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[This research was funded by the Dutch Research Council (NWO) through the Rubicon programme (project number 019.211EN.023, “Synthetic chemicals as a cause of biodiversity loss”). The authors would like to thank the following institutions for providing biological data:

o            NLWKN – Lower Saxon State Department for Waterway, Coastal and Nature Conservation, for data collected within the context of the WFD (Water Framework Directive) monitoring.

o            LUBW – Landesanstalt für Umwelt Baden-Württemberg.

o            Slovenian Environment Agency.

o            Austrian Federal Ministry for Agriculture, Forestry, Regions and Water Management (BML, Department I/2 National and International Water Management) and the Offices of the Provincial Governments for data from the Ordinance on the Monitoring of the Quality of Water Bodies (Federal Law Gazette II No. 479/2006).

o            LHW – Landesbetrieb für Hochwasserschutz und Wasserwirtschaft Sachsen-Anhalt (State Office for Flood Protection and Water Management Saxony-Anhalt)

o            LfU Bayern – Bayerisches Landesamt für Umwelt (Bavarian Environment Agency).

o            FGG Elbe – Flussgebietsgemeinschaft Elbe (Elbe River Basin Community) (www.elbe-datenportal.de).

o            ICPDR – International Commission for the Protection of the Danube River.

o            JDS3 – Joint Danube Survey 3.

o            ICPR – International Commission for the Protection of the Rhine.

o            Ministry of Economy and Sustainable Development, Croatia.

o            Landesamt für Natur, Umwelt und Verbraucherschutz (LANUV).

o            Basque Water Agency, Spain

o            Gipuzkoa Provincial Government, Spain

o            Povodí Labe, state enterprise, Czech Republic

o            Spanish Ministry for the Ecological Transition and the Demographic Challenge

o            Environment Agency (England)

Since 16 April 2025, Miguel CANEDO ARGUELLES has been seconded to the ERC Executive agency. The views expressed in this paper are purely those of the author. They do not necessarily reflect the views or official positions of the European Commission, the ERC Executive Agency or the ERC Scientific Council.]

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[I.R. Pit

Synthetic chemicals as

a cause of biodiversity loss

(file no. 019.211EN.023)

-The grant is subject both to the Terms and Conditions of Rubicon Research Grants, round Rubicon 2021-1 S, to the NWO Regulation on Granting and The Dutch General Administrative Law Act.

-http://www.nwo.nl/en

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: No

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Pit and colleagues show the heterogeneity (and homogeneity) of available European macroinvertebrate monitoring data and present the sentiments of freshwater data curators and providers toward potentially standardizing European sampling. Given the increasing importance of big data, this is substantial achievement, especially since they present FAIR data for 28 European countries! This will certainly be useful for future meta-analyses and syntheses. However, being a curator of data from Lithuania and Poland, I am surprised that these data were not included, especially since several colleagues on the manuscript are aware that such data from these countries exist. Specifically, Lithuania has macroinvertebrate data for 462 river sites collected between 2010 – 2016 (25.76% have a single year of sampling, 45.24% have 2 years sampling, 17.75% have three years, with 52 sites having five years or more of sampling), and for 253 lake sites collected between 2013 – 2016 (90.51% only have a single year of data). In Poland, macroinvertebrate data are available from 710 river sites (87.18% only have a single year of sampling, 11.55% have two years of sampling) and 26 lake sites (100% have data for a single year) for the years 2010 – 2016. Neither the Lithuanian nor the Polish data were included in Welti et al. (2024) – Scientific data, but subsets of the Lithuanian data have been included in other manuscripts, namely Sinclair et al. (2024) – Nature Ecology and Evolution and Baker et al. (2025) – Hydrobiologia. I do, however, understand and acknowledge the potential conflict of interest here. While it would allow for greater spatial representatitiveness of the current manuscipt, I do not explicitly advocate for including these data, I merely wanted to bring it to the Authors’ attention. With that said, the data inclusion criteria are unclearly stipulated within the presented manuscript and I recommend the authors spend a few sentences describing them in more detail. For example: Did the data have to be sampled in each year between 2010 and 2015? Were there a minimum number of sites required per country? Note: This comment alos pertains to a comment further down highlighting discrepensicies in data availability between countries.

