Port and coastal environments are critical contamination hotspots for both antifouling biocides and microplastics (MPs), yet their combined toxicity remains poorly understood. This study investigated the individual and mixture toxicity of the biocide DCOIT (4,5-dichloro-2-n-octyl-4-isothiazolin-3-one) and virgin polypropylene microparticles (PP-MPs, >100 µm) on the embryonic development of the mussel Mytilus galloprovincialis and the sea urchin Paracentrotus lividus. Standard embryo-larval assays revealed species-specific sensitivities. M. galloprovincialis was more sensitive to DCOIT (LOEC = 12.5 µg DCOIT L⁻¹, EC₅₀ = 112.7 µg DCOIT L⁻¹) and to PP-MPs alone (LOEC = 100.0 mg PP L⁻¹) than P. lividus (LOEC= 200.0 µg DCOIT L⁻¹, EC₅₀ = 480.0 µg DCOIT L⁻¹; PP-MPs: LOEC > 10,000.0 mg PP L⁻¹). Crucially, binary mixtures elicited opposite interactions: a strong synergism was observed for the mussel (e.g., significant effects at 3.12 µg DCOIT L⁻¹ + 25 mg PP L⁻¹), quantitatively confirmed by Bliss Independence, Loewe Additivity, and Zero Interaction Potency (ZIP) models (Combination Index < 1, positive ZIP-like scores). For the sea urchin, the mixture resulted in antagonism, likely due to PP-MP-mediated adsorption of DCOIT reducing its bioavailability. Principal Component Analysis (PCA) further delineated the mixture's distinct toxic profile. These results demonstrate that microplastics are active modulators of chemical toxicity, acting either as vectors that exacerbate risk for filter-feeders or as sinks that temporarily mitigate exposure to other species. This species-dependent interplay underscores an urgent need to incorporate mixture and particle-interaction studies into environmental risk assessments for coastal ecosystems, as current single-contaminant approaches critically underestimate ecological risk.

Metallic corrosion poses an economic problem worldwide and is often mitigated by adding corrosion inhibitors (CIs) to protective coatings. Conventional CIs, such as 2-mercaptobenzothiazole (MBT) and benzotriazole (BTA), exhibit toxicity to aquatic biota, necessitating the development of eco-friendly alternatives. ZnAl layered double hydroxides (LDHs) have been used to immobilize CIs, reducing their toxicity and providing long-term protection through controlled release. This study evaluated the toxicity of CIs (MBT, BTA, bio-based sodium gluconate, and sodium nitrite, NaNO2), their nanostructured forms (LDH-MBT, LDH-BTA, LDH-Gluconate, LDHNO2), and the base nanomaterial (LDHNO3). Adverse effects were assessed using the benthic polychaete Hediste diversicolor through a battery of biochemical biomarkers (AChE, LPO, CAT, and GST) and bioaccumulation analysis, coupled with the quantification of Al3+ and Zn2+ dissolution from the nanostructures in both aqueous and sedimentary compartments. Juveniles were individually exposed in spiked sediment at three concentrations (0.2, 2, 20 mg kg-1) for 10 days. Metal concentration in water did not differ significantly from the control levels. Conversely, there was a significant decrease in Al3+ and Zn2+ in the sediment in most treatments compared to the control, with the exception of Zn2+ at the highest LDH-Gluconate and LDH-BTA exposure levels. Biochemical biomarkers indicated neurotoxicity induced by MBT, LDH-MBT, LDH-BTA, and LDHNO3, with a decrease in AChE activity, despite no significant bioaccumulation. In contrast, the bio-based LDH-Gluconate and the bio-inspired LDHNO2 exhibited no significant toxicity, representing promising eco-friendly options for marine anticorrosive applications.

