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.

Probing Long-Term Impacts: Low-Dose Polystyrene Nanoplastics Exacerbate Mitochondrial Health and Evoke Secondary Glycolysis via Repeated and Single Dosing.

Nanoplastics (NPs) are omnipresent in the environment and contribute to human exposure. However, little is known regarding the long-term effects of NPs on human health. In this study, human intestinal Caco-2 cells were exposed to polystyrene nanoplastics (nanoPS) in an environmentally relevant concentration range (102-109 particles/mL) under two realistic exposure scenarios. In the first scenario, cells were repeatedly exposed to nanoPS every 2 days for 12 days to study the long-term effects. In the second scenario, only nanoPS was added once and Caco-2 cells were cultured for 12 days to study the duration of the initial effects of NPs. Under repeated dosing, initial subtle effects on mitochondria induced by low concentrations would accrue over consistent exposure to nanoPS and finally lead to significant impairment of mitochondrial respiration, mitochondrial mass, and cell differentiation process at the end of prolonged exposure, accompanied by significantly increased glycolysis over the whole exposure period. Single dosing of nanoPS elicited transient effects on mitochondrial and glycolytic functions, as well as increased reactive oxygen species (ROS) production in the early phase of exposure, but the self-recovery capacity of cells mitigated these effects at intermediate culture times. Notably, secondary effects on glycolysis and ROS production were observed during the late culture period, while the cell differentiation process and mitochondrial mass were not affected at the end. These long-term effects are of crucial importance for comprehensively evaluating the health hazards arising from lifetime exposure to NPs, complementing the extensively observed acute effects associated with prevalent short-term exposure to high concentrations. Our study underlines the need to study the toxicity of NPs in realistic long-term exposure scenarios such as repeated dosing.

Cellular and bioenergetic effects of polystyrene microplastic in function of cell type, differentiation status and post-exposure time.

The ubiquity of microplastics (MPs) in food sources and personal care products increasingly raises concerns on human health. However, little is known about the duration of the effects of MPs and whether effects depend on cellular differentiation status. Herein, cellular and bioenergetic effects of MPs in different exposure scenarios on four types of human cell lines derived from lung (A549 and BEAS-2B), colon (Caco-2) and liver (HepG2) were investigated. These cell lines are models for the major exposure routes in the body (inhalation, ingestion and physiological transport through the liver by the portal vein). To this aim, different scenarios were implemented by exposing undifferentiated and differentiated cells to single dosing of 2-µm polystyrene (PS) (102-105 particles/mL) for 48 h and 12 days. The undifferentiated Caco-2 cells with short exposure (48 h) showed the highest uptake rate of PS yet without significant cellular and mitochondrial responses. The biological effects, with the exception of ROS production, were not influenced by differentiation states of A549 and Caco-2 cells although differentiated cells showed much weaker ability to internalize PS. However, PS had significantly long-term impacts on cellular and mitochondrial functions even after the initial exposure period. In particular, Caco-2 cells that were post-exposed for 12 days after single PS dosing suffered higher oxidative stress and exhibited mitochondrial dysfunction than that for short exposure. Correspondingly, we observed that PS particles still remained in cell membrane and even in nuclei with high retention rate by 14-d post exposure during which metabolism and exchange of internalization and release occurred in cells. This indicates PS could induce chronic stress and even harmful effects on human cells after single intake that persists for a long time. This study paves the way for assessing the influence of PS on human health at low particle concentrations and with multiple exposure scenarios.

Assessment of road run-off and domestic wastewater contribution to microplastic pollution in a densely populated area (Flanders, Belgium).

