Deciphering the Biosynthesis and Physiological Function of 5-Methylated Pyrazinones Produced by Myxobacteria.

Myxobacteria are a prolific source of secondary metabolites with sheer chemical complexity, intriguing biosynthetic enzymology, and diverse biological activities. In this study, we report the discovery, biosynthesis, biomimetic total synthesis, physiological function, structure-activity relationship, and self-resistance mechanism of the 5-methylated pyrazinone coralinone from a myxobacterium Corallococcus exiguus SDU70. A single NRPS/PKS gene corA was genetically and biochemically demonstrated to orchestrate coralinone, wherein the integral PKS part is responsible for installing the 5-methyl group. Intriguingly, coralinone exacerbated cellular aggregation of myxobacteria grown in liquid cultures by enhancing the secretion of extracellular matrix, and the 5-methylation is indispensable for the alleged activity. We provided an evolutionary landscape of the corA-associated biosynthetic gene clusters (BGCs) distributed in the myxobacterial realm, revealing the divergent evolution for the diversity-oriented biosynthesis of 5-alkyated pyrazinones. This phylogenetic contextualization provoked us to identify corB located in the proximity of corA as a self-resistance gene. CorB was experimentally verified to be a protease that hydrolyzes extracellular proteins to antagonize the agglutination-inducing effect of coralinone. Overall, we anticipate these findings will provide new insights into the chemical ecology of myxobacteria and lay foundations for the maximal excavation of these largely underexplored resources.

[Source Apportionment of Morphine in Wastewater].

It is a new approach to identify legal or illegal use of morphine through information on municipal wastewater. However, the sources of morphine in wastewater are complex, and distinguishing the contribution of different sources has become a key issue. A total of 262 influent samples from 61 representative wastewater treatment plants in a typical city were collected from October 2022 to March 2023. The concentrations of morphine, codeine, thebaine, papaverine, noscapine, and monoacetylmorphine were analyzed in wastewater and poppy straws. Combined with the proportion of alkaloids in poppy straws, the source analysis of alkaloids in wastewater was analyzed using the ratio method and positive matrix factorization model (PMF). Only five alkaloids were detected in wastewater, and monoacetylmorphine, a metabolite of heroin, was not detected. The concentrations of morphine and codeine were significantly higher than those of noscapine, papaverine, and thebaine. By constructing the ratios of codeine/(morphine + codeine) and noscapine/(noscapine + codeine), the source of poppy straw could be qualitatively distinguished. The PMF results showed that three sources of morphine for medical use, poppy straw, and codeine contributed 44.9%, 43.7%, and 9.4%, respectively. The different sources varied in these months due to the COVID-19 and influenza A outbreaks, in which the use of drugs containing poppy straws and codeine was the main source, whereas the use of morphine analgesics remained relatively stable. Inventory analysis further demonstrated the reliability of the source contributions from the PMF model, and morphine was not abused in this city.

Workflow to facilitate the detection of new psychoactive substances and drugs of abuse in influent urban wastewater.

The complexity around the dynamic markets for new psychoactive substances (NPS) forces researchers to develop and apply innovative analytical strategies to detect and identify them in influent urban wastewater. In this work a comprehensive suspect screening workflow following liquid chromatography - high resolution mass spectrometry analysis was established utilising the open-source InSpectra data processing platform and the HighResNPS library. In total, 278 urban influent wastewater samples from 47 sites in 16 countries were collected to investigate the presence of NPS and other drugs of abuse. A total of 50 compounds were detected in samples from at least one site. Most compounds found were prescription drugs such as gabapentin (detection frequency 79%), codeine (40%) and pregabalin (15%). However, cocaine was the most found illicit drug (83%), in all countries where samples were collected apart from the Republic of Korea and China. Eight NPS were also identified with this protocol: 3-methylmethcathinone 11%), eutylone (6%), etizolam (2%), 3-chloromethcathinone (4%), mitragynine (6%), phenibut (2%), 25I-NBOH (2%) and trimethoxyamphetamine (2%). The latter three have not previously been reported in municipal wastewater samples. The workflow employed allowed the prioritisation of features to be further investigated, reducing processing time and gaining in confidence in their identification.

A collector-generator cell for in-situ detection of electrochemically produced H2.

