Taxonomic accuracy and complementarity between bulk and eDNA metabarcoding provides an alternative to morphology for biological assessment of freshwater macroinvertebrates.

Determining biological status of freshwater ecosystems is critical for ensuring ecosystem health and maintaining associated services to such ecosystems. Freshwater macroinvertebrates respond predictably to environmental disturbances and are widely used in biomonitoring programs. However, many freshwater species are difficult to capture and sort from debris or substrate and morphological identification is challenging, especially larval stages, damaged specimens, or hyperdiverse groups such as Diptera. The advent of high throughput sequencing technologies has enhanced DNA barcoding tools to automatise species identification for whole communities, as metabarcoding is increasingly used to monitor biodiversity. However, recent comparisons have revealed little congruence between morphological and molecular-based identifications. Using broad range universal primers for DNA barcode marker cox1, we compare community composition captured between morphological and molecular-based approaches from different sources - tissue-based (bulk benthic and bulk drift samples) and environmental DNA (eDNA, filtered water) metabarcoding - for samples collected along a gradient of anthropogenic disturbances. For comparability, metabarcoding taxonomic assignments were filtered by taxa included in the standardised national biological metric IBMWP. At the family level, bulk benthic metabarcoding showed the highest congruence with morphology, and the most abundant taxa were captured by all techniques. Richness captured by morphology and bulk benthic metabarcoding decreased along the gradient, whereas richness recorded by eDNA remained constant and increased downstream when sequencing bulk drift. Estimates of biological metrics were higher using molecular than morphological identification. At species level, diversity captured by bulk benthic samples were higher than the other techniques. Importantly, bulk benthic and eDNA metabarcoding captured different and complementary portions of the community - benthic versus water column, respectively - and their combined use is recommended. While bulk benthic metabarcoding can likely replace morphology using similar benthic biological indices, water eDNA will require new metrics because this technique sequences a different portion of the community.

Development of Macroinvertebrate Multimetric Index for Assessment of Large Rivers in Thailand.

Human activities are a significant threat to the health of river ecosystems, especially in developing countries. In Thailand, benthic macroinvertebrates have been widely used for bioassessment for aquatic ecosystem evaluation. However, most of them focuses on streams, which may not be applicable to large rivers. This study aimed to fill this gap by developing a macroinvertebrate multimetric index for Thailand's large rivers. Sampling was conducted in 15 rivers, and 41 sites across Thailand. Physico-chemical parameters, habitat characteristics, and macroinvertebrates were analyzed. Macroinvertebrate data were analyzed and metrics calculated. The seven selected core metrics, including Plecoptera taxa, EPT taxa, insect individuals %, Crustaceans and Mollusca individuals %, collector-gatherers taxa, intolerant taxa in BMWPThai, and Hilsenhoff Biotic Index, showed a strong response to anthropogenic disturbance. The final Thailand Large River Multimetric Index (TLMI) classified river health from "Excellent" to "Very Poor" condition. The validation of TLMI confirmed its ability to distinguish river health conditions. Overall, the TLMI can used as an assessing and monitoring ecological health tool for Thailand's large rivers, that can be applied for river management and conservation.

A Rapid and Cost-Effective Identification of Invertebrate Pests at the Borders Using MinION Sequencing of DNA Barcodes.

The rapid and accurate identification of invertebrate pests detected at the border is a challenging task. Current diagnostic methods used at the borders are mainly based on time consuming visual and microscopic examinations. Here, we demonstrate a rapid in-house workflow for DNA extraction, PCR amplification of the barcode region of the mitochondrial cytochrome oxidase subunit I (COI) gene and Oxford Nanopore Technologies (ONT) MinION sequencing of amplified products multiplexed after barcoding on ONT Flongle flow cells. A side-by-side comparison was conducted of DNA barcode sequencing-based identification and morphological identification of both large (>0.5 mm in length) and small (<0.5 mm in length) invertebrate specimens intercepted at the Australian border. DNA barcode sequencing results supported the morphological identification in most cases and enabled immature stages of invertebrates and their eggs to be identified more confidently. Results also showed that sequencing the COI barcode region using the ONT rapid sequencing principle is a cost-effective and field-adaptable approach for the rapid and accurate identification of invertebrate pests. Overall, the results suggest that MinION sequencing of DNA barcodes offers a complementary tool to the existing morphological diagnostic approaches and provides rapid, accurate, reliable and defendable evidence for identifying invertebrate pests at the border.

