Investigating the Appearance of New Psychoactive Substances in South 1 Australia using Wastewater and Forensic Data

New psychoactive substances (NPS) have increased in use and popularity worldwide. Wastewater analysis has been successfully applied to evaluate illicit drugs use within a population. However, for NPS, such an approach may be limited due to low doses of NPS combined with their ever-changing composition and usage. The dynamic nature of the NPS market means use may be opportunistic, infrequent, and with few users. Hence, the use of complementary information sources is recommended to improve the knowledge on NPS consumption. The aim of this study was to investigate the changing landscape of NPS use on a community scale by combining wastewater analysis and forensic toxicology. Forensic analysis provided specific information on NPS prevalence in post-mortem blood samples in Adelaide, South Australia over five years, while wastewater analysis showed community use over the same period. A qualitative liquid chromatography--high resolution mass spectrometry method was initially used to screen the wastewater samples. A total of 24 NPS were found: 6 in wastewater only, 13 in forensic post-mortem toxicology samples only, and 5 in both. As these results showed the presence of NPS, a targeted method was subsequently employed to quantify levels of these NPS in wastewater. Temporal trends were found in wastewater with distinct tendencies for synthetic cathinones visible over the period studied.


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A total of 85 NPS reference standards in the form of mixed standard solutions in methanol 97 were made available for use by Forensic Science SA (FSSA) for the screening method (Table   98   S1). The mixed solutions were supplied in accordance with the appropriate licencing 99 conditions at both the FSSA and the University of South Australia sites. Butylone, 100 mephedrone, methylenedioxypyrovalerone (MDPV), methedrone, methylone, naphyrone and 101 N-ethylcathinone were analysed quantitatively as in our previous work 19  conditioned with methanol (6 mL) and sodium acetate buffer (20 mM pH 5, 6 mL). The 159 cartridges were successively washed with sodium acetate buffer (6 mL), 0.1 M acetic acid (2 160 mL) and methanol (6 mL wastewater due to the lack of a reference standard at the laboratory. In total, 18 NPS were 229 found in the forensic samples, 11 in wastewater and five in both.

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All wastewater samples were screened by applying a three-step workflow using MasterView.

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The first step assumed that no standard was available and contained just the exact mass of the There are some distinct patterns in NPS use visible in Table 1. Methcathinone was detected 283 at a relatively constant concentration in all samples. Alpha-PVP and mephedrone were only 284 detected in the 2015 samples, which mirrors the screening data (Figures 1a and b). Ethylone synthetic cathinone, pentylone, was detected intermittently, but at a higher concentration in 295 more recent samples. It will be of interest to see whether these cathinones will continue to be 296 seen in future samples. the death when it is still detectable in blood, even if the compound was not the cause of death.

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When a NPS is taken by a sub-population, it may thus not appear in any forensic toxicology 304 post mortems.

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WWA is a complementary tool and can be considered as a diluted pooled urine sample, 306 allowing the measurement and estimation of a drug that is consumed in a community. In this 307 study, it was used to indicate the presence of 11 NPS. Due to dilution effects and low 308 excretion rates, infrequently used NPS may not be found in wastewater. In addition, the 309 metabolism of some NPS remains unknown and therefore a method targeting the parent drug 310 may not find the drug in wastewater. As pharmacokinetic information becomes available, this 311 limitation may be overcome. In addition to detecting community use of a NPS, WWA can show the scale of use. Since there will always only be partial overlap between the two 313 datasets, this work emphasises the complementarity of these sources.

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A temporal investigation into NPS use in South Australia from 2012 -2017 has been done 316 utilising both post mortem forensic and wastewater data. A total of 24 NPS were found: six in 317 wastewater only, 13 in forensic post mortem toxicology samples only and five in both.

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Synthetic cathinones were most prevalent, with an interesting temporal pattern of use.

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Methylone was used in the early years, followed by ethylone, while in more recent samples 320 butylone and pentylone were found. The study showed that by combining forensic and