Data Hidden in Sewage: Advanced Methods for Identification and Quantification of Synthetic Cannabinoids in Urban Wastewater

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Summary of manuscript and general comments:

 

In this manuscript, Kurzeja et al. reviewed studies that measured synthetic cannabinoids (SCs) in wastewater samples. SCs are psychoactive compounds with potential for abuse, and monitoring their levels in wastewater is important for epidemiological surveys. The manuscript fits the scope of the journal Molecules and provided a comprehensive survey of the field from 2013-2025, which coincides with the historical use of SCs. Overall, this will provide researchers an important reference on the topic. However, there are several areas that need improvement, especially how information is being presented in the tables and insights into the future. The following are suggestions for improving the manuscript.

 

Specific comments:

 

The first few paragaphs of the introduction discuss the variety of synthetic cannabinoids. Please add a figure that shows the different structural classes (e.g., JWH, AM, HU, WIN, etc.). A good source to check is a EUDA report (www.euda.europa.eu/topics/pods/synthetic-cannabinoids_en), although it is 8 years old and could be outdated.

 

Tables 1 and 2 are very detailed and dense, making them hard to read. Since the journal Molecules has diverse audience, more general information is of interest to the reader, not details like injection volume and composition of mobile phases. Therefore, please move Tables 1 and 2 into supplementary materials. In the main text, replace these tables with a single table that highlights the most important and interesting aspects from each publication like sampling location, dates, general detection method (e.g., triple quad MS/MS, HR-MS, QTOF-MS), number of samples, number of individual SCs identified, and general concentration ranges.

 

For challenges and perspectives section, please add discussion on biosensors and bioassays for potential detection of SCs (these are currently used for detecting drugs and pollutants in wastwater monitoring). Since the SCs bind strongly to cannabinoid receptors, cannabinoid receptor bioassays could detect unknown SCs or metabolites based upon their pharmacological activity, without knowing their structure. Some suggested references include dois: 10.1021/es505349s, 10.1016/j.chemosphere.2024.142255, 10.1021/cbe.4c00188, and 10.1021/acs.est.5c05367. These bioassays can be helpful in conjunction with LC-MS.

 

Line 118: Define “HLB” as hydrophilic-lipophilic balance.

 

Line 140: Define “PFP” as pentafluorophenyl.

 

Lines 80-85: Compare general concentration range of SCs (ng/L) to other illicit drugs and metabolites in wastewater (e.g., cocaine, THC, MDMA). This can put the topic into perspective for the reader.

 

Author Response

Reviewer #1

We sincerely thank the Reviewer for the time and effort devoted to the thorough evaluation of our manuscript and for the insightful and constructive comments provided. We agree with the vast majority of the suggestions and have revised the manuscript extensively in accordance with the Reviewer’s recommendations.

 

In this manuscript, Kurzeja et al. reviewed studies that measured synthetic cannabinoids (SCs) in wastewater samples. SCs are psychoactive compounds with potential for abuse, and monitoring their levels in wastewater is important for epidemiological surveys. The manuscript fits the scope of the journal Molecules and provided a comprehensive survey of the field from 2013-2025, which coincides with the historical use of SCs. Overall, this will provide researchers an important reference on the topic. However, there are several areas that need improvement, especially how information is being presented in the tables and insights into the future. The following are suggestions for improving the manuscript.

The first few paragraphs of the introduction discuss the variety of synthetic cannabinoids. Please add a figure that shows the different structural classes (e.g., JWH, AM, HU, WIN, etc.).

Reply and revision: Done accordingly. We have added Figure 1 with the different structural classes of synthetic cannabinoids.

 

Tables 1 and 2 are very detailed and dense, making them hard to read. Since the journal Molecules has diverse audience, more general information is of interest to the reader, not details like injection volume and composition of mobile phases. Therefore, please move Tables 1 and 2 into supplementary materials. In the main text, replace these tables with a single table that highlights the most important and interesting aspects from each publication like sampling location, dates, general detection method (e.g., triple quad MS/MS, HR-MS, QTOF-MS), number of samples, number of individual SCs identified, and general concentration ranges.

Reply and revision: We appreciate this comment. We agree that Table 1 is very detailed and vast. Therefore, we have replaced it with a simplified version in the revised version of the manuscript. It has a compact layout and takes up less space. The detailed version of Table 1 has been moved to supplementary materials. We have decided to keep Table 2 unchanged because it is not as extensive as Table 1. We believe that not only the number of individual SCs identified, but also their names, will be interesting to the readers of the article. Combining both tables would be problematic because they contain different references. Moreover, we intentionally maintained a clear separation between the methodological aspects (Table 1) and the results-related information (Table 2), as this distinction reflects the conceptual structure of the manuscript and facilitates reader comprehension.