Silver nanoparticles (AgNPs) synthesized from Aspergillus niger IBCLP20 exhibit strong antimicrobial properties; however, strategies to mitigate their environmental risks remain insufficiently validated across contrasting aquatic systems. Here, we assessed calcium alginate encapsulation (AgNPsIBCLP20/CA) as a safe-by-design approach, integrating silver release dynamics with short-term ecotoxicity in freshwater and marine species. In this context, safe-by-design refers to the use of calcium alginate microcapsules to modulate the release of dissolved silver from mycogenic AgNPs intended for water disinfection, thereby reducing and delaying peak exposure without altering the intrinsic toxicity of silver. Silver concentrations were quantified using ICP-MS and energy-dispersive X-ray fluorescence spectroscopy. Leachate-based assays revealed higher sensitivity in marine taxa, with Paracentrotus lividus (LOEC = 1%) and Daphnia magna (LOEC = 0.781%) were the most responsive species, while freshwater Danio rerio embryos and Raphidocelis subcapitata were unaffected. Silver release strongly depended on medium ionic strength, decreasing from 19.3 μg⋅L−1 in ultrapure water to 0.83 μg⋅L−1 in hard water. Hazard assessment yielded lower predicted no-effect concentration (PNEC) values for AgNPsIBCLP20/CA in freshwater than in seawater, indicating ecosystem-specific mitigation performance. These findings demonstrate that the ionic composition governs nanoencapsulation efficiency and that risk reduction strategies for biogenic AgNPs cannot be generalized across aquatic environments. Our results provide quantitative support for integrating encapsulation performance into environmental risk assessment frameworks for antimicrobial nanomaterials. Because the degradation of the alginate matrix may remobilize additional silver over time, encapsulation should be viewed as a hazard mitigation and exposure modulation strategy rather than a permanent elimination of silver-related risks.

Understanding more about the risks posed by hazardous pesticides is essential, particularly for ecologically and economically important species such as the Pacific oyster (Magallana gigas). Although dimethoate toxicity has been widely studied and has led to regulatory measures in certain regions, it is still used in others, such as Tunisia. Investigations on sensitive species in such contexts can provide valuable insights for better assessing and managing potential environmental risks. This study offers a novel evaluation of the toxic effects of experimental concentrations of dimethoate after a 72-h exposure period on the redox state, cholinergic system, and fatty acid composition in the gills of M. gigas oysters, as well as in terms of DNA damage on their hemocytes. Throughout the exposure period, we observed an increase in lipid and protein oxidation, along with changes in the activities of the antioxidant enzymes catalase, superoxide dismutase, and glutathione peroxidase, as well as an inhibition of the acetylcholinesterase activity in the tested oysters. Changes in fatty acid composition were detected, characterized by a decrease in saturated fatty acids and an increase in unsaturated fatty acids, particularly docosahexaenoic acid and arachidonic acid. After exposure to higher concentrations of dimethoate, genotoxic effects were observed, as evidenced by increased DNA damage in the oysters' hemolymph. Together, these biochemical and molecular alterations indicate that dimethoate exposure triggers complex physiological and behavioral adjustments in oysters when subjected to more severe stressors. These findings underscore potential threats to oyster quality and consumer safety under continued use of unregulated toxic pesticides.

Metal corrosion is a colossal technical, economic, and environmental challenge worldwide. Protective coatings containing corrosion inhibitors (CIs) are commonly used to address this natural process, particularly severe in immersed structures in seawater. However, high-performance CIs, such as benzotriazole (BTA), often exhibit toxicity towards aquatic organisms and leach prematurely. This study introduces safe and sustainable-by-design engineered nanomaterials, specifically layered double hydroxides loaded with BTA (Mg-Al LDH-BTA and Zn-Al LDH-BTA), as an innovative and eco-friendly approach compared to state-of-the-art CIs. This study aims to characterize both nanomaterials, assess their anti-corrosion performance when incorporated in polyurethane coatings, and evaluate their environmental behavior when dispersed in water, short-term acute and chronic effects on temperate marine species, and the environmental hazard. Key findings include a superior anti-corrosion performance of coatings containing Zn-Al LDH-BTA compared to BTA-coatings. Aqueous dispersions of nanomaterials exhibit instability of particle size and zeta potential over time, while concentrations of metals (Al, Zn) and nitrates reach high levels in the highest tested concentration due to partial dissolution, which may explain the observed toxicity patterns (median effect concentrations in the mg L-1 range). The tested compounds were not toxic for most tested species, apart from bacteria (Aliivibrio fischeri) and/or echinoderms (Paracentrotus lividus) and, in case of Mg-Al LDH-BTA, also on two microalgae species. The highest statistical PNEC value was observed for Mg-Al LDH-BTA (PNEC = 0.326 mg BTA per L), while the highest deterministic PNEC value was found for Zn-Al LDH-BTA (PNEC = 0.00041 mg BTA per L). These findings indicate that both nanomaterials are environmentally sound and efficient alternatives for anti-corrosion maritime applications.