A collector-generator (C-G) cell used for the in-situ detection of H2 generated through electrochemical catalysis was described. The cell was mainly assembled with two fluorine-doped tin oxide (FTO) electrodes deposited with Pt nanoparticles, the magnitude of the current generated by the electrocatalytic oxidation of H2 at the collector was used for quantitative analysis of H2 generated at the generator. When the generator potential was set at -0.5 âˆ¼ -0.6 V vs. Ag/AgCl and the collector potential at 0.4 ∼ 0.5 V vs. Ag/AgCl, the total Faradaic efficiency of the C-G cell could stabilize about 70%, the detection limit was about 45 µmol/L and the sensitivity was about 1 mA/55 µmol L-1. This dual working electrode technology could provide a convenient and rapid method for H2 determination and evaluate the performance of H2 generation catalysts that assembled on the semiconductor thin-films.

Development and validation of an in situ high-resolution technique for measuring antibiotics in sediments.

Important biogeochemical processes occur in sediments at fine scales. Sampling techniques capable of yielding information with high resolution are therefore needed to investigate chemical distributions and fluxes and to elucidate key processes affecting chemical fates. In this study, a high-resolution diffusive gradients in thin-films (DGT) technique was systematically developed and tested in a controlled sediment system to measure organic contaminants, antibiotics, for the first time. The DGT probe was used to resolve compound distributions at the mm scale. It also reflected the fluxes from the sediment pore-water and remobilization from the solid phase, providing more dynamic information. Through the fine scale detection, a reduction of re-supply was observed over time, which was concentration and location dependent. Compared to the Rhizon sampling method, antibiotic concentrations obtained by DGT probes were less than the pore-water concentrations, as DGT measures the labile fraction of the compounds. The DGT probe was also tested on an intact sediment core sampled from a lake in China and used to measure the distribution of labile antibiotics with depth in the core at the mm scale. ENVIRONMENTAL IMPLICATION: The abuse of antibiotics and widespread of their residues influences the ecosystem, induces the generation of super-bacteria, and finally poses threat to human health. Sediments adsorbs pollutants from the aquatic environment, while may also release them back to the environment. We systematically developed DGT probe approach for measuring antibiotics in sediment in situ in high resolving power, it provides information at fine scale to help us investigate biogeochemical processes take place in sediment and sediment-water interface.

Recent advances in discovery and biosynthesis of natural products from myxobacteria: an overview from 2017 to 2023.

Covering: 2017.01 to 2023.11Natural products biosynthesized by myxobacteria are appealing due to their sophisticated chemical skeletons, remarkable biological activities, and intriguing biosynthetic enzymology. This review aims to systematically summarize the advances in the discovery methods, new structures, and bioactivities of myxobacterial NPs reported in the period of 2017-2023. In addition, the peculiar biosynthetic pathways of several structural families are also highlighted.

Comparing the photodegradation of typical antibiotics in ice and in water: Degradation kinetics, mechanisms, and effects of dissolved substances.

New antibiotic contaminants have been detected in both surface waters and natural ice across cold regions. However, few studies have revealed distinctions between their ice and aqueous photochemistry. In this study, the photodegradation and effects of the main dissolved substances on the photolytic kinetics were investigated for sulfonamides (SAs) and fluoroquinolones (FQs) in ice/water under simulated sunlight. The results showed that the photolysis of sulfamethizole (SMT), sulfachloropyridazine (SCP), enrofloxacin (ENR) and difloxacin (DIF) in ice/water followed the pseudo-first-order kinetics with their quantum yields ranging from 4.93 × 10-3 to 11.15 × 10-2. The individual antibiotics experienced disparate photodegradation rates in ice and in water. This divergence was attributed to the concentration-enhancing effect and the solvent cage effect that occurred in the freezing process. Moreover, the main constituents (Cl-, HASS, NO3- and Fe(III)) exhibited varying degrees of promotion or inhibition on the photodegradation of SAs and FQs in the two phases (p < 0.05), and these effects were dependent on the individual antibiotics and the matrix. Extrapolation of the laboratory data to the field conditions provided a reasonable estimate of environmental photolytic half-lives (t1/2,E) during midsummer and midwinter in cold regions. The estimated t1/2,E values ranged from 0.02 h for ENR to 14 h for SCP, which depended on the reaction phases, latitudes and seasons. These results revealed the similarities and differences between the ice and aqueous photochemistry of antibiotics, which is important for the accurate assessment of the fate and risk of these new pollutants in cold environments.