 

For challenges and perspectives section, please add discussion on biosensors and bioassays for potential detection of SCs (these are currently used for detecting drugs and pollutants in wastwater monitoring). Since the SCs bind strongly to cannabinoid receptors, cannabinoid receptor bioassays could detect unknown SCs or metabolites based upon their pharmacological activity, without knowing their structure.

Reply and revision: We sincerely thank the Reviewer for this insightful suggestion. Following this comment, we have expanded the “Challenges and Perspectives” section to include a discussion on the potential application of biosensors and bioassays for the detection of synthetic cannabinoids in wastewater (cited below). In particular, cannabinoid receptor-based bioassays are highlighted as promising tools, as they can detect unknown SCs or metabolites based on their pharmacological activity, independent of chemical structure. This addition complements the discussion on advanced analytical methods and provides readers with a broader perspective on innovative approaches that could enhance SC monitoring and surveillance in complex wastewater matrices.

„Beyond conventional mass spectrometry based approaches, biosensors and bioassays are emerging as powerful complementary tools for the detection of illicit drugs in wastewater, offering rapid response, high sensitivity, and the potential for activity-based screening [56]. In particular, recent advances in nanomaterial-assisted bio-sensing have demonstrated the feasibility of detecting trace- and ultra-trace-level drug residues directly in complex wastewater matrices. A biosensor-based platform combining selective enrichment with signal amplification enabled the simultaneous detection of structurally diverse synthetic cannabinoids at ultra-low concentrations, addressing key analytical challenges associated with their low abundance, rapid degradation, and structural heterogeneity [23]. Comparable biosensor concepts have been successfully applied to other classes of illicit drugs in WBE [57,58,59]. These studies demonstrate that biosensors, particularly those exploiting molecular recognition elements such as aptamers or receptor-based interactions, hold substantial promise for future WBE research.”

 

Line 118: Define “HLB” as hydrophilic-lipophilic balance.

Reply and revision: Corrected accordingly.

 

Line 140: Define “PFP” as pentafluorophenyl.

Reply and revision: Corrected accordingly.

 

Reviewer 2 Report

Comments and Suggestions for Authors

In this work, the authors reviewed advanced methods of sample preparation coupled with analytical detection for identification and quantification of synthetic cannabinoids (SCs) in urban wastewater. I read with great interest. The review is generally well organized and related comments are highly appreciated. The work can provide important references for analytical detection of SCs and waste water investigation. Some revisions are required before it is possible acceptance by Molecules according to the following comments.

 

1. Figure 1. The left three words should be put sideways for easily reading.
2. Please add 2-3 figures to schematically show related advanced sample preparation procedures from typical references.
3. Several typical chemical structures of SCs can be schematically shown.
4. Line 45, why say “from China”?
5. Table 2, the qualified values of References [28], [23] and [19] are the same as that of Reference [22]? If yes, I think they should be vividly given.
6. Several recently related work can be cited to further strengthen the comprehensiveness. For example,

-About synthetic cannabinoid. Identification and Analytical Characterization of a Novel Synthetic Cannabinoid-Type Substance in Herbal Material in Europe, Molecules 2021, 26, 793; Classification of Mass Spectral Data to Assist in the Identification of Novel Synthetic Cannabinoids, Molecules 2024, 29, 4646.

-Analysis in water and wastewater. Molecularly imprinted polymers and porous organic frameworks based analytical methods for disinfection by-products in water and wastewater, Environmental Pollution, 2024, 356, 124249.

7. Please carefully check and revise the total manuscript for logical clarity and grammar accuracy. For instances,

-Title should be revised. I appreciate “From Sewage to Science” but still feel strange. -Line 179, “LODs” and “LOQs” should be added for replacement.

Author Response

Reviewer #2

We are very grateful to the Reviewer for the careful assessment of our work and for the valuable and constructive suggestions. The comments were highly helpful and have led to substantial improvements in the manuscript, which has been revised accordingly.

 

1. Figure 1. The left three words should be put sideways for easily reading.

Reply and revision: Done accordingly. We thank the Reviewer for this helpful suggestion. The orientation of the text in the figure has been corrected by placing the three leftmost words sideways to improve readability. Additionally, due to the inclusion of a new figure presenting the chemical structures of synthetic cannabinoids, the figure numbering has been updated accordingly, and the revised figure is now presented as Figure 2.

