Aerosolization of Micro- and Nanoplastics via Sea Spray: Investigating the Role of Polymer Type, Size, and Concentration, and Potential Implications for Human Exposure.

Micro- and nanoplastics (MNPs) can enter the atmosphere via sea spray aerosols (SSAs), but the effects of plastic characteristics on the aerosolization process are unclear. Furthermore, the importance of the transport of MNPs via these SSAs as a possible new exposure route for human health remains unknown. The aim of this study was two-fold: (1) to examine if a selection of factors affects aerosolization processes of MNPs, and (2) to estimate human exposure to MNPs via aerosols inhalation. A laboratory-based bubble bursting mechanism, simulating the aerosolization process at sea, was used to investigate the influence of MNP as well as seawater characteristics. To determine the potential human exposure to microplastics via inhalation of SSAs, the results of the laboratory experiments were extrapolated to the field based on sea surface microplastic concentrations and the volume of inhaled aerosols. Enrichment seemed to be influenced by MNP size, concentration and polymer type. With higher enrichment for smaller particles and denser polymers. Experiments with different concentrations showed a larger range of variability but nonetheless lower concentrations seemed to result in higher enrichment, presumably due to lower aggregation. In addition to the MNP characteristics, the type of seawater used seemed to influence the aerosolization process. Our human exposure estimate to microplastic via inhalation of sea spray aerosols shows that in comparison with reported inhaled concentrations in urban and indoor environments, this exposure route seems negligible for microplastics. Following the business-as-usual scenario on plastic production, the daily plastic inhalation in coastal areas in 2100 is estimated to increase but remain far below 1 particle per day. This study shows that aerosolization of MNPs is a new plastic transport pathway to be considered, but in terms of human exposure it seems negligible compared to other more important sources of MNPs, based on current reported environmental concentrations.

The immunostimulatory activity of sea spray aerosols: bacteria and endotoxins activate TLR4, TLR2/6, NF-κB and IRF in human cells.

Frequent exposure to sea spray aerosols (SSA) containing marine microorganisms and bioactive compounds may influence human health. However, little is known about potential immunostimulation by SSA exposure. This study focuses on the effects of marine bacteria and endotoxins in SSA on several receptors and transcription factors known to play a key role in the human innate immune system. SSA samples were collected in the field (Ostend, Belgium) or generated in the lab using a marine aerosol reference tank (MART). Samples were characterized by their sodium contents, total bacterial counts, and endotoxin concentrations. Human reporter cells were exposed to SSA to investigate the activation of toll-like receptor 4 (TLR4) in HEK-Blue hTLR4 cells and TLR2/6 in HEK-Blue hTLR2/6 cells, as well as the activation of nuclear factor kappa B (NF-κB) and interferon regulatory factors (IRF) in THP1-Dual monocytes. These responses were then correlated to the total bacterial counts and endotoxin concentrations to explore dose-effect relationships. Field SSA contained from 3.0 × 103 to 6.0 × 105 bacteria/m3 air (averaging 2.0 ± 1.9 × 105 bacteria/m3 air) and an endotoxin concentration ranging from 7 to 1217 EU/m3 air (averaging 389 ± 434 EU/m3 air). In contrast, MART SSA exhibited elevated levels of total bacterial count (from 2.0 × 105 to 2.4 × 106, averaging 7.3 ± 5.5 × 105 cells/m3 air) and endotoxin concentration from 536 to 2191 (averaging 1310 ± 513 EU/m3 air). SSA samples differentially activated TLR4, TLR2/6, NF-κB and IRF. These immune responses correlated dose-dependently with the total bacterial counts, endotoxin levels, or both. This study sheds light on the immunostimulatory potential of SSA and its underlying mechanisms, highlighting the need for further research to deepen our understanding of the health implications of SSA exposure.

Micro- and nanoplastics transfer from seawater to the atmosphere through aerosolization under controlled laboratory conditions.