From Synthesis to Mechanisms: In-Depth Exploration of the Dual-Atom Catalytic Mechanisms Toward Oxygen Electrocatalysis.

Dual-atom catalysts (DACs) hold a higher metal atom loading and provide greater flexibility in terms of the structural characteristics of their active sites in comparison to single-atom catalysts. Consequently, DACs hold great promise for achieving improved catalytic performance. This article aims to provide a focused overview of the latest advancements in DACs, covering their synthesis and mechanisms in reversible oxygen electrocatalysis, which plays a key role in sustainable energy conversion and storage technologies. The discussion starts by highlighting the structures of DACs and the differences in diatomic coordination induced by various substrates. Subsequently, the state-of-the-art fabrication strategies of DACs for oxygen electrocatalysis are discussed from several different perspectives. It particularly highlights the challenges of increasing the diatomic loading capacity. More importantly, the main focus of this overview is to investigate the correlation between the configuration and activity in DACs in order to gain a deeper understanding of their active roles in oxygen electrocatalysis. This will be achieved through density functional theory calculations and sophisticated in situ characterization technologies. The aim is to provide guidelines for optimizing and upgrading DACs in oxygen electrocatalysis. Additionally, the overview discusses the current challenges and future prospects in this rapidly evolving area of research.

A dye sensitized photosynthesis cell for stable water oxidation mediated by photo-generated bromine.

The critical step of a dye-sensitized photoelectrochemical cell (DSPEC) is water oxidation. The photoanodes suffer rapid surface carrier recombination due to slow water oxidation kinetics and this results in low solar conversion efficiency and poor stability. Here, we design a novel sensitization-oxidation photo-anode, where the [Ru(4,4'-PO3H2-bpy)(bpy)2]2+ (RuP2+, bpy = 2,2'-bipyridine) chromophore and [Ru(bda)(pic)2] (pic = picoline, bda = 2,2'-bipyridine-6,6'-dicarboxylate) water oxidation catalyst (WOC) are immobilized separately on the surface of a mesoporous semiconductor thin film. The electrode creates a mobile and strong Br3- oxidant generated by the dye-sensitized electrode, which initiates water oxidation at a remote catalytic site. Under 100 mW cm-2 illumination in pH 4.65 in 0.1 M acetate buffer solution, the photoanode could perform a stable photoelectrochemical water oxidation for 6 hours with a photocurrent density of around 0.25 mA cm-2. This dual electrode could open a new door by utilizing an electron transfer mediator to design highly stable photoelectrochemical cells.

Taste traces: Capsaicin and sweeteners as anthropogenic markers in municipal wastewater.

Dietary-derived substances possess significant potential as anthropogenic markers owing to the large consumption and different intake habit. To investigate and evaluate such markers, wastewater samples from 35 wastewater treatment plants across 29 Chinese cities were collected to analyze artificial sweeteners (acesulfame and cyclamate) and natural spicy compounds (capsaicin and dihydrocapsaicin). Acesulfame (mean: 14.6 µg/L), cyclamate (mean: 24.3 µg/L), and capsaicin (mean: 101 ng/L) can be further investigated as anthropogenic markers due to their high detection frequency at high concentrations. Spatial use patterns revealed that acesulfame (5.31 g/d/1000 inhabitants (inh)) and cyclamate (8.16 g/d/1000 inh) use in northern China notably surpassed that in southern China (1.79 g/d/1000 inh and 3.23 g/d/1000 inh, p < 0.05). Conversely, chili pepper use was significantly higher (p < 0.05) in southern China (6702 g/d/1000 inh) than in northern China (2751 g/d/1000 inh), signifying a preference for sweetness in the northern regions and a predilection for spiciness in the southern regions. The total annual use of acesulfame (1842 t), cyclamate (3110 t), and chili (18.4 million tonnes) in China was estimated by this study, which was close to the national statistical production. In addition, sweetener use was negatively associated with the elderly population ratio, suggesting that the elderly population might not consume sweet foods. This study reveals the dietary sources of anthropogenic markers, highlighting the need for further research on the environmental implications of such markers.

Surveillance of COVID-19 and influenza A(H1N1) prevalence in China via medicine-based wastewater biomarkers.

