Routine Monitoring Programs Research Articles

Performance of Storm Overflows Impacting on Shellfish Waters in England

Storm overflow (SO) discharges to shellfish growing waters are a concern for shellfish growers and may pose a health risk to consumers. We investigated the performance of permitted SOs with a shellfish water spill monitoring requirement against the design criterion of 10 spills per year (averaged over 10 years) used in England. Performance against this criterion over the period 2019–2021 differed between the five water companies whose data was analysed. Across all companies, over half of SOs spilled more frequently than this criterion (percentage greater than 10 spills: 52%, 55% and 57% for 2019, 2020 and 2021, respectively). The number of SOs with the monitoring requirement also differed considerably between the water companies and consequently some companies deal with a significantly higher ‘burden’ than others. The number of SOs spilling more than 100 times in a year also differed between water companies, from 0% (Wessex Water) to 16% (United Utilities). Discharges from SOs can lead to short-term reductions in water quality that may be missed by routine monitoring programmes using faecal indicator bacteria such as E. coli. Such discharges can lead to a higher incidence of norovirus in shellfish and thus potential illness in consumers. We conclude that site-specific impact assessments, supported by spill event-based monitoring, are required given the increased demands on sewerage networks from urban growth and climate change and the need to improve shellfish production area classifications.

Nontoxic Enantiomeric Reference Materials for Saxitoxins.

Saxitoxin (STX) is a potent neurotoxin that is biosynthesized by toxic dinoflagellates and accumulated in shellfish via the food chain. STX and its various analogues are now monitored in shellfish by the hygiene authorities in many countries with instrumental analytical methods, which require calibration with standards. Unfortunately, STX is registered as a chemical warfare agent in Schedule 1 of the Chemical Weapons Convention, and this has made it difficult to import calibration standards into some countries. We aimed to avoid violation of the Chemical Weapons Convention and facilitate analyses by preparing calibration standards based on unnatural nontoxic antipodal STXs (ent-STXs) with the same physicochemical properties as natural STXs. Our findings demonstrate that the nontoxic ent-STXs can be safely utilized as alternative reference materials of STXs in the routine monitoring program by the local authorities and consequently can lead to reduced usage of STX.

Characterization of Particulate Matter Generated at a Nickel Smelter Using Quantitative Mineralogy

Activities performed at mineral processing operations are capable of producing significant quantities of dust. To ensure that regulatory compliance is maintained throughout operation, dust levels are monitored by routine analysis of air filter samples. Determining the quantity and type of particulate matter present in dust allows for the operation to identify the sources of dust and where warranted, implement a dust suppressant strategy. Conventional methods of analysis, such as chemical assay, are unable to rigorously differentiate between phases containing the same elements and may result in ambiguity related to the identification of dust sources. By incorporating Quantitative Evaluation of Materials by Scanning Electron Microscope (QEMSCAN) into their routine monitoring programs, the Sudbury INO smelter has greatly improved characterization of the Ni and Co dust emissions generated at the operation. To determine specific activities responsible for emissions, bulk particulate matter was sampled from several key locations at the smelter that regularly produce dust. QEMSCAN was able to distinguish between critical subspecies of Ni and Co and mineralogical, and chemical signatures in the dust that are representative of locational activity were established. An apportionment of elements contributed by each source was calculated based on this information. The results of the characterization study assisted personnel in developing strategies to mitigate dust emissions that originate from the Smelter operations, and have general implications to occupational hygiene and environmental risk assessment.

Historical data provides context for recent monitoring and demonstrates 100 years of declining estuarine health

ABSTRACT Estuaries are complicated systems to manage effectively, requiring the monitoring of robust environmental indicators to assess health and detect changes. Routine estuarine monitoring programmes in New Zealand have only existed for 30 years at most, but estuaries have been significantly affected by anthropogenic pressures for at least 100 years. We reconstructed long term changes in indicators of estuarine health using historical information from sediment cores and aerial photographs, and combined this information with recent monitoring data. This study is focused on four estuaries in the Waikato region of New Zealand, but the findings are likely applicable elsewhere. Sediment accumulation rates increased by orders of magnitude c. 100 years ago, but mangrove forests only started to rapidly expand c. 50 years ago, which coincided with the intensification of agriculture and urban development, and resultant declines in freshwater quality. Over the past 20 years, state of the environment monitoring shows declines in benthic health at most monitoring sites, as well as continuing sediment accumulation and mangrove expansion in some places. This adds to the weight of evidence that environmental management has not been sufficient to safeguard estuarine health and demonstrates the magnitude of change in these systems over the past 100 years.