Sea spray has been suggested to enable the transfer of micro- and nanoplastics (MNPs) from the ocean to the atmosphere, but only a few studies support the role of sea spray aerosols (SSAs) as a source of airborne particles. We demonstrated that MNPs are aerosolized during wave action, via SSAs, under controlled laboratory conditions. We used a mini-Marine-Aerosol-Reference-Tank (miniMART), a device that mimics naturally occurring physical mechanisms producing SSAs, and assessed the aerosolization of fluorescent polystyrene beads (0.5-10 µm), in artificial seawater. The SSAs contained up to 18,809 particles/mL of aerosols for 0.5 µm beads, with an enrichment factor of 19-fold, and 1977 particles/mL of aerosols for 10 µm beads with a 2-fold enrichment factor. Our study demonstrates that the use of the miniMART is essential to assess MNPs aerosolization in a standardized way, supporting the hypothesis which states that MNPs in the surface of the ocean may be transferred to the atmosphere.

Warship wrecks and their munition cargos as a threat to the marine environment and humans: The V 1302 "JOHN MAHN" from World War II.

In addition to endangering sea traffic, cable routes, and wind farms, sunken warship wrecks with dangerous cargo, fuel, or munitions on board may emerge as point sources for environmental damage. Energetic compounds such as TNT (which could leak from these munitions) are known for their toxicity, mutagenicity, and carcinogenicity. These compounds may cause potential adverse effects on marine life via contamination of the marine ecosystem, and their entry into the marine and human food chain could directly affect human health. To ascertain the impending danger of an environmental catastrophe posed by sunken warships, the North Sea Wrecks (NSW) project (funded by the Interreg North Sea Region Program) was launched in 2018. Based on historical data (derived from military archives) including the calculated amount of munitions still on board, its known location and accessibility, the German World War II ship "Vorpostenboot 1302" (former civilian name - "JOHN MAHN") was selected as a case study to investigate the leakage and distribution of toxic explosives in the marine environment. The wreck site and surrounding areas were mapped in great detail by scientific divers and a multibeam echosounder. Water and sediment samples were taken in a cross-shaped pattern around the wreck. To assess a possible entry into the marine food chain, caged mussels were exposed at the wreck, and wild fish (pouting), a sedentary species that stays locally at the wreck, were caught. All samples were analyzed for the presence of TNT and derivatives thereof by GC-MS/MS analysis. As a result, we could provide evidence that sunken warship wrecks emerge as a point source of contamination with nitroaromatic energetic compounds leaking from corroding munitions cargo still on board. Not only did we find these explosive substances in bottom water and sediment samples around the wreck, but also in the caged mussels as well as in wild fish living at the wreck. Fortunately so far, the concentrations found in mussel meat and fish filet were only in the one-digit ng per gram range thus indicating no current concern for the human seafood consumer. However, in the future the situation may worsen as the corrosion continues. From our study, it is proposed that wrecks should not only be ranked according to critical infrastructure and human activities at sea, but also to the threats they pose to the environment and the human seafood consumer.

Sea Spray Aerosols Contain the Major Component of Human Lung Surfactant.

Marine phytoplankton influence the composition of sea spray aerosols (SSAs) by releasing various compounds. The biogenic surfactant dipalmitoylphosphatidylcholine (DPPC) is known to accumulate in the sea surface microlayer, but its aerosolization has never been confirmed. We conducted a 1 year SSA sampling campaign at the Belgian coast and analyzed the SSA composition. We quantified DPPC at a median and maximum air concentration of 7.1 and 33 pg m-3, respectively. This discovery may be of great importance for the field linking ocean processes to human health as DPPC is the major component of human lung surfactant and is used as excipient in medical aerosol therapy. The natural airborne exposure to DPPC seems too low to induce direct human health effects but may facilitate the effects of other marine bioactive compounds. By analyzing various environmental variables in relation to the DPPC air concentration, using a generalized linear model, we established that wave height is a key environmental predictor and that it has an inverse relationship. We also demonstrated that DPPC content in SSAs is positively correlated with enriched aerosolization of Mg2+ and Ca2+. In conclusion, our findings are not only important from a human health perspective but they also advance our understanding of the production and composition of SSAs.

Environmental stability of porcine respiratory coronavirus in aquatic environments.