Over 90 endangered fish and invertebrates are caught in industrial fisheries.

Industrial-scale harvest of species at risk of extinction is controversial and usually highly regulated on land and for charismatic marine animals (e.g. whales). In contrast, threatened marine fish species can be legally caught in industrial fisheries. To determine the magnitude and extent of this problem, we analyze global fisheries catch and import data and find reported catch records of 91 globally threatened species. Thirteen of the species are traded internationally and predominantly consumed in European nations. Targeted industrial fishing for 73 of the threatened species accounts for nearly all (99%) of the threatened species catch volume and value. Our results are a conservative estimate of threatened species catch and trade because we only consider species-level data, excluding group records such as 'sharks and rays.' Given the development of new fisheries monitoring technologies and the current push for stronger international mechanisms for biodiversity management, industrial fishing of threatened fish and invertebrates should no longer be neglected in conservation and sustainability commitments.

River health assessment of the Yellow River source region, Qinghai-Tibetan Plateau, China, based on tolerance values of macroinvertebrates.

For decades, the river health of the Yellow River source region (YRSR) on the Qinghai-Tibetan Plateau has been a focal issue owing to its unique geographic location and ecological functions. This study investigated the ecological status of the headwater streams, the main stem, and the tributaries of the Yellow River in the YRSR using the tolerance values of macroinvertebrates and those related to biotic indices. The macroinvertebrate assemblages of the headwater streams were characterized by lower biodiversity than the tributaries downstream, based on comparisons of taxonomical composition, functional feeding group composition, and the pollution-tolerant capacity of taxa. The headwater streams had a lower ratio (16%) of pollution-sensitive macroinvertebrate taxa than that of the tributaries downstream (30%). The biotic indices (family- and genus-level biotic indices) indicated that the ecological health of the headwater streams was comparably poorer than that of the downstream tributaries. The combined effect of vulnerable natural conditions and increasing human disturbance is likely the main cause of eco-environmental degradation in the Yellow River headwater streams.

The use of metabarcoding for meiofauna ecological patterns assessment.

Marine meiofauna comprises up to 22 phyla. Its morphological identification requires time and taxonomists' expertise, and molecular tools can make this task faster. We aim to disentangle meiofaunal diversity patterns at Araçá Bay by applying a model selection approach and estimating the effectiveness of metabarcoding (18S rDNA) and morphological methods for estimating the response of meiofauna diversity in small-scale interactions with environmental variables. A rarefaction curve indicated that ten samples were sufficient for estimating the total number of meiofauna OTUs in a tidal flat. In both approaches, richness was predicted by mean sand percentage, sediment sorting, and bacteria concentration. Nematode genera composition differed significantly between approaches, the result of taxonomic mismatch in the genetic database. The similarity between the model selected for diversity descriptors, the richness of nematode genera and meiofauna composition emphasized the utility of predictive models for metabarcoding estimates to detect small-scale interactions of these organisms.

Ecological thresholds of hypoxia and sedimentary H2S in coastal soft-bottom habitats: A macroinvertebrate-based assessment.

Ecological thresholds of dissolved oxygen (DO) and sedimentary hydrogen sulfide (H2S) for macrozoobenthos were examined during a 30-month monitoring of two stations in a highly eutrophic canal in inner Tokyo Bay, Japan. Bottom DO and H2S concentrations fluctuated seasonally, and were significantly correlated with water and sediment temperatures. Red tide-derived phytodetritus was a major source of sediment organic matters in the canal bottom, and the sediment became highly reduced and sulfidic condition in warmer months (sedimentary H2S; up to 8.5 mM). Dominant opportunistic taxa, including polychaetes and amphipods, were eliminated under low DO and high H2S conditions (i.e., population thresholds), and devastation of community structure occurred at 2.4-3.3 mg l-1 DO and 1.8-2.7 mM H2S (i.e., community thresholds). To maintain ecosystem function in anthropogenically degraded habitats and ensure colonization by macrozoobenthos throughout the year, DO and H2S levels should be maintained below these thresholds.

Sorting specimen-rich invertebrate samples with cost-effective NGS barcodes: Validating a reverse workflow for specimen processing.

