Innovative approaches in aquatic monitoring: a comparative study between eDNA analysis and morphological techniques in a river ecosystem

Innovative approaches in aquatic monitoring: a comparative study between eDNA analysis and morphological techniques in a river ecosystem | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Innovative approaches in aquatic monitoring: a comparative study between eDNA analysis and morphological techniques in a river ecosystem Florencia Lucila Zilli, Magdalena Licursi, María Virginia Parachú Marcó, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8787248/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Freshwater biomonitoring requires rapid and reliable diagnostic tools to minimize ecological impacts. This study compares morphological identification with environmental DNA (eDNA) metabarcoding data to evaluate which method more efficiently assesses macroinvertebrate communities across contrasting land uses. Benthic macroinvertebrates were collected in the Paraná River basin for morphological analysis, while water and biofilm samples were obtained for eDNA metabarcoding. A total of 125 amplicon sequence variants (ASVs) – high-resolution DNA sequence clusters used as taxonomic units - were detected, most of which were identified to the species or morphospecies level. The eDNA biofilm-derived recovered a higher number of ASVs than water samples, where Annelida, Arthropoda, and Mollusca are the most diverse groups. Diversity metric (richness and community composition) and ecological status classifications were generally comparable between morphological and molecular approaches. The primers employed demonstrated good taxonomic resolution, supporting their usefulness in assessing river quality and enabling the application of region-specific biotic indices. Although metabarcoding performed similarly to morphological methods in assessing diversity patterns and ecological status, 70% of the taxa identified morphologically were not detected molecularly. These limitations, particularly the under-detection of key benthic phyla, indicate that in tropical rivers, an integrative framework combining both methods yields the most accurate evaluation of biodiversity and environmental health. Biological sciences/Ecology Earth and environmental sciences/Ecology Earth and environmental sciences/Environmental sciences Biological sciences/Zoology Macroinvertebrates Freshwater Biomonitoring Land use Metabarcoding Anthropogenic impacts Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Rivers are vital resources for human society, supplying essential goods and services, yet they are deteriorating as a result of human activities, including urban expansion, agricultural runoff, industrial effluents, and hydrological alterations 1 . Macroinvertebrates are essential to river ecosystems, enhancing decomposition, material exchange, and water self-purification, and supporting vital processes like nutrient cycling and energy flow 2 . Their biodiversity is crucial for ecosystem structure and function, but recent declines in their richness and abundance, driven by water pollution, agricultural inputs such as fertilizers and insecticides, habitat loss from deforestation, hydrological alteration, and climate change, pose significant threats to these processes 3 . Biological monitoring is one of the most effective means of detecting the effects of point and diffuse pollution and alterations in watersheds 4 . In this regard, many biological indicators are based on macroinvertebrates. Their advantages as bioindicators include their ubiquity, abundance, diversity, and variable sensitivity to environmental disturbances 5 . Currently, macroinvertebrates have been widely adopted by countries around the world to indicate the ecological status of rivers 6 . In the La Plata Basin, the Intergovernmental Coordinating Committee established Strategic Action Programs 7 for the management of wetlands, their conservation, and the sustainable use of biodiversity, aimed at preventing and controlling environmental degradation processes, fragmentation, and loss of connectivity that can be exacerbated by climate change effects, to avoid the loss of goods and services due to environmental risks. However, to date, monitoring programs that integrate information from multiple aquatic communities remain scarce. In Brazil, monitoring has been conducted through the analysis of phytoplankton, zooplankton, and benthic macroinvertebrate communities 7 , while in Argentina, an integrated monitoring program that includes benthic macroinvertebrates, diatoms, and fish, along with water and sediment quality, is regionally restricted to the Matanza Riachuelo Basin 8 . Specifically, regarding benthic macroinvertebrates, in Argentina, there are numerous regional biomonitoring projects based on these organisms, with varying degrees of temporal regularity across different basins 3 . Nonetheless, most current projects for macroinvertebrate monitoring worldwide still rely on traditional morphological methods, which involve collecting specimens in the field, sorting them from sediments, and subsequently identifying them at the lowest practical level based on their morphological characteristics 9 . This process requires significant taxonomic expertise, especially for immature life stages (e.g., larvae and nymphs), where diagnostic morphological features are not fully developed, making species-level identification particularly challenging 10 . Additionally, the high diversity of macroinvertebrates, which includes numerous species from different phyla, demands expert knowledge for accurate species identification. This approach is time-consuming, costly, and resource-intensive; implementing efficient macroinvertebrate monitoring projects a significant challenge 9 . More recently, environmental DNA (eDNA) metabarcoding has been applied to monitoring biota in riverine systems, offering a potentially more time- and cost-efficient alternative to traditional morphological assessments (compiled by Poyntz-Wright et al. 9 ). Recent advances in DNA-based technology provide new opportunities to enhance the throughput, accuracy, and standardization of macroinvertebrate monitoring, as this method identifies species based on a standard fragment of their DNA (DNA barcode) rather than their morphological traits 11 . This approach is particularly relevant for taxonomic groups that are difficult or unfeasible to identify using routine protocols 12 . Metabarcoding studies of macroinvertebrate communities are typically conducted on DNA extracted from bulk samples of collected and sorted specimens, as well as directly from preservative ethanol, water, or sediments to detect freshwater macroinvertebrates. Besides, research has successfully assessed macroinvertebrate eDNA from biofilms, demonstrating that biofilms can trap DNA, leading to the temporary or permanent removal of DNA particles from the water column 13 . Although eDNA-based methodologies are becoming increasingly widespread, there is still insufficient evidence to directly apply the eDNA datasets to assess the river’s ecological status, particularly in tropical and subtropical river systems. These systems exhibit unique climatic and geomorphological characteristics compared to temperate regions, which necessitate further research to validate and adapt eDNA approaches for such a context. In this study, we used traditional morphological analyses and DNA-based technology to monitor aquatic environmental quality based on macroinvertebrates in the Paraná River floodplain channels and a tributary. To the best of our knowledge, this is the first study to comparatively assess the suitability of metabarcoding and classical morphological techniques for water quality bioindication based on macroinvertebrates in South America, particularly in the Paraná River basin. Besides, it provides a natural habitat for macroinvertebrates and other aquatic species 14 . Between 2019 and 2022, the La Plata Basin experienced moderate to extreme droughts, driven by the El Niño-South Oscillation (ENSO) and the Southern Annular Mode (SAM). Additionally, significant land-use changes, including deforestation and intensive agriculture, and the impacts of dams have shaped its three major sub-basins—Paraguay, Paraná, and Uruguay—over recent decades 15 . Our objectives were to assess the detection capacity of macroinvertebrate taxa using morphological features and metabarcoding from eDNA in different matrices (water and biofilm) in rivers with varying land uses located in the Paraná River basin and to compare and analyze the consistency between the eDNA-based metrics and the morphological-based metrics to assess the environmental quality of sites with different land use in the Middle Paraná Basin. Results Environmental parameters At the time of the study, the sites exhibited very low water flow, ranging from nearly lentic conditions with high transparency (A1 and A6) to sites with relatively higher flow and lower transparency (U6 and R1), as well as intermediate flow and transparency conditions (R5 and R6) (Table 1 ). Channel widths ranged from 13 to 48 meters. Table 1 Environmental parameters measured at each site during the study period in the Middle Paraná River. R1 R5 R6 A1 A6 U6 Relative water flow High Medium Medium Low (almost lentic) Low (almost lentic) High Channel width (m) 43.73 47.61 42.56 12.89 21.5 47 Transparency value (m) 0.15 0.18 0.19 0.26 0.6 0.105 Deph (m) 7.7 3.6 9.3 1.6 0.6 1.7 Temperature (°C) 19.1 22.8 20.7 12 14.7 16.1 Dissolved oxygen (mg/L) 6.71 5.79 5.31 7.03 8.73 7.98 Conductivity (µScm − 1 ) 1000 1002 1001 345 1754 195.3 pH 7.22 7.12 7.2 7.44 8.57 7.69 Granulometry of bottom sediments Silt-clayed in the banks and with fine sand in the center Sandy with a low proportion of silt and clay in the banks and sandy in the center Sandy with a low proportion of silt and clay in the banks and sandy in the center Silt-clayed Silt-clayed with gravels Sandy with a thin layer of silt and clay in the banks and sandy in the center BPOM quality Low content; mainly fine detritus, and macrophyte roots remain in the litoral zones Low content; mainly fine detritus, and macrophyte roots remain in the litoral zones Low content; mainly fine detritus, and macrophyte roots remain in the litoral zones Low content of fine detritus Relatively high fine detritus and vegetation remains With minimum detritus content in the center and coarse and fine remains of vegetation in the banks Macrophyte composition Paspalum spp and Pontederia crassipes Paspalum spp and Pontederia crassipes Paspalum spp and Pontederia crassipes . Less Azolla sp and Polygonum sp Without riverine or floating macrophytes Ludwigia peploides and Juncus sp Paspalum spp and Pontederia crassipes NO 3 --N (ug L − 1 ) 185.95 198.39 165.14 3.56 6755.15* 206.35 NO 2 ---N (ug L − 1 ) 5.91 4.49 3.88 0.52 543.23* 1.25 Water temperature was relatively higher at the floodplain sites compared to the streams. The sites had relatively well-oxygenated waters, with the highest oxygen levels observed at A1, A6, and U6, while conductivity was lowest at U6. pH values were approximately neutral, with the highest readings recorded at U6 and A6. The sites ranged from sandy to silt-clay and with low to high organic matter content, determined by granulometry and detritus quality; additionally, the floodplain sites featured stands of floating macrophytes and rooted grasses. Similar intermediate values were observed for all nitrogen forms, SRP, BOD 5 , and COD in R1, R5, and R6. All nitrogen forms peaked at A6, followed by U6 due to ammonium, with the lowest values recorded at A1. The SRP ranged from 200 to 300 across all sites, with the minimum observed at U6. A6 exhibited the highest BOD 5 , while A1 had the highest COD. Additionally, the BOD 5 /COD ratio indicated a high availability of biodegradable material only at A6. Based on the nitrogen forms, BOD 5 , and BOD 5 /COD ratio, the sites can be classified along a gradient from very good conditions (U6) through good conditions with low pollution (R1, R5, R6, and A1) to relatively polluted conditions (A6). The pollution observed at A1 is likely associated with the high COD, resulting from raw sewage discharges, primarily of municipal origin. eDNA metabarcoding The eDNA obtained extracts showed good quality and concentration (Table 2). On average, the concentration values were higher in water samples than in biofilm samples. Purity was similar between samples, as was observed in the 260/280 ratio (range between 1,6 and 1,95). Table 2 DNA extracts obtained quality and concentrations Sample Source Concentration (ng/ul) Absorbance (260/280) R1 Water 76.39 1.67 R5 Biofilm 19.8 1.75 R6 Water 72.76 1.6 A1 Water 62.41 1.61 A6 Water 127.22 1.86 A6 Biofilm 17.02 1.78 U6 Water 25.53 1.95 U6 Biofilm 17.99 1.84 Invertebrate composition Among all the ASVs detected by MolBio, a higher percentage corresponded to fungi, invertebrates (both micro and macro), algae, and protists, while chordates and plants showed a very low rate of representation (Supplementary Fig. S1). In the case of MolWat, the highest proportion was represented by protists, chordates, invertebrates, and algae, with fungi and plants represented to a lesser extent. A total of 125 invertebrate ASVs were detected using DNA-based technology at the study sites, classified into 4 phyla, 6 classes, 10 orders, 12 families, and 23 genera (Supplementary Table S1, Supplementary Fig. S2). The eDNA detection technique in biofilm allowed for the identification and recovery of a greater number of ASVs compared to those found in water. Most of the ASVs were assigned to the species or morphospecies level (90%), while fewer were classified at the family or order levels using both techniques. At all taxonomic levels, from Phylla to Genera and species or morphospecies, the highest number of taxonomic units was identified using Morpho, followed by MolBio detection and, finally, MolWat (Supplementary Table S1). The MolBio was the only technique to detect the families Aelosomatidae, Psychodidae, and Candonidae, as well as the genera Candona , Cypridopsis ( C. vidua ), and Stenopcypris ( S. chidesteri ), Culex ( C. chidesteri ), Callibaetis ( C. ferrugineus , C. pretiosus ), Cladopelma ( C. virescens , C. forcipis ), Hydra ( H. vulgaris ), which were not recorded by the other techniques. Cnidaria was identified through both MolWat and Morpho. Nematoda and Platyhelminthes were recorded only using Morpho. Except for Phyla, at all the taxonomic levels analyzed, only a minimal number of the total taxonomic units were shared between Morpho and Mol, with a relatively higher overlap observed between MolBio and MolWat, particularly at the genus level (Fig. 1a-e). Additionally, except in the case of Genera, the MolWat technique did not identify any taxa exclusively (all show 0%). All phyla, classes, and orders were detected at more than 65% of the sites, except for Cnidaria, Ostracoda, Stylomatophora, and Podocopida (MolWat) and Decapoda (MolBio) (Fig. 2a-c). Morpho and Mol yielded comparable results for Annelida (Naididae) and Arthropoda (Chironomidae, Culicidae, and Ephemeroptera). The most frequently recorded families (Chironomidae, Cochliopidae, and Naididae) were recorded in over 60% of the sites for each method (Fig. 2d). MolWat consistently recorded lower frequencies for all taxa. Biotic indices Except for the ICBrio index, all other indexes increase as water quality improves. In general, for all indices, Morpho classified the sites as being of higher quality compared to MolBio and MolWat (Table 3). Table 3: Values of the biotic indices calculated according to different techniques for the sites in the Paraná River basin during the study period. Different colors indicate the classifications obtained in each case, as detailed below. Score R1 Morpho R1 MolWat R5 Morpho R5 MolBio R6 Morpho R6 MolWat U6 Morpho U6 MolBio U6 MolWat A1 Morpho A1 MolWat A6 Morpho A6 MolBio A6 MolWat BMWP 41 2 29 10 47 6 54 3 1 9 6 29 10 6 BMWP´ 38 2 26 15 47 6 58 6 1 12 9 29 14 6 BMWP” 53 2 35 10 50 6 63 9 1 9 6 32 10 6 ASPT 4.556 2 3.625 2.5 4.7 2 4.909 1.5 1 2.25 2 4.143 2.5 2 IMRP 15.5 2.6 13.9 6.9 15 3.6 15.9 3.9 1 4.9 4.6 8.9 8.2 3.6 ICBrio 1.5 4 1.25 2.75 1.25 4.333 1.25 3.333 5 3.667 3.667 2.333 2 4 IBIS 31.02 14.29 43.22 26.21 65.44 10.9 90.14 26 17.14 27.66 5.16 12.03 12.48 13.42 Classes BMWP Good (100-150) Excellent (> 150) Regular (50-99) Poor (25-49) Very Poor ( 150) Contaminated (36-60) Very contaminated (16-35) Strongly contaminated ( 150) Doubtfully contaminated (36-60) Critical (16-35) Strongly Critical (<15) ASPT Good (5.01-6) Very Good (6.01-7) Excellent (≥7.01) Moderate pollution (4.01-5) Bad (3.01-4) Very Bad (2-3) IMRP Good (12.1-20) Acceptable (8-12) Low contamination (4-7.9) Moderately contaminated (2.6-3.9) Very Contaminated (1.1-2.5) Strongly contaminated (<1) ICBrio Excellent (1) Good (2) Regular (3) Bad (4) Very Bad (5) IBIS Good (≥75) Moderate (50-74) Poor (25- 49) Bad (< 25) Across the different techniques, the floodplain secondary channels exhibited more variable classifications than the tributary stream. Based on Morpho's classification, the average gradient was U6-R6-R1-R5-A6-A1. For MolBio, the average gradient was R5-A6-U6, while according to MolWat, all sites were generally classified as being in poor condition, except for A6, A1, R1, U6, and R6 under the IMRP and ICBRio indices. Comparing techniques, the differences were greater between MolWat and Morpho than between MolBio and Morpho, and the only significant numerical differences, albeit weak, were observed among sites for the IMRP between molecular and morphological sets (Fig. 3). Considering the classifications obtained, the indices BMWP, BMWP”, BMWP´, and ASPT generally classified the sites as degraded (except for BMWP” in U6), while the other indices displayed greater variability across sites (Table 3). The first group of indices was both numerically and qualitatively consistent, as was the case with IMRP and ICBRio. IBIS was numerically and qualitatively different from the other two groups of indices. Discussion Numerically, most indices did not show significant differences between techniques at the sites, except for the IMRP. However, the use of biotic indices is grounded in the definition of qualitative ranges for the environmental status of sites. This is particularly evident when comparing the classifications from IMRP and ICBRio, which yielded similar qualitative outcomes despite the ANOVA results. The indices BMWP, BMWP”, BMWP’, and ASPT consistently classified the sites as degraded (except for BMWP” in U6), while the other indices exhibited greater variability across sites (see Table 3). On one hand, these indices depend exclusively on the presence of sensitive or tolerant families rather than the relative abundance of taxa. On the other hand, unlike the IMRP, which is also based on tolerance values, the BMWP and ASPT indices were developed for streams in Europe and Brazil. Consequently, although they are affected by the taxonomic resolution achieved through different methods, they reflect less local variability between sites compared to indices specifically