Coronaviruses (CoVs) are a family of viruses that are best known as the causative agents of human diseases like the common cold, Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS) and COVID-19. CoVs spread by human-to-human transmission via droplets or direct contact. There is, however, concern about potential waterborne transmission of SARS-CoV-2, the virus responsible for COVID-19, as it has been found in wastewater facilities and rivers. To date, little is known about the stability of SARS-CoV-2 or any other free coronavirus in aquatic environments. The inactivation of terrestrial CoVs in seawater is rarely studied. Here, we use a porcine respiratory coronavirus (PRCV) that is commonly found in animal husbandry as a surrogate to study the stability of CoVs in natural water. A series of experiments were conducted in which PRCV (strain 91V44) was added to filtered and unfiltered fresh- and saltwater taken from the river Scheldt and the North Sea. Virus titres were then measured by TCID50-assays using swine testicle cell cultures after various incubation times. The results show that viral inactivation of PRCV in filtered seawater can be rapid, with an observed 99% decline in the viral load after just two days, which may depend on temperature and the total suspended matter concentration. PRCV degraded much slower in filtered water from the river Scheldt, taking over 15 days to decline by 99%, which was somewhat faster than the PBS control treatment (T99 = 19.2 days). Overall, the results suggest that terrestrial CoVs are not likely to accumulate in marine environments. Studies into potential interactions with exudates (proteases, nucleases) from the microbial food web are, however, recommended.

Phycotoxin-Enriched Sea Spray Aerosols: Methods, Mechanisms, and Human Exposure.

To date, few studies have examined the role of sea spray aerosols (SSAs) in human exposure to harmful and beneficial marine compounds. Two groups of phycotoxins (brevetoxins and ovatoxins) have been reported to induce respiratory syndromes during harmful algal blooms. The aerosolization and coastal air concentrations of other common marine phycotoxins have, however, never been examined. This study provides the first (experimental) evidence and characterization of the aerosolization of okadaic acid (OA), homoyessotoxin, and dinophysistoxin-1 using seawater spiked with toxic algae combined with the realistic SSA production in a marine aerosol reference tank (MART). The potential for aerosolization of these phycotoxins was highlighted by their 78- to 1769-fold enrichment in SSAs relative to the subsurface water. To obtain and support these results, we first developed an analytical method for the determination of phycotoxin concentrations in SSAs, which showed good linearity (R2 > 0.99), recovery (85.3-101.8%), and precision (RSDs ≤ 17.2%). We also investigated natural phycotoxin air concentrations by means of in situ SSA sampling with concurrent aerosolization experiments using natural seawater in the MART. This approach allowed us to indirectly quantify the (harmless) magnitude of OA concentrations (0.6-51 pg m-3) in Belgium's coastal air. Overall, this study provides new insights into the enriched aerosolization of marine compounds and proposes a framework to assess their airborne exposure and effects on human health.

Harmful algal blooms and their effects in coastal seas of Northern Europe.