The simultaneous monitoring of individual or multiple diseases can be achieved by selecting therapeutic medicines used to treat the primary symptoms of the condition as biomarkers in wastewater. This study proposes a novel approach to monitor the prevalence of COVID-19 and influenza A (H1N1) by selecting nine medicines to serve as biomarkers, including three antipyretics, three antivirals, and three cough suppressants. To verify our approach, wastewater samples were collected from seventeen urban and five rural wastewater treatment plants (WWTPs) in a Chinese city over a period of one year. The use of antipyretics increased notably during the COVID-19 pandemic, while the consumption of antivirals for influenza A (H1N1) rose in the post-COVID-19 pandemic period, indicating a minor spike in the occurrence of influenza A (H1N1) after the COVID-19 pandemic. Fever is a significant symptom of COVID-19 and can serve as a reliable indicator of disease prevalence. Our research found that the prevalence of COVID-19 in urban areas was significantly higher (at 78.5 %, 95 % CI: 73.4 % - 83.9 %) than in rural areas (with a prevalence of 48.1 %, 95 % CI: 42.4 % - 53.8 %). The prevalence of COVID-19 in urban areas in this study was consistent with the data reported by the Chinese center for Disease Control and Prevention (82.4 %). Continuous monitoring of WWTPs in urban areas with fluctuating populations and complex demographics can provide early disease warning. Our results demonstrate the feasibility of evaluating community disease prevalence by selecting major therapeutic medicines as biomarkers in wastewater.

Dummy molecularly imprinted polymers-agarose gel mixed matrix membrane for extraction of amphetamine-type stimulants in wastewater and urine.

Dummy molecularly imprinted polymers (DMIPs) with high selectivity for amphetamine-type stimulants (ATSs) were synthesized using synephrine molecule as a dummy template. The polymers were irregularly massive with a specific surface area of 330 m2g-1. Adsorption experiments found that the imprinting factors for five ATSs (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, and 3,4-methylenedioxy-N-ethylamphetamine) were 2.3∼3.7. The DMIPs-agarose gel mixed matrix membranes (MMMs) were further prepared by incorporating DMIPs in the agarose matrix. MMMs were used to extract five ATSs from wastewater and urine samples. Extraction conditions such as membrane matrix, sample pH, dissolved organic matter content, extraction time, and elution reagent were optimized. Under optimal conditions, the developed MMMs-HPLC-MS/MS method exhibited low limits of detection (0.1∼3.0ng L-1), satisfactory recoveries (91.7∼100%), and good repeatability (RSD<7%, n=3). It was then successfully applied to ATSs analysis in wastewater and urine samples. Recoveries of ATSs in spiked wastewater and urine were 82.0∼98.4% and 82.3∼95.7%, respectively. Moreover, compared with other methods, the present method possessed the advantages of high quantitative ability, suitable for typical environmental conditions, and low application cost. The above results suggested that the developed MMMs-HPLC-MS/MS method could be used as a feasible strategy to extract and determine trace ATSs in wastewater and urine samples.

Three years of wastewater surveillance for new psychoactive substances from 16 countries.

The proliferation of new psychoactive substances (NPS) over recent years has made their surveillance complex. The analysis of raw municipal influent wastewater can allow a broader insight into community consumption patterns of NPS. This study examines data from an international wastewater surveillance program that collected and analysed influent wastewater samples from up to 47 sites in 16 countries between 2019 and 2022. Influent wastewater samples were collected over the New Year period and analysed using validated liquid chromatography - mass spectrometry methods. Over the three years, a total of 18 NPS were found in at least one site. Synthetic cathinones were the most found class followed by phenethylamines and designer benzodiazepines. Furthermore, two ketamine analogues, one plant based NPS (mitragynine) and methiopropamine were also quantified across the three years. This work demonstrates that NPS are used across different continents and countries with the use of some more evident in particular regions. For example, mitragynine has highest mass loads in sites in the United States, while eutylone and 3-methylmethcathinone increased considerably in New Zealand and in several European countries, respectively. Moreover, 2F-deschloroketamine, an analogue of ketamine, has emerged more recently and could be quantified in several sites, including one in China, where it is considered as one of the drugs of most concern. Finally, some NPS were detected in specific regions during the initial sampling campaigns and spread to additional sites by the third campaign. Hence, wastewater surveillance can provide an insight into temporal and spatial trends of NPS use.

Enrichment Characteristics of Hazardous Trace Elements in Feed Coal and Coal Ash in Huaibei Area under Leaching.