tailored for our region (i.e., IMRP, IBIS, ICBRio). Adjustments to the original BMWP index made for the Iberian and Brazilian regions also affect variations in our classifications, as they incorporate Families with higher or lower scores that can be found in our regions as well (e.g., Gomphidae, Aeshnidae, Libellulidae, Caenidae, Polycentropodidae, Hydroptilidae, Coenagrionidae, Ancylidae, Corixidae, Dytiscidae, Hydrophilidae, Curculionidae, Polymitacidae, Elmidae, Tricladida, etc. 16,17,18 . Similar to the aforementioned indices, regionalized indices also predominantly use oligochaetes and dipterans (Chironomidae, Ceratopogonidae, Culicidae, etc.) as indicators of degraded conditions, while mayflies, caddisflies, hydrozoans, crustaceans, and mollusks indicate better environmental conditions1 19.20, 21 . A deeper understanding of the ecology of genera and species within these common groups in our region 14 allows for the development of indices that more accurately reflect site characteristics and disturbances. For instance, the IMRP enhances the resolution of the considered tolerant and sensitive taxa by assigning scores on a continuous scale from 0 to 2 19 . The ICBRio incorporates genus and species-level measurements, such as the dominance of tubificids without capillary setae, Tubifex , and Chironomus as indicators of degraded environmental conditions, and the Tanytarsini/Chironomidae ratio as an indicator of good environmental conditions 20 . IBIS relies on the relative abundance of Limnodrilus hoffmeisteri and Pristina . Furthermore, both IBIS and ICBRio consider emergent attributes of communities, potentially enabling a more comprehensive assessment of benthos response to local conditions 22 . Additionally, IBIS provides insights into the functional structure of communities by integrating metrics that link ecosystem degradation to impairments in ecosystem function and service delivery, which is crucial for improving aquatic ecosystem management 23 . As a result, multimetric indices, particularly IBIS, supported a more accurate classification of sites- or at least a classification more aligned with environmental parameters and characterization- reflecting the gradient of conditions from the least impacted site (U6) to the most impacted sites (A1, A6). The primers used in this study exhibited good taxonomic resolution, offering dual advantages: they enable the selection of specific subsets of invertebrates with known sensitivity for river quality assessment and support the application of regionally adapted indices such as ICBRio or IBIS, which were particularly effective in capturing environmental variability. For most indices, Morpho classified the sites as having higher quality compared to MolBio and MolWat. The differences between MolWat and Morpho were more pronounced than those between MolBio and Morpho, as indicated by the analysis of shared taxa at various taxonomic levels. The likelihood of detecting a greater number of sensitive taxa—particularly at the species, genus, or family levels, which are frequently used in biotic indices—depends on factors such as sampling effort, the specific techniques employed for sorting or extraction, taxonomic expertise, and the presence of life stages that are often not identifiable at the species level 25 . In our study, DNA metabarcoding as a monitoring method did not detect the complete assemblages of local communities, on average, detecting fewer taxa than traditional methods, a finding also observed in other studies worldwide 9, 25, 26 . Comparatively, the highest richness of ASVs was found in the biofilm matrix. Within the biofilm, intracellular signals containing a significant amount of high-quality DNA likely prevail over extra-organismal DNA, which has a higher proportion of degraded DNA potentially originating from the biofilm itself, adjacent river habitats, and upstream environments. This provides a more accurate reflection of local conditions than the water matrix due to the habitat and life forms of the studied organisms 13 . In our study, we detected small chironomids and very small oligochaetes, such as aelosomatids, as potential inhabitants of the biofilm - taxa typically not detected in morphological studies. Additionally, many species that may inhabit biofilms and are also found in the benthos and pleuston of the region were identified 14 (Supplementary Table S1). However, the detection of taxa known to be absent from biofilms and to inhabit other environments suggests the capacity of biofilms to trap DNA from certain crustaceans (e.g., Trychodactyllus borellianus , Hyalella curvispina , ostracods) as well as from mollusks (e.g., Heleobia ), oligochaetes (e.g., Aulodrilus piguetti ), and cnidarians (e.g., Hydra vulgaris ). In the case of some insects (e.g., Baetidae, Culicidae), which are unlikely to inhabit biofilms as larvae, detection may instead arise from the DNA of their eggs 13 . In contrast, traditional morphological techniques do not account for eggs, which may introduce bias when interpreting results across methods. Furthermore, in water samples, genetic material transported from upstream within watersheds may result in false positives. This may explain the high ASV richness observed at sites such as R6 and A1 with MolWat (Supplementary Fig. S2). Indeed, Deiner & Altermatt 27 demonstrated that DNA from invertebrates can be detected up to 12 km downstream from their population source, with predictions extending this distance to 15-50 km before detection drops below a 5% threshold. Biofilm may thus have an advantage as an indicator matrix, as it could provide a more accurate synthesis of local conditions. Particularly for invertebrates, the number of ASVs generated through metabarcoding significantly differed from the taxa observed morphologically in the same environmental samples, resulting in either overestimations or underestimations 24 . In our study, 74% of the taxonomic entities recorded using morphological techniques were not detected by molecular techniques. These findings align with other surveys where even important indicator taxa went undetected 28 , with an average of more than 30% of the existing taxa not identified 12 . Although eDNA offers great potential as a more easily applied method for monitoring invertebrates, the inability of current approaches to detect large numbers of riverine taxa (particularly from Arthropoda) and even complete phyla (Nematoda, Platyhelminthes, and Nematomorpha) with the currently applied markers and DNA primers highlights significant limitations. These issues must be addressed before eDNA can be used as a robust method for the general monitoring of riverine macroinvertebrates 9 . Nonetheless, the techniques were consistent in detecting all phyla, classes, orders, and families (Chironomidae, Cochliopidae, and Naididae) in over 60% of the sites. Thus, the differences between indices could partially stem from the incompleteness of local reference libraries and the lack of barcoded taxa from our region. Indeed, species from tropical zones are known to be poorly represented in public reference barcoding libraries, with many barcodes being kept private 13 . Even in well-studied regions like Europe or North America, the coverage of reference libraries is far from complete 29,30 . For instance, for European taxa, COI DNA information was available for less than 60% of Annelida, Mollusca, and insect taxa (Diptera and Ephemeroptera), whereas it is available for over 80% of Odonata, Trichoptera, Hemiptera, and Crustacea species 30 . Similarly, for many North American freshwater invertebrates, COI sequence reference information is unavailable; for instance, more than half of Crustacea lack COI sequence information 31 . In our datasets, the different methods detected a higher number of Arthropoda compared to other phyla, although a substantial number of Arthropoda were also consistently missed by eDNA (approximately 80 taxa, Supplementary Table S1). This may be due to variations in the amount of DNA released into the water by different species, which is influenced by factors such as size, biomass, age classes, biological activity, feeding and reproductive behavior, and migration patterns 32 . Specifically, for arthropods, the presence of sclerotized exoskeletons may limit DNA release compared to other organisms, as these structures reduce the shedding of DNA 33 . Overall, sequences for Annelida, Nematoda, and Platyhelminthes, which are commonly used in bioindication, are severely underrepresented in public libraries (<5%) 30 . This limitation explains why our study recorded comparatively fewer oligochaete species and did not detect nematodes or platyhelminths with metabarcoding, but they were identified using morphological techniques. Generally, primer specificity is regarded as the primary factor influencing detection limits. However, incomplete reference databases and biological processes, such as recent divergence, may also contribute to the reduced number of genetically identified taxa 24 . In contrast, in our study, the percentage of ASVs identified at the genus and species levels was significantly higher than the percentage typically achieved using longer sequences of the COI gene. These longer sequences tend to degrade more rapidly and are more challenging to obtain 34,35 . For instance, when using eDNA from biofilm, several taxa were identified at the species level, including mayflies, dipterans, cnidarians, ostracods, and oligochaetes (Supplementary Table S1), which are likely to have been inhabiting the study sites during the sampling period. Despite its strong performance with macroinvertebrates, the primer pair used in this study also detected a considerable proportion of non-macroinvertebrate taxa (73%), consistent with findings by Rivera et al. 13 (Supplementary Fig. S1). Water and biofilms host a rich biodiversity, including eukaryotic algae, fungi, invertebrate larvae, and their eggs, protists, and DNA from different sources such as vertebrates and plants, as observed in our results. In this context, the techniques employed in this study, particularly the use of biofilms as a sampling matrix, demonstrate significant potential for future assessment of ecological integrity. These methods could offer faster and more cost-effective alternatives to traditional approaches, enabling broader and more efficient monitoring efforts. None of our indices relies on absolute abundance for their calculation, so comparisons between different methodologies are valid, as they are based solely on relative abundance. It is noteworthy that taxa or individuals with high biovolume, particularly fish and macroinvertebrates, are often overrepresented in metabarcoding results compared to morphological counts 12,36 . Besides, while some studies have found strong, positive relationships between organism abundance or biomass and eDNA concentrations under field conditions (e.g., Salter et al. 37 ), others report that organismal abundance or biomass is often only weakly associated with in situ eDNA concentrations or detectability (e.g. Yates et al. 38 ). In our study, we detected larger taxa such as trichodactylid crabs, but the majority of identified taxa were smaller organisms (e.g., dipterans, worms, ostracods). Despite the challenges, some research suggests that the relative abundance of certain taxa aligns consistently between molecular and morphological datasets, making it possible to use read abundance to calculate biotic indices effectively 24 . Environmental factors such as water temperature, organic material content, UV levels, DNA-degrading microorganisms, salinity (marine vs. freshwater), and lower pH can affect DNA persistence 39 . However, the variability of these parameters in our environments was not evident enough to create a marked gradient or generate significant noise for detections. Furthermore, degraded conditions at sites A1 and A6, reflected by high levels of dissolved nitrogen forms, BOD 5 , and COD, may indicate unfavorable conditions for preserving eDNA even for species present in situ . Nevertheless, at these sites, both classical techniques and metabarcoding showed very low richness and abundance, with high consistency among the indices across both methods. Thus, based on our findings, the primary challenges may be related to the incompleteness of reference libraries rather than the use of read abundance. Finally, the life stage at the time of sampling, which is influenced by seasonality, affects the likelihood of detecting species 24 . For example, in our region, the highest reproductive activity of many invertebrates, such as mollusks and insects, occurs during spring and summer 14 , potentially leading to their overrepresentation in samples. Consequently, we believe that sampling during the autumn, as conducted in this study, may be more suitable for monitoring local conditions, as it likely provides a more equitable representation of all taxa, as evidenced by the results from the morphological approach. However, if the goal is to detect specific groups or species, selecting seasons with higher abundances of these taxa would be advisable. In biomonitoring, rapid diagnostics are essential for mitigating impacts on ecosystems. Compared to traditional morphological techniques for invertebrates, eDNA sampling and processing are faster, more cost-effective, and require significantly fewer resources 24 . eDNA monitoring is non-invasive, does not require taxonomic expertise during sample collection, and enables the simultaneous amplification and identification of multiple taxa through high-throughput sequencing 40 . This approach addresses the limitations of conventional biomonitoring, which often fails to account for many taxonomic groups due to challenges in morphological identification. Besides, eDNA allows for a holistic analysis of ecosystems, providing insights into both target organisms and broader biotic communities, which can enhance ecological integrity assessment. Our results indicated that molecular techniques, particularly eDNA from biofilm, produced outputs more closely aligned with morphological assessments, suggesting that this methodology should be prioritized when selecting molecular approaches. While metabarcoding shows great promise, especially in identifying certain arthropods, its current constraints in detecting key phyla emphasize the importance of integrating it with traditional morphological methods. A combined approach that leverages the strengths of both techniques will provide a more comprehensive and accurate assessment of biodiversity and environmental health in riverine ecosystems and can assist decision-makers in the management and conservation of water resources. Methods Study area and sites The study area is located in the Paraná River basin in Argentina (31° 13.856'S - 60° 37.523'W and 31° 45.979'S - 60° 23.178'W; Fig. 4). In the study region, two main environmental zones are recognized 41 : to the west is the more Mediterranean area, exhibiting characteristics of both the Chaco and Pampas environments, featuring gently undulating to flat terrain, depressed near streams and rivers, with low forests, grasslands dominated by Panicum prionitis , and Ludwigia peploides , Juncus spp. , and Typha spp. primarily associated with water bodies. To the east, the defining environmental factor is the vast wetland system of the Middle Paraná River. The vegetation of the Paraná River floodplain forms a mosaic, ranging from forests characterized by pioneer or mature communities to gallery forests with greater structural complexity, characterized by a diverse arboreal layer, where medium to large evergreen trees like Albizia inundata , Inga verna , and Nectandra angustifolia form the forest canopy. In floodplain channels, herbaceous and shrub vegetation develops that tolerates varying degrees and frequencies of flooding. This vegetation can be categorized as either rooted (such as Panicum elephantipes and Polygonum spp .), rooted stem-floating (like Ludwigia peploides and Paspalum repens ), or free-floating (including Eichhornia crassipes, E. azurea, Azolla filiculoides, and Limnobium laevigatum ) 42 . Our dataset consists of six sites with samples collected during the autumn of the Southern Hemisphere in May 2021. The sampling took place during a severe to extreme drought of the La Plata Basin 15 . Four of the study sites are secondary channels located in the Middle Paraná River floodplain (U6, R1, R5, and R6), and the remaining two sites (A1 and A6) are embedded in an agricultural matrix (Fig. 4). The land quality in this basin is predominantly low or very low, primarily involving livestock activities, extensive grain farming, and horticulture 41 . Field collection and laboratory processing of samples At each sampling site, invertebrate samples were collected for classical morphological studies (Morpho) using the multihabitat approach 43 , as well as water and biofilm samples for studies and comparisons through molecular determinations. Molecular data were obtained from biofilm samples (MolBio) at 3 sites, and water samples (MolWat) at 5 sites. Additionally, one sample was collected to determine bottom organic matter content, and another sample was collected for granulometric analysis, using grabs suitable for the depth and substrate characteristics of each patch 44 . Besides, environmental variables were measured in situ at all sites, and physicochemical analyses were conducted in the laboratory. The biological material was collected under the provincial permit granted by Res. Prov. N⁰ 025/20 del Ministerio de Ambiente y Cambio Climático, Gobierno de la Provincia de Santa Fe, Argentina, for non-commercial purposes. Environmental parameters Rivers were categorized into low, medium, and high discharge (Qm500m 3 s -1 , respectively) while channel widths were measured using satellite images taken on the sampling dates 45 . Limnological parameters were measured in situ , including dissolved oxygen (O2 in ppm), pH, conductivity (C in µS/cm), and temperature with a HQ40D – Multi/2 Channels system (Hach®). Water transparency (Se in meters) and depth (Z in meters) were measured with a Secchi disk and a measuring tape. Bottom sediment granulometry was categorized by observation as sandy (>50% sand), silt-clayey (20-50% sand), or sand-silt-clay (<20% sand) 46 . Benthic organic matter in the sediments (BPOM) was also assessed by observation in the field. Water samples were taken from each site to assess concentrations of inorganic dissolved nutrients, biological oxygen demand (BOD 5 ), and chemical oxygen demand (COD). These samples were immediately transported to the laboratory under cold and dark conditions. The combined nitrate and nitrite (NO 3 - + NO 2 - –N) were analyzed by reducing nitrate with hydrazine sulfate, followed by nitrite determination through diazotization with sulfanilamide. Ammonium (NH 4 + -N) was measured using the indophenol blue method 47 , and soluble reactive phosphorus (SRP) was determined by the ascorbic acid method 48 . BOD 5 (mg L-1) was measured using an LDO BOD probe (model LBOD 10101, HACH), with oxygen consumption during the 5-day incubation period