 

2. Please add 2-3 figures to schematically show related advanced sample preparation procedures from typical references.

Reply and revision: We thank the Reviewer for this valuable suggestion. Sample preparation procedures indeed play a crucial role in the analysis of synthetic cannabinoids in wastewater. As emphasized in the manuscript, solid-phase extraction (SPE) represents the most frequently applied and most relevant sample preparation technique in the reviewed studies and is therefore discussed in detail in the main text: „Solid-phase extraction (SPE) using mixed-mode sorbents such as Oasis MCX or HLB (hydrophilic-lipophilic balance) cartridges represented the most common sample preparation approach [14–19].”

To avoid redundancy and to maintain the focus of the manuscript on advanced analytical identification and quantification strategies rather than procedural repetition, schematic representations of sample preparation workflows were not included in the main text of the article. However, to address the Reviewer’s comment and to provide additional visual support for interested readers, a representative schematic figure (Figure S1) illustrating the advanced sample preparation procedure of SPE has been added to the revised version of the manuscript.

 

3. Several typical chemical structures of SCs can be schematically shown.

Reply and revision: Corrected accordingly. We have added Figure 1 with the different structural classes of synthetic cannabinoids.

 

4. Line 45, why say “from China”?

Reply and revision: We thank the Reviewer for pointing out this issue. The phrase “from China” was used to reflect the well-documented role of China as a major source of synthetic cannabinoids and their precursors supplying the European illicit drug market. We acknowledge that the original version of the manuscript did not include an explicit reference to support this statement. This has now been corrected by adding appropriate and relevant literature source to substantiate the claim, and the text has been revised accordingly.

Old version: „Most of these products are distributed to the European market from China.”

New version: „Most of these products are distributed to the European market from China [3].”

 

5. Table 2, the qualified values of References [28], [23] and [19] are the same as that of Reference [22]? If yes, I think they should be vividly given.

Reply and revision: We thank the Reviewer for carefully examining Table 2 and for raising this important point. The qualified values reported for Reference [22] were indeed determined in that study. In contrast, the corresponding parameters were not investigated in References [28], [23], and [19]. We acknowledge that the absence of hyphens in these rows may have led to confusion and an incorrect interpretation, suggesting that identical qualified values were reported across these references. We apologize for this oversight. The table has now been corrected by explicitly indicating missing data with hyphens, thereby clearly distinguishing between reported and non-reported values.

 

6. Several recently related work can be cited to further strengthen the comprehensiveness. For example,

- About synthetic cannabinoid. Identification and Analytical Characterization of a Novel Synthetic Cannabinoid-Type Substance in Herbal Material in Europe, Molecules 2021, 26, 793; Classification of Mass Spectral Data to Assist in the Identification of Novel Synthetic Cannabinoids, Molecules 2024, 29, 4646.

- Analysis in water and wastewater. Molecularly imprinted polymers and porous organic frameworks based analytical methods for disinfection by-products in water and wastewater, Environmental Pollution, 2024, 356, 124249.

Reply and revision: We thank the Reviewer for this valuable suggestion and for pointing out relevant recent literature that could further strengthen the comprehensiveness of the review. The article “Identification and Analytical Characterization of a Novel Synthetic Cannabinoid-Type Substance in Herbal Material in Europe” (Molecules 2021, 26, 793) has now been incorporated into the Introduction section to support the paragraph describing the forms and marketing of synthetic cannabinoids, including their availability as herbal smoking mixtures and alternative formulations. The inclusion of this relatively recent study strengthens the timeliness and contextual relevance of the manuscript. The indicated paragraph is quoted below:

„Products of SCs are marketed as herbal smoking mixtures in attractive packages with appealing names such as „K2” (in North America), „Spice” (in Europe), or „Youcatan”, „Chill”, or „Black Mamba”. SCs may also be available in alternative formulations, including powders, tablets, and e-liquids for vaping. They are sold mostly over the Internet or on the street market [2–4].”

In addition, inspired by the second suggested reference „Analysis in water and wastewater. Molecularly imprinted polymers and porous organic frameworks based analytical methods for disinfection by-products in water and wastewater” (Environmental Pollution, 2024, 356, 124249), a new sentence has been added to the manuscript in section “Challenges and perspectives of analysis of SCs in wastewater” to broaden the discussion on future perspectives in synthetic cannabinoid research, particularly with respect to pre-analytical and data-driven approaches. This addition allowed us to expand the outlook section and provide a more comprehensive and forward-looking perspective on emerging trends in SC analysis. The new sentences are quoted below:

„Advanced functional materials such as molecularly imprinted polymers and metal–organic frameworks have been shown to enhance selective extraction and pre-concentration of trace contaminants in complex aqueous matrices, addressing challenges of low abundance and matrix interference [55]. Their further development and integration into WBE workflows are expected to play an increasingly important role in the reliable monitoring of ultra-trace level substances, including SCs.”