Biologists frequently sort specimen-rich samples to species. This process is daunting when based on morphology, and disadvantageous if performed using molecular methods that destroy vouchers (e.g., metabarcoding). An alternative is barcoding every specimen in a bulk sample and then presorting the specimens using DNA barcodes, thus mitigating downstream morphological work on presorted units. Such a "reverse workflow" is too expensive using Sanger sequencing, but we here demonstrate that is feasible with an next-generation sequencing (NGS) barcoding pipeline that allows for cost-effective high-throughput generation of short specimen-specific barcodes (313 bp of COI; laboratory cost <$0.50 per specimen) through next-generation sequencing of tagged amplicons. We applied our approach to a large sample of tropical ants, obtaining barcodes for 3,290 of 4,032 specimens (82%). NGS barcodes and their corresponding specimens were then sorted into molecular operational taxonomic units (mOTUs) based on objective clustering and Automated Barcode Gap Discovery (ABGD). High diversity of 88-90 mOTUs (4% clustering) was found and morphologically validated based on preserved vouchers. The mOTUs were overwhelmingly in agreement with morphospecies (match ratio 0.95 at 4% clustering). Because of lack of coverage in existing barcode databases, only 18 could be accurately identified to named species, but our study yielded new barcodes for 48 species, including 28 that are potentially new to science. With its low cost and technical simplicity, the NGS barcoding pipeline can be implemented by a large range of laboratories. It accelerates invertebrate species discovery, facilitates downstream taxonomic work, helps with building comprehensive barcode databases and yields precise abundance information.

Biases encountered in long-term monitoring studies of invertebrates and microflora: Australian examples of protocols, personnel, tools and site location.

Monitoring forms the basis for understanding ecological change. It relies on repeatability of methods to ensure detected changes accurately reflect the effect of environmental drivers. However, operator bias can influence the repeatability of field and laboratory work. We tested this for invertebrates and diatoms in three trials: (1) two operators swept invertebrates from heath vegetation, (2) four operators picked invertebrates from pyrethrum knockdown samples from tree trunk and (3) diatom identifications by eight operators in three laboratories. In each trial, operators were working simultaneously and their training in the field and laboratory was identical. No variation in catch efficiency was found between the two operators of differing experience using a random number of net sweeps to catch invertebrates when sequence, location and size of sweeps were random. Number of individuals and higher taxa collected by four operators from tree trunks varied significantly between operators and with their 'experience ranking'. Diatom identifications made by eight operators were clustered together according to which of three laboratories they belonged. These three tests demonstrated significant potential bias of operators in both field and laboratory. This is the first documented case demonstrating the significant influence of observer bias on results from invertebrate field-based studies. Examples of two long-term trials are also given that illustrate further operator bias. Our results suggest that long-term ecological studies using invertebrates need to be rigorously audited to ensure that operator bias is accounted for during analysis and interpretation. Further, taxonomic harmonisation remains an important step in merging field and laboratory data collected by different operators.

Genetic assessment of meiobenthic community composition and spatial distribution in coastal sediments along northern Gulf of Mexico.

Meiobenthic (meiofauna and micro-eukaryotes) organisms are important contributors to ecosystem functioning in aquatic environments through their roles in nutrient transport, sediment stability, and food web interactions. Despite their ecological importance, information pertaining to variation of these communities at various spatial and temporal scales is not widely known. Many studies in the Gulf of Mexico (GOM) have focused either on deep sea or continental shelf areas, while little attention has been paid to bays and coastal regions. Herein, we take a holistic approach by using high-throughput sequencing approaches to examine spatial variation in meiobenthic communities within Alabama bays and the coastal northern GOM region. Sediment samples were collected along three transects (Mississippi Sound: MS, FOCAL: FT, and Orange Beach: OB) from September 2010 to April 2012 and community composition was determined by metabarcoding the V9 hypervariable region of the nuclear18S rRNA gene. Results showed that Stramenopiles (diatoms), annelids, arthropods (copepods), and nematodes were the dominate groups within samples, while there was presence of other phyla throughout the dataset. Location played a larger role than time sampled in community composition. However, samples were collected over a short temporal scale. Samples clustered in reference to transect, with the most eastern transect (OB) having a distinct community composition in comparison to the other two transects (MS and FT). Communities also differed in reference to region (Bay versus Shelf). Bulk density and percent inorganic carbon were the only measured environmental factors that were correlated with community composition.

Trophic status and meiofauna biodiversity in the Northern Adriatic Sea: Insights for the assessment of good environmental status.