calculated by the difference between initial and final values 49 . For COD estimation, water samples were digested for two hours at 150 °C with K 2 Cr 2 O 7 in an acidic medium. The colorimetric determination was carried out at 420 nm, with results expressed in mg L −1 49 . Morphological taxonomy (Morpho) For classical morphological analyses, a multi-habitat sampling approach was employed 43 . Six samples of invertebrates from bottom sediments (infaunal benthos, episammon, epilithon, epipelon) and associated with macrophytes (pleuston, epiphyton) were collected from various available habitats, including littoral and limnetic bottom sediments as well as from stands of macrophytes (Table 1). The plant species were identified according to Schneider et al. (2021) 50 , and the reference collection deposited in the Laboratorio de Macrófitas, Instituto Nacional de Limnología (INALI-CONICET-UNL). All samples were sieved through a 200 µm mesh size and preserved in the field with 10% formaldehyde. In the laboratory, invertebrates were hand-picked from the samples using a stereomicroscope and subsequently stored in 70% ethanol for identification and counting. Molecular eDNA Taxonomy (MolBio and MolWat) At each site, 100 ml of water was collected with a sterile bottle in the littoral zone near the bottom without disturbing the sediments. All samples were kept cool (4 °C) until processing. In the laboratory, each water sample was poured into a beaker. The beaker was decontaminated with 10% bleach. All water samples were vacuum filtered using an MSI, Acetate Plus, Supported Plain Membrane Filter with a 0.22-μm pore size, including a negative control consisting of clean water. The filtration system was cleaned with commercial chlorine-based 10% bleach between samples to avoid contamination between sampling sites. Lastly, the filters were placed into 2 ml Eppendorf tubes using sterile forceps and stored at −80 °C until DNA extraction. The filters were processed following the method described by Amavet et al. 51 . Protocols for biofilm sampling were adjusted following Rivera et al. 13 . Integrated samples were taken from the bottom sediments and aquatic macrophytes. In the field, subsamples of available patches (i.e. epipelon, episammon, epiphyton) were identified and collected. Epipelon and epissamon were collected from the most superficial layer of bottom sediments with a core and then stored in a Falcon previously rinsed with alcohol. Epiphyton samples were collected from the submerged parts of the most representative macrophyte species, which were cut using scissors and sterile forceps (rinsed with hypochlorite in the field) and placed in sterile jars. In the laboratory, the collected epiphyton samples were sonicated for 3 cycles of 2 minutes in a TestLabr ® ultrasonic washer after ultrapure water was added. Epiphyton, epipelon, and/or episammon were integrated and centrifuged at 500 rpm for 10 minutes, eliminating the supernatant. The pellet was homogenized and fractionated into 1.1 ml samples, then centrifuged at 8000 g for 10 minutes at 4 ºC in a Sartorius® refrigerated centrifuge. The supernatant was removed, and the samples were stored at -80 ºC in an ultra-cold freezer until DNA extraction. To extract genetic material from biofilm samples, we used the PURO Soil Kit (Productos Bio-Lógicos®) following the standardized methods. The quality and concentration of the obtained DNA extracts were analyzed by electrophoresis on 0.8% agarose gels and a spectrophotometer Gene Quant II (Pharmacia Biotech®) using the ratio of absorbance at 260 nm and 280 nm. Then, the DNA extracts were analyzed by EcoMol Consultoria (Piracicaba, Brazil) using two primer pairs to amplify two fragments from the COI mtDNA gene: the pair 1 -fwhF2/fwhR2n- 34 to amplify a fragment of 205 bp and the pair 2 -MG2_LCO1490_F/MG2_univ_R- 52,53 to amplify a fragment of 133 bp. The eDNA samples were purified using magnetic beads (Agencourt AMPure XP® – Beckman Coulter) and then amplified. PCR conditions were: 40 cycles consisting of 95 °C for 30 seconds, 55 °C for 30 seconds, 72 °C for 2 minutes, with initial denaturation of 95°C for 5 minutes and final extension of 72 °C for 10 minutes. The master mix included 0.8 μl of each primer 10 μM and 2 μl of DNA in 20 μl of final volume, using negative controls. The PCR products were purified and amplified using the primer pair, together with adaptors from Nextera Index kit® (Illumina). Once purified and normalized, these PCR products were pooled into a single sequencing pool. Using real-time PCR, performed with the KAPA Biosystems Quantification Kit reagent (Illumina), the pool was quantified, diluted to a concentration of 2nM, and quantified again to confirm the final concentration. The final solution was diluted and loaded onto the iSeq100® equipment (Illumina), using the iSeq100 v2 sequencing kit, 300 cycles (2x150bp), and 30% phiX. Raw sequencing data were demultiplexed into forward and reverse read files. The sequences generated for each primer pair were combined in all possible orientations: forward, complement, reverse, and reverse complement. Using the Cutadapt 54 and DADA2 55 programs, several cleaning procedures were performed. The pipeline was organized in R 56 . The reads were subjected to the removal of sequences containing undefined bases (Ns), as well as the removal of primer sequences, and sequencing-related errors were also identified and removed. From the remaining sequences of this quality control, unique sequences present in the samples were identified. These sequences are called ASVs (Amplicon Sequencing Variants) and represent the amplification products of the DNAs used in the construction of the library, which were subjected to taxonomic identification. Using the DADA2 and phyloseq packages 57 , each of the ASVs was assigned a count relative to its frequency in the library, as well as a classification based on the association with sequences from the public reference banks NCBI and BOLD SYSTEMS (https://www.ncbi.nlm.nih.gov/; https://www.boldsystems.org/ ). These associations were made using the classifier developed by Wang et al. 58 . To define the final taxonomic classification at each sampling site, we followed these criteria: Species level: identity (sequence similarity with the reference sequence in the database) above 98% (ID > 98%) and relative incompleteness (number of reads generated concerning the total generated for the sample) greater than 0.02 (relative abundance > 2%); Genus level: sequence identity above 95% (ID > 95%); Family and Order levels: sequence identity with the reference sequence in the database less than 95% and greater than 80% (80% < ID < 95%). Data Analysis The following biotic indices were calculated using the metrix package 59 : Biological Monitoring Working Party (BMWP) 16 ; Average Score Per Taxon (ASPT) 16 ; Biological Monitoring Working Party prime (BMWP’) 17 ; Biological Monitoring Working Party double prime (BMWP”) 18 ; Índice da Comunidade Bentônica para rios (ICBrio) 20 ; Índice de Macroinvertebrados en Ríos Pampeanos (IMRP) 19 . Additionally, the Index of Benthic Invertebrates in Saline Rivers (IBIS) was calculated 21 . To determine whether there were significant differences between the indices based on the type of analysis at the sites, a factorial analysis of variance (ANOVA) was performed, followed by post hoc pairwise comparison with Bonferroni correction (p<0.05). All analyses were conducted in R and R Studio 56 . Declarations Acknowledgements This research was financially supported by Ministerio de Ciencia y Técnica (MinCyT, Argentina), Proyecto PICT 2018 03744 and Agencia Santafesina de Ciencia y Tecnología (Sectei, Provincia de Santa Fe, Argentina), Proyecto PEICi+d 2021037. Funding Declaration This research was financially supported by Ministerio de Ciencia y Técnica (MinCyT, Argentina), Proyecto PICT 2018 03744 and Agencia Santafesina de Ciencia y Tecnología (Sectei, Provincia de Santa Fe, Argentina), Proyecto PEICi+d 2021037. Author Contributions Statement F.Z., M.V.P.M., M.L., and P.A. obtained samples and performed analysis. J.C. obtained biological indices. All authors reviewed the manuscript. Data availability statement The datasets generated and/or analysed during the current study are available in the CONICET Institutional Repository, https://ri.conicet.gov.ar/handle/XXXXX/XXXXX. Competing Interest statement The authors have no competing interests to declare. References Almond, R. E., Grooten, M. & Peterson, T. Living Planet Report 2020 – Bending the curve of biodiversity loss. World Wildlife Fund. Gland, Switzerland: World Wildlife Fund. ISBN 978-2-940529-99-5 (2020). Wallace, J. B. & Webster, J. R. The role of macroinvertebrates in stream ecosystem function. Annu. Rev. Entomol. 41 , 115–139 (1996). Dominguez, E. et al. Biomonitoreo en ríos de Argentina: un camino por recorrer. Ecol. Austral . 8 , 934–949 (2021). Bonada, N., Prat, N., Resh, V. H. & Statzner, B. Developments in aquatic insect biomonitoring: a comparative analysis of recent approaches. Annu. Rev. Entomol. 51 , 495–523 (2006). Bashnin, T. et al. Relationship between pesticide accumulation in transplanted zebra mussels ( Dreissena polymorpha ) and community structure of aquatic macroinvertebrates. Environ. Pollut . 252 , 591–598 (2019). Feeley, H. B. et al. A national macroinvertebrate dataset collected for the biomonitoring of Ireland’s river network, 2007–2018. Sci. Data . 7 , 280. https://doi.org/10.1038/s41597-020-00618-8 (2020). CIC (Comité Intergubernamental Coordinador de los Países de la Cuenca del Plata). Programa de Acción Estratégica para la Cuenca del Plata. Buenos Aires, Argentina. 188 pp. (2017). Autoridad de Cuenca Matanza Riachuelo (ACUMAR). Informe de Monitoreo Ambiental de la Cuenca Matanza-Riachuelo. Autoridad de Cuenca Matanza-Riachuelo, Buenos & Aires Argentina. (2019). https://www.acumar.gob.ar/monitoreo-ambiental/ . Poyntz-Wright, I. P., Harrison, X. A., Pedersen, S. & Tyler, C. R. Effectiveness of eDNA for monitoring riverine macroinvertebrates. Sci. Total Environ. 941 , 173621. https://doi.org/10.1016/j.scitotenv.2024.173621 (2024). Sweeney, B. W., Battle, J. M., Jackson, J. K. & Dapkey, T. Can DNA barcodes of stream macroinvertebrates improve descriptions of community structure and water quality? J. N Am. Benthol Soc. 30 , 195–216 (2011). Taberlet, P., Coissac, E., Hajibabaei, M., Rieseberg, L. H. & Environmental, D. N. A. Mol. Ecol. 21 , 2155–2160 (2012). Elbrecht, V., Vamos, E. E., Meissner, K., Aroviita, J. & Leese, F. Assessing strengths and weaknesses of DNA metabarcoding-based macroinvertebrate identification for routine stream monitoring. Methods Ecol. Evol. 8 , 1265–1275 (2017). Rivera, S. et al. Fish eDNA metabarcoding from aquatic biofilm samples: Methodological aspects. Mol. Ecol. Res. 22 (4), 1440–1453 (2021). Ezcurra de Drago, I. E., Marchese, M., Montalto, L. & Benthic invertebrates in The Middle Paraná River: Limnology of a Subtropical Wetland (ed. Iriondo, M.H., Paggi, J.C. & Parma, M.J) 251–275Springer,. (2007). Naumann, G. et al. The 2019–2021 extreme drought episode in La Plata Basin in Publications Office of the European Union (2021). https://doi.org/10.2760/773 Armitage, P. D., Moss, D., Wright, J. F. & Furse, M. T. The performance of a new biological water quality score system based on macroinvertebrates over a wide range of unpolluted running-water sites. Water Res. 17 , 333–347 (1983). Alba-Tercedor, J. & Sánchez-Ortega, A. Un método rápido y simple para evaluar la calidad biológica de las aguas corrientes basado en el de Hellawell Limnetica. 4, 51–56 (1988). (1978). Loyola, R. G. N. Atual estágio do IAP no uso de índices biológicos de qualidade. Anais do V Simpósio de Ecossistemas Brasileiros: Conservação . 10 , 46–52 (2000). Rodrigues Capítulo, A. Los macroinvertebrados como indicadores de calidad de ambientes lóticos en el área pampeana. Rev. Soc. Entom Arg . 58 , 208–217 (1999). Kuhlmann, M., Imbimbo, H. V. & Ogura, L. L. Protocolo para el biomonitoreo con las comunidades bentónicas de ríos y reservorios del estado de São Paulo (CESTEB, 2012). Capeletti, J., Marchese, M. R. & Zilli, F. L. Evaluating macroinvertebrate metrics for ecological assessment of large saline rivers (Argentina). Environ. Sci. Pollut Res. 28 , 66464–66476 (2021). Stoddard, J. L. et al. A process for creating multimetric indices for large-scale aquatic surveys. J. N Am. Benthol Soc. 27 , 878–889 (2008). Barquín, J. et al. Coupling virtual watersheds with ecosystem services assessment: A 21st-century platform to support river research and management. Wiley Interdiscip Rev. : Water . 2 , 609–621 (2015). Pawlowski, J. et al. The future of biotic indices in the ecogenomic era: integrating (e)DNA metabarcoding in biological assessment of aquatic ecosystems. Sci. Total Environ. 637 , 1295–1310 (2018). Craine, J. M. et al. Surveying tropical faunal diversity via airborne DNA analyses. Sci. Rep. 15 , 36701. https://doi.org/10.1038/s41598-025-20767-3 (2025). Kim, G., Kang, Y. & Song, Y. Multi-primer-based environmental DNA survey for vertebrate biodiversity monitoring in protected island wetlands. Glob Ecol. Conserv. 58 , e03474. https://doi.org/10.1016/j.gecco.2025.e03474 (2025). Deiner, K. & Altermatt, F. Transport Distance of Invertebrate Environmental DNA in a Natural River. PLOS ONE . 9 (2), e88786. https://doi.org/10.1371/journal.pone.0088786 (2014). Deiner, K., Walser, J. C., Mächler, E. & Altermatt, F. Choice of capture and extraction methods affect detection of freshwater biodiversity from environmental DNA. Biol. Conserv. 183 , 53–63 (2015). Leray, M., Knowlton, N., Ho, S. L., Nguyen, B. N. & Machida, R. J. GenBank is a reliable resource for 21st-century biodiversity research. Proc. Nat. Acad. Sci. U.S.A. 116, 22651–22656 (2019). Weigand, H. et al. DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work. Sci. Total Environ. 678 , 499–524 (2019). Curry, C. J., Gibson, J. F., Shokralla, S., Hajibabaei, M. & Baird, D. J. Identifying North American freshwater invertebrates using DNA barcodes: Are existing COI sequence libraries fit for purpose? Freshw. Sci. 37 , 178–189 (2018). Barnes, M. A. & Turner, C. R. The ecology of environmental DNA and implications for conservation genetics. Conserv. Genet. 17 , 1–17 (2016). Taberlet, P., Bonin, A., Zinger, L., Coissac, E. & Environmental, D. N. A. For biodiversity research and monitoring (Oxford University Press, 2018). Vamos, E., Elbrecht, V. & Leese, F. Short COI markers for freshwater macroinvertebrate metabarcoding. Metabarcoding Metagenom . 1 , e14625. https://doi.org/10.3897/mbmg.1.14625 (2017). Leese, F. et al. Improved freshwater macroinvertebrate detection from environmental DNA through minimized nontarget amplification. Environ. DNA . 3 , 261–276 (2021). Elbrecht, V. & Leese, F. Can DNA-based ecosystem assessments quantify species abundance? Testing primer bias and biomass-sequence relationships with an innovative metabarcoding protocol. PLOS ONE . 10 , e0130324. https://doi.org/10.1371/journal.pone.0130324 (2015). Salter, I., Joensen, M., Kristiansen, R., Steingrund, P. & Vestergaard, P. Environmental DNA concentrations are correlated with regional biomass of Atlantic cod in oceanic waters. Commun. Biol. 2 , 461. https://doi.org/10.1038/s42003-019-0696-8 (2019). Yates, M. C. et al. The relationship between eDNA particle concentration and organism abundance in nature is strengthened by allometric scaling. Mol. Ecol. 30 , 3068–3082 (2021). Van Bochove, K. et al. Organic matter reduces the amount of detectable environmental DNA in freshwater. Ecol. Evol. 10 , 3647–3654 (2020). Ruppert, K. M., Kline, R. J. & Rahman, M. S. Past, present, and future perspectives of environmental DNA (eDNA) metabarcoding: A systematic review in methods, monitoring, and applications of global eDNA. Global Ecol. Conserv. 17 , e00547. https://doi.org/10.1016/j.gecco.2019.e00547 (2019). Biasatti, N. R. et al. Las ecoregiones, su conservación y las áreas naturales protegidas de la provincia de Santa Fe (Ministerio de Medio Ambiente, 2016). Marchetti, Z. Y., Latrubesse, E. M., Pereira, M. S. & Ramonell, C. G. Vegetation and its relationship with geomorphologic units in the Paraná River floodplain, Argentina. J. S Am. Earth Sci. 46 , 122–136 (2013). Barbour, M. T., Gerritsen, J., Blaine, S. D. & Stribling, J. B. in Rapid Bioassessment Protocols for Use in Streams and Wadeable Rivers: Periphyton, Benthic Macroinvertebrates, and Fish . (eds (2ª) (U.S. Environmental Protection Agency, Office of Water, 1999). Anderson, J. T. et al. Sampling and processing aquatic and terrestrial invertebrates in wetlands. in Wetland Techniques, Volume 2: Organisms (eds Anderson, J. T. & Craig) A. D) 143–195. (Springer, (2013). Google, E. Pro; earth.google.com/web/ (2024). de Bertoldi, H. Características limnológicas del Río Paraná y sus principales tributarios en el tramo Goya-Diamante. Los sedimentos de fondo y su fauna. Natura Neotropicalis , 1 (17) (1986). Koroleff, F. Revised version of Direct determination of ammonia in natural waters as indophenol blue, Int. Con. Explor. Sea, C. M. 1969/C:9. ICES Information on Techniques and Methods for Sea Water Analysis. Interlab. Rep . 3, 19–22 (1970). Murphy, J. & Riley, J. P. A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta . 27 , 31–36 (1962). APHA. Standard Methods for the Examination of Water and Wastewater (23rd ed.). American Public Health Association. (2017). Schneider, B., Hadad, H. & Rodriguez, E. Plantas acuáticas del río Paraná Medio. Colección Cátedra, Universidad Nacional del Litoral; (2022). https://hdl.handle.net/11185/6737 Amavet, P., Poletta, G., Odetti, L., Marcó, M. V. P. & Siroski, P. Detection of the maned wolf, a cryptic and vulnerable species, through environmental DNA studies. J. Nat. Conserv. 74 , 1–5 (2023). Gillet, F. et al. A new method to identify the endangered Pyrenean desman ( Galemys pyrenaicus ) and to study its diet, using next generation sequencing from faeces. Mamm. Biol. 80 , 505–509 (2015). Tournayre, O. et al. In silico and empirical evaluation of twelve metabarcoding primer sets for insectivorous diet analyses. Ecol. Evol. 10 , 6310–6332 (2020). Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBL , 17, 10–12 (2011). Callahan, B. J. et al. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods . 13 , 581–583 (2016). R Core Team. R: A Language and Environment for Statistical Computing_. R Foundation for Statistical Computing, Vienna, Austria. < (2023). https://www.R-project.org/ McMurdie, P. J. & Holmes, S. Phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLOS ONE . 8 , e61217. https://doi.org/10.1371/journal.pone.0061217 (2013). Wang, Q., Garrity, G. M., Tiedje, J. M. & Cole, J. R. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73 , 5261–5267 (2007). Cabrera, J. M. & Capeletti, J. Metrix': An R package for assessing the biological quality of water. Ecol. Modell . 484 , 110473. https://doi.org/10.1016/j.ecolmodel.2023.110473 (2023). Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8787248","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":600751713,"identity":"bf94e618-6e08-4e97-8321-3c2023bff256","order_by":0,"name":"Florencia Lucila Zilli","email":"","orcid":"","institution":"Instituto Nacional de Limnología, Consejo Nacional de Investigaciones Científicas y Técnicas-Universidad Nacional del Litoral","correspondingAuthor":false,"prefix":"","firstName":"Florencia","middleName":"Lucila","lastName":"Zilli","suffix":""},{"id":600751714,"identity":"6c2358e8-7538-4374-9014-6c780f388ec2","order_by":1,"name":"Magdalena Licursi","email":"","orcid":"","institution":"Instituto Nacional de Limnología, Consejo Nacional de Investigaciones Científicas y Técnicas-Universidad Nacional del Litoral","correspondingAuthor":false,"prefix":"","firstName":"Magdalena","middleName":"","lastName":"Licursi","suffix":""},{"id":600751715,"identity":"b9e582f3-8f84-4fb1-8931-f8524ff495e4","order_by":2,"name":"María Virginia Parachú Marcó","email":"","orcid":"","institution":"Consejo Nacional de Investigaciones Científicas y Técnicas-Universidad Nacional del Litoral","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Virginia Parachú","lastName":"Marcó","suffix":""},{"id":600751718,"identity":"be96b637-74b4-45f0-bc44-f43a9d79c8e1","order_by":3,"name":"Julieta Capeletti","email":"","orcid":"","institution":"Instituto Nacional de Limnología, Consejo Nacional de Investigaciones Científicas y Técnicas-Universidad Nacional del Litoral","correspondingAuthor":false,"prefix":"","firstName":"Julieta","middleName":"","lastName":"Capeletti","suffix":""},{"id":600751720,"identity":"d489b723-afbe-401f-a5ea-40c5047c761b","order_by":4,"name":"Patricia Amavet","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYBADOfYZEIYMEBsQo8PAmOcGhMVDtJbEHqK1mLOfTvzMU/EnvUe6x/ADY9sdHgb25m0SDDX3cGqx7MndLM1zxiC3R+aMsQRj2zMeBp5jZRIMx4pxu+hA7gbJmW0GufslcswYGNsO8zAAGRIMbAm4tZx/u/nnzH8G6TxwLfJvgFr+4dFyI3ebxMcGgwSEFgkeM6ALcWuxnPF2m8WHY8aGPRJpxRIJ5w7zsPGkFVsk9uHWYs6fu/lGQo2cPI9E8sYPH8oOy/GzH95448M3PA5D4YHUscEYxGkZBaNgFIyCUYANAABVsktWDxyC8QAAAABJRU5ErkJggg==","orcid":"","institution":"Universidad Nacional del Litoral-Consejo Nacional de Investigaciones Científicas y Técnicas","correspondingAuthor":true,"prefix":"","firstName":"Patricia","middleName":"","lastName":"Amavet","suffix":""}],"badges":[],"createdAt":"2026-02-04 13:40:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8787248/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8787248/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104247501,"identity":"e27c411e-159c-4226-8411-0425f21964f2","added_by":"auto","created_at":"2026-03-09 15:36:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":205244,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the eDNA monitoring datasets and the morphological records at a) Phyla, b) Class, c) Order, d) Family, e) Genera levels in the Paraná River.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8787248/v1/4aaf9933000a10121731ccec.png"},{"id":104247503,"identity":"1c7ab059-4a29-4ef6-b152-115d5241158c","added_by":"auto","created_at":"2026-03-09 15:36:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":155599,"visible":true,"origin":"","legend":"\u003cp\u003eRelative frequency of occurrence of taxa at the a) Phyla, b) Class, c) Order, and d) Family levels detected by classical morphological techniques (Morpho) and metabarcoding in water (MolWat), and biofilm (MolBio) in the Paraná River.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8787248/v1/44ca03b6aaa5838d6d6517a7.png"},{"id":104247502,"identity":"c2a1d8b5-d3f7-4702-9791-18b82ed77ec8","added_by":"auto","created_at":"2026-03-09 15:36:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":78693,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplots of the water quality evaluation, based on the a) BMWP, b) BMWP’, c) BMWP”, d) ASPT, e) ICBrio, f) IMRP, and g) IBIS obtained by classical morphological techniques (Morpho) and metabarcoding in water (MolWat), and biofilm (MolBio) in the Paraná River. Significance codes of ANOVA: 0 ‘*’, 0.001 ‘*’, 0.01 ‘’, 0.05 ‘.’, 0.1 ‘ ’. Friedman´s results are displayed only for significant ANOVAs for overall comparisons.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8787248/v1/3d49d8f5b9b4a3b5cc1d5a28.png"},{"id":104247505,"identity":"038b862a-64f2-4d7d-b203-7a76ae03ab1f","added_by":"auto","created_at":"2026-03-09 15:36:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":680199,"visible":true,"origin":"","legend":"\u003cp\u003eMap of the study area. a) Location of the area in South America; b) Location of the area in Santa Fe, province, Argentina; c) Sample sites within the Paraná River Basin; d) and e) A1 and A6 location: situated in the Aguiar stream and are embedded in an agricultural matrix; f) U6 location: drains into a rural-urban area where vegetables and strawberries are cultivated upstream; g), h) and i) R1, R4, and R5 locations: located downstream of Santa Fe and Santo Tomé cities\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8787248/v1/95b40d2ce9850ee259d8e8e2.png"},{"id":109296835,"identity":"954296d0-88b7-4fcd-9f0e-f63a4fc6623e","added_by":"auto","created_at":"2026-05-15 08:51:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1615723,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8787248/v1/68804df3-791c-4b3e-a745-bc6534e4c3c6.pdf"},{"id":104247504,"identity":"74d8eb29-43a9-4850-bc48-ce6e35fee62d","added_by":"auto","created_at":"2026-03-09 15:36:06","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":67270,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8787248/v1/9fb8c88a4b379f7be4425297.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Innovative approaches in aquatic monitoring: a comparative study between eDNA analysis and morphological techniques in a river ecosystem","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRivers are vital resources for human society, supplying essential goods and services, yet they are deteriorating as a result of human activities, including urban expansion, agricultural runoff, industrial effluents, and hydrological alterations\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Macroinvertebrates are essential to river ecosystems, enhancing decomposition, material exchange, and water self-purification, and supporting vital processes like nutrient cycling and energy flow\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Their biodiversity is crucial for ecosystem structure and function, but recent declines in their richness and abundance, driven by water pollution, agricultural inputs such as fertilizers and insecticides, habitat loss from deforestation, hydrological alteration, and climate change, pose significant threats to these processes\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBiological monitoring is one of the most effective means of detecting the effects of point and diffuse pollution and alterations in watersheds\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In this regard, many biological indicators are based on macroinvertebrates. Their advantages as bioindicators include their ubiquity, abundance, diversity, and variable sensitivity to environmental disturbances\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Currently, macroinvertebrates have been widely adopted by countries around the world to indicate the ecological status of rivers\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In the La Plata Basin, the Intergovernmental Coordinating Committee established Strategic Action Programs\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e for the management of wetlands, their conservation, and the sustainable use of biodiversity, aimed at preventing and controlling environmental degradation processes, fragmentation, and loss of connectivity that can be exacerbated by climate change effects, to avoid the loss of goods and services due to environmental risks. However, to date, monitoring programs that integrate information from multiple aquatic communities remain scarce. In Brazil, monitoring has been conducted through the analysis of phytoplankton, zooplankton, and benthic macroinvertebrate communities\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, while in Argentina, an integrated monitoring program that includes benthic macroinvertebrates, diatoms, and fish, along with water and sediment quality, is regionally restricted to the Matanza Riachuelo Basin\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSpecifically, regarding benthic macroinvertebrates, in Argentina, there are numerous regional biomonitoring projects based on these organisms, with varying degrees of temporal regularity across different basins\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Nonetheless, most current projects for macroinvertebrate monitoring worldwide still rely on traditional morphological methods, which involve collecting specimens in the field, sorting them from sediments, and subsequently identifying them at the lowest practical level based on their morphological characteristics\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. This process requires significant taxonomic expertise, especially for immature life stages (e.g., larvae and nymphs), where diagnostic morphological features are not fully developed, making species-level identification particularly challenging\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Additionally, the high diversity of macroinvertebrates, which includes numerous species from different phyla, demands expert knowledge for accurate species identification. This approach is time-consuming, costly, and resource-intensive; implementing efficient macroinvertebrate monitoring projects a significant challenge\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMore recently, environmental DNA (eDNA) metabarcoding has been applied to monitoring biota in riverine systems, offering a potentially more time- and cost-efficient alternative to traditional morphological assessments (compiled by Poyntz-Wright et al.\u003csup\u003e9\u003c/sup\u003e). Recent advances in DNA-based technology provide new opportunities to enhance the throughput, accuracy, and standardization of macroinvertebrate monitoring, as this method identifies species based on a standard fragment of their DNA (DNA barcode) rather than their morphological traits\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This approach is particularly relevant for taxonomic groups that are difficult or unfeasible to identify using routine protocols\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Metabarcoding studies of macroinvertebrate communities are typically conducted on DNA extracted from bulk samples of collected and sorted specimens, as well as directly from preservative ethanol, water, or sediments to detect freshwater macroinvertebrates. Besides, research has successfully assessed macroinvertebrate eDNA from biofilms, demonstrating that biofilms can trap DNA, leading to the temporary or permanent removal of DNA particles from the water column\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Although eDNA-based methodologies are becoming increasingly widespread, there is still insufficient evidence to directly apply the eDNA datasets to assess the river\u0026rsquo;s ecological status, particularly in tropical and subtropical river systems. These systems exhibit unique climatic and geomorphological characteristics compared to temperate regions, which necessitate further research to validate and adapt eDNA approaches for such a context.\u003c/p\u003e \u003cp\u003eIn this study, we used traditional morphological analyses and DNA-based technology to monitor aquatic environmental quality based on macroinvertebrates in the Paran\u0026aacute; River floodplain channels and a tributary. To the best of our knowledge, this is the first study to comparatively assess the suitability of metabarcoding and classical morphological techniques for water quality bioindication based on macroinvertebrates in South America, particularly in the Paran\u0026aacute; River basin. Besides, it provides a natural habitat for macroinvertebrates and other aquatic species\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Between 2019 and 2022, the La Plata Basin experienced moderate to extreme droughts, driven by the El Ni\u0026ntilde;o-South Oscillation (ENSO) and the Southern Annular Mode (SAM). Additionally, significant land-use changes, including deforestation and intensive agriculture, and the impacts of dams have shaped its three major sub-basins\u0026mdash;Paraguay, Paran\u0026aacute;, and Uruguay\u0026mdash;over recent decades\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur objectives were to assess the detection capacity of macroinvertebrate taxa using morphological features and metabarcoding from eDNA in different matrices (water and biofilm) in rivers with varying land uses located in the Paran\u0026aacute; River basin and to compare and analyze the consistency between the eDNA-based metrics and the morphological-based metrics to assess the environmental quality of sites with different land use in the Middle Paran\u0026aacute; Basin.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEnvironmental parameters\u003c/h2\u003e \u003cp\u003eAt the time of the study, the sites exhibited very low water flow, ranging from nearly lentic conditions with high transparency (A1 and A6) to sites with relatively higher flow and lower transparency (U6 and R1), as well as intermediate flow and transparency conditions (R5 and R6) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Channel widths ranged from 13 to 48 meters.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEnvironmental parameters measured at each site during the study period in the Middle Paran\u0026aacute; River.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eU6\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRelative water flow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLow (almost lentic)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLow (almost lentic)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChannel width\u003c/p\u003e \u003cp\u003e(m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTransparency value\u003c/p\u003e \u003cp\u003e(m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.105\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDeph\u003c/p\u003e \u003cp\u003e(m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemperature\u003c/p\u003e \u003cp\u003e(\u0026deg;C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDissolved oxygen (mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConductivity\u003c/p\u003e \u003cp\u003e(\u0026micro;Scm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e345\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1754\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e195.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGranulometry of bottom sediments\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSilt-clayed in the banks and with fine sand in the center\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSandy with a low proportion of silt and clay in the banks and sandy in the center\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSandy with a low proportion of silt and clay in the banks and sandy in the center\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSilt-clayed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSilt-clayed with gravels\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSandy with a thin layer of silt and clay in the banks and sandy in the center\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBPOM quality\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLow content; mainly fine detritus, and macrophyte roots remain in the litoral zones\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLow content; mainly fine detritus, and macrophyte roots remain in the litoral zones\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLow content; mainly fine detritus, and macrophyte roots remain in the litoral zones\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLow content of fine detritus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRelatively high fine detritus and vegetation remains\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWith minimum detritus content in the center and coarse and fine remains of vegetation in the banks\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMacrophyte composition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePaspalum spp\u003c/em\u003e and \u003cem\u003ePontederia crassipes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePaspalum spp\u003c/em\u003e and \u003cem\u003ePontederia crassipes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePaspalum spp\u003c/em\u003e and \u003cem\u003ePontederia crassipes\u003c/em\u003e. Less \u003cem\u003eAzolla sp\u003c/em\u003e and \u003cem\u003ePolygonum sp\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWithout riverine or floating macrophytes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eLudwigia peploides\u003c/em\u003e and \u003cem\u003eJuncus sp\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003ePaspalum spp\u003c/em\u003e and \u003cem\u003ePontederia crassipes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNO\u003csub\u003e3\u003c/sub\u003e--N\u003c/p\u003e \u003cp\u003e(ug L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e185.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e198.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e165.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6755.15*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e206.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNO\u003csub\u003e2\u003c/sub\u003e---N\u003c/p\u003e \u003cp\u003e(ug L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e543.23*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWater temperature was relatively higher at the floodplain sites compared to the streams. The sites had relatively well-oxygenated waters, with the highest oxygen levels observed at A1, A6, and U6, while conductivity was lowest at U6. pH values were approximately neutral, with the highest readings recorded at U6 and A6. The sites ranged from sandy to silt-clay and with low to high organic matter content, determined by granulometry and detritus quality; additionally, the floodplain sites featured stands of floating macrophytes and rooted grasses.\u003c/p\u003e \u003cp\u003eSimilar intermediate values were observed for all nitrogen forms, SRP, BOD\u003csub\u003e5\u003c/sub\u003e, and COD in R1, R5, and R6. All nitrogen forms peaked at A6, followed by U6 due to ammonium, with the lowest values recorded at A1. The SRP ranged from 200 to 300 across all sites, with the minimum observed at U6. A6 exhibited the highest BOD\u003csub\u003e5\u003c/sub\u003e, while A1 had the highest COD. Additionally, the BOD\u003csub\u003e5\u003c/sub\u003e/COD ratio indicated a high availability of biodegradable material only at A6. Based on the nitrogen forms, BOD\u003csub\u003e5\u003c/sub\u003e, and BOD\u003csub\u003e5\u003c/sub\u003e/COD ratio, the sites can be classified along a gradient from very good conditions (U6) through good conditions with low pollution (R1, R5, R6, and A1) to relatively polluted conditions (A6). The pollution observed at A1 is likely associated with the high COD, resulting from raw sewage discharges, primarily of municipal origin.