Population size is crucial when estimating population-normalized drug consumption (PNDC) from wastewater-based drug epidemiology (WBDE). Three conceptually different population estimates can be used: de jure (common census, residence), de facto (all persons within a sewer catchment), and chemical loads (contributors to the sampled wastewater). De facto and chemical loads will be the same where all households contribute to a central sewer system without wastewater loss. This study explored the feasibility of determining a de facto population and its effect on estimating PNDC in an urban community over an extended period. Drugs and other chemicals were analyzed in 311 daily composite wastewater samples. The daily estimated de facto population (using chemical loads) was on average 32% higher than the de jure population. Consequently, using the latter would systemically overestimate PNDC by 22%. However, the relative day-to-day pattern of drug consumption was similar regardless of the type of normalization as daily illicit drug loads appeared to vary substantially more than the population. Using chemical loads population, we objectively quantified the total methodological uncertainty of PNDC and reduced it by a factor of 2. Our study illustrated the potential benefits of using chemical loads population for obtaining more robust PNDC data in WBDE.

Wastewater analysis, or wastewater-based epidemiology, has become a common tool to monitor trends of illicit drug consumption around the world. In this study, we examined trends in cocaine, 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine consumption by measuring their residues in wastewater from two wastewater treatment plants in Australia (specifically, an urban and a rural catchment, both in South East Queensland) between 2009 and 2015. With direct injection of the samples, target analytes were identified and quantified using liquid chromatography-mass spectrometry. Cocaine and MDMA residues and metabolites were mainly quantifiable in the urban catchment while methamphetamine residues were consistently detected in both urban and rural catchments. There was no consistent trend in the population normalised mass loads observed for cocaine and MDMA at the urban site between 2009 and 2015. In contrast, there was a five-fold increase in methamphetamine consumption over this period in this catchment. For methamphetamine consumption, the rural area showed a very similar trend as the urban catchment starting at a lower baseline. The observed increase in per capita loads of methamphetamine via wastewater analysis over the past six years in South East Queensland provides objective evidence for increased methamphetamine consumption in the Australian population while the use of other illicit stimulants remained relatively stable.

Pyrrolizidine alkaloids (PAs) are a diverse group of plant secondary metabolites with known varied toxicity. Consumption of 1,2-unsaturated PAs has been linked to acute and chronic liver damage, carcinogenicity and death, in livestock and humans, making their presence in food of concern to food regulators in Australia and internationally. In this survey, honey samples sourced from markets and shops in Queensland (Australia), were analysed by high-resolution Orbitrap UHPLC-MS/MS for 30 common PAs. Relationships between the occurrence of pyrrolizidine alkaloids and the botanical origin of the honey are essential as pyrrolizidine alkaloid contamination at up to 3300 ng/g were detected. In this study, the predominant alkaloids detected were isomeric PAs, lycopsamine, indicine and intermedine, exhibiting identical MS/MS spectra, along with lesser amounts of each of their N-oxides. Crucially, chromatographic UHPLC conditions were optimised by operation at low temperature (5 • C) to resolve these key isomeric PAs. Such separation of these isomers by UHPLC, enabled the relative proportions of these PAs present in honey to be compared to alkaloid levels in suspect source plants. Overall plant pyrrolizidine alkaloid profiles were compared to those found in honey samples to help identify the most important plants responsible for honey contamination. The native Australian vines of Parsonsia spp. are proposed as a likely contributor to high levels of lycopsamine in many of the honeys surveyed. Botanical origin information such as this, gained via low temperature chromatographic resolution of isomeric PAs, will be very valuable in identifying region of origin for honey samples. Key Contribution:This study demonstrated the occurrence of indicine and the isomeric lycopsamine as predominant pyrrolizidine alkaloids in Australian honey; and established a HRAM LC-MS/MS method that chromatographically separated these stereoisomers; enabling the investigation of botanical origin of honey contamination by the respective alkaloids.