Passive Sampling Helps the Appraisal of Contaminant Bioaccumulation in Norwegian Fish Used for Regulatory Chemical Monitoring.

Hexachlorobenzene (HCB), listed on the Stockholm Convention on persistent organic pollutants and regulated as a hazardous priority pollutant by the Water Framework Directive (WFD), is ubiquitously distributed in the environment and assumed to mildly biomagnify in aquatic foodwebs. The proposal to include trophic magnification factors (TMFs) in the procedure for comparing contaminant levels in biota at different trophic levels (TLs) with WFD environmental quality standards requires adequate selection of TMFs. In the first step of our study, we compared two independently obtained datasets of pentachlorobenzene (PeCB) and HCB concentration ratios from passive sampling (PS) in water and in fish through routine monitoring programs in Norway to evaluate possible biomagnification. In this procedure, PeCB is used for benchmarking the bioconcentration in fish, and the observed HCB/PeCB ratios in fish are compared with ratios expected in the case of (i) HCB bioconcentration or (ii) biomagnification using published TMF values. Results demonstrate that it is not possible to confirm that HCB biomagnifies in fish species that would be used for WFD monitoring in Norway and challenges the proposed monitoring procedures for such compounds in Norwegian or European waters. In the second step, fish-water chemical activity ratios for HCB and PeCB as well as for polychlorinated biphenyls where biota and PS were conducted alongside were calculated and found to rarely exceed unity for cod (Gadus morhua), a fish species with a TL of approximately 4.

An automated analyzer for the simultaneous determination of silicate and phosphate in seawater

Accurate and automated determination methods for silicate and phosphate are in high demand to improve understanding of biogeochemistry and ecology in dynamic and complex estuarine-coastal ecosystems. Here, a portable automated analyzer is reported for the simultaneous determination of silicate and phosphate in water samples with varying salinity. After comprehensive optimization of the chemical reactions and flow manifold, the system demonstrated limits of detection of 0.09 and 0.05 μmol L−1 for silicate and phosphate, respectively, exhibiting linearity at concentrations up to 400 and 200 μmol L−1 with automated dilution, achieving relative standard deviations (n = 11) of 0.27% (20 μmol L−1 silicate), 0.51% (5 μmol L−1 phosphate) and 0.80% (1 μmol L−1 phosphate). Compared with similar automated flow analyzers, the system exhibited advantages, such as low consumption of reagents (10–20 μL/sample), portability (4.8 kg), rapid start-up (5 min), reliability (automated analysis of quality control sample) and applicability within a broad salinity range (from 0 to 35) allowing analysis of dynamic estuarine and seawater samples. The system was successfully applied to a routine monitoring program by a national marine station and is potentially suitable for installation on different observation platforms for on-line and real-time underway analysis of nutrients in coastal areas.

Using null models and species traits to optimize phytoplankton monitoring: An application across oceans and ecosystems

Phytoplankton assemblages are privileged descriptors of the ecological status of marine ecosystems regularly included in routine monitoring programmes. The high spatial and temporal variability of phytoplankton and the intrinsic difficulties of species identifications, however, combine in making reiterate assessments of this component of marine biota particularly demanding. Coarse levels of taxonomic resolution (e.g., genus, family) or morpho-functional categories have been proposed to reduce identification efforts or to ease the analysis of phytoplankton assemblages for monitoring purposes, although with contrasting outcomes. A major issue is that, in the absence of control for the loss of information associated to these alternative approaches, their application may lead to poor representations of genuine spatial and/or temporal patterns of assemblages in relation to natural and anthropogenic sources of variation. We provided a new approach to reduce the efforts required to analyse phytoplankton assemblages that integrate morpho-functional classification of phytoplankton with the use of null models to estimate the consequent loss of information on species-level community patterns. Null models for information loss were built by randomly grouping the original species variables into a progressively decreasing number of groups, in order to identify the minimum number of aggregate variables needed to detect community patters as at species level. Aggregate variables were then defined as morpho-functional groups, by grouping species on the basis of a combination of morpho-functional traits, including general taxonomy, cell size, shape, elongation and complexity. We applied the approach to six case studies investigating the response of phytoplankton assemblages from marine and transitional water ecosystems under different environmental settings in areas spanning the world’s ocean, including coral atolls, mangroves, estuaries, coastal lagoons and inlets. The approach allowed obtaining parsimonious sets of morpho-functional groups, which were suitable to detect changes in phytoplankton assemblage structure as at species level in all case studies. Trait-based approaches to phytoplankton research and monitoring are crucial to shed light on processes underlying phytoplankton community assembly and dynamics in the face of global change. In this perspective, our framework incorporates cost-effectiveness, instances from traditional monitoring programmes aiming at the detection of community patterns, and the current need for a deeper understanding of functional responses of phytoplankton to environmental drivers.