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eeDNA metabarcoding\u003c/h3\u003e\n\u003cp\u003eThe eDNA obtained extracts showed good quality and concentration (Table\u0026nbsp;2). On average, the concentration values were higher in water samples than in biofilm samples. Purity was similar between samples, as was observed in the 260/280 ratio (range between 1,6 and 1,95).\u003c/p\u003e\n\u003cdiv\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eDNA extracts obtained quality and concentrations\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConcentration\u003c/p\u003e\n \u003cp\u003e(ng/ul)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAbsorbance (260/280)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWater\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e76.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiofilm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWater\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e72.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWater\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e62.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWater\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e127.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiofilm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWater\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiofilm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eInvertebrate composition\u003c/p\u003e\n\u003cp\u003eAmong all the ASVs detected by MolBio, a higher percentage corresponded to fungi, invertebrates (both micro and macro), algae, and protists, while chordates and plants showed a very low rate of representation (Supplementary Fig. S1). In the case of MolWat, the highest proportion was represented by protists, chordates, invertebrates, and algae, with fungi and plants represented to a lesser extent.\u003c/p\u003e\n\u003cp\u003eA total of 125 invertebrate ASVs were detected using DNA-based technology at the study sites, classified into 4 phyla, 6 classes, 10 orders, 12 families, and 23 genera (Supplementary Table S1, Supplementary Fig. S2). The eDNA detection technique in biofilm allowed for the identification and recovery of a greater number of ASVs compared to those found in water. Most of the ASVs were assigned to the species or morphospecies level (90%), while fewer were classified at the family or order levels using both techniques. At all taxonomic levels, from Phylla to Genera and species or morphospecies, the highest number of taxonomic units was identified using Morpho, followed by MolBio detection and, finally, MolWat (Supplementary Table S1). The MolBio was the only technique to detect the families Aelosomatidae, Psychodidae, and Candonidae, as well as the genera \u003cem\u003eCandona\u003c/em\u003e, \u003cem\u003eCypridopsis\u003c/em\u003e (\u003cem\u003eC. vidua\u003c/em\u003e), and \u003cem\u003eStenopcypris\u003c/em\u003e (\u003cem\u003eS. chidesteri\u003c/em\u003e), \u003cem\u003eCulex\u003c/em\u003e (\u003cem\u003eC. chidesteri\u003c/em\u003e), \u003cem\u003eCallibaetis\u003c/em\u003e (\u003cem\u003eC. ferrugineus\u003c/em\u003e, \u003cem\u003eC. pretiosus\u003c/em\u003e), \u003cem\u003eCladopelma\u003c/em\u003e (\u003cem\u003eC. virescens\u003c/em\u003e, \u003cem\u003eC. forcipis\u003c/em\u003e), \u003cem\u003eHydra\u003c/em\u003e (\u003cem\u003eH. vulgaris\u003c/em\u003e), which were not recorded by the other techniques. Cnidaria was identified through both MolWat and Morpho. Nematoda and Platyhelminthes were recorded only using Morpho.\u003c/p\u003e\n\u003cp\u003eExcept for Phyla, at all the taxonomic levels analyzed, only a minimal number of the total taxonomic units were shared between Morpho and Mol, with a relatively higher overlap observed between MolBio and MolWat, particularly at the genus level (Fig. 1a-e). Additionally, except in the case of Genera, the MolWat technique did not identify any taxa exclusively (all show 0%).\u003c/p\u003e\n\u003cp\u003eAll phyla, classes, and orders were detected at more than 65% of the sites, except for Cnidaria, Ostracoda, Stylomatophora, and Podocopida (MolWat) and Decapoda (MolBio) (Fig. 2a-c). Morpho and Mol yielded comparable results for Annelida (Naididae) and Arthropoda (Chironomidae, Culicidae, and Ephemeroptera). The most frequently recorded families (Chironomidae, Cochliopidae, and Naididae) were recorded in over 60% of the sites for each method (Fig. 2d). MolWat consistently recorded lower frequencies for all taxa.\u003c/p\u003e\n\u003cp\u003eBiotic indices\u003c/p\u003e\n\u003cp\u003eExcept for the ICBrio index, all other indexes increase as water quality improves. In general, for all indices, Morpho classified the sites as being of higher quality compared to MolBio and MolWat (Table 3).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTable 3:\u0026nbsp;\u003c/strong\u003eValues of the biotic indices calculated according to different techniques for the sites in the Paran\u0026aacute; River basin during the study period. Different colors indicate the classifications obtained in each case, as detailed below.\u003c/p\u003e\n\u003ctable\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:47.35pt;border:solid windowtext 1.0pt;background:white;padding:0in 3.5pt 0in 3.5pt;height:13.8pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eScore\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border:solid windowtext 1.0pt;border-left:none;padding:0in 3.5pt 0in 3.5pt;height:13.8pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eR1\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eMorpho\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border:solid windowtext 1.0pt;border-left:none;padding:0in 3.5pt 0in 3.5pt;height:13.8pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eR1\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eMolWat\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border:solid windowtext 1.0pt;border-left:none;padding:0in 3.5pt 0in 3.5pt;height:13.8pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eR5\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eMorpho\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border:solid windowtext 1.0pt;border-left:none;padding:0in 3.5pt 0in 3.5pt;height:13.8pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eR5\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eMolBio\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border:solid windowtext 1.0pt;border-left:none;padding:0in 3.5pt 0in 3.5pt;height:13.8pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eR6\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eMorpho\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border:solid windowtext 1.0pt;border-left:none;padding:0in 3.5pt 0in 3.5pt;height:13.8pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eR6\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eMolWat\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border:solid windowtext 1.0pt;border-left:none;padding:0in 3.5pt 0in 3.5pt;height:13.8pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eU6\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eMorpho\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border:solid windowtext 1.0pt;border-left:none;padding:0in 3.5pt 0in 3.5pt;height:13.8pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eU6\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eMolBio\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border:solid windowtext 1.0pt;border-left:none;padding:0in 3.5pt 0in 3.5pt;height:13.8pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eU6\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eMolWat\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border:solid windowtext 1.0pt;border-left:none;padding:0in 3.5pt 0in 3.5pt;height:13.8pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eA1\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eMorpho\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border:solid windowtext 1.0pt;border-left:none;padding:0in 3.5pt 0in 3.5pt;height:13.8pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eA1\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eMolWat\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border:solid windowtext 1.0pt;border-left:none;padding:0in 3.5pt 0in 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style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#FFC000;padding:0in 3.5pt 0in 3.5pt;height:18.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:47.35pt;border:solid windowtext 1.0pt;border-top:none;padding:0in 3.5pt 0in 3.5pt;height:18.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eIBIS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#FFC000;padding:0in 3.5pt 0in 3.5pt;height:18.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e31.02\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:red;padding:0in 3.5pt 0in 3.5pt;height:18.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e14.29\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#FFC000;padding:0in 3.5pt 0in 3.5pt;height:18.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e43.22\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#FFC000;padding:0in 3.5pt 0in 3.5pt;height:18.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e26.21\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:yellow;padding:0in 3.5pt 0in 3.5pt;height:18.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e65.44\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:red;padding:0in 3.5pt 0in 3.5pt;height:18.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e10.9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#00B050;padding:0in 3.5pt 0in 3.5pt;height:18.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e90.14\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#FFC000;padding:0in 3.5pt 0in 3.5pt;height:18.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e26\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:red;padding:0in 3.5pt 0in 3.5pt;height:18.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e17.14\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#FFC000;padding:0in 3.5pt 0in 3.5pt;height:18.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e27.66\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:red;padding:0in 3.5pt 0in 3.5pt;height:18.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e5.16\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:red;padding:0in 3.5pt 0in 3.5pt;height:18.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e12.03\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:red;padding:0in 3.5pt 0in 3.5pt;height:18.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e12.48\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:47.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:red;padding:0in 3.5pt 0in 3.5pt;height:18.6pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e13.42\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ctable style=\"border: none;border-collapse: collapse;width: 933px;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width:699.7pt;border:solid windowtext 1.0pt;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eClasses\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:62.25pt;border:solid windowtext 1.0pt;border-top:none;padding: 0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eBMWP\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:62.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#00B050;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eGood\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(100-150)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eExcellent\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(\u0026gt; 150)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:67.6pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:yellow;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eRegular\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(50-99)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:67.6pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#FFC000;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003ePoor\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(25-49)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width:440.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:red;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eVery Poor (\u0026lt;25)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:62.25pt;border:solid windowtext 1.0pt;border-top:none;padding: 0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eBMWP\u0026acute;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:62.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#00B050;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eAcceptable (61-100)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eGood\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(101-121)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eVery Good\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(\u0026gt; 150)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:67.6pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:yellow;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eContaminated\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(36-60)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:67.6pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#FFC000;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eVery contaminated\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(16-35)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width:440.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:red;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eStrongly contaminated\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(\u0026lt;15)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:62.25pt;border:solid windowtext 1.0pt;border-top:none;padding: 0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eBMWP\u0026rdquo;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:62.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#00B050;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eAcceptable\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(61-100)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eGood\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(101-121)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eVery Good\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(\u0026gt; 150)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:67.6pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:yellow;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eDoubtfully contaminated\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(36-60)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:67.6pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#FFC000;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eCritical\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(16-35)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width:440.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:red;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eStrongly Critical\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(\u0026lt;15)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:62.25pt;border:solid windowtext 1.0pt;border-top:none;padding: 0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eASPT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:62.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#00B050;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eGood\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(5.01-6)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eVery Good (6.01-7)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eExcellent (\u0026ge;7.01)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:67.6pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:yellow;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eModerate pollution (4.01-5)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:67.6pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#FFC000;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eBad\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(3.01-4)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width:440.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:red;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eVery Bad\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(2-3)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:62.25pt;border:solid windowtext 1.0pt;border-top:none;padding: 0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eIMRP\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:62.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#00B050;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eGood\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(12.1-20)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width:135.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#FFD966;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eAcceptable\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(8-12)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:74.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:yellow;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eLow contamination\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(4-7.9)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#FFC000;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eModerately contaminated\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(2.6-3.9)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:red;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eVery Contaminated\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(1.1-2.5)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:red;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eStrongly contaminated (\u0026lt;1)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:62.25pt;border:solid windowtext 1.0pt;border-top:none;padding: 0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eICBrio\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:62.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#00B050;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eExcellent\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(1)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width:135.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#FFD966;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eGood\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(2)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:74.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:yellow;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eRegular\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(3)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#FFC000;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eBad (4)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"border-top:none;border-left:none;border-bottom: solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background: red;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eVery Bad\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(5)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:62.25pt;border:solid windowtext 1.0pt;border-top:none;padding: 0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eIBIS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:62.