Harmful algal blooms (HAB) are recurrent phenomena in northern Europe along the coasts of the Baltic Sea, Kattegat-Skagerrak, eastern North Sea, Norwegian Sea and the Barents Sea. These HABs have caused occasional massive losses for the aquaculture industry and have chronically affected socioeconomic interests in several ways. This status review gives an overview of historical HAB events and summarises reports to the Harmful Algae Event Database from 1986 to the end of year 2019 and observations made in long term monitoring programmes of potentially harmful phytoplankton and of phycotoxins in bivalve shellfish. Major HAB taxa causing fish mortalities in the region include blooms of the prymnesiophyte Chrysochromulina leadbeateri in northern Norway in 1991 and 2019, resulting in huge economic losses for fish farmers. A bloom of the prymesiophyte Prymnesium polylepis (syn. Chrysochromulina polylepis) in the Kattegat-Skagerrak in 1988 was ecosystem disruptive. Blooms of the prymnesiophyte Phaeocystis spp. have caused accumulations of foam on beaches in the southwestern North Sea and Wadden Sea coasts and shellfish mortality has been linked to their occurrence. Mortality of shellfish linked to HAB events has been observed in estuarine waters associated with influx of water from the southern North Sea. The first bloom of the dictyochophyte genus Pseudochattonella was observed in 1998, and since then such blooms have been observed in high cell densities in spring causing fish mortalities some years. Dinoflagellates, primarily Dinophysis spp., intermittently yield concentrations of Diarrhetic Shellfish Toxins (DST) in blue mussels, Mytilus edulis, above regulatory limits along the coasts of Norway, Denmark and the Swedish west coast. On average, DST levels in shellfish have decreased along the Swedish and Norwegian Skagerrak coasts since approximately 2006, coinciding with a decrease in the cell abundance of D. acuta. Among dinoflagellates, Alexandrium species are the major source of Paralytic Shellfish Toxins (PST) in the region. PST concentrations above regulatory levels were rare in the Skagerrak-Kattegat during the three decadal review period, but frequent and often abundant findings of Alexandrium resting cysts in surface sediments indicate a high potential risk for blooms. PST levels often above regulatory limits along the west coast of Norway are associated with A. catenella (ribotype Group 1) as the main toxin producer. Other Alexandrium species, such as A. ostenfeldii and A. minutum, are capable of producing PST among some populations but are usually not associated with PSP events in the region. The cell abundance of A. pseudogonyaulax, a producer of the ichthyotoxin goniodomin (GD), has increased in the Skagerrak-Kattegat since 2010, and may constitute an emerging threat. The dinoflagellate Azadinium spp. have been unequivocally linked to the presence of azaspiracid toxins (AZT) responsible for Azaspiracid Shellfish Poisoning (AZP) in northern Europe. These toxins were detected in bivalve shellfish at concentrations above regulatory limits for the first time in Norway in blue mussels in 2005 and in Sweden in blue mussels and oysters (Ostrea edulis and Crassostrea gigas) in 2018. Certain members of the diatom genus Pseudo-nitzschia produce the neurotoxin domoic acid and analogs known as Amnesic Shellfish Toxins (AST). Blooms of Pseudo-nitzschia were common in the North Sea and the Skagerrak-Kattegat, but levels of AST in bivalve shellfish were rarely above regulatory limits during the review period. Summer cyanobacteria blooms in the Baltic Sea are a concern mainly for tourism by causing massive fouling of bathing water and beaches. Some of the cyanobacteria produce toxins, e.g. Nodularia spumigena, producer of nodularin, which may be a human health problem and cause occasional dog mortalities. Coastal and shelf sea regions in northern Europe provide a key supply of seafood, socioeconomic well-being and ecosystem services. Increasing anthropogenic influence and climate change create environmental stressors causing shifts in the biogeography and intensity of HABs. Continued monitoring of HAB and phycotoxins and the operation of historical databases such as HAEDAT provide not only an ongoing status report but also provide a way to interpret causes and mechanisms of HABs.

Future HAB science: Directions and challenges in a changing climate.

There is increasing concern that accelerating environmental change attributed to human-induced warming of the planet may substantially alter the patterns, distribution and intensity of Harmful Algal Blooms (HABs). Changes in temperature, ocean acidification, precipitation, nutrient stress or availability, and the physical structure of the water column all influence the productivity, composition, and global range of phytoplankton assemblages, but large uncertainty remains about how integration of these climate drivers might shape future HABs. Presented here are the collective deliberations from a symposium on HABs and climate change where the research challenges to understanding potential linkages between HABs and climate were considered, along with new research directions to better define these linkages. In addition to the likely effects of physical (temperature, salinity, stratification, light, changing storm intensity), chemical (nutrients, ocean acidification), and biological (grazer) drivers on microalgae (senso lato), symposium participants explored more broadly the subjects of cyanobacterial HABs, benthic HABs, HAB effects on fisheries, HAB modelling challenges, and the contributions that molecular approaches can bring to HAB studies. There was consensus that alongside traditional research, HAB scientists must set new courses of research and practices to deliver the conceptual and quantitative advances required to forecast future HAB trends. These different practices encompass laboratory and field studies, long-term observational programs, retrospectives, as well as the study of socioeconomic drivers and linkages with aquaculture and fisheries. In anticipation of growing HAB problems, research on potential mitigation strategies should be a priority. It is recommended that a substantial portion of HAB research among laboratories be directed collectively at a small sub-set of HAB species and questions in order to fast-track advances in our understanding. Climate-driven changes in coastal oceanographic and ecological systems are becoming substantial, in some cases exacerbated by localized human activities. That, combined with the slow pace of decreasing global carbon emissions, signals the urgency for HAB scientists to accelerate efforts across disciplines to provide society with the necessary insights regarding future HAB trends.