We believe that these additions have improved the completeness and scientific depth of the manuscript.

 

7. Please carefully check and revise the total manuscript for logical clarity and grammar accuracy. For instances,

- Title should be revised. I appreciate “From Sewage to Science” but still feel strange.

Reply and revision: In line with the Reviewer’s suggestion, we have reformulated the title to adopt a more conventional and journal-appropriate phrasing, while preserving the original conceptual link between sewage-derived information and advanced analytical science. The revised title now reads: “Data Hidden in Sewage: Advanced Methods for Identification and Quantification of Synthetic Cannabinoids in Urban Wastewater”. While “Data Hidden in Sewage” serves as an engaging introductory phrase, the second part of the title explicitly defines the advanced analytical methods applied for the identification and quantification of synthetic cannabinoids in urban wastewater. We believe that this structure is consistent with current trends in high-impact analytical and environmental journals, where informative yet engaging titles are encouraged. The title was deliberately formulated to combine scientific precision with accessibility to a broader interdisciplinary audience. The revised title continues to clearly define the scientific scope of the work, explicitly highlighting the applied methodologies, target compounds, and environmental matrix, thereby maintaining both academic style and logical clarity.

- Line 179, “LODs” and “LOQs” should be added for replacement.

Reply and revision: Corrected accordingly.

Reviewer 3 Report

Comments and Suggestions for Authors

I reviewed the manuscript entitled From Sewage to Science: Advanced Methods for Identification and Quantification of Synthetic Cannabinoids in Urban Wastewater.

 

I agree to accept this manuscript after major revision. 

 

1)  the European Union Drug Agency (EUDA) has been monitoring 247 synthetic cannabinoids,  since the abbreviation has already been defined earlier, it can be used directly here. This sentence should be revised as: the European Union Drug Agency (EUDA) has been monitoring 247 SCs,

2) 2. Methodology of the literature search, the initial letter of each substantive word in a level-one heading must be capitalized. Please review and correct similar issues throughout the entire text.

3) Sampling strategies involved mainly 24-hour composite, 24-hour should change to 24 h; International unit symbols, rather than words, should be used. Please check and correct similar issues throughout the entire text.

4) The samples were typically stored at -20 °C, -20 °C should change to -20°C. A space should be inserted between numbers and units, except for the degree Celsius symbol (°C) and the percentage sign (%).Please check and correct similar issues throughout the entire text.

5) Magnetic solid-phase extraction (MSPE) and supramolecular solvent (SUPRAS) extraction were also described as a new approach to wastewater sample preparation for SC analysis [22,23].  Magnetic solid-phase extraction (MSPE), supramolecular solvent (SUPRAS), abbreviations should only be used when the term appears three or more times; otherwise, they are unnecessary, as excessive abbreviations may confuse readers. Please check and correct similar issues throughout the entire text.

6) C18 phase columns are preferred, 18 should be subscripted. Please check and correct similar issues throughout the entire text.

7) Abstract Section: The time range of the literature search was not mentioned in the abstract and should be added.

8) Why was the literature search timeframe limited to 2013–September 2025? Does this lead to the omission of the latest studies published after September 2025? Please conduct a new literature search to verify if any omissions exist.

9) The paper states that the majority of the studies are short-term monitoring, with only two classified as long-term. Could the specific definition of "long-term" (e.g., >1 month) be clarified?

10) Table 3 shows significant variations in the stability of different SCs. Can it be summarized which structural types of SCs tend to be more stable?

11) There is a considerable variation in the LOD/LOQ values reported across different studies (e.g., 0.1 ng/L vs. 6.3 ng/L). Can the influencing factors (such as instrumentation and sample pretreatment methods) be analyzed?

12) Table 2 indicates that China reports the highest diversity of detected SCs. Is this related to cultural, regulatory, or analytical methodological differences?

13) Do all studies employ 24-hour composite sampling? Have there been studies comparing the impact of different sampling strategies on the results?

14) Different studies use different storage temperatures (e.g., -20°C, -80°C). Does this affect the stability and comparability of SCs?

15) For structurally similar SC isomers, is a PFP column consistently superior to a C18 column?

16) Population normalization: Have all the studies applied the “mass load per 1000 inhabitants per day” correction? Are there studies that did not perform this normalization?