Plastics are omnipresent in our daily life. Unfortunately, the produced plastics will partly end up in the environment including aquatic ecosystems. People often refer to littering or illegal waste dumping as sources of plastic emission to the environment. However, daily-life sources could also, unknowingly, contribute considerably to the total microplastic pollution in the ecosystem. Hence, there is an urgent need to study these potential sources. In this research, two common sources, i.e. domestic wastewater and road run-off from tire and road wear particles, were studied in detail to quantify the relative contribution of both domestic sources towards microplastic pollution in freshwater ecosystems in Flanders, Belgium. This assessment shows that every person (in studied area) emits on average 1145 microplastics (25-1000 µm) daily through domestic wastewater, resulting in a yearly discharge of 418,000 microplastic particles per person. The road run-off samples contained between 0.02 and 9.2 mg tire wear particles per litre per day, which corresponds to an emission of 10.8 mg tire wear particles per driven vehicle km. The gross and net emissions of both above mentioned microplastic sources were extrapolated to the whole Flanders region using an emission model. From the yearly gross microplastic pollution in the domestic wastewater, 623 kg (20%) will be discharged in the freshwater. The highest losses originated from the households that have a private drain or are not (yet) connected to an active wastewater treatment plant. In Flanders, the yearly net microplastic emission into the aquatic environment of tire wear particles is estimated to be 246 tonnes (38%), mainly from the direct run-off from the road surface. Based on the results, specific mitigation measures can be installed to reduce the emission of microplastics towards the freshwater ecosystem. Other sources should be quantified in a similar way for a more holistic strategy to counteract plastic pollution.

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.

Distribution of microplastics in freshwater systems in an urbanized region: A case study in Flanders (Belgium).

Microplastics (MPs) are an emerging pollutant of concern in all known aquatic ecosystems. However, studies at a regional scale on MP pollution in freshwater systems and the necessary risk assessments are limited. Therefore, in this study, we examined microplastic concentrations, size distributions, and polymer types in surface waters and sediments in the geographic region Flanders (Belgium), as a case study for a densely populated region and one of the most developed parts of Europe. Samples have been taken on nine different locations, of which five were repeated in a different weather condition. In total 43 aqueous and nine sediment samples have been collected. The quantity and identity of the microplastics in the samples were determined with µFTIR spectroscopy in the range of 25-1000 µm. The MPs' abundances in surface waters and sediments ranged from 0 to 4.8 MP L-1 (average = 0.48 MP L-1) and from 0 to 9558 MP kg-1 dry weight (average = 2774.57 ± 2317.93 MP kg-1 DW), respectively. Polystyrene and polypropylene were the most common polymer compositions found. No correlations were observed between microplastic concentrations in the sediment/the surface water samples and the measured environmental variables rainfall, conductivity, pH, dissolved oxygen content, waterway flow rate and width, and surrounding land use. Risk assessment results for the measured surface water concentrations through the risk quotient (RQ) method and the probabilistic risk assessment framework suggest that most of the sampled sites in Flanders posed negligible risks to freshwater biota, while this was not the case for some of the sediment concentrations. Our results illustrate the need to urgently develop analytical methods that can routinely measure the full size range of MP in environmental samples to adequately assess risks for the environment.

Toward a Better Understanding of the Contribution of Wastewater Treatment Plants to Microplastic Pollution in Receiving Waterways.

Microplastics (1 µm-5 mm), are ubiquitous in daily-use products and regularly end up in the wastewater. The main part of the wastewater is treated in wastewater treatment plants (WWTPs), which allow for at least partial removal of microplastics. The present study aimed to understand the contribution of domestic wastewater to microplastic pollution in Flanders (Belgium) via two main discharge routes of microplastics: (1) the effluent, and (2) removed fractions. Furthermore the effect of effluent discharge on the microplastic contamination in the waterway was studied in both surface water and sediment samples of upstream and downstream locations of the discharge from three WWTPs. On average, 12.64 ± 20.20 microplastic/L entered a WWTP (10 µm-5 mm). The effluent contained on average 0.41 ± 0.91 microplastic/L, resulting in an average removal efficiency of 97.46% ± 2.33%, which is comparable with various (non-)European countries. Removal efficiencies are both polymer- and size-specific, and data suggest that smaller particles are less efficiently removed from the wastewater, which also causes an increased input of smaller particles to the environment. The sludge is the most efficient treatment process to remove microplastics. Despite the high removal efficiencies, still 1.11 × 107 ± 3.07 × 107 microplastics end up in the nearby waterway daily. Nonetheless, based on the results gathered in the present study, this does not seem to impact the microplastic concentration in the waterway significantly. In summary, the present study offers a holistic approach in the research on the impact of wastewater on microplastic pollution in the ecosystem, integrating different discharge routes and measuring the impact on environmental microplastic pollution. Environ Toxicol Chem 2023;42:642-654. © 2022 SETAC.

