Prevalence and emergence of steroidal and non-steroidal anabolic agents in the Australian community measured through wastewater analysis

Abstract

Develop and validate a method for the detection of performance- and image-enhancing drugs (PIEDs) in wastewater. Investigate prevalence of steroidal and non-steroidal anabolic agents in around 45% of the Australian population during Census week in August 2021. Explore changes and patterns in use over a 13-year period (2009–2021) and emergence of non-steroidal anabolic agents, e.g., selective androgen receptor modulators (SARMs), within one Australian wastewater treatment plant (WWTP) catchment. Performance- and image-enhancing drugs include, inter alia, steroidal (anabolic-androgenic steroids) and non-steroidal anabolic agents [e.g., SARMs, peroxisome proliferator-activated receptor delta (PPAR-δ) agonists, growth hormone secretagogues]. Despite known serious negative health side effects, these substances are misused by athletes and non-athletes to boost physical performance as well as appearance. Due to their misuse, often at concentrations significantly higher than for medical purposes, easy access through the internet, and lack of quality controls, PIED use has become a public health problem. The extent of this issue is difficult to quantify and is currently monitored through the combination of multiple data sources such as surveys, seizures, and anti-doping. To fully understand the prevalence of these substances, especially among the general population, a broader monitoring tool is needed to complement current sources of data. In the past two decades, wastewater-based epidemiology has been developed to investigate exposure to, use, and prevalence of chemicals within entire populations and has potential to be a useful additional tool for PIED monitoring. A solid-phase extraction (SPE) liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the detection and quantification of around 50 anabolic agents and metabolites in wastewater. This method was applied to wastewater samples collected in August 2021 from 51 WWTPs across Australia, covering roughly 11.6 million people. This method was also used for temporal analysis of anabolic agents in wastewater from one catchment collected from 2009 to 2021. Limits of quantification (LOQs) were, with few exceptions, in the low ng/L with steroids and their metabolites generally having higher LOQs than the non-steroidal anabolic agents. Spatial wastewater analysis showed the use of multiple types of PIEDs within the Australian population in August 2021. Results of temporal samples indicated no clear trends for detected steroidal anabolic agents. However, they showed that the first non-steroidal anabolic agent was detected as early as 2011, with others following in later years. After their first detection, all non-steroidal anabolic agent concentrations increased almost linearly until 2021, often with a peak in concentration in 2018. LOQs are suitable for the detection of trace amounts of PIEDs in wastewater, especially for SARMs and other non-steroidal anabolic agents. As the method is semi-quantitative, concentrations only give an indication of anabolic agent levels, however, these estimates are likely conservative. The analysis of wastewater across 51 Australian WWTPs revealed widespread use of multiple types of steroidal and non-steroidal anabolic agents. The steady increase of concentrations of non-steroidal anabolic agents in the temporal wastewater samples demonstrates the growing popularity of these substances within the general community. A suitable SPE LC-MS/MS method was developed for the analysis of steroidal and non-steroidal anabolic agents in wastewater influent and was successfully applied to spatial and temporal wastewater samples. Many non-steroidal anabolic agents were detected in wastewater for the first time. Spatial analysis showed the prevalence of anabolic agents and temporal analysis revealed the first detections and subsequent emergence of non-steroidal anabolic agents within an Australian WWTP catchment. Results demonstrated that wastewater analysis is a useful complementary tool to further our understanding of PIED use within the general population and that wastewater data may be useful to public health and PIED researchers.