The present communication reports the first record of the exotic polychaete Boccardiella hamata (Webster, 1879) for Spain and the Iberian Peninsula. Individuals were collected between April and August 2023 along the Piedras River estuary (Huelva, SW Spain). Despite the fact that the sampling was carried out in soft bottom and on oyster beds formed by Magallana spp., B. hamata was found only on the surface of Magallana spp. every sampling month, co-occurring with its congener Boccardiella ligerica (Ferronnière, 1898). The Atlantic coast of North America and both sides of the Pacific Ocean have been proposed as the native area of B. hamata, but recently it has been introduced to Northwestern Europe. The present record in Southwestern Europe supports the expansion of this species across Europe. However, previous re-examinations of old collections suggested that the presence of B. hamata European waters could be earlier due to misidentifications with B. ligerica. The present communication contributes to the updating of the introduced range of B. hamata, so this record should be taken into consideration for early detection and exotic species management plans.

Microplastics have become ubiquitous in the environment, posing significant threats to ecosystems and organisms across the globe. Accurate risk assessment of these pollutants hinges on the availability of comprehensive qualitative and quantitative data. This paper demonstrates the potential of photoacoustic spectroscopy (PAS) coupled with an external cavity quantum cascade laser (EC-QCL) for identifying various plastic polymers within the mid-infrared (mid-IR) range. To simulate the presence of microplastics in aqueous environments, the particles were prepared under controlled conditions through cryogenic milling of commercial polymer beads, followed by dispersion in ultrapure water. Finally, these simulated samples underwent filtration using gold-coated polycarbonate filters. The microplastics deposited on these filters were successfully identified by analyzing the photoacoustic (PA) spectra of individual particles or small clusters. The resulting absorption spectra were compared with both attenuated total reflectance (ATR) library spectra and transmission (TRX) spectra. While the PA spectra closely matched the transmission spectra, they exhibited shifts in absorption peak positions compared to ATR spectra. This study serves as a proof of concept, highlighting the capability of photoacoustic spectroscopy in identifying microplastic particles in environmental samples and paving the way for more accurate and efficient microplastic detection methodologies.

This study investigates the environmental impact and corrosion inhibition of novel double-chain arginine-based cationic surfactants developed as antimicrobial agents. The research focuses on asymmetric double-chain surfactants (LANHCx) with a 12-carbon alkyl chain and a second chain of 3-10 carbons, linked to the amino acid polar head group via amide bonds. The study assessed how alkyl chain length affects the ecological properties. Aerobic biodegradability (CO2 headspace test) and aquatic toxicity (short-term exposure on Daphnia magna, Aliivibrio fischeri, Tetraselmis chuii, and Phaeodactylum tricornutum) were evaluated. Corrosion inhibition efficiency was determined using electrochemical impedance spectroscopy (EIS). Alkyl chain length significantly influenced biodegradation rates (1-63 % at 12 mg C/L), with the C6 homologue showing minimal degradation, though it improved at lower concentrations. Biodegradation correlated with antimicrobial potency. These compounds exhibited over one order of magnitude lower aquatic toxicity than conventional quaternary ammonium surfactants (QACs). The arginine-based surfactant LANHC6 effectively inhibited carbon steel corrosion in neutral conditions (80 % at 0.5 mM), outperforming conventional DTAC at a 32-fold lower concentration. Overall, these new amino acid-derived antimicrobial agents demonstrate higher biodegradation rates and lower toxicity compared to conventional QACs, making them promising alternatives as environmentally preferable corrosion inhibitors.