Explorative study on scale cortisol accumulation in wild caught common dab (Limanda limanda).

BACKGROUND: Flatfish live in a diverse marine ecosystem that is changing due to natural variations and anthropogenic influences. These changes can evoke a stress response mainly resulting in production of the glucocorticoid cortisol, which mediates effects on various levels of biological organization. The finding that cortisol accumulates in fish scales, offering a retrospective view on cortisol production, provides opportunities to use this matrix for chronic stress assessment. The present study is the first to gather information on scale cortisol concentration in wild-caught common dab (Limanda limanda), based on a two-pronged approach using (1) field measurements and (2) a laboratory in vivo-study where wild-caught dab were fed by cortisol-spiked feed during 30 or 90 days to demonstrate the possible accumulation of cortisol in the scales and to evaluate its impact on fish health. RESULTS: Based on the field measurements, the average scale cortisol concentration in wild-caught fish was 0.0034 ± 0.0046 µg kg-1 scale (n = 67). This indicates that wild common dab is indeed able to incorporate cortisol in the scales. Based on the experimental data, the cortisol-fed fish showed an increased plasma cortisol concentration (80.16 ± 82.58 µg L-1) compared to the control group (4.54 ± 9.57 µg L-1) after 30 days of cortisol feeding. The increase in plasma cortisol concentration was positively correlated with an increased cortisol concentration in the scale after 30 days of cortisol-spiked feeding. This correlation was, however, no longer observed after 90 days of cortisol-spiked feeding. Interestingly, cortisol concentration of the scales on the pigmented side was significantly higher compared to the non-pigmented side. Some health parameters such as epidermal thickness, body condition and Ichthyobodo sp. parasitic infection showed a correlation with scale cortisol concentration after 30 days. CONCLUSIONS: We have demonstrated that common dab is able to accumulate cortisol in its scales. This seems to occur proportionally to circulating concentrations of plasma cortisol in fish fed with cortisol supplemented feed after 30 days.

INFLUENCE OF PATHOGENS, FISH-RELATED CHARACTERISTICS, AND ENVIRONMENTAL FACTORS ON THE DEVELOPMENT OF SKIN ULCERATIONS IN WILD COMMON DAB (LIMANDA LIMANDA) FROM THE NORTH SEA.

Environmental changes or stressors can result in the development of diseases. Through regular fish disease surveys in the Belgian part of the North Sea, attention was drawn to a sudden increase of skin ulceration prevalence between 2011 and 2014 in common dab (Limanda limanda). Information on prevalence, ulceration, bacteriology, fish-related (e.g., length, age, and sex) and (spatial and temporal) environmental factors, and fishing intensity were gathered. This detailed investigation was framed within a long-term monitoring program, executed every spring-autumn from 2000 to present. Ulcerations were observed in 1.3% of fish (n=3,999). Spatial and temporal differences were evident, and highest prevalence was found in summer. Vibrio was the dominant cultivated bacterial genus present in the lesions. Skin ulcerations appeared to be correlated with length and body condition of the fish, as well as with temperature and pH of the seawater and fishing vessel density. Our research suggested the involvement of multiple factors in the development of skin ulcerations in common dab and endorsed the effects of changing environment and human influence on the marine ecosystem through activities such as fishing.

Pinpointing the role of Aeromonas salmonicida in the development of skin ulcerations in common dab (Limanda limanda).

Aeromonas salmonicida was isolated from ulcerations in common dab (Limanda limanda). An experiment was performed to pinpoint its role in ulceration development, considering the importance of the skin barrier and the pigmented and non-pigmented sides. The skin of dab was treated in three zones, one where scales and epidermis were removed, one where mucus was discarded and one non-treated zone. Fish were tagged to allow individual identification and challenged with A. salmonicida. Mortality and severity of the developing lesions were recorded for 21 days post-inoculation. Starting 12 days post-inoculation, mortality occurred gradually in challenged fish; however, no direct cause could be established. Both control fish and challenged fish developed ulcerations containing A. salmonicida. Sequencing of vapA gene revealed that isolates retrieved from both groups were distinct, suggesting the presence of A. salmonicida prior to the trial. Most ulcerations developed in zones where skin was removed, suggesting that abrasion might be a predisposing factor in ulceration development. Ulcerations were also observed at the insertion site of the tag, where exposed muscle tissue might have favoured the development of ulcerations. In conclusion, A. salmonicida seems to be involved in the development of skin ulcerations in dab, although the exact pathogenesis needs to be elucidated.