The Descriptor 5 (Eutrophication) of the EU Marine Strategy Framework Directive aims at preventing the negative effects of eutrophication. However, in coastal systems all indicators based on water column parameters fail in identifying the trophic status and its effects on biodiversity and ecosystem functioning. We investigated benthic trophic status, in terms of sedimentary organic matter quantity, composition and quality, along with meiofaunal abundance, richness of taxa and community composition in three coastal sites (N Adriatic Sea) affected by different levels of anthropogenic stressors. We show that, on the basis of organic matter quantity and composition, the investigated areas can be classified from oligo-to mesotrophic, whereas using meiofauna as a descriptor, their environmental quality ranged from sufficient to moderately impacted. Our results show that the benthic trophic status based on organic matter variables, is not sufficient to provide a sound assessment of the environmental quality in marine coastal ecosystems. However, data reported here indicate that the integration of the meiofaunal variable allows providing robust assessments of the marine environmental status.

Impact assessment of agricultural driven stressors on benthic macroinvertebrates using simulated data.

Agricultural land use poses a significant threat to the ecological integrity of rivers in Europe. Particularly in the Mediterranean, water abstraction and nutrient application are anthropogenic pressures that have a significant impact on aquatic habitats and biodiversity. In this article, we assessed the effects of agricultural management practices on benthic macroinvertebrates in a large river basin of central Greece using simulated data based on the application of SWAT (Soil Water Assessment Tool) model. Physicochemical and hydrological output variables of the model were used as predictors of the ASPT (Average Score Per Taxon) metric based on a correlated component regression analysis (CCR) built on empirical data. The estimation of ASPT was performed for the wet and dry seasons within a 20-year period for a total of 47 subbasins under the baseline conditions and after implementing three management scenarios that reduced: a) irrigation water applied to crops by 30%, b) chemical fertilization applied to crops by 30% and c) both irrigation and fertilization by 30%. The results revealed that application of the reduced irrigation resulted to a slight increase of the simulated dissolved inorganic nitrogen concentration (DIN), which in turn decreased the mean ASPT in 21 of the 47 subbasins implying a negative effect on the macroinvertebrate communities. On the contrary, the reduction of fertilization as well as the combined scenario decreased both the simulated DIN and phosphate concentration causing an increase of the mean ASPT for a total of 40 of the 47 subbasins. Based on these results, we suggest that the best management option is a combined practice of deficit irrigation and fertilization reduction since it improved water quality, increased ASPT values and saved a considerable amount of water. Overall, this work demonstrates a simple methodology that can efficiently assess the effects of agricultural management practices on biotic indicators.

Soil invertebrate community change over fuel-contaminated sites on a subantarctic island: An ecological field-based line of evidence for site risk assessment.

A number of fuel spills, of both recent and historic origins, have occurred on World Heritage-listed subantarctic Macquarie Island. Sites contaminated by mainly diesel fuels are undergoing remediation by the Australian Antarctic Division. The risks posed by these sites are being managed using a "weight of evidence" approach, for which this study provides a preliminary line of evidence for the ecological assessment component of this site management decision framework. This knowledge is pertinent, given the absence of environmental guidelines for fuel contaminants in subantarctic ecosystems. We provide a field-based, site-specific ecological risk assessment for soil invertebrate communities across the fuel spill sites, before the commencement of in situ remediation activities. Springtails (Collembola) were the most abundant taxa. Springtail community patterns showed only limited correlations with the level of fuel contamination at the soil surface, even when elevated levels occurred in the substratum layers. Of the environmental variables measured, community patterns were most strongly correlated with vegetation cover. We identify a suite of 6 species that contribute most to the community dynamics across these sites. A subset of these we propose as useful candidates for future development of single-species toxicity tests: Folsomotoma punctata, Cryptopygus caecus, Cryptopygus antarcticus and Parisotoma insularis. Findings from this study advance our understanding of soil invertebrate community dynamics within these contaminated sites, directly contributing to the improved management and restoration of the sites. Not only does this study provide an important line of evidence for the island's ecological risk assessment for fuel contaminants, it also enhances our understanding of the potential impact of fuels at other subantarctic islands.

Ecotoxicological risk assessment of chemical pollution in four Iberian river basins and its relationship with the aquatic macroinvertebrate community status.