Examining the Relevance of the Microplastic-Associated Additive Fraction in Environmental Compartments

Plastic contamination is ubiquitous in the environment and has been related to increasing global plastic usage since the 1950s. Considering the omnipresence of additives in plastics, the risk posed by this contamination is related not only to the physical effects of plastic particles but also to their additive content. Until now, most routine environmental monitoring programs involving additives have not considered the presence of these additives still associated with the plastic they were added to during their production. Understanding environmental additive speciation is essential to address the risk they pose through their bioavailability and plastic-associated transport. Here, we present and apply a theoretical framework for sampling and analytical procedures to characterize the speciation of hydrophobic nonionized additives in environmental compartments. We show that this simple framework can help develop sampling and sample treatment procedures to quantify plastic-associated additives and understand additive distribution between plastics and organic matter. When applied to concrete cases, internal consistency checks with the model allowed for identifying plastic-associated additives in a sample. In other cases, the plastic-organic carbon ratio and additive concentration in the matrix are key factors affecting the ability to identify plastic-associated additives. The effect of additive dissipation through diffusion out of plastic particles is also considered.

A generic and rapid analytical method for comprehensive determination of veterinary drugs and other contaminants in raw honey

A generic, rapid, simple and low-cost analytical method for simultaneously screening and confirming 400 veterinary drugs and other contaminants in raw honey was developed. The method was based on one-step extraction and ultra-high performance liquid chromatography coupled with electrospray ionization and tandem mass spectrometry. A well-designed extraction method results in the complete precipitation of proteins and separation of analytes from carbohydrates and reduces the time and cost of analysis by covering polar to non-polar analytes. Optimized pretreatment processes lead to no significant interference on analysis from complicated sample matrix. Competent linearity was found for all of target compounds with linear regression coefficients (R) higher than 0.99. Detection limits ranged from 0.05 µg/kg to 10 µg/kg. Average recovery rates spiked in raw honey and ranged from 57.3% to 139.8%, and associated relative standard deviation values ranged from 0.4% to 25.2% under selected conditions. The extraction sensitivity, linearity, recovery, and precision of the method were validated. The results clearly demonstrated the feasibility of using this approach in raw honey purchase. The application of this method, which improved efficiency and the coverage of residues, may considerably reduce effort and time in routine monitoring programs.

Assessing ecological status in karstic lakes through the integration of phytoplankton functional groups, morphological approach and environmental DNA metabarcoding

Phytoplankton is one of the key Biological Quality Elements within the Water Framework Directive, used to assess the ecological status of surface water bodies. Water samples for phytoplankton identification were collected from April to September at a total of eight sampling sites in all six Croatian natural karstic lakes with an area greater than 0.5 km2. The main objective was to show the comparability of environmental DNA metabarcoding (Illumina sequencing using the hypervariable V9 region of the eukaryotic SSU rRNA gene) with morphologically based assessment and its applicability in assessing the ecological status of lakes. The value of Hungarian lake phytoplankton index (HLPI) indicating the final ecological status was calculated for both datasets using biomass and composition metrics. Chlorophyll a concentration measured using Ultra-High-Performance Liquid Chromatography and Spectrophotometer giving two biomass metrics along with the functional group approach (FG) as the composition metric for the complete taxa/operational taxonomic units (OTUs) lists as well as for the taxa/OTUs that contributed more than 5% to the total biomass/number of amplicons gave four to four HLPI values per sample. HLPI values from both approaches were highly correlated (Pearson's r > 0.92) and classified into good or high ecological status, although different compositions and proportions of FGs were recorded, thus giving the important role to the equal or similar factors assignment to different FGs with similar ecological demands and favourable habitats. In 89% of the samples, HLPI values indicate an equal range of ecological status and most differences were found due to different methods of Chlorophyll a measurement. Different composition metrics between approaches showed significant differences (p < 0.05) only in lakes Prošće and Crniševo. This study showed the applicability of the V9 region of 18S rRNA in ecological status assessment for oligotrophic and mesotrophic lakes due to the comparable results between approaches, but further development and standardization of eDNA metabarcoding are needed for the implementation in routine monitoring programs.

