Ecotoxicological Effects of Potassium Dichromate on the Tadpole Shrimp Triops longicaudatus.

The tadpole shrimp Triops longicaudatus is a freshwater crustacean with fast embryonic and larval development, short life cycle, and high fecundity. They are very active swimmers of a reasonable size, easy to spot and record. Such characteristics make it a promising candidate as an experimental model in ecotoxicology to evaluate the effects of aquatic pollutants, particularly using its locomotor behavior as an endpoint. To evaluate the sensitivity of T. longicaudatus and develop endpoints of interest, we conducted exposure experiments with lethal and sub-lethal concentrations of potassium dichromate, a compound known for its ecotoxicological importance and as a hexavalent chromium source. The endpoints evaluated were mortality, growth, sexual maturation, reproductive output, cholinesterase activity and locomotor/swimming behavior. The 96 h median lethal concentration was found to be 65 µg/L. Furthermore, exposure to potassium dichromate at higher concentrations had a significant negative impact on the growth rate of T. longicaudatus in terms of both body mass and length. The time for maturation was also delayed at higher concentrations. In addition, locomotor behavior allowed for the discrimination of all tested chromium concentrations and the control group and from each other, proving to be the most sensitive endpoint. Overall, the data support the potential of T. longicaudatus as a model for ecotoxicity testing, using apical endpoints with impact at the population level; in particular, results suggest that behavior assessments in this species might be useful for detecting hazardous compounds in environmental monitoring of freshwater ecosystems.

Toxic Relationships: Prediction of TBT's Affinity to the Ecdysteroid Receptor of Triops longicaudatus.

Tributyltin (TBT) is a biocide introduced in the 1960s in antifouling paints. Despite legislation banning its use, its persistence in the environment still causes significant harm to organisms. Tributyltin is a ligand of retinoid X receptors (RXR) and ecdysteroid receptors (EcRs), which in arthropods act as homologs of RXR. Focusing on Metazoan species, this study used genomic and proteomic information from different sources to compare their three-dimensional structure, phylogenetic distribution, and amino acid sequence alterations. The objective was to identify possible patterns that relate organisms' sensitivity to TBT using the species Triops longicaudatus as the basis for the comparisons. The results showed great conservation of this protein across several species when comparing the interaction amino acids described to RXR (an EcR analog) in Homo sapiens. The three-dimensional comparison of RXR showed little conformational variation between different sequences by maintaining the interaction pocket. As for the Species Sensitivity Distribution (SSD) curve, an HC05 = 0.2649 [0.0789-0.7082] µg/L was obtained with no specific distribution between the different taxa. Protein-ligand docking analysis was then used to confirm the SSD curve ranking of species. Still, the results showed an opposite trend that may be related, for example, to differences in the LC50 values used in the calculations. This study serves as the first step for applying bioinformatics techniques to produce information that can be used as an alternative to animal or cellular experimentation. These techniques could be adapted to various chemicals and proteins, allowing for observations in a shorter timeframe and providing information on a broader spectrum.

Cytotoxicity and maternal toxicity attributed to exposure to Momordica charantia L. (Cucurbitaceae) dry leaf extract.

Momordica charantia L. (Cucurbitaceae), popularly known as "bitter melon" or "bitter gourd," is a climbing plant well-adapted to tropical countries. This plant is used traditionally to treat several conditions including diabetes mellitus, inflammation, liver dysfunctions, and cancer. Given the widespread ethnopharmacological use, this study aimed to examine the cytogenetic, maternal, and developmental toxicity attributed to exposure to dry extract of M. charantia leaves using Allium cepa and Wistar rats as test models. First, phytochemical characterization of the dry extract by high performance liquid chromatography (HPLC) analyses was performed. Then, Allium cepa roots were exposed to three different concentrations of the dry extract (0.25, 0.5, or 1 mg/ml) to determine the mitotic index, frequency of chromosomal aberrations, and nuclear abnormalities. In addition, pregnant Wistar rats were administered either 500; 1,000 or 2,000 mg/kg dry extract during the gestational period (GD) days 6-15, and subsequently possible toxic effect on the dams and fetuses were recorded. HPLC analyses confirmed rutin as the main secondary metabolite present in the dry extract. In the Allium cepa test, the dry extract was cytotoxic. In Wistar rats, dry extract administration reduced water and feed intake and mean body mass gain, indicating maternal toxicity during the organogenesis period. However, the dry extract did not markedly affect reproductive outcome parameters evaluated. Regarding developmental toxicity assessment, the dry extract treatment did not significantly alter number of skeletal malformations in the offspring. Data demonstrated that the dry extract of M. charantia leaves presents cytotoxicity and low maternal toxicity, indicating indiscriminate use needs to be avoided.