Aerosolizable Marine Phycotoxins and Human Health Effects: In Vitro Support for the Biogenics Hypothesis.

Respiratory exposure to marine phycotoxins is of increasing concern. Inhalation of sea spray aerosols (SSAs), during harmful Karenia brevis and Ostreopsis ovata blooms induces respiratory distress among others. The biogenics hypothesis, however, suggests that regular airborne exposure to natural products is health promoting via a downregulation of the mechanistic target of rapamycin (mTOR) pathway. Until now, little scientific evidence supported this hypothesis. The current explorative in vitro study investigated both health-affecting and potential health-promoting mechanisms of airborne phycotoxin exposure, by analyzing cell viability effects via cytotoxicity assays and effects on the mTOR pathway via western blotting. To that end, A549 and BEAS-2B lung cells were exposed to increasing concentrations (ng·L-1 - mg·L-1) of (1) pure phycotoxins and (2) an extract of experimental aerosolized homoyessotoxin (hYTX). The lowest cell viability effect concentrations were found for the examined yessotoxins (YTXs). Contradictory to the other phycotoxins, these YTXs only induced a partial cell viability decrease at the highest test concentrations. Growth inhibition and apoptosis, both linked to mTOR pathway activity, may explain these effects, as both YTXs were shown to downregulate this pathway. This proof-of-principle study supports the biogenics hypothesis, as specific aerosolizable marine products (e.g., YTXs) can downregulate the mTOR pathway.

Marine biogenics in sea spray aerosols interact with the mTOR signaling pathway.

Sea spray aerosols (SSAs) have profound effects on our climate and ecosystems. They also contain microbiota and biogenic molecules which could affect human health. Yet the exposure and effects of SSAs on human health remain poorly studied. Here, we exposed human lung cancer cells to extracts of a natural sea spray aerosol collected at the seashore in Belgium, a laboratory-generated SSA, the marine algal toxin homoyessotoxin and a chemical inhibitor of the mammalian target of rapamycin (mTOR) pathway. We observed significant increased expression of genes related to the mTOR pathway and Proprotein convertase subtilisin/kexin type 9 (PCSK9) after exposure to homoyessotoxin and the laboratory-generated SSA. In contrast, we observed a significant decrease in gene expression in the mTOR pathway and of PCSK9 after exposure to the natural SSA and the mTOR inhibitor, suggesting induction of apoptosis. Our results indicate that marine biogenics in SSAs interact with PCSK9 and the mTOR pathway and can be used in new potential pharmaceutical applications. Overall, our results provide a substantial molecular evidence base for potential beneficial health effects at environmentally relevant concentrations of natural SSAs.

Risk assessment of microplastics in the ocean: Modelling approach and first conclusions.

We performed an environmental risk assessment for microplastics (<5 mm) in the marine environment by estimating the order of magnitude of the past, present and future concentrations based on global plastic production data. In 2100, from 9.6 to 48.8 particles m-3 are predicted to float around in the ocean, which is a 50-fold increase compared to the present-day concentrations. From a meta-analysis with effect data available in literature, we derived a safe concentration of 6650 buoyant particles m-3 below which adverse effects are not likely to occur. Our risk assessment (excluding the potential role of microplastics as chemical vectors) suggests that on average, no direct effects of free-floating microplastics in the marine environment are to be expected up to the year 2100. Yet, even today, the safe concentration can be exceeded in sites that are heavily polluted with buoyant microplastics. In the marine benthic compartment between 32 and 144 particles kg-1 dry sediment are predicted to be present in the beach deposition zone. Despite the scarcity of effect data, we expect adverse ecological effects along the coast as of the second half of the 21st century. From then ambient concentrations will start to outrange the safe concentration of sedimented microplastics (i.e. 540 particles kg-1 sediment). Additional ecotoxicological research in which marine species are chronically exposed to realistic environmental microplastic concentration series are urgently needed to verify our findings.