Maritime corrosion is a global problem often retarded through protective coatings containing corrosion inhibitors (CIs). ZnAl layered double hydroxides (LDH) have been used to immobilize CIs, which can reduce their early leaching and, thus, foster long-term corrosion protection. However, the environmental behavior of these nanomaterials remains largely unknown, particularly in the context of global changes. The present study aims to assess the environmental behavior of four anti-corrosion nanomaterials in an ocean acidification scenario (IPCC SSP3-7.0). Three different concentrations of the nanostructured CIs (1.23, 11.11, and 100 mg L−1) were prepared and maintained at 20 °C and 30 °C in artificial salt water (ASW) at two pH values, with and without the presence of organic matter. The nanomaterials’ particle size and the release profiles of Al3+, Zn2+, and anions were monitored over time. In all conditions, the hydrodynamic size of the dispersed nanomaterials confirmed that the high ionic strength favors their aggregation/agglomeration. In the presence of organic matter, dissolved Al3+ increased, while Zn2+ decreased, and increased in the ocean acidification scenario at both temperatures. CIs were more released in the presence of humic acid. These findings demonstrate the influence of the tested parameters in the nanomaterials’ environmental behavior, leading to the release of metals and CIs.

Anthropogenic activities have introduced various contaminants into freshwater and marine ecosystems. Microplastics (MPs) are persistent and ubiquitous contaminants threatening natural ecosystems and impairing organisms at different biological levels of organization. Their durability and degradation rate pose a great concern in the scientific community, and thus, several techniques have been used to detect MPs effectively. The present study critically reviews the most commonly used techniques (FTIR, Raman, and fluorescence) and others considered novel regarding MP detection and characterisation, namely LIBS. Despite the effectiveness of such methodologies, none are free from drawbacks. The scientific community must join efforts to create, for example, innovative real-time (bio)sensing methodologies for MPs to overcome this gap.

Metallic corrosion is an unsolved economic and environmental problem, mitigated with coatings often containing toxic corrosion inhibitors. This study aims to extensively assess two eco-friendly and efficient anti-corrosion nanomaterials for maritime applications, specifically layered double hydroxides containing nitrite (Mg-Al LDH-NO2 and Zn-Al LDH-NO2) in terms of anti-corrosion performance, ecotoxicity, and environmental behavior and risk, along with a novel polyurethane coating. In aqueous dispersions, both LDHs behave as stimuli-responsive materials: unstable, tendency to rapidly aggregate and sink and release chemicals through anionic exchange and partial dissolution. Both nanomaterials presented negligible toxicity to most species, except microalgae Isochrysis galbana. Zn-Al LDH-NO2 also caused effects on the gastropod Phorcus lineatus and the echinoderm Paracentrotus lividus, being the most hazardous compound. Considering the environmental concentration of NO2 -, reported in the literature for well-oxygenated open ocean waters, and the statistically predicted no effect concentrations, tested chemicals potentially represent no environmental risk. The novel coating containing Zn-Al LDH-NO2 showed improved corrosion protection of carbon steel when benchmarked with state-of-the-art coatings. Leachates from these coatings also caused no/low ecotoxicological effects, attributed to the low release of chemicals over four weeks of immersion. These breakthroughs demonstrate that these novel ENMs are environmentally promising anti-corrosion nanoadditives for maritime coatings.