Antibiotics removal from aquaculture effluents by ozonation: chemical and toxicity descriptors.

Antibiotics are often applied in aquaculture to prevent fish diseases. These substances can cause disturbances on receiving waters, when not properly eliminated from the aquaculture effluents. In this work, ozone (O3) was investigated as a possible oxidizing agent to remove fishery antibiotics from aquaculture effluents: florfenicol (FF), oxytetracycline (OTC), sulfadimethoxine (SDM), sulfamethoxazole (SMX), and trimethoprim (TMP). Batch experiments were performed using ultrapure water and aquaculture effluents spiked with a mixture of target antibiotics at relatively high concentrations (10 mg L-1 each). OTC, SMX and TMP were fully removed (< 30 min) regardless of the tested conditions, mainly by O3 direct attack. In contrast, FF was partially removed in 30 min (∼ 10 and 60%, in aquaculture effluents and ultrapure water, respectively), but only in the presence of hydroxyl radicals (HO•), the FF concentrations reaching levels below the detection limits in ultrapure water after 60 min. In the case of SDM, its degradation was highly influenced by the selected water matrix, but with removals always higher than 68%. In continuous-flow experiments applying more environmentally relevant antibiotic concentrations (100 ng L-1 each) and low O3 doses (1.5 mg L-1), ozonation highly removed (> 98%) all tested antibiotics from aquaculture effluents with a hydraulic retention time (HRT) of 10 min, except FF (68%). Although by-products were detected in treated samples, zebrafish (Danio rerio) embryotoxicity tests did not show a toxicity increase by applying this ozonation treatment. Ozonation is thus a possible solution to remove antibiotics from aquaculture effluents. Still, full-scale studies in aquaculture farms are needed, and generation of HO• may be favoured to readily oxidize the FF antibiotic.

Environmental diagnosis with Raman Spectroscopy applied to diatoms.

Freshwater quality has been changing due to the ever greater use of water resources and the contamination load resulting from human activities. Management of these systems, thus, requires constant diagnose of water quality with fast and efficient methodologies. The conventional methods adopted are, however, time-consuming, often very expensive, and require specialised expertise. Raman spectroscopy (RS) is a simple, fast and label-free technique that can be applied to environmental diagnosis using diatoms. Here, we developed a diagnostic method based on Raman spectroscopy applied to freshwater diatoms. For this, Raman spectra were recorded from diatoms of three lakes of a natural city park. The data acquired was analysed by chemometrics methods to describe the data (Partial Least Squares Regression), infer relationships in the dataset (Cluster Analysis) and produce classification models (Artificial Neural Network). The classification models developed diagnosed the lakes with excellent accuracy (89%) without requiring taxonomic information about the diatom species recorded. This study provides a proof-of-concept for the application of diatom Raman spectroscopy to diagnosing water quality, laying an important foundation for future environmental studies aiming at assessing freshwater systems, to be replicated at larger scales and to varied geographic settings.

Dietary tryptophan supplementation does not affect growth but increases brain serotonin level and modulates the expression of some liver genes in zebrafish (Danio rerio).