This research focused on the hazardous elements in the main coal seam of Huaibei coalfield, China. Through collecting 20 feed coal samples from different coal seams of nine coal mines in the region, and combining with XRF, XRD, ICP-MS, and sequential chemical extraction, the mineral composition and the contents of major elements and HEs for feed coal were analyzed. Compared with previous research results, the enrichment characteristics of HEs in feed coal were revealed. The leaching behaviors of Se, Hg, and Pb in feed coal and coal ash under different leaching conditions were analyzed in depth by using a leaching device independently developed. Results showed that, compared with Chinese coals and World coals, the content of other elements, except Se, Sb, Hg, and Pb, in feed coal of Huaibei coalfield were at the "Normal" level, and no "Low" level elements were found; as the acidity of leaching solution decreased, the relative leaching rate of Se (LSe) was gradually increasing, while the LHg and LPb were not obvious; the LSe in feed coal and coal ash had a great relationship with the modes of occurrence of Se. The difference in the Hg content in the ion exchange state in feed coal may be an important reason for the difference in Hg leaching behavior. However, the content of Pb in feed coal had little influence on its leaching behavior. The modes of occurrence of Pb determined that the LPb in feed coal and coal ash was not high. The LSe increased with the increase in acidity of leaching solution and leaching time. The leaching time was the main influencing factor of the LHg and LPb.

Direct observation of highly effective hydrogen isotope separation at active metal sites by in situ DRIFT spectroscopy.

In situ diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy was developed for the first time to observe the hydrogen isotope separation behavior at active CuI sites within CuI-MFU-4l, and clear evidence of the preferential adsorption of D2 over H2 was directly captured. More importantly, our results show direct spectral proof to clarify the chemical affinity quantum sieving mechanism of hydrogen isotope separation within porous adsorbents.

Constructing a Myxobacterial Natural Product Database to Facilitate NMR-Based Metabolomics Bioprospecting of Myxobacteria.

Myxobacteria are fascinating prokaryotes featuring a potent capacity for producing a wealth of bioactive molecules with intricate chemical topology as well as intriguing enzymology, and thus it is critical to developing an efficient pipeline for bioprospecting. Herein, we construct the database MyxoDB, the first public compendium solely dedicated to myxobacteria, which enabled us to provide an overview of the structural diversity and taxonomic distribution of known myxobacterial natural products. Moreover, we demonstrated that the cutting-edge NMR-based metabolomics was effective to differentiate the biosynthetic priority of myxobacteria, whereby MyxoDB could greatly streamline the dereplication of multifarious known compounds and accordingly speed up the discovery of new compounds. This led to the rapid identification of a class of linear di-lipopeptides (archangimins) and a rare rearranged sterol (corasterol) that were endowed with unique chemical architectures and/or biosynthetic enzymology. We also showcased that NMR-based metabolomics, MyxoDB, and genomics can also work concertedly to accelerate the targeted discovery of a polyketidic compound pyxipyrrolone C. All in all, this study sets the stage for the discovery of many more novel natural products from underexplored myxobacterial resources.

Factors affecting the toxicity and oxidative stress of layered double hydroxide-based nanomaterials in freshwater algae.

Layered double hydroxide (LDH) nanomaterials are utilized extensively in numerous fields because of their distinctive structural properties. It is critical to understand the environmental behavior and toxicological effects of LDHs to address potential concerns caused by their release into the environment. In this work, the toxicological effects of two typical LDHs (Mg-Al-LDH and Zn-Al-LDH) on freshwater green algae (Scenedesmus obliquus) and the main affecting factors were examined. The Zn-Al-LDH exhibited a stronger growth inhibition toxicity than the Mg-Al-LDH in terms of median effect concentration. This toxicity difference was connected to the stability of particle dispersion in water and the metallic composition of LDHs. The contribution of the dissolved metal ions to the overall toxicity of the LDHs was lower than that of their particulate forms. Moreover, the joint toxic action of different dissolved metal ions in each LDH belonged to additive effects. The Mg-Al-LDH induced a stronger oxidative stress effect in algal cells than the Zn-Al-LDH, and mitochondrion was the main site of LDH-induced production of reactive oxygen species. Scanning electron microscope observation indicated that both LDHs caused severe damage to the algal cell surface. At environmentally relevant concentrations, the LDHs exhibited joint toxic actions with two co-occurring contaminants (oxytetracycline and nano-titanium dioxide) on S. obliquus in an additive manner mainly. These findings emphasize the impacts of the intrinsic nature of LDHs, the aqueous stability of LDHs, and other environmental contaminants on their ecotoxicological effects.