Blue heliotrope (Heliotropium amplexicaule) is an invasive environmental weed that is widely naturalized in eastern Australia and has been implicated as a source of pyrrolizidine alkaloid (PA) poisoning in livestock. Less welldocumented is the potential of such carcinogenic alkaloids to contaminate honey from bees foraging on this plant species. In this study, the PA profile of H. amplexicaule plant material, determined by HRAM LC-MS/MS, revealed the presence of nine PAs and PA-N-oxides, including several PAs and PA-N-oxides of the indicine class, which have not previously been reported. The predominant alkaloid, indicine, represents 84% of the reduced PA content, with minor alkaloids identified as intermedine and the newly reported helioamplexine, constituting 7 and 9%, respectively. NMR analysis confirmed the identity of helioamplexine as a previously unreported indicine homologue. This is the first report of the isolation of intermedine, helioamplexine, and 3′-O-angelylindicine from H. amplexicaule. Also described is the identification of N-chloromethyl analogues of the major alkaloids as isolation-derived artifacts from reactions with dichloromethane. Analysis of regional-market honey samples revealed a number of honey samples with PA profiles analogous to that seen in H. amplexicaule, with measured PA contents of up to 2.0 μg of PAs per gram of honey. These results confirm the need for honey producers to be aware of H. amplexicaule as a potential PA source, most particularly in products where honey is sourced from a single location.

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Recent media reports claimed that a large proportion of honey sold in Australian supermarkets was adulterated, although no specific details of the nature of the adulteration were made public.To investigate the basis of these claims, the current study was conducted with samples of Australian honey obtained from supermarkets or direct from beekeepers/small scale producers.Without evidence to the contrary, it was assumed that the beekeeper honey would be unadulterated. Analysis of these samples using the official AOAC method (998.12) found that a proportion of both supermarket and beekeeper honeys had apparent C4 sugar contents greater than 7% and this proportion was similar to the failure rates reported by popular media. The AOAC test measures the difference in carbon isotopic composition between the honey (mostly sugar) and protein precipitated from the honey and relies on the assumption that nectar (the source of sugar) comes from the same plant species as pollen (the source of protein). Initial results found that most Australian honey failed the AOAC test because the protein was depleted in 13 C with respect to the sugar although the sugar was in the normal range (δ 13 C more negative than -24‰). A series of experiments investigated the nature of the precipitate obtained from Australian honey and found the weight of precipitate was greater than the amount of protein measured in the honey and the precipitate contained non-protein material. Based on these findings a modification to the AOAC method is proposed whereby samples, after addition of sodium tungstate and acid, are left unheated for 48 hours for a floc to form. This procedure produced a precipitate that was mostly comprised of protein. It is also recommended that the acceptance criteria for Australian honey is increased from 1‰ to 2‰ difference between the carbon isotopic composition of the honey and the precipitate. Using the modified method and acceptance criteria the failure rate for Australian honey was no more than 8%, compared to 37% using the official method.

Honey is a widely available natural sweetener containing sugars, and small quantities ofvitamins and minerals, proteins, amino acids and fatty acids. Owing to its nutritious components,commercial honeys are sold in bulk blends or as trendy and premium products. Meanwhile, honeybees are considered as environmental monitors and have the potential to transfer environmentalcontaminants, if present, to honey. In high density urban and industrial environments polycyclicaromatic hydrocarbons (PAHs) and heavy metals can be prevalent, whilst pesticides and mineraland trace elements are ubiquitous. Honey hives are traditionally located in rural and forested areas,but there is a growing trend to locate hives in urban areas. This project has investigated the presenceof environmental contaminants in honey samples from high density urban, peri-urban as well asrural areas. Australian honey samples (n = 211) were purchased between 2016 and 2018, including52 honeys claiming to be of urban origin purchased online. Stingless bee honeys (n = 36) fromQueensland and Malaysia were compared. Processed samples were analysed by UHPLC-MS/MS(herbicides), GC-MS/MS (pesticides and PAHs) and ICP-MS and ICP-OES (elemental analyses). Theresults showed low or negligible pesticide, herbicide, and PAH contamination, and that these lowresults were similar regardless of urban or rural origins. Wide variations of essential trace element(Fe, Zn, Cu, Mo, Co, Mn, Cr) and mineral levels (K, Na, P, Mg, Ca) were found in honey products,which are a good dietary source of K and Zn. Relatively low levels of toxic heavy metals were foundin honeys.

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