This study aimed at assessing the effects of the dietary tryptophan (Trp) supplementation on growth and feed utilization, brain serotonin content, and expression of selected liver genes (involved in the liver serotonin pathway, protein synthesis degradation, and antioxidant activity) in zebrafish. A growth trial was conducted with zebrafish juveniles fed five experimental isoproteic (40%DM) and isolipidic (8%DM) fishmeal-based diets containing graded levels of Trp: a Trp-non-supplemented diet (diet Trp0, with 0.22% Trp) and four Trp-supplemented diets containing 2-16 times higher Trp content (diets Trp2, Trp4, Trp8, and Trp16 with 0.40, 0.91, 2.02, and 3.34% Trp, respectively). Diets were tested in quadruplicate, with fish being fed twice a day, 6 days a week for 6 weeks to apparent visual satiation. At the end of the trial, growth performance and feed utilization were assessed, and fish from all experimental groups were sampled for whole-body composition analysis. In addition, fish fed low (Trp0), medium (Trp4), and high (Trp16) Trp diets were also sampled for analysis of brain serotonin content and liver gene expression. Tested tryptophan levels did not influence growth performance nor feed intake. However, values of energy and nitrogen retention as well as body energy content indicate a better feed utilization with diets containing around 0.9% and 2.0% DM Trp. Brain serotonin content increased with increasing dietary tryptophan levels. In addition, regarding liver genes, dietary treatment had a modulatory effect on the expression of Htr1aa and Htr2cl1 genes (encoding for serotonin receptors), TPH1a gene (encoding for tryptophan hydroxylase, the rate-limiting enzyme in the synthesis of serotonin from tryptophan), TOR gene (involved in protein synthesis), and Keap1 gene (involved in antioxidant responses).

Biomaterials and Adipose-Derived Mesenchymal Stem Cells for Regenerative Medicine: A Systematic Review.

The use of biological templates for the suitable growth of adipose-derived mesenchymal stem cells (AD-MSC) and "neo-tissue" construction has exponentially increased over the last years. The bioengineered scaffolds still have a prominent and biocompatible framework playing a role in tissue regeneration. In order to supply AD-MSCs, biomaterials, as the stem cell niche, are more often supplemented by or stimulate molecular signals that allow differentiation events into several strains, besides their secretion of cytokines and effects of immunomodulation. This systematic review aims to highlight the details of the integration of several types of biomaterials used in association with AD-MSCs, collecting notorious and basic data of in vitro and in vivo assays, taking into account the relevance of the interference of the cell lineage origin and handling cell line protocols for both the replacement and repairing of damaged tissues or organs in clinical application. Our group analyzed the quality and results of the 98 articles selected from PubMed, Scopus and Web of Science. A total of 97% of the articles retrieved demonstrated the potential in clinical applications. The synthetic polymers were the most used biomaterials associated with AD-MSCs and almost half of the selected articles were applied on bone regeneration.

Insights on a new sulfonamide chalcone with potential antineoplastic application.

Chalcones (E)-1,3-diphenyl-2-propene-1-ones, a class of biosynthetic precursor molecules of flavonoids, have a wide variety of biological applications. Besides the natural products, many synthetic derivatives and analogs became an object of continued interest in academia and industry. In this work, a synthesis and an extensive structural study were performed on a sulfonamide chalcone 1-Benzenesulfonyl-3-(4-bromobenzylidene)-2-(2-chlorophenyl)-2,3-dihydro-1H-quinolin-4-one with potential antineoplastic application. In addition, in silico experiments have shown that the sulfonamide chalcone fits well in the ligand-binding site of EGFR with seven µ-alkyl binding energy interactions on the ligand-binding site. Finally, the kinetic stability and the pharmacophoric analysis for EGFR indicated the necessary spatial characteristics for potential activity of sulfonamide chalcone as an antagonist.

Microplastics as a vehicle of exposure to chemical contamination in freshwater systems: Current research status and way forward.

Contamination by microplastics is increasing steadily worldwide, affecting all environments. Additionally, aquatic organisms are often exposed to mixtures of other contaminants, including various chemicals. Numerous studies reported adsorption of chemicals to microplastics, raising concern about their possible role as vehicles of exposure through transfer to biota. Nevertheless, until recently, the studies on the topic were mostly focused on the marine environment. In the past five years, however, plenty of publications contributed empirical data about freshwater ecosystems, raising the need for a critical appraisal of the information. Herein the scientific literature was reviewed and multivariate data analysis was done. The analysed studies employed widely different experimental designs, endpoints, test species, shapes and concentrations of various polymer types and chemicals, often not relevant for the freshwater environment. Our integrated analytical approach revealed unfathomable research gaps, given the theoretical knowledge available and lessons learned from research about the marine environment. Greater harmonization of laboratory studies investigating this topic is needed, as well as testing conditions reflecting real exposure scenarios. Furthermore, standardized testing protocols are urgently required to guide such experiments and improve the comparability of the results obtained.