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#00B050;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eGood\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(\u0026ge;75)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width:135.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:yellow;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eModerate\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(50-74)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:74.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background:#FFC000;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003ePoor\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(25- 49)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"border-top:none;border-left:none;border-bottom: solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;background: red;padding:0in 3.5pt 0in 3.5pt;height:15.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eBad\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:14.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:13px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e(\u0026lt; 25)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAcross the different techniques, the floodplain secondary channels exhibited more variable classifications than the tributary stream. Based on Morpho's classification, the average gradient was U6-R6-R1-R5-A6-A1. For MolBio, the average gradient was R5-A6-U6, while according to MolWat, all sites were generally classified as being in poor condition, except for A6, A1, R1, U6, and R6 under the IMRP and ICBRio indices. Comparing techniques, the differences were greater between MolWat and Morpho than between MolBio and Morpho, and the only significant numerical differences, albeit weak, were observed among sites for the IMRP between molecular and morphological sets (Fig. 3).\u003c/p\u003e\n\u003cp\u003eConsidering the classifications obtained, the indices BMWP, BMWP”, BMWP´, and ASPT generally classified the sites as degraded (except for BMWP” in U6), while the other indices displayed greater variability across sites (Table 3). The first group of indices was both numerically and qualitatively consistent, as was the case with IMRP and ICBRio. IBIS was numerically and qualitatively different from the other two groups of indices.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNumerically, most indices did not show significant differences between techniques at the sites, except for the IMRP. However, the use of biotic indices is grounded in the definition of qualitative ranges for the environmental status of sites. This is particularly evident when comparing the classifications from IMRP and ICBRio, which yielded similar qualitative outcomes despite the ANOVA results. The indices BMWP, BMWP”, BMWP’, and ASPT consistently classified the sites as degraded (except for BMWP” in U6), while the other indices exhibited greater variability across sites (see Table 3). On one hand, these indices depend exclusively on the presence of sensitive or tolerant families rather than the relative abundance of taxa. On the other hand, unlike the IMRP, which is also based on tolerance values, the BMWP and ASPT indices were developed for streams in Europe and Brazil. Consequently, although they are affected by the taxonomic resolution achieved through different methods, they reflect less local variability between sites compared to indices specifically tailored for our region (i.e., IMRP, IBIS, ICBRio). Adjustments to the original BMWP index made for the Iberian and Brazilian regions also affect variations in our classifications, as they incorporate Families with higher or lower scores that can be found in our regions as well (e.g., Gomphidae, Aeshnidae, Libellulidae, Caenidae, Polycentropodidae, Hydroptilidae, Coenagrionidae, Ancylidae, Corixidae, Dytiscidae, Hydrophilidae, Curculionidae, Polymitacidae, Elmidae, Tricladida, etc.\u003csup\u003e16,17,18\u003c/sup\u003e. Similar to the aforementioned indices, regionalized indices also predominantly use oligochaetes and dipterans (Chironomidae, Ceratopogonidae, Culicidae, etc.) as indicators of degraded conditions, while mayflies, caddisflies, hydrozoans, crustaceans, and mollusks indicate better environmental conditions1\u003csup\u003e19.20, 21\u003c/sup\u003e. A deeper understanding of the ecology of genera and species within these common groups in our region\u003csup\u003e14\u003c/sup\u003e allows for the development of indices that more accurately reflect site characteristics and disturbances. For instance, the IMRP enhances the resolution of the considered tolerant and sensitive taxa by assigning scores on a continuous scale from 0 to 2\u003csup\u003e19\u003c/sup\u003e. The ICBRio incorporates genus and species-level measurements, such as the dominance of tubificids without capillary setae, \u003cem\u003eTubifex\u003c/em\u003e, and \u003cem\u003eChironomus\u003c/em\u003e as indicators of degraded environmental conditions, and the Tanytarsini/Chironomidae ratio as an indicator of good environmental conditions\u003csup\u003e20\u003c/sup\u003e. IBIS relies on the relative abundance of \u003cem\u003eLimnodrilus hoffmeisteri\u003c/em\u003e and \u003cem\u003ePristina\u003c/em\u003e. Furthermore, both IBIS and ICBRio consider emergent attributes of communities, potentially enabling a more comprehensive assessment of benthos response to local conditions\u003csup\u003e22\u003c/sup\u003e. Additionally, IBIS provides insights into the functional structure of communities by integrating metrics that link ecosystem degradation to impairments in ecosystem function and service delivery, which is crucial for improving aquatic ecosystem management\u003csup\u003e23\u003c/sup\u003e. As a result, multimetric indices, particularly IBIS, supported a more accurate classification of sites- or at least a classification more aligned with environmental parameters and characterization- reflecting the gradient of conditions from the least impacted site (U6) to the most impacted sites (A1, A6).\u003c/p\u003e\n\u003cp\u003eThe primers used in this study exhibited good taxonomic resolution, offering dual advantages: they enable the selection of specific subsets of invertebrates with known sensitivity for river quality assessment and support the application of regionally adapted indices such as ICBRio or IBIS, which were particularly effective in capturing environmental variability.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor most indices, Morpho classified the sites as having higher quality compared to MolBio and MolWat. The differences between MolWat and Morpho were more pronounced than those between MolBio and Morpho, as indicated by the analysis of shared taxa at various taxonomic levels. The likelihood of detecting a greater number of sensitive taxa—particularly at the species, genus, or family levels, which are frequently used in biotic indices—depends on factors such as sampling effort, the specific techniques employed for sorting or extraction, taxonomic expertise, and the presence of life stages that are often not identifiable at the species level\u003csup\u003e25\u003c/sup\u003e. In our study, DNA metabarcoding as a monitoring method did not detect the complete assemblages of local communities, on average, detecting fewer taxa than traditional methods, a finding also observed in other studies worldwide\u003csup\u003e9, 25, 26\u003c/sup\u003e. Comparatively, the highest richness of ASVs was found in the biofilm matrix. Within the biofilm, intracellular signals containing a significant amount of high-quality DNA likely prevail over extra-organismal DNA, which has a higher proportion of degraded DNA potentially originating from the biofilm itself, adjacent river habitats, and upstream environments. This provides a more accurate reflection of local conditions than the water matrix due to the habitat and life forms of the studied organisms\u003csup\u003e13\u003c/sup\u003e. In our study, we detected small chironomids and very small oligochaetes, such as aelosomatids, as potential inhabitants of the biofilm - taxa typically not detected in morphological studies. Additionally, many species that may inhabit biofilms and are also found in the benthos and pleuston of the region were identified\u003csup\u003e14\u0026nbsp;\u003c/sup\u003e(Supplementary Table S1). However, the detection of taxa known to be absent from biofilms and to inhabit other environments suggests the capacity of biofilms to trap DNA from certain crustaceans (e.g., \u003cem\u003eTrychodactyllus borellianus\u003c/em\u003e, \u003cem\u003eHyalella curvispina\u003c/em\u003e, ostracods) as well as from mollusks (e.g., \u003cem\u003eHeleobia\u003c/em\u003e), oligochaetes (e.g., \u003cem\u003eAulodrilus piguetti\u003c/em\u003e), and cnidarians (e.g., \u003cem\u003eHydra vulgaris\u003c/em\u003e). In the case of some insects (e.g., Baetidae, Culicidae), which are unlikely to inhabit biofilms as larvae, detection may instead arise from the DNA of their eggs\u003csup\u003e13\u003c/sup\u003e. In contrast, traditional morphological techniques do not account for eggs, which may introduce bias when interpreting results across methods. Furthermore, in water samples, genetic material transported from upstream within watersheds may result in false positives. This may explain the high ASV richness observed at sites such as R6 and A1 with MolWat (Supplementary Fig. S2). Indeed, Deiner \u0026amp; Altermatt\u003csup\u003e27\u003c/sup\u003e demonstrated that DNA from invertebrates can be detected up to 12 km downstream from their population source, with predictions extending this distance to 15-50 km before detection drops below a 5% threshold. Biofilm may thus have an advantage as an indicator matrix, as it could provide a more accurate synthesis of local conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eParticularly for invertebrates, the number of ASVs generated through metabarcoding significantly differed from the taxa observed morphologically in the same environmental samples, resulting in either overestimations or underestimations\u003csup\u003e24\u003c/sup\u003e. In our study, 74% of the taxonomic entities recorded using morphological techniques were not detected by molecular techniques. These findings align with other surveys where even important indicator taxa went undetected\u003csup\u003e28\u003c/sup\u003e, with an average of more than 30% of the existing taxa not identified\u003csup\u003e12\u003c/sup\u003e. Although eDNA offers great potential as a more easily applied method for monitoring invertebrates, the inability of current approaches to detect large numbers of riverine taxa (particularly from Arthropoda) and even complete phyla (Nematoda, Platyhelminthes, and Nematomorpha) with the currently applied markers and DNA primers highlights significant limitations. These issues must be addressed before eDNA can be used as a robust method for the general monitoring of riverine macroinvertebrates\u003csup\u003e9\u003c/sup\u003e. Nonetheless, the techniques were consistent in detecting all phyla, classes, orders, and families (Chironomidae, Cochliopidae, and Naididae) in over 60% of the sites. Thus, the differences between indices could partially stem from the incompleteness of local reference libraries and the lack of barcoded taxa from our region. Indeed, species from tropical zones are known to be poorly represented in public reference barcoding libraries, with many barcodes being kept private\u003csup\u003e13\u003c/sup\u003e. Even in well-studied regions like Europe or North America, the coverage of reference libraries is far from complete\u003csup\u003e29,30\u003c/sup\u003e. For instance, for European taxa, COI DNA information was available for less than 60% of Annelida, Mollusca, and insect taxa (Diptera and Ephemeroptera), whereas it is available for over 80% of Odonata, Trichoptera, Hemiptera, and Crustacea species\u003csup\u003e30\u003c/sup\u003e. Similarly, for many North American freshwater invertebrates, COI sequence reference information is unavailable; for instance, more than half of Crustacea lack COI sequence information\u003csup\u003e31\u003c/sup\u003e. In our datasets, the different methods detected a higher number of Arthropoda compared to other phyla, although a substantial number of Arthropoda were also consistently missed by eDNA (approximately 80 taxa, Supplementary Table S1). This may be due to variations in the amount of DNA released into the water by different species, which is influenced by factors such as size, biomass, age classes, biological activity, feeding and reproductive behavior, and migration patterns\u003csup\u003e32\u003c/sup\u003e. Specifically, for arthropods, the presence of sclerotized exoskeletons may limit DNA release compared to other organisms, as these structures reduce the shedding of DNA\u003csup\u003e33\u003c/sup\u003e. Overall, sequences for Annelida, Nematoda, and Platyhelminthes, which are commonly used in bioindication, are severely underrepresented in public libraries (\u0026lt;5%)\u003csup\u003e30\u003c/sup\u003e. This limitation explains why our study recorded comparatively fewer oligochaete species and did not detect nematodes or platyhelminths with metabarcoding, but they were identified using morphological techniques.\u003c/p\u003e\n\u003cp\u003eGenerally, primer specificity is regarded as the primary factor influencing detection limits. However, incomplete reference databases and biological processes, such as recent divergence, may also contribute to the reduced number of genetically identified taxa\u003csup\u003e24\u003c/sup\u003e. In contrast, in our study, the percentage of ASVs identified at the genus and species levels was significantly higher than the percentage typically achieved using longer sequences of the COI gene. These longer sequences tend to degrade more rapidly and are more challenging to obtain\u003csup\u003e34,35\u003c/sup\u003e. For instance, when using eDNA from biofilm, several taxa were identified at the species level, including mayflies, dipterans, cnidarians, ostracods, and oligochaetes (Supplementary Table S1), which are likely to have been inhabiting the study sites during the sampling period. Despite its strong performance with macroinvertebrates, the primer pair used in this study also detected a considerable proportion of non-macroinvertebrate taxa (73%), consistent with findings by Rivera et al. \u003csup\u003e13\u003c/sup\u003e (Supplementary Fig. S1). Water and biofilms host a rich biodiversity, including eukaryotic algae, fungi, invertebrate larvae, and their eggs, protists, and DNA from different sources such as vertebrates and plants, as observed in our results. In this context, the techniques employed in this study, particularly the use of biofilms as a sampling matrix, demonstrate significant potential for future assessment of ecological integrity. These methods could offer faster and more cost-effective alternatives to traditional approaches, enabling broader and more efficient monitoring efforts.\u003c/p\u003e\n\u003cp\u003eNone of our indices relies on absolute abundance for their calculation, so comparisons between different methodologies are valid, as they are based solely on relative abundance. It is noteworthy that taxa or individuals with high biovolume, particularly fish and macroinvertebrates, are often overrepresented in metabarcoding results compared to morphological counts\u003csup\u003e12,36\u003c/sup\u003e. Besides, while some studies have found strong, positive relationships between organism abundance or biomass and eDNA concentrations under field conditions (e.g., Salter et al.\u003csup\u003e37\u003c/sup\u003e), others report that organismal abundance or biomass is often only weakly associated with \u003cem\u003ein situ\u003c/em\u003e eDNA concentrations or detectability (e.g. Yates et al. \u003csup\u003e38\u003c/sup\u003e). In our study, we detected larger taxa such as trichodactylid crabs, but the majority of identified taxa were smaller organisms (e.g., dipterans, worms, ostracods). Despite the challenges, some research suggests that the relative abundance of certain taxa aligns consistently between molecular and morphological datasets, making it possible to use read abundance to calculate biotic indices effectively\u003csup\u003e24\u003c/sup\u003e. \u0026nbsp;Environmental factors such as water temperature, organic material content, UV levels, DNA-degrading microorganisms, salinity (marine vs. freshwater), and lower pH can affect DNA persistence\u003csup\u003e39\u003c/sup\u003e. However, the variability of these parameters in our environments was not evident enough to create a marked gradient or generate significant noise for detections. Furthermore, degraded conditions at sites A1 and A6, reflected by high levels of dissolved nitrogen forms, BOD\u003csub\u003e5\u003c/sub\u003e, and COD, may indicate unfavorable conditions for preserving eDNA even for species present \u003cem\u003ein situ\u003c/em\u003e. Nevertheless, at these sites, both classical techniques and metabarcoding showed very low richness and abundance, with high consistency among the indices across both methods. Thus, based on our findings, the primary challenges may be related to the incompleteness of reference libraries rather than the use of read abundance. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, the life stage at the time of sampling, which is influenced by seasonality, affects the likelihood of detecting species\u003csup\u003e24\u003c/sup\u003e. \u0026nbsp;For example, in our region, the highest reproductive activity of many invertebrates, such as mollusks and insects, occurs during spring and summer\u003csup\u003e14\u003c/sup\u003e, potentially leading to their overrepresentation in samples. Consequently, we believe that sampling during the autumn, as conducted in this study, may be more suitable for monitoring local conditions, as it likely provides a more equitable representation of all taxa, as evidenced by the results from the morphological approach. However, if the goal is to detect specific groups or species, selecting seasons with higher abundances of these taxa would be advisable.