Discrimination between the exogenous and endogenous origin of thiouracil in farm animals, the final chapter?

ABSTRACT Thiouracil (2-thiouracil) is a thyreostatic compound that can be used as an illegal growth promoter. In bovine, porcine and other farm animals, low concentrations of thiouracil are detected in urine. There is much debate on which concentrations can be considered to originate from feed (‘natural’) and which concentrations are caused by the illegal administration of thiouracil for growth-promoting purposes. Currently, a threshold value of 10 µg/L in urine is applied. The threshold value is based on epidemiological data. Data on thiouracil from animals treated with thiouracil is scarce. We conducted a study whereby animals were fed with rapeseed, rapeseed with thiouracil, or regular feed with thiouracil (low and high concentration). It was determined that administration of thiouracil leads to concentrations higher than the current 10 µg/L threshold of thiouracil and its metabolites in urine during treatment. Animals fed with rapeseed showed higher thiouracil concentrations than the control group, mostly above 10 µg/L and in some cases above 30 µg/L. In the discovery study, several biomarkers for thiouracil treatment were tentatively identified and confirmed with reference standards. One metabolite was identified as indicative for thiouracil abuse, namely 6-methyl-thiouracil. Another metabolite, 4-thiouracil, was indicative for endogenous formation and did not increase during 2-thiouracil treatment. 6-Methyl-thiouracil was not found in urine samples from the Dutch routine control programmes that contained (endogenous) 2-thiouracil above the threshold value. However, 4-thiouracil was found at high concentrations in the same samples when 2-thiouracil was present. This study’s overall conclusion is that the threshold value for thiouracil in bovine urine samples should be set at 10 µg/L and for porcine urine samples at 30 µg/L. Also, confirmation of 6-methyl-thiouracil and 4-thiouracil should be used as indicators for exogenous or endogenous origin in routine control monitoring programmes.

Applications of nDATA for screening, quantitation, and identification of pesticide residues in fruits and vegetables using UHPLC/ESI Q-Orbitrap all ion fragmentation and data independent acquisition.

High sample throughput and effective multiresidue methods for screening, quantitation, and identification are desired for the analysis of a large number of pesticides in routine monitoring programs for food safety. This study was designed to explore the use of an UHPLC/ESI Q-Orbitrap nontarget data acquisition for target analysis (nDATA) workflow for screening 655 pesticides and quantifying a small group of 46 most likely incurred pesticide residues in fruits and vegetables in a single analysis. High-resolution mass spectrometers such as the Q-Orbitrap offer unique applications for pesticide analysis using full MS scan with data independent acquisition (DIA) or all ion fragmentation (AIF) scan. The experiments were designed to achieve a balance between selectivity and cycle time by considering parameter settings such as mass resolution and the number of mass isolation windows or isolation window widths. Coupled with ultra-high performance liquid chromatography (UHPLC), both full MS/DIA and full MS/AIF nDATA workflows were evaluated for screening, quantification, and identification in a single analysis. In general, UHPLC/ESI full MS/vDIA detected more fragment ions per pesticide than AIF when one to four fragments were compared. UHPLC/ESI full MS/vDIA and AIF generated comparable quantitative results, but the latter provided slightly better repeatability likely due to its shorter cycle time and more scans across a chromatographic peak. UHPLC/ESI full MS/vDIA may be preferable for screening, quantitation and identification when the testing scope covers a few hundreds of pesticides in a single analysis.