17) There are significant variations in validation parameters across different studies. Could a set of minimum validation standards applicable to WBE studies be proposed?

18) This review should clearly distinguish between a "methodology review" and a "monitoring results review," and balance the two to enhance the systematic nature and practical utility of the paper.

19) The paper mentions "scarcity of metabolism studies," "lack of standardization," and "insufficient long-term monitoring." Can a clear "future research roadmap" be proposed based on these points?

20) Ethical and privacy considerations: As a population-level monitoring tool, should WBE be discussed in greater depth within the paper regarding its ethical boundaries, data anonymization, and privacy protection?

21)The author also needs to check and revise all the references according to the requirements of the journal.

22) This study reviews current literature on the detection and quantification of synthetic cannabinoids (SCs) in untreated urban wastewater via wastewater-based epidemiology (WBE). Analytical methodologies are compared, and global occurrence data are summarized, reflecting the evolving illicit SC market. Key challenges across experimental stages—including metabolite identification, analyte stability, and extraction efficiency—are examined. Optimized analytical workflows are needed to improve SC monitoring. Future research should prioritize metabolic studies, enhanced stability assessments, and refined extraction techniques tailored to complex wastewater matrices.

Author Response

Reviewer #3

We would like to express our sincere appreciation to the Reviewer for the considerable time and effort invested in the detailed review of our manuscript. The insightful comments and suggestions were extremely valuable and have significantly contributed to improving the quality and clarity of the revised version.

 

1)  the European Union Drug Agency (EUDA) has been monitoring 247 synthetic cannabinoids,  since the abbreviation has already been defined earlier, it can be used directly here. This sentence should be revised as: the European Union Drug Agency (EUDA) has been monitoring 247 SCs,

Reply and revision: Corrected accordingly.

 

2) Methodology of the literature search, the initial letter of each substantive word in a level-one heading must be capitalized. Please review and correct similar issues throughout the entire text.

Reply and revision: Corrected accordingly.

 

3) Sampling strategies involved mainly 24-hour composite, 24-hour should change to 24 h; International unit symbols, rather than words, should be used. Please check and correct similar issues throughout the entire text.

Reply and revision: Corrected accordingly.

 

4) The samples were typically stored at -20 °C, -20 °C should change to -20°C. A space should be inserted between numbers and units, except for the degree Celsius symbol (°C) and the percentage sign (%). Please check and correct similar issues throughout the entire text.

Reply and revision: Corrected accordingly.

 

5) Magnetic solid-phase extraction (MSPE) and supramolecular solvent (SUPRAS) extraction were also described as a new approach to wastewater sample preparation for SC analysis [22,23]. Magnetic solid-phase extraction (MSPE), supramolecular solvent (SUPRAS), abbreviations should only be used when the term appears three or more times; otherwise, they are unnecessary, as excessive abbreviations may confuse readers. Please check and correct similar issues throughout the entire text.

Reply and revision: We thank the Reviewer for this comment regarding the use of abbreviations. Magnetic solid-phase extraction (MSPE) is mentioned three times and supramolecular solvent (SUPRAS) extraction is mentioned six times throughout the manuscript, meeting the commonly accepted threshold for the introduction of abbreviations. Moreover, given the length and technical complexity of these terms, the use of standardized abbreviations significantly improves readability and avoids unnecessary repetition, particularly in method-focused sections. Nevertheless, we have carefully rechecked the entire manuscript to ensure that abbreviations are introduced only when appropriate and are consistently defined at first occurrence. No unnecessary or excessive abbreviations remain in the revised version.

 

6) C18 phase columns are preferred, 18 should be subscripted. Please check and correct similar issues throughout the entire text.

Reply and revision: Corrected accordingly.

 

7) Abstract Section: The time range of the literature search was not mentioned in the abstract and should be added.

Reply and revision: We thank the Reviewer for this helpful comment. The time range of the literature search has now been explicitly added to the Abstract section in the revised version of the manuscript.

Old version: „In this review article, we summarized the available literature on the detection and quantification of SCs in raw wastewater samples.”

New version: „In this review article, we summarized the available literature on the detection and quantification of SCs in raw wastewater samples published between 2013 and 2025.”

 

8) Why was the literature search timeframe limited to 2013–September 2025? Does this lead to the omission of the latest studies published after September 2025? Please conduct a new literature search to verify if any omissions exist.