\u003c/p\u003e\n\u003cp\u003eIn biomonitoring, rapid diagnostics are essential for mitigating impacts on ecosystems. Compared to traditional morphological techniques for invertebrates, eDNA sampling and processing are faster, more cost-effective, and require significantly fewer resources\u003csup\u003e24\u003c/sup\u003e. eDNA monitoring is non-invasive, does not require taxonomic expertise during sample collection, and enables the simultaneous amplification and identification of multiple taxa through high-throughput sequencing\u003csup\u003e40\u003c/sup\u003e. This approach addresses the limitations of conventional biomonitoring, which often fails to account for many taxonomic groups due to challenges in morphological identification. Besides, eDNA allows for a holistic analysis of ecosystems, providing insights into both target organisms and broader biotic communities, which can enhance ecological integrity assessment. Our results indicated that molecular techniques, particularly eDNA from biofilm, produced outputs more closely aligned with morphological assessments, suggesting that this methodology should be prioritized when selecting molecular approaches. While metabarcoding shows great promise, especially in identifying certain arthropods, its current constraints in detecting key phyla emphasize the importance of integrating it with traditional morphological methods. A combined approach that leverages the strengths of both techniques will provide a more comprehensive and accurate assessment of biodiversity and environmental health in riverine ecosystems and can assist decision-makers in the management and conservation of water resources.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eStudy area and sites\u003c/p\u003e\n\u003cp\u003eThe study area is located in the Paraná River basin in Argentina (31° 13.856'S - 60° 37.523'W and 31° 45.979'S - 60° 23.178'W; Fig. 4). In the study region, two main environmental zones are recognized\u003csup\u003e41\u003c/sup\u003e: to the west is the more Mediterranean area, exhibiting characteristics of both the Chaco and Pampas environments, featuring gently undulating to flat terrain, depressed near streams and rivers, with low forests, grasslands dominated by \u003cem\u003ePanicum prionitis\u003c/em\u003e, and \u003cem\u003eLudwigia peploides\u003c/em\u003e, \u003cem\u003eJuncus spp.\u003c/em\u003e, and \u003cem\u003eTypha spp.\u0026nbsp;\u003c/em\u003eprimarily associated with water bodies. To the east, the defining environmental factor is the vast wetland system of the Middle Paraná River. The vegetation of the Paraná River floodplain forms a mosaic, ranging from forests characterized by pioneer or mature communities to gallery forests with greater structural complexity, characterized by a diverse arboreal layer, where medium to large evergreen trees like \u003cem\u003eAlbizia inundata\u003c/em\u003e, \u003cem\u003eInga verna\u003c/em\u003e, and \u003cem\u003eNectandra angustifolia\u003c/em\u003e form the forest canopy. In floodplain channels, herbaceous and shrub vegetation develops that tolerates varying degrees and frequencies of flooding. This vegetation can be categorized as either rooted (such as \u003cem\u003ePanicum elephantipes\u003c/em\u003e and \u003cem\u003ePolygonum spp\u003c/em\u003e.), rooted stem-floating (like \u003cem\u003eLudwigia peploides\u003c/em\u003e and \u003cem\u003ePaspalum repens\u003c/em\u003e), or free-floating (including \u003cem\u003eEichhornia crassipes, E. azurea, Azolla filiculoides,\u003c/em\u003e and \u003cem\u003eLimnobium laevigatum\u003c/em\u003e)\u003csup\u003e42\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOur dataset consists of six sites with samples collected during the autumn of the Southern Hemisphere in May 2021. The sampling took place during a severe to extreme drought of the La Plata Basin\u003csup\u003e15\u003c/sup\u003e. Four of the study sites are secondary channels located in the Middle Paraná River floodplain (U6, R1, R5, and R6), and the remaining two sites (A1 and A6) are embedded in an agricultural matrix (Fig. 4). The land quality in this basin is predominantly low or very low, primarily involving livestock activities, extensive grain farming, and horticulture\u003csup\u003e41\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eField collection and laboratory processing of samples\u003c/p\u003e\n\u003cp\u003eAt each sampling site, invertebrate samples were collected for classical morphological studies (Morpho) using the multihabitat approach\u003csup\u003e43\u003c/sup\u003e, as well as water and biofilm samples for studies and comparisons through molecular determinations. Molecular data were obtained from biofilm samples (MolBio) at 3 sites, and water samples (MolWat) at 5 sites. Additionally, one sample was collected to determine bottom organic matter content, and another sample was collected for granulometric analysis, using grabs suitable for the depth and substrate characteristics of each patch\u003csup\u003e44\u003c/sup\u003e. \u0026nbsp;Besides, environmental variables were measured \u003cem\u003ein situ\u003c/em\u003e at all sites, and physicochemical analyses were conducted in the laboratory. The biological material was collected under the provincial permit granted by Res. Prov. N⁰ 025/20 del Ministerio de Ambiente y Cambio Climático, Gobierno de la Provincia de Santa Fe, Argentina, for non-commercial purposes.\u003c/p\u003e\n\u003cp\u003eEnvironmental parameters\u003c/p\u003e\n\u003cp\u003eRivers were categorized into low, medium, and high discharge (Qm\u0026lt;100 m\u003csup\u003e3\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e, Qm 100-500 m\u003csup\u003e3\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e, Qm\u0026gt;500m\u003csup\u003e3\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e, respectively) while channel widths were measured using satellite images taken on the sampling dates\u003csup\u003e45\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLimnological parameters were measured \u003cem\u003ein situ\u003c/em\u003e, including dissolved oxygen (O2 in ppm), pH, conductivity (C in µS/cm), and temperature with a HQ40D – Multi/2 Channels system (Hach®). Water transparency (Se in meters) and depth (Z in meters) were measured with a Secchi disk and a measuring tape.\u003c/p\u003e\n\u003cp\u003eBottom sediment granulometry was categorized by observation as sandy (\u0026gt;50% sand), silt-clayey (20-50% sand), or sand-silt-clay (\u0026lt;20% sand)\u003csup\u003e46\u003c/sup\u003e. Benthic organic matter in the sediments (BPOM) was also assessed by observation in the field.\u003c/p\u003e\n\u003cp\u003eWater samples were taken from each site to assess concentrations of inorganic dissolved nutrients, biological oxygen demand (BOD\u003csub\u003e5\u003c/sub\u003e), and chemical oxygen demand (COD). These samples were immediately transported to the laboratory under cold and dark conditions. The combined nitrate and nitrite (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e + NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e–N) were analyzed by reducing nitrate with hydrazine sulfate, followed by nitrite determination through diazotization with sulfanilamide. Ammonium (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N) was measured using the indophenol blue method\u003csup\u003e47\u003c/sup\u003e, and soluble reactive phosphorus (SRP) was determined by the ascorbic acid method\u003csup\u003e48\u003c/sup\u003e. BOD\u003csub\u003e5\u003c/sub\u003e (mg L-1) was measured\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eusing an LDO BOD probe (model LBOD 10101, HACH), with oxygen consumption during the 5-day incubation period calculated by the difference between initial and final values\u003csup\u003e49\u003c/sup\u003e. For COD estimation, water samples were digested for two hours at 150 °C with K\u003csub\u003e2\u003c/sub\u003eCr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e in an acidic medium. The colorimetric determination was carried out at 420 nm, with results expressed in mg L\u003csup\u003e−1 49\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eMorphological taxonomy (Morpho)\u003c/p\u003e\n\u003cp\u003eFor classical morphological analyses, a multi-habitat sampling approach was employed\u003csup\u003e43\u003c/sup\u003e. Six samples of invertebrates from bottom sediments (infaunal benthos, episammon, epilithon, epipelon) and associated with macrophytes (pleuston, epiphyton) were collected from various available habitats, including littoral and limnetic bottom sediments as well as from stands of macrophytes (Table 1). The plant species were identified according to Schneider et al. (2021)\u003csup\u003e50\u003c/sup\u003e, and the reference collection deposited in the Laboratorio de Macrófitas, Instituto Nacional de Limnología (INALI-CONICET-UNL).\u003c/p\u003e\n\u003cp\u003eAll samples were sieved through a 200 µm mesh size and preserved in the field with 10% formaldehyde. In the laboratory, invertebrates were hand-picked from the samples using a stereomicroscope and subsequently stored in 70% ethanol for identification and counting.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMolecular eDNA Taxonomy (MolBio and MolWat)\u003c/p\u003e\n\u003cp\u003eAt each site, 100 ml of water was collected with a sterile bottle in the littoral zone near the bottom without disturbing the sediments. All samples were kept cool (4 °C) until processing. \u0026nbsp;In the laboratory, each water sample was poured into a beaker. The beaker was decontaminated with 10% bleach. All water samples were vacuum filtered using an MSI, Acetate Plus, Supported Plain Membrane Filter with a 0.22-μm pore size, including a negative control consisting of clean water. The filtration system was cleaned with commercial chlorine-based 10% bleach between samples to avoid contamination between sampling sites. Lastly, the filters were placed into 2 ml Eppendorf tubes using sterile forceps and stored at −80 °C until DNA extraction. The filters were processed following the method described by Amavet et al.\u003csup\u003e51\u003c/sup\u003e. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProtocols for biofilm sampling were adjusted following Rivera et al.\u003csup\u003e13\u003c/sup\u003e. Integrated samples were taken from the bottom sediments and aquatic macrophytes. In the field, subsamples of available patches (i.e. epipelon, episammon, epiphyton) were identified and collected. Epipelon and epissamon were collected from the most superficial layer of bottom sediments with a core and then stored in a Falcon previously rinsed with alcohol. Epiphyton samples were collected from the submerged parts of the most representative macrophyte species, which were cut using scissors and sterile forceps\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(rinsed with hypochlorite in the field) and placed in sterile jars. In the laboratory, the collected epiphyton samples were sonicated for 3 cycles of 2 minutes in a TestLabr ® ultrasonic washer after ultrapure water was added. Epiphyton, epipelon, and/or episammon were integrated and centrifuged at 500 rpm for 10 minutes, eliminating the supernatant. The pellet was homogenized and fractionated into 1.1 ml samples, then centrifuged at 8000 g for 10 minutes at 4 ºC in a Sartorius® refrigerated centrifuge. The supernatant was removed, and the samples were stored at -80 ºC in an ultra-cold freezer until DNA extraction. To extract genetic material from biofilm samples, we used the PURO Soil Kit (Productos Bio-Lógicos®) following the standardized methods.\u003c/p\u003e\n\u003cp\u003eThe quality and concentration of the obtained DNA extracts were analyzed by electrophoresis on 0.8% agarose gels and a spectrophotometer Gene Quant II (Pharmacia Biotech®) using the ratio of absorbance at 260 nm and 280 nm.\u003c/p\u003e\n\u003cp\u003eThen, the DNA extracts were analyzed by EcoMol Consultoria (Piracicaba, Brazil) \u0026nbsp;using two primer pairs to amplify two fragments from the COI mtDNA gene: \u0026nbsp;the pair 1 -fwhF2/fwhR2n-\u003csup\u003e34\u003c/sup\u003e to amplify a fragment of 205 bp and the pair 2 -MG2_LCO1490_F/MG2_univ_R- \u003csup\u003e52,53\u003c/sup\u003e to amplify a fragment of 133 bp.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe eDNA samples were purified using magnetic beads (Agencourt AMPure XP® – Beckman Coulter) and then amplified. PCR conditions were: 40 cycles consisting of 95 °C for 30 seconds, 55 °C for 30 seconds, 72 °C for 2 minutes, with initial denaturation of 95°C for 5 minutes and final extension of 72 °C for 10 minutes. The master mix included 0.8 μl of each primer 10 μM and 2 μl of DNA in 20 μl of final volume, using negative controls. The PCR products were purified and amplified using the primer pair, together with adaptors from Nextera Index kit® (Illumina). Once purified and normalized, these PCR products were pooled into a single sequencing pool. Using real-time PCR, performed with the KAPA Biosystems Quantification Kit reagent (Illumina), the pool was quantified, diluted to a concentration of 2nM, and quantified again to confirm the final concentration. The final solution was diluted and loaded onto the iSeq100® equipment (Illumina), using the iSeq100 v2 sequencing kit, 300 cycles (2x150bp), and 30% phiX.\u003c/p\u003e\n\u003cp\u003eRaw\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003esequencing data were demultiplexed into forward and reverse read files. The sequences generated for each primer pair were combined in all possible orientations: forward, complement, reverse, and reverse complement. Using the Cutadapt\u003csup\u003e54\u003c/sup\u003e and DADA2\u003csup\u003e55\u003c/sup\u003e programs, several cleaning procedures were performed. The pipeline was organized in R\u003csup\u003e56\u003c/sup\u003e. The reads were subjected to the removal of sequences containing undefined bases (Ns), as well as the removal of primer sequences, and sequencing-related errors were also identified and removed. From the remaining sequences of this quality control, unique sequences present in the samples were identified. These sequences are called ASVs (Amplicon Sequencing Variants) and represent the amplification products of the DNAs used in the construction of the library, which were subjected to taxonomic identification. Using the DADA2 and phyloseq packages\u003csup\u003e57\u003c/sup\u003e, each of the ASVs was assigned a count relative to its frequency in the library, as well as a classification based on the association with sequences from the public reference banks NCBI and BOLD SYSTEMS (https://www.ncbi.nlm.nih.gov/;\u0026nbsp;\u003ca href=\"https://www.boldsystems.org/\"\u003ehttps://www.boldsystems.org/\u003c/a\u003e). These associations were made using the classifier developed by Wang et al.\u003csup\u003e58\u003c/sup\u003e. To define the final taxonomic classification at each sampling site, we followed these criteria: Species level: identity (sequence similarity with the reference sequence in the database) above 98% (ID \u0026gt; 98%) and relative incompleteness (number of reads generated concerning the total generated for the sample) greater than 0.02 (relative abundance \u0026gt; 2%); \u0026nbsp; Genus level: sequence identity above 95% (ID \u0026gt; 95%); \u0026nbsp;Family and Order levels: sequence identity with the reference sequence in the database less than 95% and greater than 80% (80% \u0026lt; ID \u0026lt; 95%).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Data Analysis\u003c/p\u003e\n\u003cp\u003eThe following biotic indices were calculated using the metrix package\u003csup\u003e59\u003c/sup\u003e: Biological Monitoring Working Party (BMWP)\u003csup\u003e16\u003c/sup\u003e; Average Score Per Taxon (ASPT)\u003csup\u003e16\u003c/sup\u003e; Biological Monitoring Working Party prime (BMWP’)\u003csup\u003e17\u003c/sup\u003e; Biological Monitoring Working Party double prime (BMWP”)\u003csup\u003e18\u003c/sup\u003e;\u0026nbsp;\u003cem\u003eÍndice da Comunidade Bentônica para rios\u003c/em\u003e (ICBrio)\u003csup\u003e20\u003c/sup\u003e;\u0026nbsp;\u003cem\u003eÍndice de Macroinvertebrados en Ríos Pampeanos\u003c/em\u003e (IMRP)\u003csup\u003e19\u003c/sup\u003e. Additionally, the Index of Benthic Invertebrates in Saline Rivers (IBIS) was calculated\u003csup\u003e21\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine whether there were significant differences between the indices based on the type of analysis at the sites, a factorial analysis of variance (ANOVA) was performed, followed by post hoc pairwise comparison with Bonferroni correction (p\u0026lt;0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll analyses were conducted in R and R Studio\u003csup\u003e56\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was financially supported by Ministerio de Ciencia y Técnica (MinCyT, Argentina), Proyecto PICT 2018 03744 and Agencia Santafesina de Ciencia y Tecnología (Sectei, Provincia de Santa Fe, Argentina), Proyecto PEICi+d 2021037.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was financially supported by Ministerio de Ciencia y Técnica (MinCyT, Argentina), Proyecto PICT 2018 03744 and Agencia Santafesina de Ciencia y Tecnología (Sectei, Provincia de Santa Fe, Argentina), Proyecto PEICi+d 2021037.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eF.Z., M.V.P.M., M.L., and P.A. obtained samples and performed analysis. J.C. obtained biological indices. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are available in the CONICET Institutional Repository, https://ri.conicet.gov.ar/handle/XXXXX/XXXXX.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlmond, R. E., Grooten, M. \u0026amp; Peterson, T. Living Planet Report 2020 \u0026ndash; Bending the curve of biodiversity loss. World Wildlife Fund. Gland, Switzerland: World Wildlife Fund. ISBN 978-2-940529-99-5 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWallace, J. B. \u0026amp; Webster, J. R. The role of macroinvertebrates in stream ecosystem function. \u003cem\u003eAnnu. Rev. Entomol.\u003c/em\u003e \u003cb\u003e41\u003c/b\u003e, 115\u0026ndash;139 (1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDominguez, E. et al. Biomonitoreo en r\u0026iacute;os de Argentina: un camino por recorrer. \u003cem\u003eEcol. Austral\u003c/em\u003e. \u003cb\u003e8\u003c/b\u003e, 934\u0026ndash;949 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonada, N., Prat, N., Resh, V. H. \u0026amp; Statzner, B. Developments in aquatic insect biomonitoring: a comparative analysis of recent approaches. \u003cem\u003eAnnu. Rev. Entomol.\u003c/em\u003e \u003cb\u003e51\u003c/b\u003e, 495\u0026ndash;523 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBashnin, T. et al. Relationship between pesticide accumulation in transplanted zebra mussels (\u003cem\u003eDreissena polymorpha\u003c/em\u003e) and community structure of aquatic macroinvertebrates. \u003cem\u003eEnviron. Pollut\u003c/em\u003e. \u003cb\u003e252\u003c/b\u003e, 591\u0026ndash;598 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeeley, H. B. et al. A national macroinvertebrate dataset collected for the biomonitoring of Ireland\u0026rsquo;s river network, 2007\u0026ndash;2018. \u003cem\u003eSci. Data\u003c/em\u003e. \u003cb\u003e7\u003c/b\u003e, 280. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41597-020-00618-8\u003c/span\u003e\u003cspan address=\"10.1038/s41597-020-00618-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCIC (Comit\u0026eacute; Intergubernamental Coordinador de los Pa\u0026iacute;ses de la Cuenca del Plata). Programa de Acci\u0026oacute;n Estrat\u0026eacute;gica para la Cuenca del Plata. Buenos Aires, Argentina. 