Rivers are a vital resource for human wellbeing. To reduce human impact on water bodies, the European Union has established an essential regulatory framework for protection and sustainable management (WFD; 2000/60/EC). In this strategy, reliable and economic bioindicators are a fundamental component. Benthic macroinvertebrates are the group most commonly used as bioindicators through all European countries. However, their conventional assessment currently entails serious cost-efficiency limitations. In this study, we have tested the reliability of metabarcoding as a tool to record river macroinvertebrates using samples from a mock community (in vitro validation) and eDNA extracted for field validation from water from six sites within a north Iberian river (River Nalón, Asturias, Spain). Two markers (V4 region within the nuclear 18S rDNA and a fragment of the mitochondrial COI gene) were amplified and sequenced using an Illumina platform. The molecular technique has proven to be more sensitive than the visual one. A cost-benefit analysis shows that the metabarcoding approach is more expensive than conventional techniques for determining macroinvertebrate communities but requires fewer sampling and identification efforts. Our results suggest metabarcoding is a useful tool for alternative assessment of freshwater quality.

Background: Bioassessment of rivers is a fundamental method to determine surface water quality. One of the groups most commonly employed as bioindicators of aquatic ecosystems are benthic macroinvertebrates. Their conventional assessment is based on morphological identification and entails several limitations, such as being time‐consuming and requires trained experts for taxonomic identification. The use of genetic tools to solve these limitations offers an alternative way to evaluate rivers status. The use of environmental DNA (eDNA) metabarcoding has increased in recent years for different purposes, but its use in water quality evaluation is yet to be tested. Here, morphological and eDNA based inventories of macroinvertebrates were compared from the same seven sampling sites in the Upper Nalón River Basin (Asturias, Spain).Materials & Methods: High‐Throughput Sequencing (HTS) of the cytochrome oxidase subunit 1 (COI) gene was carried out on DNA from water samples using an Ion Torrent platform. Biotic water quality indices were calculated from morphological and molecular data and compared with independent physico‐chemical habitat assessment to validate eDNA based approach.Results: Highly positive and significant correlation was found between eDNA metabarcoding and morphological methods (Morphological and eDNA indices, r = 0.798, 5 degrees of freedom d.f., P = 0.031;) and a highly significant negative correlation was found between molecular and habitat quality indices (Stress score & eDNA, ρ = ‐0.878 and P = 0.009; Stress score & Visual, ρ = ‐0.949 and P = 0.0002).Discussion: The similarity of results from the two approaches and the correlation of eDNA metabarcoding data with the habitat quality indices, suggest that eDNA performs as well as conventional methods for calculating biotic indices in this system, positioning eDNA metabarcoding of macroinvertebrate communities to transform how river bioassessment can be achieved.Conclusion: The usefulness of eDNA metabarcoding to assess rivers water quality based on macroinvertebrates assessment has been demonstrated in a dammed river basin.

Background: Salmonids are native from the North Hemisphere but have been introduced for aquaculture and sport fishing in the South Hemisphere and inhabit most rivers and lakes in temperate and cold regions worldwide. Five species are included in the Global Invasive Species Database: rainbow troutOncorhynchus mykiss, Atlantic salmonSalmo salar, brown troutSalmo trutta, brook troutSalvelinus fontinalis, and lake troutSalvelinus namaycush. In contrast, other salmonids are endangered in their native settings.Methods: Here we have developed a method to identify salmonid species directly from water samples, focusing on the Iberian Peninsula as a case study. We have designed nested Salmonidae-specific primers within the 16S rDNA region. From these primers and a PCR-RFLP procedure the target species can be unequivocally identified from DNA extracted from water samples.Results: The method was validated in aquarium experiments and in the field with water from watersheds with known salmonid populations. Finally, the method was applied to obtain a global view of the Salmonidae community in Nalón River (north coast of Spain).Discussion: This new powerful, very sensitive (identifying the species down to 10 pg DNA/ml water) and economical tool can be applied for monitoring the presence of salmonids in a variety of situations, from checking upstream colonization after removal of river barriers to monitoring potential escapes from fish farms.

Ballast water is one of the main vectors of transport of nuisance species among marine ports. Neither treatment nor interchange completely reduces the risk of ballast water containing DNA from harmful species, being a signal of potential threat. However, although there are some efficient treatments, they are not available on all ships and there might be some technological/economical constrains for their active and routine usage. Understanding what routes lead to a higher risk of contamination is important for designing targeted surveillance. We analysed ballast water from seven ships arriving in Gijon port (south Bay of Biscay, Spain). DNA metabarcoding was employed for identification of exotic species and harmful algae. One ship carried DNA of 20 risk species in the ballast water. Three ships contained DNA of only one risk species, and three ships had none. Seventy two algae species were found, 22.2% are exotic to the Bay of Biscay and 11.1% are catalogued as harmful. The results demonstrated the importance of continuous surveillance of ballast water.