Dissolved Organic Matter and Lignin Modulate Aquatic Toxicity and Oxidative Stress Response Activated by Layered Double Hydroxides Nanomaterials.

Advanced nanomaterials can be released into the environment and can coexist with natural organic matter (NOM). However, evidence on the impacts of NOM on the environmental behavior and toxicity of advanced nanomaterials is still scarce. Here, we investigated the behavior and toxic effects of two layered double hydroxides (LDHs) nanomaterials with different metallic constituents (Mg-Al-LDH and Zn-Al-LDH) at relatively low exposure concentrations on a freshwater green alga (Chlorella pyrenoidosa) in the absence and presence of two types of NOM, namely dissolved organic matter (DOM) and dealkaline lignin (DL). The DOM or DL interaction with the LDHs at different mixture levels was shown to be an antagonistic effect on the growth inhibition toxicity to C. pyrenoidosa mainly. The estimation of the index of Integrated Biological Responses version 2 indicated that the joint interaction of the LDHs with DOM or DL occurred in the following order of frequency synergism > antagonism > additivity. Furthermore, the physicochemical characteristics of LDHs were crucial for illuminating the mechanism by which the DOM or DL modified the LDH-induced oxidative stress response. These findings highlighted the important role of NOM in the behavior and effect of LDHs as a representative of a new class of multifunctional nanomaterials in the freshwater environment.

Evaluation of eight psychoactive drugs used in Chinese cities by wastewater-based epidemiology.

With rapid economic development, an increasing number of people suffer from mental health diseases, which are gradually receiving the attention of society. However, basic data from surveys of mental disorders are limited. Composite influent samples were collected from 26 wastewater treatment plants in 23 major cities in China. The concentrations of the psychoactive drugs diphenhydramine, fluoxetine, doxepin, imipramine, sulpiride, zolpidem, carbamazepine, and flunitrazepam in the wastewater were determined. The detection frequency of diphenhydramine, sulpiride, and carbamazepine was close to 100 %, whereas that of the compounds was lower than 35 %. Carbamazepine had the highest mean consumption (31.1 mg/d/1000 people), followed by diphenhydramine (10.4 mg/d/1000 people) and sulpiride (11.3 mg/d/1000 people). Wastewater-based epidemiology (WBE) estimates of the average use of the three drugs were lower than those from the drug statistics data. Consumption of diphenhydramine in northern China was higher than that in southern China. A correlation analysis of psychotropic and illicit drugs revealed a correlation between sulpiride and heroin use, which may be related to the adverse effects of sulpiride treatment after heroin withdrawal. Psychotropic drug use is associated with both economic and social factors. We found associations between the use of the three drugs and age, occupation, and obesity, which are risk factors for mental disorders. The results showed that the monitoring of psychotropic drug using WBE has a certain reference value for public health care and for improving the understanding of mental disorders.

Refining the estimation of amphetamine consumption by wastewater-based epidemiology.

Consumption of amphetamine and methamphetamine, two common illicit drugs, has been monitored by wastewater-based epidemiology (WBE) in many countries over the past decade. There is potential for the estimated amount of amphetamine used to be skewed at locations where methamphetamine is also consumed, because amphetamine is also excreted to wastewater following methamphetamine consumption. The present study aims to review the available data in the literature to identify an average ratio of amphetamine/methamphetamine (AMP/METH) that is excreted to wastewater after methamphetamine consumption. This ratio could then be used to refine the estimation of amphetamine consumption in catchments where there is both amphetamine and methamphetamine use. Using data from more than 6000 wastewater samples from Australia where methamphetamine is the dominant illicit amphetamine-type substance on the market, we were able to subtract the contribution of legal sources of amphetamine contribution and obtain the median AMP/METH ratio in wastewater of 0.09. Using this value, the amphetamine derived from methamphetamine consumption can be calculated and subtracted from the total amphetamine mass loads in wastewater samples. Without considering the contribution of amphetamine from methamphetamine use, selected European catchments with comparable consumption of amphetamine and methamphetamine showed up to 83% overestimation of amphetamine use. For catchments with AMP/METH ratio greater than 1.00, the impact of amphetamine from methamphetamine would be negligible; for catchments with AMP/METH ratio in the range of 0.04-0.19, it will be difficult to accurately estimate amphetamine consumption.