Taxon-specific sensitivities to flow intermittence reveal macroinvertebrates as potential bioindicators of intermittent rivers and streams

As complex mosaics of lotic, lentic, and terrestrial habitats, intermittent rivers and ephemeral streams (IRES) support high biodiversity. Despite their ecological importance, IRES are poorly represented in routine monitoring programs, but recent recognition of their considerable—and increasing—spatiotemporal extent is motivating efforts to better represent IRES in ecological status assessments. We examine response patterns of aquatic macroinvertebrate communities and taxa to flow intermittence (FI) across three European climatic regions. We used self-organizing map (SOM) to ordinate and classify sampling sites based on community structure in regions with continental, Mediterranean and oceanic climates. The SOM passively introduced FI, quantified as the mean annual % flow, and visualized its variability across classified communities, revealing a clear association between community structure and FI in all regions. Indicator species analysis identified taxa indicative of low, intermediate and high FI. In the continental region, the amphipod Niphargus was indicative of high FI and was associated with groundwater-fed IRES, whereas indicators of Mediterranean IRES comprised Odonata, Coleoptera and Heteroptera taxa, which favor lentic conditions. In the oceanic region, taxa indicative of relatively high FI included leuctrid stoneflies and a limnephilid caddisfly, likely reflecting the colonization of IRES by aerial adults from nearby perennial reaches. The Diptera families Chironomidae and Simuliidae showed contrasting FI preferences among regions, reflecting environmental heterogeneity between regions and the coarse taxonomic resolution to which these organisms were identified. These region-specific community and taxon responses of aquatic biota to FI highlight the need to adapt standard biotic indices to enable effective ecological status assessments in IRES.

Risk-based prioritization of suspects detected in riverine water using complementary chromatographic techniques

Surface waters are widely used as drinking water sources and hence their quality needs to be continuously monitored. However, current routine monitoring programs are not comprehensive as they generally cover only a limited number of known pollutants and emerging contaminants. This study presents a risk-based approach combining suspect and non-target screening (NTS) to help extend the coverage of current monitoring schemes. In particular, the coverage of NTS was widened by combining three complementary separations modes: Reverse phase (RP), Hydrophilic interaction liquid chromatography (HILIC) and Mixed-mode chromatography (MMC). Suspect lists used were compiled from databases of relevant substances of very high concern (e.g., SVHCs) and the concentration of detected suspects was evaluated based on ionization efficiency prediction. Results show that suspect candidates can be prioritized based on their potential risk (i.e., hazard and exposure) by combining ionization efficiency-based concentration estimation, in vitro toxicity data or, if not available, structural alerts and QSAR.based toxicity predictions. The acquired information shows that NTS analyses have the potential to complement target analyses, allowing to update and adapt current monitoring programs, ultimately leading to improved monitoring of drinking water sources.

Recommendations for the use of active personal dosemeters (APDs) in interventional workplaces in hospitals.

Occupational radiation doses from interventional procedures have the potential to be relatively high. The requirement to optimise these doses encourages the use of electronic or active personal dosimeters (APDs) which are now increasingly used in hospitals. They are typically used in tandem with a routine passive dosimetry monitoring programme, with APDs used for real-time readings, for training purposes and when new imaging technology is introduced. However, there are limitations when using APDs. A survey in hospitals to identify issues related to the use of APDs was recently completed, along with an extensive series of APD tests by the EURADOS Working Group 12 on Dosimetry for Medical Imaging. The aim of this review paper is to summarise the state of the art regarding the use of APDs. We also used the results of our survey and our tests to develop a set of recommendations for the use of APDs in the clinical interventional radiology/cardiology settings, and draw attention to some of the current challenges.

Ganciclovir therapeutic drug monitoring in transplant recipients.