Raman spectroscopy applied to diatoms (microalgae, Bacillariophyta): Prospective use in the environmental diagnosis of freshwater ecosystems.

Diatom species are good pollution bioindicators due to their large distribution, fast response to changes in environmental parameters and different tolerance ranges. These organisms are used in ecological water assessment all over the world using autoecological indices. Such assessments commonly rely on the taxonomic identification of diatom species-specific shape and frustule ornaments, from which cell counts, species richness and diversity indices can be estimated. Taxonomic identification is, however, time-consuming and requires years of expertise. Additionally, though the diatom autoecological indices are region-specific, they are often applied indiscriminately across regions. Raman spectroscopy is a simpler, fast and label-free technique that can be applied to environmental diagnosis with diatoms. However, this approach has been poorly explored. This work reviews Raman spectroscopy studies involving the structure, location and conformation of diatom cell components and their variation under different conditions. A critical appreciation of the pros and cons of its application to environmental diagnosis is also given. This knowledge provides a strong foundation for the development of environmental protocols using Raman spectroscopy in diatoms. Our work aims at stimulating further research on the application of Raman spectroscopy as a tool to assess physiological changes and water quality under a changing climate.

Influência das atividades de lazer na qualidade de vida de idosos institucionalizados / Influence of leisure activities on the quality of life of institutionalized elderly / Influencia de las actividades de ocio en la calidad de vida de las personas mayores institucionalizadas

Introdução: As alterações sociodemográficas que acarretaram o aumento do número de idosos mundialmente têm sido responsáveis pelas pesquisas cada vez mais constantes na área de gerontologia e qualidade de vida (QV), em busca de avanços e melhorias nos serviços de promoção, prevenção e assistência aos longevos. Objetivos: Avaliar a qualidade de vida dos idosos institucionalizados, caracterizar de forma sociodemográfica as pessoas idosas investigadas e apresentar quais das atividades oferecidas interferem significativamente nos escores da QV dos mesmos. Método: Amostra obtida de 13 idosos residentes em Instituição de Longa Permanência (ILPI), entre novembro de 2017 a janeiro de 2018, os quais foram entrevistados através de questionários, sendo dois destes de Avaliação de QV da Organização Mundial da Saúde, e o terceiro de caracterização sociodemográfica e de atividades. Resultados: A maior parte dos idosos são do gênero masculino, têm boa adesão e participação nas atividades oferecidas e possuem QV satisfatórias quando comparadas a outros estudos em idosos institucionalizados. Houve maiores escores nos domínios Social e Morte e Morrer, e menores nos Físico e Autonomia. Conclusão: É necessário fortalecer e incentivar atividades que promovam melhorias físicas autonômicas aos idosos institucionalizados, os quais são afetados pelas idades mais avançadas e pelo desuso ocasionado na institucionalização. Estes idosos constituem parte importante de nossa sociedade, logo, cabe aos gestores dos serviços que prestam atenção em gerontologia, às universidades e à sociedade promover campanhas, projetos, medidas educativas capazes de melhorar a QV e proporcionar maior autonomia e independência aos nossos idosos.

Introduction: The sociodemographic changes that have led to the increase in the number of elderly people worldwide have been responsible for the increasingly constant research in the area of Gerontology and quality of life (QOL), in search of advances and improvements in promotion services, prevention and assistance to longevity. Objectives: To evaluate the quality of life of institutionalized elderly people, to characterize in a sociodemographic way the elderly people investigated and to present which of the activities offered significantly interfere in their QOL scores. Method: Sample obtained from 13 elderly residents of Long Stay Institution (ILPI), between November 2017 to January 2018, who were interviewed through questionnaires, two of them from World Health Organization's QOL Assessment, and the third of socio-demographic characterization and activities. Results: Most of the elderly are male, have good adherence and participation in the activities offered and have satisfactory QOL when compared to other studies in institutionalized elderly. There were higher scores in the Social and Death and Death domains, and lower scores in the Physical and Autonomy domains. Conclusion: It is necessary to strengthen and encourage activities that promote autonomic physical improvements to institutionalized elderly, who are affected by advanced ages and disuse caused by institutionalization. These elderly people are an important part of our society, so it is up to the managers of the services that pay attention to Gerontology, universities and society to promote campaigns, projects, educational measures capable of improving the QOL and providing greater autonomy and independence to our elderly.(AU)