The authors open the floor to an interesting topic, namely the potential of a “strict EU-wide protocol” for invertebrate sampling. I must admit that I too have my reservations about its feasibility due to, amongst other things, the impact it might have on the homogeneity of long-term monitoring programs at the national level (there were already observed shifts in German monitoring around 2004) but it is nonetheless an important conversation to have. Moreover, the authors advocate for collecting species level data where possible but offer no suggestions on how to improve traditional (i.e., non-DNA-based) identification, as it remains time consuming and difficult, especially for mega-diverse groups like Diptera. DNA-based identification methods can be used as a complement to traditional identification, but I also believe that the development of an EU-defined standardized operational list is just as pertinent and should be added to the discussion. The Water Framework Directive seeks harmonized biomonitoring approaches across Europe but lacks explicit taxonomic requirements for biological quality elements. This gap allows member states to independently define macroinvertebrate operational taxonomic units, creating taxonomic inconsistencies across national and regional scales. While the authors acknowledge that freshwaterecology.info is a possible tool, one I use regularly myself, it still contains incongruencies and is optimized for western and central European fauna.

Other comments (Note: The lack of line and page numbers makes pinpointing exact locations within the manuscript difficult):

Material and Methods, section 2.1: It is unclear why the WISE database was used during the initial phase of data aggregation, as it only provides the final EQR value. Please elaborate as to why it was used if the goal was to aggregate raw data.

Material and Methods, section 3.2: You state that no data are available from Slovakia, but later you present data from Slovakia. Please correct accordingly.

Results, section 3.3: These data are highly dependent on the number of institutions in any given country that have access to it and that conduct macroinvertebrate surveys. The more institutions, the greater the likelihood of more sampling points. It is therefore unclear how many institutions in each country were selected, and whether comparability can truly be achieved. For instance, very few data points are available for Latvia, with these points not accurately reflecting the monitoring network across the entire country. The data exists. Therefore, statements like “For many other countries (e.g., Portugal, Switzerland, and Slovakia), only a limited amount of stream orders is represented and smaller streams (orders 1 and 2) tend to be neglected” are not well supported since the data exists, but the authors just do not have it (e.g., see recent paper by Šamulková et al. 2025 – Metabarcoding & Metagenomics).

Material and Methods, section 2.3: Another avenue for exploring the data may be to examine the time of year in which countries collect their samples (e.g., sampling season). This would provide an additional layer for the discussion around Strahler order and land use, as different countries conduct their sampling at different times of year. I assume that there will also be discrepancies here that could limit comparisons between countries.

All things considered, I do recommend this manuscript for publication in PLOS ONE after the minor points are addressed. Further, to mitigate any potential conflicts of interest, I waive my anonymity.

Sincerely,

Nathan Jay Baker

Reviewer #2: The manuscript PONE-D-25-54149 titled “Availability and representativeness of freshwater macroinvertebrate data to enable Europe-wide assessment of biodiversity trends” studied macroinvertebrate diversity trends in freshwater ecosystem over a period of 15 years. The authors stressed the importance of harmonizing data standardization in relation to taxonomic resolution and sampling efforts that will facilitate biodiversity conservation efforts.

The manuscript addresses a true challenge in research - data harmonization and has worked to provide suggestions regarding the same. It is a herculean task and the authors have done a commendable job indeed.

However, as pointed by the authors in the manuscript, not everyone ma be willing to share data, and even ion they do so, may not provide permission to make it accessible to the public. As mentioned, close to almost half the data used in this work was acquired by direct contact with the contributors. The question of harmonization comes into the circle only when the data is available.

Moreover, there are often barriers - institutional or otherwise including, but not limited to funding agencies - regarding data distribution. These are some real world challenges that require answering.

On the varying taxonomic level, the detail to which data is often collected may not be properly reflected - for example, only families may be mentioned and not genus or species. The main problem in harmonizing these type of raw data lies in the inconsistency of resolution.

Sampling effort and protocol, seasonality are some of the facets to consider while collecting the data. It affects the data density (as reported in table 2) and can create inaccuracies regarding the reliability.

These are a few concerns that came to mind when reading the manuscript, and may be beneficial to add/justify them.

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what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes:  Nathan Jay Baker

Reviewer #2: No

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Availability and representativeness of freshwater macroinvertebrate data to enable Europe-wide assessment of biodiversity trends

PONE-D-25-54149R1

Dear Dr. Pit,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Mattias Gaglio, PhD

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: No

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Pit and colleagues have comprehensively addressed my comments and concerns. I have no further comments and recommend that this work be accepted in its current form. Thank you for giving me the opportunity to review this timely piece of work.

Reviewer #2: All the comments from my side have been properly addressed. I have no objection to the publication of this manuscript.

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what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review?  For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: Yes:  Nathan Jay Baker

Reviewer #2: No

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