BackgroundThe use of (val)ganciclovir is complicated by toxicity, slow response to treatment and acquired resistance.ObjectivesTo evaluate a routine therapeutic drug monitoring (TDM) programme for ganciclovir in a transplant patient population.MethodsAn observational study was performed in transplant recipients from June 2018 to February 2020. Dose adjustments were advised by the TDM pharmacist as part of clinical care. For prophylaxis, a trough concentration (Cmin) of 1–2 mg/L and an AUC24h of >50 mg·h/L were aimed for. For treatment, a Cmin of 2–4 mg/L and an AUC24h of 80–120 mg·h/L were aimed for.ResultsNinety-five solid organ and stem cell transplant patients were enrolled. Overall, 450 serum concentrations were measured; with a median of 3 (IQR = 2–6) per patient. The median Cmin and AUC24h in the treatment and prophylaxis groups were 2.0 mg/L and 90 mg·h/L and 0.9 mg/L and 67 mg·h/L, respectively. Significant intra- and inter-patient patient variability was observed. The majority of patients with an estimated glomerular filtration rate of more than 120 mL/min/1.73 m2 and patients on continuous veno-venous haemofiltration showed underexposure. The highest Cmin and AUC24h values were associated with the increase in liver function markers and decline in WBC count as compared with baseline.ConclusionsThis study revealed that a standard weight and kidney function-based dosing regimen resulted in highly variable ganciclovir Cmin and under- and over-exposure were observed in patients on dialysis and in patients with increased renal function. Clearly there is a need to explore the impact of concentration-guided dose adjustments in a prospective study.

Occurrence of Human Enteric Viruses in Shellfish along the Production and Distribution Chain in Sicily, Italy.

Contamination of bivalve mollusks with human pathogenic viruses represents a recognized food safety risk. Thus, monitoring programs for shellfish quality along the entire food chain could help to finally preserve the health of consumers. The aim of the present study was to provide up-to-date data on the prevalence of enteric virus contamination along the shellfish production and distribution chain in Sicily. To this end, 162 batches of mollusks were collected between 2017 and 2019 from harvesting areas, depuration and dispatch centers (n = 63), restaurants (n = 6) and retail stores (n = 93) distributed all over the island. Samples were processed according to ISO 15216 standard method, and the presence of genogroup GI and GII norovirus (NoV), hepatitis A and E viruses (HAV, HEV), rotavirus and adenovirus was investigated by real-time reverse transcription polymerase chain reaction (real-time-RT PCR), nested (RT)-PCR and molecular genotyping. Our findings show that 5.56% of samples were contaminated with at least one NoV, HAV and/or HEV. Contaminated shellfish were sampled at production sites and retail stores and their origin was traced back to Spain and several municipalities in Italy. In conclusion, our study highlights the need to implement routine monitoring programs along the whole food chain as an effective measure to prevent foodborne transmission of enteric viruses.

Beyond taxonomy: Validating functional inference approaches in the context of fish-farm impact assessments.

Characterization of microbial assemblages via environmental DNA metabarcoding is increasingly being used in routine monitoring programs due to its sensitivity and cost-effectiveness. Several programs have recently been developed which infer functional profiles from 16S rRNA gene data using hidden-state prediction (HSP) algorithms. These might offer an economic and scalable alternative to shotgun metagenomics. To date, HSP-based methods have seen limited use for benthic marine surveys and their performance in these environments remains unevaluated. In this study, 16S rRNA metabarcoding was applied to sediment samples collected at 0 and ≥1,200m from Norwegian salmon farms, and three metabolic inference approaches (Paprica, Picrust2 and Tax4Fun2) evaluated against metagenomics and environmental data. While metabarcoding and metagenomics recovered a comparable functional diversity, the taxonomic composition differed between approaches, with genera richness up to 20× higher for metabarcoding. Comparisons between the sensitivity (highest true positive rates) and specificity (lowest true negative rates) of HSP-based programs in detecting functions found in metagenomic data ranged from 0.52 and 0.60 to 0.76 and 0.79, respectively. However, little correlation was observed between the relative abundance of their specific functions. Functional beta-diversity of HSP-based data was strongly associated with that of metagenomics (r ≥ 0.86 for Paprica and Tax4Fun2) and responded similarly to the impact of fish farm activities. Our results demonstrate that although HSP-based metabarcoding approaches provide a slightly different functional profile than metagenomics, partly due to recovering a distinct community, they represent a cost-effective and valuable tool for characterizing and assessing the effects of fish farming on benthic ecosystems.