Corrosion significantly affects the maritime industry. To address this issue, corrosion inhibitors are incorporated into polymeric coatings. However, some state-of-the-art inhibitors are toxic, prone to spontaneous leaching, and interact with coating components. Accordingly, benzotriazole (BTA) and gluconate (GCN) have recently emerged as promising alternatives. Their immobilization into layered double hydroxides (LDHs) prevents direct contact with the polymeric matrix. It also allows a controlled release of BTA or GCN in response to specific stimuli (e.g., seawater, pH changes), thus providing long-lasting protection with hypothetical environmental benefits. The present study tests this hypothesis by assessing and comparing the sub-lethal effects of two novel anti-corrosion nanomaterials (Zn-Al LDH-BTA and Zn-Al LDH-GCN) and their soluble counterparts (BTA and GCN) in the edible bivalve Ruditapes philippinarum. Marine clams were exposed to sub-lethal concentrations (1.23, 11.11, and 100 mg BTA or GCN/L) of soluble and nanostructured BTA and GCN for 96 h. Subsequently, effects on health condition status, oxidative stress, neurotoxicity, and genotoxicity were assessed. Both nanoadditives (Zn-Al LDH-BTA and Zn-Al LDH-GCN) and soluble GCN caused no significant sub-lethal effects. However, exposure to soluble BTA induced GPx activity at 1.23 mg/L and DNA damage at 1.23 and 100 mg/L. This suggests that both nanoadditives are promising alternatives with high anti-corrosion performance and low ecotoxicity. GCN forms may be safer, as the biomolecule caused no sub-lethal effects in the tested species. This study presents a step forward in the development of and holistic assessment of eco-friendly anti-corrosion nanoadditives with enhanced performance.

This study reports the development of anticorrosive bilayer coatings consisting of in situ grown layered double hydroxides (LDH) covered with a polyurethane layer on a steel substrate. This design aims at providing corrosion protection via the controlled release of gluconate from LDH near the substrate, while at the same time contributing to the improvement of the polyurethane layer coating adhesion. The CaAl-LDH thin film was initially grown directly on AISI 1080/1010 carbon steel and modified with environmentally friendly gluconate molecules through an ion-exchange reaction. The effect of polyurethane treatments on the LDHs thin film was systematically explored: gluconate is either intercalated in LDHs or dispersed in polyurethane coatings, and the two systems are studied to understand the role of inhibitors in bilayer coating systems at defined conditions. The structural characteristics of the developed coatings were evaluated by scanning electrochemical microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (ATR-FTIR), and glow discharge optical microscopy (GDOES). The findings of electrochemical impedance spectroscopy (EIS) measurements on coated carbon steel substrates in NaCl solution demonstrated the significance of the bilayer film design for long-term corrosion protection, by combining active corrosion protection provided by the LDH conversion film with the passive barrier effect against electrolyte species rendered by the organic polyurethane layer. Additionally, improving the polyurethane coating’s wet adhesion to the substrate when applied onto CaAl-LDH opens new directions toward the co-development of surface treatments with organic coatings.

Engineered nanomaterials (ENMs), such as silica mesoporous nanocapsules (SiNC), have emerged as a powerful tool for the controlled delivery and release of active compounds in various fields. However, the environmental impact of SiNC on marine biota, particularly when they enter the marine environment through wastewater effluents or direct release from maritime coatings, remains poorly understood. Studying their effects is thus crucial for environmental and human health protection, the development of safe-by-design ENMs, and informed policy-making. This study aims to assess the ecotoxicological effects and internalization of industrially-relevant SiNC in marine phytoplankton, namely on the microalgae Tetraselmis chuii, Nannochloropsis gaditana, and Isochrysis galbana, and diatoms Phaeodactylum tricornutum, and Chaetoceros calcitrans. For this purpose, a fluorescent nanocarrier (SiNC-UMB) is developed by labeling the SiNC with the fluorescent natural dye umbelliferone (UMB). UV-vis and fluorescence spectroscopic analyses confirmed the successful loading of UMB into SiNC. Phytoplankton can internalize these ENMs, even at low concentrations, although adsorption to the cell wall can also occur. This confirms the internal exposure and growth inhibition observed in the microalgae. These findings highlight the potential of using SiNC-UMB as a valuable tool for tracking their uptake and assessing their effects on marine biota and beyond. Silica mesoporous nanocapsules (SiNC) labeled with the fluorescent dye umbelliferone can be internalized by marine microalgae even at low concentrations, although adsorption to the cell wall may also occur. Ecotoxicological findings on five marine microalgae species suggest there is space for optimization to be safely used for tracking purposes and/or assessment of SiNC effects on biota. image