Ecotoxicological risk assessment of chemical pollution in four Iberian river basins (Llobregat, Ebro, Júcar and Guadalquivir) was performed. The data set included more than 200 emerging and priority compounds measured at 77 sampling sites along four river basins studied. The toxic units (TU) approach was used to assess the risk of individual compounds and the concentration addition model (CA) to assess the site specific risk. Link between chemical pollution and aquatic macroinvertebrate communities in situ was examined by using four biological indexes; SPEAR ("Species at Risk Index") as the indicator of decline of sensitive species in relation to general organic (SPEARorganic) and pesticides (SPEARpesticides) pollution; and Shannon and Margalef biodiversity indexes. The results of the study suggested that organic chemicals posed the risk of acute effects at 42% of the sampling sites and the risk of chronic effects at all the sites. Metals posed the acute risk at 44% of the sites. The main drivers of the risk were mainly pesticides and metals. However, several emerging contaminants (e.g. the antidepressant drug sertraline and the disinfectant triclosan) were contributing to the chronic effects risk. When risk associated with metals and organic chemicals was compared, the latter dominated in 2010, mainly due to the presence of highly toxic pesticides, while metals did in 2011. Compounds that are not regulated on the European level were posing the risk of chronic effects at 23% of the sites. The decline of sensitive macroinvertebrate taxa expressed in terms of SPEAR index was correlated with the increase of toxic stress related to organic compounds Biodiversity indexes were negatively correlated with the metals and the urban land use type in the catchment.

Molecular Assessment of Bacterial Community Dynamics and Functional End Points during Sediment Bioaccumulation Tests.

Whole sediment toxicity tests play an important role in environmental risk assessment of organic chemicals. It is not clear, however, to what extent changing microbial community composition and associated functions affect sediment test results. We assessed the development of bacterial communities in artificial sediment during a 28 day bioaccumulation test with polychlorinated biphenyls, chlorpyrifos, and four marine benthic invertebrates. DGGE and 454-pyrosequencing of PCR-amplified 16S rRNA genes were used to characterize bacterial community composition. Abundance of total bacteria and selected genes encoding enzymes involved in important microbially mediated ecosystem functions were measured by qPCR. Community composition and diversity responded most to the time course of the experiment, whereas organic matter (OM) content showed a low but significant effect on community composition, biodiversity and two functional genes tested. Moreover, OM content had a higher influence on bacterial community composition than invertebrate species. Medium OM content led to the highest gene abundance and is preferred for standard testing. Our results also indicated that a pre-equilibration period is essential for growth and stabilization of the bacterial community. The observed changes in microbial community composition and functional gene abundance may imply actual changes in such functions during tests, with consequences for exposure and toxicity assessment.

Assessment of ecological quality of the Tajan River in Iran using a multimetric macroinvertebrate index and species traits.

The objectives of this study were to assess the biological water of the Iranian Tajan River using different metrics, i.e., a Multimetric Macroinvertebrate Index (MMI) and a traits-based method. Twenty-eight physico-chemical parameters, 10 habitat factors, and abundance of macroinvertebrates were obtained for 17 sites. The Shahid-Rajaie dam divides the Tajan River into an up- and downstream part, with different land uses. Eighteen metrics were used to represent four components of ecosystem quality, including tolerance (Hilsenhoff, SIGNAL), diversity (Margalef, Shannon-Wiener, Simpson, and Evenness), abundance (total number of taxa, individuals, Ephemeroptera, Plecoptera, Trichoptera, EPT, and Insects), and composition of assemblages (% Ephemeroptera, % Plecoptera, % Trichoptera, and % EPT Taxa). The integrated MMI was calculated by averaging the obtained scores of all indices. In the next step, we gathered information on 22 biological traits of macroinvertebrates to evaluate whether (group of) traits could be identified that are indicative for specific or general stress. Result showed a decrease in MMI from upstream (very good water quality) to downstream (bad) due to human activities. Industrial activities like pulping and papermaking operations or sand mining in the downstream part had more effects than agriculture and fish ponds in the upstream part. A redundancy analysis biplot showed the variation between the modalities of trait of macroinvertebrates and their correlation with physico-chemical parameters in Tajan River. The findings show that traits can be indicative for different kind of stress but that more effort has to be put in gathering data sets to disentangle the effect of habitat quality, pollution, and the physico-chemical properties of high- versus lowland rivers.

Ecological risk assessment for residual coal fly ash at Watts Bar Reservoir, Tennessee: Limited alteration of riverine-reservoir benthic invertebrate community following dredging of ash-contaminated sediment.