Performance and cost-efficiency of eDNA and eRNA capture methodologies: experimental assessment using cultured microalgae

Following the recent leap in biotechnologies and particularly in high-throughput sequencing techniques, environmental DNA and RNA (eDNA and eRNA) are increasingly being used for biodiversity assessments and monitoring of complex ecosystems – lakes, streams and coastal waters. Growing interest and affordability of eDNA/eRNA based tools have led to the emergence of manifold protocols for capturing genetic material from variable biological matrices, e.g. soil, water, feces, biofilms, etc. The variability in eDNA and eRNA material (ranging from free-floating molecules to cellular complexes to intact organisms) and its patchy distribution in the environment may substantially affect effective capture from the environment. Therefore, it is extremely challenging for stakeholders to standardize or choose optimal protocols, impeding incorporation of eDNA/eRNA methods into routine monitoring and surveillance programs. Although there is still no consensus on the standardized workflow for processing eDNA/eRNA samples, the common practice for water samples is to concentrate nucleic acids (NA) via filtration, ranging from 0.22 to 20-micron pore size. Although using the smallest pore is assumed efficient for NA capture from a wide range of material (including sub-cellular particles), a trade-off between detection of the meaningful molecular signal and time(cost)-efficiency is needed. Using finer pore membranes increases the likelihood of clogging and prohibits processing larger volumes of water, thus reducing the chances of detecting rare biodiversity. Moreover, large sample volumes may be accompanied by increased concentrations of inhibitory substances (e.g. humic compounds), suppressing target molecular signal. Comparative studies involving formal cost-efficiency assessments are lacking, restricting informed decision-making around the optimized sampling approach for addressing a particular research or surveillance question. Identifying the optimal combination of time effort and signal detection efficiency is particularly crucial for targeted surveillance, e.g. detection and monitoring of nuisance organisms, endangered and indicator taxa or other species of particular economic or cultural importance. Although it has been previously shown that larger pore filters can be as efficient for species detection from waterborne eDNA, more data are needed on the amount and type of NA material capture and loss. Here, we present a comparison study using an easily cultured microalgal species (Alexandrium pacificum) as a proxy to test the effectiveness of different filter membranes (cellulose acetate membranes of 5 μm, 1.2 μm or 0.45 μm pore size, and positively charged nylon membrane with 1.2 μm pore size) in the context of targeted species detection. We performed an efficiency analysis to identify the method that delivered the optimal use of resources. A tiered experimental design was applied to: i) assess the impact of membranes on capturing various fractions of target eDNA/eRNA (intact cells, partially lysed cells, naked NAs) spiked into pre-filtered and ambient environmental seawater and ii) establish efficiency and utility of different membranes in terms of optimizing the performance – maximized output (capture of target eDNA and eRNA) balanced against minimized time and cost input. The results showed no statistically significant difference between membranes for capturing DNA signal from intact and partially lysed cell treatments. However, positively charged nylon membranes were more efficient in capturing naked NAs, as well as RNA from partially lysed cells. In terms of time effort and volume processed, higher efficacy was reported for the larger pore size cellulose membranes. However positively charged nylon consistently performed better for capturing RNA signal across treatments.

Towards a standard protocol in coastal aquaculture biomonitoring: an interlaboratory study to assess reproducibility of the wet lab protocol and of Illumina sequencing

Salmon aquaculture is one of the fastest growing food sectors in the world. Because aquaculture can have a significant impact on the benthic environment, frequent monitoring is required by law. Recently, eDNA (environmental DNA) metabarcoding of benthic bacterial communities emerged as a very powerful tool to infer ecological quality based on amplicon sequence variants (ASVs) and their analyses. To exploit this monitoring approach and enable implementation into routine monitoring programs and official regulations, a high reproducibility of a standardized protocol among different laboratories is a prerequisite. Therefore, sediment samples from a compliance monitoring of a Scottish salmon farm were split into aliquots and distributed to two different laboratories for the wet lab procedure prior to independent Illumina sequencing of the two datasets. We then conducted pattern matching analyses and used supervised machine learning to predict ecological quality for each sample in the two datasets. Results were compared among the two datasets and to the reference data (ecological quality status as obtained from traditional macrofauna-based benthic monitoring). The obtained results demonstrate the high degree of congruence between the ecological quality assessments from the two laboratories. We conclude that the standard wet lab protocol used in our study is sufficiently robust across different laboratories, which meets a milestone criterion for practical routine application in industry.