The high toxicity and occurrence of ochratoxin A (OTA) in grains and foods has been a growing concern due to the impacts on health and the economy in many countries. In this sense, simplified devices with high sensitivity and specificity for local monitoring are enthusiastically pursued. In this work, we report for the first time the detection of ochratoxin A in coffee samples using a spoon-shaped waveguide immunosensor. The biosensor was built with the surface of the spoon-shaped waveguide covered by a 60 nm layer of gold to enable the SPR phenomenon. The measurements indicated a linear relationship between the change in the SPR phenomenon values and the OTA concentration in the range from 0.2 ppt to 5 ppt. When analyzed in coffee samples, the biosensor was highly selective and did not suffer matrix interference. The developed biosensor represents a promising analytical device for coffee quality analyses, as it is portable, simple, and suitable for onsite detection of target analytes.

Dimethoate (DMT) is one of the most harmful and commonly used organophosphate pesticides in agricultural lands to control different groups of parasitic insects. However, this pesticide is considered a dangerous pollutant for aquatic organisms following its infiltration in coastal ecosystems through leaching. Yet, our investigation aimed to gain new insights into the toxicity mechanism of DMT in the muscles of the green crab Carcinus aestuarii, regarding oxidative stress, neurotransmission impairment, histological aspects, and changes in lipid composition, assessed for the first time on the green crab’s muscle. Specimens of C. aestuarii were exposed to 50, 100, and 200 µg DMT L−1 for 24 h. Compared to the negative control group, the higher the DMT concentration, the lower the saturated fatty acids (SFA), and the higher the monounsaturated fatty acids (MUFA). The significant increase in polyunsaturated fatty acid n-6 (PUFA n-6) was related to the high release, mainly, of linoleic acid (LA, C18: 2n6) and arachidonic acid (ARA, C20: 4n6) levels. Biochemical biomarkers showed that DMT exposure promoted oxidative stress, highlighted by increased levels of hydrogen peroxide (H2O2), malondialdehyde (MDA), advanced oxidation protein product levels (AOPP), and protein carbonyl (PCO). Furthermore, the antioxidant defense system was activated, as demonstrated by the significant changes in the enzymatic activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and reduced glutathione (GSH) levels associated with an adaptation process of C. aestuarii to cope with the DMT exposure. This pesticide significantly impairs the neurotransmission process, as evidenced by the inhibition of acetylcholinesterase (AChE) activity. Finally, several histopathological changes were revealed in DMT-treated crabs, including vacuolation, and muscle bundle loss. This research offered new insights into the toxic mechanism of DMT, pointing to the usefulness of fatty acid (FA) composition as a sensitive biomarker in littoral crabs.

DCOIT (4,5-Dichloro-2-octylisothiazol-3(2H)-one) is a widely used antifouling biocide that emerged after the ban on tributyltin. It has been immobilized in nanostructured silica (SiNC–DCOIT) to reduce its hazard in maritime coatings. This study aimed to compare the bioaccumulation, trophic transfer, and biomagnification of DCOIT in its soluble and nanostructured forms on mussels Mytilus galloprovincialis, using three different uptake routes: aqueous exposure (i.e., contaminated seawater), dietary exposure (i.e., microalgae Tetraselmis chuii as a contaminated food), and both contaminated food and seawater. DCOIT was determined on water and tissues after 1, 3, and 24 h of uptake and after 72 h of depuration. Briefly, mussels were able to rapidly uptake and metabolize DCOIT and SiNC–DCOIT. Both compounds were non-bioaccumulative, as their bioconcentration and bioaccumulation factor values were lower than 2000. However, the predator–prey biomagnification factors indicated that both forms could be transferred across the trophic web. Therefore, while our findings provide further insight into the environmental risk assessment of DCOIT and SiNC–DCOIT, they do not rule out the possibility of long-term DCOIT bioaccumulation, particularly in areas with constant DCOIT influx. Further studies are thus needed using larger experimental designs and under continuous exposure scenarios to increase the sustainability of the innovative nanomaterial.