High resolution mass spectrometry-based screening reveals lipophilic toxins in multiple trophic levels from the North Sea.

Lipophilic marine biotoxins, which are mainly produced by small dinoflagellates, are increasingly detected in coastal waters across the globe. As these producers are consumed by zooplankton and shellfish, the toxins are introduced, bioaccumulated and possibly biomagnified throughout marine food chains. Recent research has demonstrated that ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) is an excellent tool to detect marine toxins in algae and seafood. In this study, UHPLC-HRMS was used to screen lipophilic marine biotoxins in organisms from different trophic levels of the Belgian coastal zone ecosystem. A total of 20 tentatively identified lipophilic compounds was detected. Hereby, the trophic transfer of lipophilic marine biotoxins to the upper trophic level was considered to be rather limited. Furthermore, 36% of the compounds was clearly transferred between different organisms. A significant biotransformation of compounds from the okadaic acid and spirolide toxin groups was observed (64%), mainly in filter feeders. Through a multi-targeted approach, this study showed that marine organisms in the Belgian coastal zone are exposed to a multi-toxin mixture. Further research on both single compound and interactive toxic effects of the frequently detected lipophilic marine toxin ester metabolites throughout the food chain is therefore needed. As a future perspective, confirmatory identification of potential toxins by studying their fragmentation spectra (using new tools such as hybrid quadrupole Q-Exactive™ Orbitrap-MS) is designated.

Three-Dimensional Reconstruction of the Tissue-Specific Multielemental Distribution within Ceriodaphnia dubia via Multimodal Registration Using Laser Ablation ICP-Mass Spectrometry and X-ray Spectroscopic Techniques.

In this work, the three-dimensional elemental distribution profile within the freshwater crustacean Ceriodaphnia dubia was constructed at a spatial resolution down to 5 µm via a data fusion approach employing state-of-the-art laser ablation-inductively coupled plasma-time-of-flight mass spectrometry (LA-ICP-TOFMS) and laboratory-based absorption microcomputed tomography (µ-CT). C. dubia was exposed to elevated Cu, Ni, and Zn concentrations, chemically fixed, dehydrated, stained, and embedded, prior to µ-CT analysis. Subsequently, the sample was cut into 5 µm thin sections that were subjected to LA-ICP-TOFMS imaging. Multimodal image registration was performed to spatially align the 2D LA-ICP-TOFMS images relative to the corresponding slices of the 3D µ-CT reconstruction. Mass channels corresponding to the isotopes of a single element were merged to improve the signal-to-noise ratios within the elemental images. In order to aid the visual interpretation of the data, LA-ICP-TOFMS data were projected onto the µ-CT voxels representing tissue. Additionally, the image resolution and elemental sensitivity were compared to those obtained with synchrotron radiation based 3D confocal µ-X-ray fluorescence imaging upon a chemically fixed and air-dried C. dubia specimen.

Quantitative Determination and Subcellular Imaging of Cu in Single Cells via Laser Ablation-ICP-Mass Spectrometry Using High-Density Microarray Gelatin Standards.

This manuscript describes the development and characterization of a high-density microarray calibration standard, manufactured in-house and designed to overcome the limitations in precision, accuracy, and throughput of current calibration approaches for the quantification of elemental concentrations on the cellular level using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICPMS). As a case study, the accumulation of Cu in the model organism Scrippsiella trochoidea resulting from transition metal exposure (ranging from 0.5 to 100 µg/L) was evaluated. After the Cu exposure, cells of this photosynthetic dinoflagellate were treated with a critical point drying protocol, transferred to a carbon stub, and sputter-coated with a Au layer for scanning electron microscopy (SEM) analysis. In subsequent LA-ICPMS analysis, approximately 100 cells of each population were individually ablated. This approach permitted the evaluation of the mean concentration of Cu in the cell population across different exposure levels and also allowed the examination of the cellular distribution of Cu within the populations. In a cross-validation exercise, subcellular LA-ICPMS imaging was demonstrated to corroborate synchrotron radiation confocal X-ray fluorescence (SR-XRF) microimaging of single cells investigated under in vivo conditions.

