operators. Daily consumption of NIC was estimated to be approximately 2.39±1.47mg/d/capita. Monte Carlo simulation was used to analyze uncertainty and variability in the number of cigarettes consumed by smokers in the range of 9.8 to 31.4 per day with a median of 16.9. The data of tobacco consumption in this study coordinated strongly with a traditional survey on the consumption of tobacco in China, indicating sewage epidemiology with NH4-N equivalent population estimation may provide a suitable and useful tool for tobacco use monitoring.