188 pp. (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAutoridad de Cuenca Matanza Riachuelo (ACUMAR). Informe de Monitoreo Ambiental de la Cuenca Matanza-Riachuelo. Autoridad de Cuenca Matanza-Riachuelo, Buenos \u0026amp; Aires Argentina. (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.acumar.gob.ar/monitoreo-ambiental/\u003c/span\u003e\u003cspan address=\"https://www.acumar.gob.ar/monitoreo-ambiental/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoyntz-Wright, I. P., Harrison, X. A., Pedersen, S. \u0026amp; Tyler, C. R. Effectiveness of eDNA for monitoring riverine macroinvertebrates. \u003cem\u003eSci. Total Environ.\u003c/em\u003e \u003cb\u003e941\u003c/b\u003e, 173621. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scitotenv.2024.173621\u003c/span\u003e\u003cspan address=\"10.1016/j.scitotenv.2024.173621\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSweeney, B. W., Battle, J. M., Jackson, J. K. \u0026amp; Dapkey, T. Can DNA barcodes of stream macroinvertebrates improve descriptions of community structure and water quality? \u003cem\u003eJ. N Am. Benthol Soc.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e, 195\u0026ndash;216 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaberlet, P., Coissac, E., Hajibabaei, M., Rieseberg, L. H. \u0026amp; Environmental, D. N. A. \u003cem\u003eMol. Ecol.\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e, 2155\u0026ndash;2160 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElbrecht, V., Vamos, E. E., Meissner, K., Aroviita, J. \u0026amp; Leese, F. Assessing strengths and weaknesses of DNA metabarcoding-based macroinvertebrate identification for routine stream monitoring. \u003cem\u003eMethods Ecol. Evol.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 1265\u0026ndash;1275 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRivera, S. et al. Fish eDNA metabarcoding from aquatic biofilm samples: Methodological aspects. \u003cem\u003eMol. Ecol. Res.\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e (4), 1440\u0026ndash;1453 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEzcurra de Drago, I. E., Marchese, M., Montalto, L. \u0026amp; Benthic invertebrates in \u003cem\u003eThe Middle Paran\u0026aacute; River: Limnology of a Subtropical Wetland\u003c/em\u003e (ed. Iriondo, M.H., Paggi, J.C. \u0026amp; Parma, M.J) 251\u0026ndash;275Springer,. (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNaumann, G. et al. The 2019\u0026ndash;2021 extreme drought episode in La Plata Basin in \u003cem\u003ePublications Office of the European Union\u003c/em\u003e (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2760/773\u003c/span\u003e\u003cspan address=\"10.2760/773\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArmitage, P. D., Moss, D., Wright, J. F. \u0026amp; Furse, M. T. The performance of a new biological water quality score system based on macroinvertebrates over a wide range of unpolluted running-water sites. \u003cem\u003eWater Res.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 333\u0026ndash;347 (1983).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlba-Tercedor, J. \u0026amp; S\u0026aacute;nchez-Ortega, A. Un m\u0026eacute;todo r\u0026aacute;pido y simple para evaluar la calidad biol\u0026oacute;gica de las aguas corrientes basado en el de Hellawell \u003cem\u003eLimnetica.\u003c/em\u003e 4, 51\u0026ndash;56 (1988). (1978).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLoyola, R. G. N. Atual est\u0026aacute;gio do IAP no uso de \u0026iacute;ndices biol\u0026oacute;gicos de qualidade. \u003cem\u003eAnais do V Simp\u0026oacute;sio de Ecossistemas Brasileiros: Conserva\u0026ccedil;\u0026atilde;o\u003c/em\u003e. \u003cb\u003e10\u003c/b\u003e, 46\u0026ndash;52 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodrigues Cap\u0026iacute;tulo, A. Los macroinvertebrados como indicadores de calidad de ambientes l\u0026oacute;ticos en el \u0026aacute;rea pampeana. \u003cem\u003eRev. Soc. Entom Arg\u003c/em\u003e. \u003cb\u003e58\u003c/b\u003e, 208\u0026ndash;217 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuhlmann, M., Imbimbo, H. V. \u0026amp; Ogura, L. L. \u003cem\u003eProtocolo para el biomonitoreo con las comunidades bent\u0026oacute;nicas de r\u0026iacute;os y reservorios del estado de S\u0026atilde;o Paulo\u003c/em\u003e (CESTEB, 2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCapeletti, J., Marchese, M. R. \u0026amp; Zilli, F. L. Evaluating macroinvertebrate metrics for ecological assessment of large saline rivers (Argentina). \u003cem\u003eEnviron. Sci. Pollut Res.\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e, 66464\u0026ndash;66476 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStoddard, J. L. et al. A process for creating multimetric indices for large-scale aquatic surveys. \u003cem\u003eJ. N Am. Benthol Soc.\u003c/em\u003e \u003cb\u003e27\u003c/b\u003e, 878\u0026ndash;889 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarqu\u0026iacute;n, J. et al. Coupling virtual watersheds with ecosystem services assessment: A 21st-century platform to support river research and management. \u003cem\u003eWiley Interdiscip Rev. : Water\u003c/em\u003e. \u003cb\u003e2\u003c/b\u003e, 609\u0026ndash;621 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePawlowski, J. et al. The future of biotic indices in the ecogenomic era: integrating (e)DNA metabarcoding in biological assessment of aquatic ecosystems. \u003cem\u003eSci. Total Environ.\u003c/em\u003e \u003cb\u003e637\u003c/b\u003e, 1295\u0026ndash;1310 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCraine, J. M. et al. Surveying tropical faunal diversity via airborne DNA analyses. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, 36701. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-025-20767-3\u003c/span\u003e\u003cspan address=\"10.1038/s41598-025-20767-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, G., Kang, Y. \u0026amp; Song, Y. Multi-primer-based environmental DNA survey for vertebrate biodiversity monitoring in protected island wetlands. \u003cem\u003eGlob Ecol. Conserv.\u003c/em\u003e \u003cb\u003e58\u003c/b\u003e, e03474. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.gecco.2025.e03474\u003c/span\u003e\u003cspan address=\"10.1016/j.gecco.2025.e03474\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeiner, K. \u0026amp; Altermatt, F. Transport Distance of Invertebrate Environmental DNA in a Natural River. \u003cem\u003ePLOS ONE\u003c/em\u003e. \u003cb\u003e9\u003c/b\u003e (2), e88786. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0088786\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0088786\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeiner, K., Walser, J. C., M\u0026auml;chler, E. \u0026amp; Altermatt, F. Choice of capture and extraction methods affect detection of freshwater biodiversity from environmental DNA. \u003cem\u003eBiol. Conserv.\u003c/em\u003e \u003cb\u003e183\u003c/b\u003e, 53\u0026ndash;63 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeray, M., Knowlton, N., Ho, S. L., Nguyen, B. N. \u0026amp; Machida, R. J. GenBank is a reliable resource for 21st-century biodiversity research. \u003cem\u003eProc. Nat. Acad. Sci. U.S.A.\u003c/em\u003e 116, 22651\u0026ndash;22656 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeigand, H. et al. DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work. \u003cem\u003eSci. Total Environ.\u003c/em\u003e \u003cb\u003e678\u003c/b\u003e, 499\u0026ndash;524 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCurry, C. J., Gibson, J. F., Shokralla, S., Hajibabaei, M. \u0026amp; Baird, D. J. Identifying North American freshwater invertebrates using DNA barcodes: Are existing COI sequence libraries fit for purpose? \u003cem\u003eFreshw. Sci.\u003c/em\u003e \u003cb\u003e37\u003c/b\u003e, 178\u0026ndash;189 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarnes, M. A. \u0026amp; Turner, C. R. The ecology of environmental DNA and implications for conservation genetics. \u003cem\u003eConserv. Genet.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 1\u0026ndash;17 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaberlet, P., Bonin, A., Zinger, L., Coissac, E. \u0026amp; Environmental, D. N. A. \u003cem\u003eFor biodiversity research and monitoring\u003c/em\u003e (Oxford University Press, 2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVamos, E., Elbrecht, V. \u0026amp; Leese, F. Short COI markers for freshwater macroinvertebrate metabarcoding. \u003cem\u003eMetabarcoding Metagenom\u003c/em\u003e. \u003cb\u003e1\u003c/b\u003e, e14625. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3897/mbmg.1.14625\u003c/span\u003e\u003cspan address=\"10.3897/mbmg.1.14625\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeese, F. et al. Improved freshwater macroinvertebrate detection from environmental DNA through minimized nontarget amplification. \u003cem\u003eEnviron. DNA\u003c/em\u003e. \u003cb\u003e3\u003c/b\u003e, 261\u0026ndash;276 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElbrecht, V. \u0026amp; Leese, F. Can DNA-based ecosystem assessments quantify species abundance? Testing primer bias and biomass-sequence relationships with an innovative metabarcoding protocol. \u003cem\u003ePLOS ONE\u003c/em\u003e. \u003cb\u003e10\u003c/b\u003e, e0130324. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0130324\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0130324\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalter, I., Joensen, M., Kristiansen, R., Steingrund, P. \u0026amp; Vestergaard, P. Environmental DNA concentrations are correlated with regional biomass of Atlantic cod in oceanic waters. \u003cem\u003eCommun. Biol.\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e, 461. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s42003-019-0696-8\u003c/span\u003e\u003cspan address=\"10.1038/s42003-019-0696-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYates, M. C. et al. The relationship between eDNA particle concentration and organism abundance in nature is strengthened by allometric scaling. \u003cem\u003eMol. Ecol.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e, 3068\u0026ndash;3082 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Bochove, K. et al. Organic matter reduces the amount of detectable environmental DNA in freshwater. \u003cem\u003eEcol. Evol.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 3647\u0026ndash;3654 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuppert, K. M., Kline, R. J. \u0026amp; Rahman, M. S. Past, present, and future perspectives of environmental DNA (eDNA) metabarcoding: A systematic review in methods, monitoring, and applications of global eDNA. \u003cem\u003eGlobal Ecol. Conserv.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, e00547. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.gecco.2019.e00547\u003c/span\u003e\u003cspan address=\"10.1016/j.gecco.2019.e00547\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBiasatti, N. R. et al. \u003cem\u003eLas ecoregiones, su conservaci\u0026oacute;n y las \u0026aacute;reas naturales protegidas de la provincia de Santa Fe\u003c/em\u003e (Ministerio de Medio Ambiente, 2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarchetti, Z. Y., Latrubesse, E. M., Pereira, M. S. \u0026amp; Ramonell, C. G. Vegetation and its relationship with geomorphologic units in the Paran\u0026aacute; River floodplain, Argentina. \u003cem\u003eJ. S Am. Earth Sci.\u003c/em\u003e \u003cb\u003e46\u003c/b\u003e, 122\u0026ndash;136 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarbour, M. T., Gerritsen, J., Blaine, S. D. \u0026amp; Stribling, J. B. in \u003cem\u003eRapid Bioassessment Protocols for Use in Streams and Wadeable Rivers: Periphyton, Benthic Macroinvertebrates, and Fish\u003c/em\u003e. (eds (2\u0026ordf;) (U.S. Environmental Protection Agency, Office of Water, 1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnderson, J. T. et al. Sampling and processing aquatic and terrestrial invertebrates in wetlands. in Wetland Techniques, Volume 2: Organisms (eds Anderson, J. T. \u0026amp; Craig) A. D) 143\u0026ndash;195. (Springer, (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoogle, E. Pro; earth.google.com/web/ (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Bertoldi, H. Caracter\u0026iacute;sticas limnol\u0026oacute;gicas del R\u0026iacute;o Paran\u0026aacute; y sus principales tributarios en el tramo Goya-Diamante. Los sedimentos de fondo y su fauna. \u003cem\u003eNatura Neotropicalis\u003c/em\u003e, \u003cb\u003e1\u003c/b\u003e(17) (1986).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoroleff, F. Revised version of Direct determination of ammonia in natural waters as indophenol blue, Int. Con. Explor. Sea, C. M. 1969/C:9. ICES Information on Techniques and Methods for Sea Water Analysis. \u003cem\u003eInterlab. Rep\u003c/em\u003e. 3, 19\u0026ndash;22 (1970).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurphy, J. \u0026amp; Riley, J. P. A modified single solution method for the determination of phosphate in natural waters. \u003cem\u003eAnal. Chim. Acta\u003c/em\u003e. \u003cb\u003e27\u003c/b\u003e, 31\u0026ndash;36 (1962).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAPHA. Standard Methods for the Examination of Water and Wastewater (23rd ed.). American Public Health Association. (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchneider, B., Hadad, H. \u0026amp; Rodriguez, E. Plantas acu\u0026aacute;ticas del r\u0026iacute;o Paran\u0026aacute; Medio. Colecci\u0026oacute;n C\u0026aacute;tedra, Universidad Nacional del Litoral; (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://hdl.handle.net/11185/6737\u003c/span\u003e\u003cspan address=\"https://hdl.handle.net/11185/6737\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmavet, P., Poletta, G., Odetti, L., Marc\u0026oacute;, M. V. P. \u0026amp; Siroski, P. Detection of the maned wolf, a cryptic and vulnerable species, through environmental DNA studies. \u003cem\u003eJ. Nat. Conserv.\u003c/em\u003e \u003cb\u003e74\u003c/b\u003e, 1\u0026ndash;5 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGillet, F. et al. A new method to identify the endangered Pyrenean desman (\u003cem\u003eGalemys pyrenaicus\u003c/em\u003e) and to study its diet, using next generation sequencing from faeces. \u003cem\u003eMamm. Biol.\u003c/em\u003e \u003cb\u003e80\u003c/b\u003e, 505\u0026ndash;509 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTournayre, O. et al. In silico and empirical evaluation of twelve metabarcoding primer sets for insectivorous diet analyses. \u003cem\u003eEcol. Evol.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 6310\u0026ndash;6332 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. \u003cem\u003eEMBL\u003c/em\u003e, 17, 10\u0026ndash;12 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCallahan, B. J. et al. DADA2: High-resolution sample inference from Illumina amplicon data. \u003cem\u003eNat. Methods\u003c/em\u003e. \u003cb\u003e13\u003c/b\u003e, 581\u0026ndash;583 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR Core Team. R: A Language and Environment for Statistical Computing_. R Foundation for Statistical Computing, Vienna, Austria. \u0026lt; (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.R-project.org/\u003c/span\u003e\u003cspan address=\"https://www.R-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcMurdie, P. J. \u0026amp; Holmes, S. Phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. \u003cem\u003ePLOS ONE\u003c/em\u003e. \u003cb\u003e8\u003c/b\u003e, e61217. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0061217\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0061217\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Q., Garrity, G. M., Tiedje, J. M. \u0026amp; Cole, J. R. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. \u003cem\u003eAppl. Environ. Microbiol.\u003c/em\u003e \u003cb\u003e73\u003c/b\u003e, 5261\u0026ndash;5267 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCabrera, J. M. \u0026amp; Capeletti, J. Metrix': An R package for assessing the biological quality of water. \u003cem\u003eEcol. Modell\u003c/em\u003e. \u003cb\u003e484\u003c/b\u003e, 110473. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ecolmodel.2023.110473\u003c/span\u003e\u003cspan address=\"10.1016/j.ecolmodel.2023.110473\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Macroinvertebrates, Freshwater, Biomonitoring, Land use, Metabarcoding, Anthropogenic impacts","lastPublishedDoi":"10.21203/rs.3.rs-8787248/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8787248/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFreshwater biomonitoring requires rapid and reliable diagnostic tools to minimize ecological impacts. This study compares morphological identification with environmental DNA (eDNA) metabarcoding data to evaluate which method more efficiently assesses macroinvertebrate communities across contrasting land uses. Benthic macroinvertebrates were collected in the Paran\u0026aacute; River basin for morphological analysis, while water and biofilm samples were obtained for eDNA metabarcoding. A total of 125 amplicon sequence variants (ASVs) \u0026ndash; high-resolution DNA sequence clusters used as taxonomic units - were detected, most of which were identified to the species or morphospecies level. The eDNA biofilm-derived recovered a higher number of ASVs than water samples, where Annelida, Arthropoda, and Mollusca are the most diverse groups. Diversity metric (richness and community composition) and ecological status classifications were generally comparable between morphological and molecular approaches. The primers employed demonstrated good taxonomic resolution, supporting their usefulness in assessing river quality and enabling the application of region-specific biotic indices. Although metabarcoding performed similarly to morphological methods in assessing diversity patterns and ecological status, 70% of the taxa identified morphologically were not detected molecularly. These limitations, particularly the under-detection of key benthic phyla, indicate that in tropical rivers, an integrative framework combining both methods yields the most accurate evaluation of biodiversity and environmental health.\u003c/p\u003e","manuscriptTitle":"Innovative approaches in aquatic monitoring: a comparative study between eDNA analysis and morphological techniques in a river ecosystem","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-09 15:35:56","doi":"10.21203/rs.3.rs-8787248/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7358e3b7-82ed-459a-9608-2afbe4ecfaaf","owner":[],"postedDate":"March 9th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-05-15T05:43:31+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":63931255,"name":"Biological sciences/Ecology"},{"id":63931256,"name":"Earth and environmental sciences/Ecology"},{"id":63931257,"name":"Earth and environmental sciences/Environmental sciences"},{"id":63931258,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2026-05-15T05:55:24+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-09 15:35:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8787248","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8787248","identity":"rs-8787248","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}