Reply and revision: We thank the Reviewer for raising this point. The rationale for limiting the literature search to the period between 2013 and September 2025 is stated in the manuscript: „We limited our search to articles published between 2013 and 2025, because the first study in this field was published in 2013”. The year 2013 marks the publication of the first study addressing the analysis of synthetic cannabinoids in wastewater, and thus represents the starting point of systematic research in this field. Accordingly, the literature search was initiated at this time to ensure comprehensive coverage from the emergence of the topic to the most recent studies available at the time of manuscript preparation. The upper limit (September 2025) reflects the final date of the primary literature search before submission. In response to the Reviewer’s comment, the literature search was repeated in December 2025 to verify whether any relevant studies published after September 2025 had been omitted. This additional search did not identify any new publications meeting the inclusion criteria. Therefore, the selected timeframe captures the complete development and current state of the field without omitting any foundational or relevant work.

 

9) The paper states that the majority of the studies are short-term monitoring, with only two classified as long-term. Could the specific definition of "long-term" (e.g., >1 month) be clarified?

Reply and revision: We sincerely thank the Reviewer for this comment. In the context of this review, studies were classified as long-term when wastewater sampling was conducted over a period exceeding one month. The definition of long-term monitoring is provided in the revised version of the manuscript: “It is also important to note that the majority of studies conducted to date have been limited to short-term monitoring periods. Only two investigations could be classified as long-term, as they encompassed wastewater samples collected over more than one month [10,29].”

 

10) Table 3 shows significant variations in the stability of different SCs. Can it be summarized which structural types of SCs tend to be more stable?

Reply and revision: We sincerely thank the Reviewer for this insightful comment and for carefully examining Table 3. The relationship between structural features of synthetic cannabinoids and their stability has been addressed in the manuscript. In particular, the text summarizes which structural types of SCs tend to exhibit higher stability, based on their core scaffolds and functional group composition. For clarity, this information is explicitly discussed in the corresponding section of the revised manuscript, where the observed stability trends are linked to structural characteristics: „First generation of SCs, indole- and indazole-based SCs, including JWH-type compounds (e.g., JWH-018, JWH-210, JWH-398) and AM-series analogues (e.g., AM-2201, AM-2233), exhibited significant degradation under ambient and refrigerated conditions [15]. They degraded over time, even when frozen at -20°C, with responses decreasing up to 67% after a week [15], likely due to adsorption to suspended solids or hydrolysis of ester or amide linkages inherent to many SC structures. Studies focusing on newer fluorinated indazole- and indole-derived SCs demonstrated improved short-term stability, particularly when samples were stored at 4°C or below and treated with chemical preservatives (Table 3) [9,16,37]”.

 

11) There is a considerable variation in the LOD/LOQ values reported across different studies (e.g., 0.1 ng/L vs. 6.3 ng/L). Can the influencing factors (such as instrumentation and sample pretreatment methods) be analyzed?

Reply and revision: We thank the Reviewer for raising this issue. The major analytical challenge associated with the analysis of synthetic cannabinoids in wastewater is related to their low concentrations. Therefore, the LOD/LOQ values are crucial parameters determining the frequency of their detection in sewage. The factors influencing LOD/LOQ values can be analyzed in order to obtain methods enabling detection and quantification of synthetic cannabinoids the lowest concentrations. Inspired by the Reviewer’s comment, we have added the following sentence in the revised version of the manuscript (in the Challenges and perspectives of analysis of SCs in wastewater section): “To achieve the lowest LOD and LOQ values, the influencing factors, such as sample pretreatment methods and instrumentation, should be analyzed.”

 

12) Table 2 indicates that China reports the highest diversity of detected SCs. Is this related to cultural, regulatory, or analytical methodological differences?

Reply and revision: We thank the Reviewer for this thoughtful question. China is a geographically vast and highly populated country, and in this respect is more comparable to the European continent than to individual European states. Consequently, wastewater-based studies conducted across different regions of China may capture a broader spectrum of synthetic cannabinoids, reflecting regional heterogeneity in production, distribution, and market introduction. Moreover, China has historically been a major site of synthesis and early emergence of novel synthetic cannabinoids and their precursors, which has stimulated intensified analytical surveillance and methodological development. Differences in regulatory frameworks and early-warning monitoring systems may further contribute to increased analytical coverage. Overall, the observed diversity is therefore more likely to reflect differences in analytical focus, geographic scale, and monitoring intensity rather than true differences in consumption patterns.

 

13) Do all studies employ 24-hour composite sampling? Have there been studies comparing the impact of different sampling strategies on the results?