Scrutinizing the triad of Vibrio tapetis, the skin barrier and pigmentation as determining factors in the development of skin ulcerations in wild common dab (Limanda limanda).

Recently, Vibrio tapetis was isolated for the first time from skin ulcerations in wild-caught common dab (Limanda limanda). To further examine its role in the development of these skin lesions, an in vivo experiment was performed. The significance of the skin barrier and in addition the difference between pigmented and non-pigmented side were investigated. Hence, the skin of common dab was treated in three different ways on both the pigmented and non-pigmented side. On a first "treatment zone", the scales and overlying epidermal tissue were removed whereas in a second zone only the mucus was discarded. The third zone served as a non-treated zone. Thereafter, fish were challenged with V. tapetis. The control group was sham treated. Mortality, clinical signs, severity and size of the developing lesions were recorded. All animals were sacrificed and sampled 21 days post-inoculation. Significantly more fish of the group challenged with V. tapetis died compared to the control group with the highest incidence occurring 4 days post-inoculation. Fish challenged with V. tapetis developed more severe skin ulcerations. In zones where scales and epidermal tissue were removed, the ulcerations were more severe compared to zones where only mucus was eliminated. Ulcerations occurred more frequently, were more severe and larger on the pigmented side. Our data represents prove of V. tapetis as causative agent of ulcerative skin lesions although prior damage of the skin seems to be a major contributing factor. Furthermore, the pigmented side seemed predisposed to the development of skin ulcerations.

Biogeography of the fish pathogen Aeromonas salmonicida inferred by vapA genotyping.

A recently described typing system based on sequence variation in the virulence array protein (vapA) gene, encoding the A-layer surface protein array, allows unambiguous subtyping of Aeromonas salmonicida. In the present study, we compile A-layer typing results from a total of 675 A. salmonicida isolates, recovered over a 59-year period from 50 different fish species in 26 countries. Nine novel A-layer types (15-23) are identified, several of which display a strong predilection towards certain fish hosts, including e.g. Cyprinidae and Pleuronectidae species. Moreover, we find indications that anthropogenic transport of live fish may have aided the near global dissemination of two cyprinid-associated A-layer types. Comparison of whole genome phylogeny and A-layer typing for a subset of strains further resulted in compatible tree topologies, indicating the utility of vapA as a phylogenetic as well as an epizootiological marker in A. salmonicida. A Microreact project (microreact.org/project/r1pcOAx9m) has been created, allowing public access to the vapA analyses and relevant metadata. In sum, the results generated provide valuable insights into the global population structure of A. salmonicida, particularly in relation to its piscine host spectrum and the geographic distribution of these hosts.

Experimental infection model for vibriosis in Dover sole (Solea solea) larvae as an aid in studying its pathogenesis and alternative treatments.

Severe economic losses due to diseases in marine larviculture may be linked to vibriosis. To better understand the pathogenesis of vibriosis and evaluate new ways to prevent and combat this important disease, there is a great need for reliable and reproducible experimental infection models. The present study aimed at developing a challenge model for vibriosis in Dover sole larvae and testing its applicability to study the effect of the probiotic treatment. For that purpose, larvae were challenged at 10 days post hatching with Vibrio anguillarum WT, V. anguillarum HI610 or V. harveyi WT. Following administration of V. anguillarum WT via immersion at 1 × 107 colony forming units/mL, a larval mortality of 50% was observed at 17 days post-inoculation. In a next step, the probiotic potential of 371 isolates retrieved from Dover sole was assessed by screening for their inhibitory effects against Vibrio spp. and absence of haemolytic activity. One remaining isolate (V. proteolyticus) and V. lentus, known for its protective characteristics in seabass larvae, were further tested in vivo by means of the pinpointed experimental infection model. Neither isolate provided via the water or feed proved to be protective for the Dover sole larvae against challenge with V. anguillarum WT. This developed challenge model constitutes a firm basis to expedite basic and applied research regarding the pathogenesis and treatment of vibriosis as well as for studying the impact of (a)biotic components on larval health.