The encapsulation of antifouling compounds, such as DCOIT (4,5-Dichloro-2-octylisothiazol-3(2H)-one), in mesoporous silica nanocapsules (SiNC) has recently been demonstrated to be an eco-friendly alternative to decrease biocide toxicity towards marine non-target species. However, the lack of information on the chronic effects of such nanomaterials on non-target tropical species is critical for a more comprehensive environmental risk assessment. Thus, the present study aimed to assess the chronic toxicity and hazard of the soluble and encapsulated forms of DCOIT on neotropical marine species. Chronic tests were conducted with six ecologically relevant species. No effect concentration (NOEC) values were combined with NOEC values reported for tropical species to assess the hazard using the probabilistic approach to derive each predicted no effect concentration (PNEC). The SiNC-DCOIT was three- to ten-fold less toxic than soluble DCOIT. Probabilistic-based PNECs were set at 0.0001 and 0.0097 µg DCOIT L−1 for the biocide soluble and nanostructured forms, respectively. The immobilization of DCOIT into SiNC led to an 84-fold hazard decrease, confirming that the encapsulation of DCOIT into SiNC is a promising eco-friendly alternative technique, even in a chronic exposure scenario. Therefore, the present study will contribute to better management of the environmental risk of such innovative products in the tropical marine environment.

Histological techniques are a traditional and essential tool for adequately comprehending cellular morphology and physiology in several scientific fields, including ecotoxicological studies. Although bioassays generally recommend using small test organisms, based on advantages such as low maintenance costs and the possibility of a higher number of replicates, the histological process of such organisms is still challenging due to the difficulty of handling them. The present study proposes an accessible protocol for performing the histology of a biological sample of 8 mm maximum length and using histopathological biomarkers in Senegal sole larvae for ecotoxicological tests. Eight-day old larvae of Solea senegalensis obtained from breeders' natural spawns in cultivation conditions were submitted to histological processing by an adjusted protocol comprising fixation, agarose array pre-embedding, dehydration, paraffin embedding, slides' preparation, and image analysis by light optical microscope coupled to a digital camera. Low-cost and easy-to-use alternatives were applied in the technique execution. The histological steps were detailed, and S. senegalensis morphological features were presented. This can be considered valuable background information for ecotoxicological studies where changes in cell morphology and physiology are expected.

Layered double hydroxides (LDHs) are stimuli-responsive anionic nanoclays. The vast possibilities of using LDHs can lead to their existence in the ecosystem, raising a question of potential ecological concern. However, little is known about the effect of these nanomaterials on freshwater organisms. The present study aimed to assess the ecotoxicological effects of Zinc-Aluminium LDH-nitrate (Zn-Al LDH-NO3) in zebrafish (Danio rerio) early life stages. The endpoints measured were mortality, malformations and hatching rate after exposure of D. rerio embryos and larvae to Zn-Al LDH-NO3 following the OECD 236 guideline. The behavioral, biochemical (markers of oxidative stress and neurotoxicity), and molecular (at DNA level) alterations were also assessed using sub-lethal concentrations. No observable acute effects were detected up to 415.2 mg LDH/L while the 96 h-LC50 was estimated as 559.9 mg/L. Tested LDH caused malformations in D. rerio embryos, such as pericardial edema, incomplete yolk sac absorption and tail deformities (96 h-EC50 = 172.4 mg/L). During the dark periods, the locomotor behavior in zebrafish larvae was affected upon Zn-Al LDH-NO3 exposure. However, no significant biochemical and molecular changes were recorded. The present findings suggest that Zn-Al LDH-NO3 can be regarded as a non-toxic nanomaterial towards D. rerio (E/LC50 > > 100 mg/L) although impairment of the locomotion behavior on zebrafish embryos can be expected at concentrations below 100 mg/L.