Reply and revision: Fluctuations in influent wastewater composition over 24 h periods may affect representativeness. Therefore, 24 h composite sampling seems to be the best strategy in monitoring the consumption of illicit drugs. This strategy was applied in most of the reviewed studies - 12 out of 16. To the best of our knowledge, no studies have systematically compared the impact of different sampling strategies on the detection and quantification of synthetic cannabinoids in wastewater. Inspired by the Reviewer’s comment, we have added a sentence to the revised manuscript in the Challenges and perspectives of analysis of SCs in wastewater  section highlighting this knowledge gap and emphasizing the need for future comparative studies addressing sampling strategy effects: „Future studies should address the impact of different sampling strategies on the qualitative and quantitative data related to SC occurrence in wastewater.”

 

14) Different studies use different storage temperatures (e.g., -20°C, -80°C). Does this affect the stability and comparability of SCs?

Reply and revision: We sincerely thank the Reviewer for this important and technically relevant question. Differences in storage temperature can indeed influence the stability of synthetic cannabinoids, particularly for compounds prone to hydrolysis, oxidation, or other degradation processes. Lower storage temperatures (e.g., -80°C) generally provide enhanced preservation of analyte integrity compared to -20°C, especially during prolonged storage periods. However, the wide structural diversity of synthetic cannabinoids poses an additional challenge. Compounds with different core scaffolds and functional groups may respond differently to storage conditions, making direct quantitative comparisons across studies difficult. In the context of the reviewed literature, most stability assessments were conducted under controlled conditions within individual investigations, and comparisons were primarily made within, rather than across, studies. Consequently, while variations in storage temperature may affect absolute stability values, they are unlikely to substantially compromise the overall qualitative trends discussed in this review. Where relevant, such methodological differences are acknowledged in the manuscript and represent an inherent limitation when comparing data across heterogeneous studies.

 

15) For structurally similar SC isomers, is a PFP column consistently superior to a C18 column?

Reply and revision: The separation of structurally similar synthetic cannabinoid (SC) isomers presents a significant analytical challenge due to their closely related physicochemical properties. Pentafluorophenyl (PFP) stationary phases provide unique selectivity compared to conventional C₁₈ columns, arising from π–π interactions and the high polarizability of fluorine atoms, which can enhance the resolution of positional and geometric isomers. PFP phases have been reported to improve retention and separation for compounds containing aromatic fragments, heteroatoms, or halogen substituents, characteristics commonly observed in synthetic cannabinoids. In contrast, C₁₈ reversed-phase columns predominantly exploit hydrophobic interactions, which may be insufficient to resolve SC isomers with minimal differences in hydrophobicity. The rationale and discussion of these chromatographic properties are also addressed in the manuscript, providing readers with additional context for the selection of stationary phases. Nonetheless, from a theoretical standpoint, PFP columns are expected to provide enhanced selectivity for SC isomers that differ in aromatic substitution patterns or positional isomerism, whereas C₁₈ phases remain suitable for general hydrophobic separations. The choice of stationary phase should therefore be guided by the structural characteristics of the target analytes, as well as the goals of the chromatographic method, including resolution requirements, analysis time, and compatibility with detection techniques.

 

16) Population normalization: Have all the studies applied the “mass load per 1000 inhabitants per day” correction? Are there studies that did not perform this normalization?

Reply and revision: We thank the Reviewer for this comment. Table 2 summarizes the main findings of studies aimed at the detection and quantification of synthetic cannabinoids and their metabolites in wastewater. Not all cited studies applied the population normalization for the determined concentration values of synthetic cannabinoids (daily average wastewater flow rate and the population served by the given wastewater treatment plant should be considered). Only two studies cited in Table 2 reported values expressed as mg/day/1000 inhabitants. Moreover, some studies were limited to only qualitative analysis and showed the results of the performed screening of synthetic cannabinoids in wastewater samples (as indicated in Table 2). This demonstrates that determination of synthetic cannabinoids in wastewater is still in its infancy and further, intensified are needed to obtain more detailed picture of synthetic cannabinoids use in communities.

We have added the following sentences to the revised version of the manuscript (in the chapter 4. Occurrence of SCs in wastewater): “It should be noted that not all studies cited in Table 2 applied the population normalization for the determined concentration values of SCs. Only two studies per-formed by Reid et al. [15] and O'Rourke and Bikram Subedi [17] reported values ex-pressed as mg/day/1000 inhabitants. Moreover, some studies were limited to only qualitative analysis and showed the results of the performed screening of SCs in wastewater samples (Table 2). This demonstrates that determination of SCs in wastewater is still in its infancy and further, intensified investigations are needed to obtain more detailed picture of SC use in communities.”

 

17) There are significant variations in validation parameters across different studies. Could a set of minimum validation standards applicable to WBE studies be proposed?