Benthic invertebrate communities were assessed after the December 2008 release of approximately 4.1 million m(3) coal fly ash from a disposal dredge cell at the Tennessee Valley Authority (TVA) Kingston Fossil Plant on Watts Bar Reservoir in Roane County, Tennessee, USA. Released ash filled the adjacent embayments and the main channel of the Emory River, migrating into reaches of the Emory, Clinch, and Tennessee Rivers. Dredging was completed in summer 2010, and the benthic community sampling was conducted in December 2010. This study is part of a series that supported an Ecological Risk Assessment for the Kingston site. Benthic invertebrate communities were sampled at transects spread across approximately 20 miles of river that includes both riverine and reservoirlike conditions. Community composition was assessed on a grab sample and transect basis across multiple cross-channel transects to gain an understanding of the response of the benthic community to a fly ash release of this magnitude. This assessment used invertebrate community metrics, similarity analysis, geospatial statistics, and correlations with sediment chemistry and habitat. The community composition was reflective of a reservoir system, with dominant taxa being insect larva, bivalves, and aquatic worms. Most community metric results were similar for ash-impacted areas and upstream reference areas. Variation in the benthic community was correlated more with habitat than with sediment chemistry or residual ash. Other studies have reported that a benthic community can take several years to a decade to recover from ash or ash-related constituents. Although released ash undoubtedly had some initial impacts on the benthic community in this study, the severity of these effects appears to be limited to the initial smothering of the organisms followed by a rapid response and the initial start of recovery postdredging.

Repeated rapid assessment surveys reveal contrasting trends in occupancy of marinas by non-indigenous species on opposite sides of the western English Channel.

Rapid assessment surveys of non-indigenous species (NIS) of sessile invertebrates were made at seven marinas in NW France and 10 marinas in SW England in 2010, and repeated in 2013. Fourteen NIS were recorded, 12 of which were seen on both coasts. Site occupancy differed between the opposite sides of the western English Channel. In Brittany, most species occurred at most sites in both 2010 and 2013. In 2010, site occupancy in Devon & Cornwall was distinctly lower; by 2013, the difference compared to Brittany had narrowed considerably, largely because of rapid colonisation of additional sites by species that were infrequent in 2010. Three more of the recent NIS are present in Devon & Cornwall but have still not become widespread. It is concluded that the recently introduced fouling animals studied here are longer established in NW France than in SW England, and have probably spread northwards across the Channel.

Ecological risk assessment for residual coal fly ash at Watts Bar Reservoir, Tennessee: Site setting and problem formulation.

A baseline ecological risk assessment (BERA) was performed for residual ash in the Watts Bar Reservoir following a release of fly ash from the Tennessee Valley Authority (TVA) Kingston Fossil Plant. The site consists of parts of 3 rivers in eastern Tennessee comprising over 32 river kilometers. The purpose of the assessment was to determine if residual ash negatively impacts maintenance and reproduction of balanced communities or populations of potentially exposed ecological receptor groups in these rivers. This introductory article summarizes the site and environmental setting, assessment and measurement endpoints, risk characterization methods, and the study approach. Subsequent articles describe ecological risks to fish, benthic invertebrates, aquatic- and riparian-feeding wildlife, and aerial-feeding insectivores; and the role ecological risk characterization played in determining the most effective management of the residual ash, setting project remediation objectives and targets, and designing long-term monitoring to measure the effectiveness of the selected removal action.

An alert system for triggering different levels of coastal management urgency: Tunisia case study using rapid environmental assessment data.

Rapid environmental assessment (REA) involves scoring abundances of ecosystems/species groups and magnitude of pressures, concurrently, using the same logarithmic (0-6) assessment scale. We demonstrate the utility of REA data for an alert system identifying different levels of coastal management concern. Thresholds set for abundances/magnitudes, when crossed, trigger proposed responses. Kerkennah, Tunisia, our case study, has significant natural assets (e.g. exceptional seagrass and invertebrate abundances), subjected to varying levels of disturbance and management concern. Using REA thresholds set, fishing, green algae/eutrophication and oil occurred at 'low' levels (scores 0-1): management not (currently) necessary. Construction and wood litter prevailed at 'moderate' levels (scores 2-4): management alerted for (further) monitoring. Solid waste densities were 'high' (scores 5-6): management alerted for action; quantities of rubbish were substantial (20-200 items m⁻¹ beach) but not unprecedented. REA is considered a robust methodology and complementary to other rapid assessment techniques, environmental frameworks and indicators of ecosystem condition.