Methodological challenges of optical tweezers-based X-ray fluorescence imaging of biological model organisms at synchrotron facilities.

Recently, a radically new synchrotron radiation-based elemental imaging approach for the analysis of biological model organisms and single cells in their natural in vivo state was introduced. The methodology combines optical tweezers (OT) technology for non-contact laser-based sample manipulation with synchrotron radiation confocal X-ray fluorescence (XRF) microimaging for the first time at ESRF-ID13. The optical manipulation possibilities and limitations of biological model organisms, the OT setup developments for XRF imaging and the confocal XRF-related challenges are reported. In general, the applicability of the OT-based setup is extended with the aim of introducing the OT XRF methodology in all research fields where highly sensitive in vivo multi-elemental analysis is of relevance at the (sub)micrometre spatial resolution level.

Quantification and profiling of lipophilic marine toxins in microalgae by UHPLC coupled to high-resolution orbitrap mass spectrometry.

During the last decade, a significant increase in the occurrence of harmful algal blooms (HABs), linked to repetitive cases of shellfish contamination has become a public health concern and therefore, accurate methods to detect marine toxins in different matrices are required. In this study, we developed a method for profiling lipophilic marine microalgal toxins based on ultra-high-performance liquid chromatography coupled to high-resolution Orbitrap mass spectrometry (UHPLC-HR-Orbitrap MS). Extraction of selected toxins (okadaic acid (OA), dinophysistoxin-1 (DTX-1), pectenotoxin-2 (PTX-2), azaspiracid-1 (AZA-1), yessotoxin (YTX) and 13-desmethyl spirolide C (SPX-1)) was optimized using a Plackett-Burman design. Three key algal species, i.e., Prorocentrum lima, Protoceratium reticulatum and Alexandrium ostenfeldii were used to test the extraction efficiency of OA, YTXs and SPXs, respectively. Prorocentrum micans, fortified with certified reference solutions, was used for recovery studies. The quantitative and confirmatory performance of the method was evaluated according to CD 2002/657/EC. Limits of detection and quantification ranged between 0.006 and 0.050 ng mL(-1) and 0.018 to 0.227 ng mL(-1), respectively. The intra-laboratory reproducibility ranged from 6.8 to 11.7 %, repeatability from 6.41 to 11.5 % and mean corrected recoveries from 81.9 to 119.6 %. In addition, algae cultures were retrospectively screened for analogues and metabolites through a homemade database. Using the ToxID software programme, 18 toxin derivates were detected in the extract of three toxin producing microalgae species. In conclusion, the generic extraction and full-scan HRMS approach offers an excellent quantitative performance and simultaneously allows to profile analogues and metabolites of marine toxins in microalgae. Graphical Abstract Optimization of extraction, detection and quantification of lipophilic marine toxins in microalgae by UHPLC-HR Orbitrap MS.

In vivo X-ray elemental imaging of single cell model organisms manipulated by laser-based optical tweezers.

We report on a radically new elemental imaging approach for the analysis of biological model organisms and single cells in their natural, in vivo state. The methodology combines optical tweezers (OT) technology for non-contact, laser-based sample manipulation with synchrotron radiation confocal X-ray fluorescence (XRF) microimaging for the first time. The main objective of this work is to establish a new method for in vivo elemental imaging in a two-dimensional (2D) projection mode in free-standing biological microorganisms or single cells, present in their aqueous environment. Using the model organism Scrippsiella trochoidea, a first proof of principle experiment at beamline ID13 of the European Synchrotron Radiation Facility (ESRF) demonstrates the feasibility of the OT XRF methodology, which is applied to study mixture toxicity of Cu-Ni and Cu-Zn as a result of elevated exposure. We expect that the new OT XRF methodology will significantly contribute to the new trend of investigating microorganisms at the cellular level with added in vivo capability.