Reply and revision: We sincerely thank the Reviewer for this comment. It is indeed challenging to propose a uniform set of validation parameters, as different studies have applied diverse experimental designs, analytical conditions, and target compounds, resulting in significant variability in reported validation metrics. Based on our review and current best practices in wastewater-based epidemiology, a set of minimum validation standards that could serve as a “gold standard” for SC monitoring can include assessment of linearity across the expected concentration range, determination of limits of detection (LOD) and limits of quantification (LOQ), evaluation of accuracy and precision, recovery studies and matrix effect. We believe that highlighting these parameters in the discussion provides readers with guidance on best practices and helps frame future methodological improvements in WBE studies.

 

18) This review should clearly distinguish between a "methodology review" and a "monitoring results review," and balance the two to enhance the systematic nature and practical utility of the paper.

Reply and revision: We thank the Reviewer for this insightful comment. The structure of the manuscript was carefully developed based on three comprehensive tables that clearly delineate the main sections: (I) Analytical methodologies for the analysis of SCs in wastewater, (II) Occurrence of SCs in wastewater, and (III) Challenges and perspectives of analysis of SCs in wastewater. This organization was intended to provide readers with a clear and logical progression from methodological approaches, through global occurrence data, to the critical discussion of current limitations and future directions. By structuring the review around these tables, we aimed to highlight both the state-of-the-art analytical strategies and the ongoing challenges, while facilitating easy reference for researchers and stakeholders in the field.

 

19) The paper mentions "scarcity of metabolism studies," "lack of standardization," and "insufficient long-term monitoring." Can a clear "future research roadmap" be proposed based on these points?

Reply and revision: The aspects discussed are already visualized in Figure 2. However, we acknowledge that the figure may not have been sufficiently clear in its original form. To address this, Figure 2 has been modified to improve readability and clarity, ensuring that these key research gaps and priorities are more easily interpretable for the reader.

 

20) Ethical and privacy considerations: As a population-level monitoring tool, should WBE be discussed in greater depth within the paper regarding its ethical boundaries, data anonymization, and privacy protection?

Reply and revision: We thank the Reviewer for highlighting this important aspect. We fully agree that ethical and privacy considerations are crucial in wastewater-based epidemiology. In response, we have expanded the “Challenges and perspectives of analysis of SCs in wastewater” section (cited below) to address ethical boundaries, data anonymization, and privacy protection. We are grateful for this suggestion, as it has allowed us to strengthen the discussion and provide a more comprehensive perspective.

„WBE provides a powerful population-level monitoring tool for assessing drug use and environmental contaminants. However, the collection and analysis of wastewater data raise important ethical and privacy considerations that warrant careful discussion. Although WBE does not target individuals, aggregated data may still carry implications for community-level privacy, stigmatization, and public perception. To address these issues, ethical boundaries, data anonymization procedures, and safeguards for privacy protection should be clearly defined and implemented. Several recent studies have highlighted these concerns and proposed frameworks for ethical WBE practices [60–63]. For example, guidelines emphasize the importance of structured ethical review processes, transparency in data reporting, and careful communication of findings to avoid unintended social consequences. By incorporating these ethical considerations, WBE studies can maximize public health benefits while minimizing potential risks related to privacy and social harm.”

 

21)The author also needs to check and revise all the references according to the requirements of the journal.

Reply and revision: We have carefully double-checked all references and revised them to ensure they fully comply with the journal’s formatting requirements. We appreciate this comment as it allowed us to further improve the accuracy and consistency of the manuscript.

 

22) This study reviews current literature on the detection and quantification of synthetic cannabinoids (SCs) in untreated urban wastewater via wastewater-based epidemiology (WBE). Analytical methodologies are compared, and global occurrence data are summarized, reflecting the evolving illicit SC market. Key challenges across experimental stages—including metabolite identification, analyte stability, and extraction efficiency—are examined. Optimized analytical workflows are needed to improve SC monitoring. Future research should prioritize metabolic studies, enhanced stability assessments, and refined extraction techniques tailored to complex wastewater matrices.

Reply and revision: We very much appreciate these comments on our manuscript. We sincerely thank the Reviewer for this comprehensive and insightful summary of the study’s scope and key findings. We greatly appreciate the thoughtful recognition of the challenges addressed in the manuscript and the constructive suggestions for future research. The comments underscore the relevance and timeliness of our review and will help guide further improvements in the field of synthetic cannabinoid monitoring via wastewater-based epidemiology.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The author replied my questions and concerns, so I agree to receive it in the current form.