Introducción: Los cambios sociodemográficos que han propiciado un incremento en el número de personas mayores a nivel mundial han sido responsables de una investigación cada vez más constante en el campo de la gerontología y la calidad de vida (CV), en busca de avances y mejoras en los servicios de promoción, prevención y asistencia a los longevos. Objetivo: Evaluar la calidad de vida de ancianos institucionalizados, caracterizar sociodemográficamente a los ancianos investigados y presentar cuáles de las actividades ofrecidas interfieren significativamente en sus puntuaciones de CV. Método: Muestra obtenida de 13 ancianos residentes en el Centro de Cuidados a Largo Plazo (ILPI), entre noviembre de 2017 y enero de 2018...(AU)

Occurrence of Levonorgestrel in Water Systems and Its Effects on Aquatic Organisms: A Review.

Levonorgestrel is one of the active ingredients of oral contraceptives detected in the aquatic environment at concentrations in the order of ng/L. During the past decade, a wealth of new information about levonorgestrel has been produced, with several studies having reported negative effects in the reproduction and growth of aquatic organisms after exposure to this emerging contaminant of concern. In the present study, the data about its levels in water and its effects on aquatic organisms were integrated and used to perform an updated preliminary aquatic risk assessment for levonorgestrel based on the guideline for Environmental Risk Assessment of Medicinal Products for Human Use from the European Medicines Agency. The aim was to investigate if this pharmaceutical has a risk for adverse effects on aquatic organisms (i.e. for organisms residing in surface water and groundwater). The results evidenced that levonorgestrel is likely to pose an environmental risk to surface water (risk quotient >1). Based on these results, a more refined risk assessment for this pharmaceutical is needed. Besides, our findings highlight the need for investigation under the adverse outcome pathway (AOP) framework, as well as for further studies about toxicological interactions between levonorgestrel and other synthetic steroids.

Effects of norfluoxetine and venlafaxine in zebrafish larvae: Molecular data.

The data presented herein relates to the article entitled "Norfluoxetine and venlafaxine in zebrafish larvae: single and combined toxicity of two pharmaceutical products relevant for risk assessment" [1]. Recent studies have shown the occurrence of active metabolites of human and veterinary pharmaceuticals in surface and wastewaters. Besides their biological activity, some are predicted to interact with the same molecular targets of their parental compounds, thus showing the potential to elicit detrimental effects on animals. Despite this, limited investigation on their effects on aquatic animals has been done. Genomic material resulting from zebrafish (Danio rerio) larvae exposed to the psychoactive compounds norfluoxetine (main fluoxetine metabolite), venlafaxine, or their mixture was collected for gene expression analysis of a determined pool of genes potentially involved in their mode-of-action and metabolism. Molecular parameters are a cost-effective and reliable way to understand modes-of-action and the potential risk of micropollutants, such as pharmaceutical products, in non-target organisms. Moreover, gene expression patterns can provide crucial complementary information to improve risk assessment, and monitoring of affected systems. The data reported in this article was used to depict the effects of single or combined exposure to norfluoxetine and venlafaxine and identify biomarkers of exposure to these compounds of interest to diagnose exposure and routine monitoring.

Assessing the ecological status of small Mediterranean rivers using benthic macroinvertebrates and macrophytes as indicators.

Rivers are amongst the most threatened ecosystems in Europe. To prevent further degradation and to improve their ecological status, effective mitigation and restoration actions are needed. Those actions are primarily based on the precision of the ecological assessment results. This study aims to assess the ecological status of two small Mediterranean rivers-the Âncora (AR) and the Ferreira (FR) rivers-through the analysis of biological (benthic macroinvertebrates and macrophytes), physico-chemical and hydromorphological quality elements. Specific objectives were to analyse variations of biotic and abiotic parameters amongst rivers, and amongst seasons and sites within each river, to confirm adequate temporal windows to develop the monitoring surveys. Results showed that only one AR site achieved a good ecological status in spring and summer, while the other sites presented a moderate ecological status in all seasons. FR sites had a moderate to bad ecological status in all seasons. Both rivers showed high levels of nutrients, particularly during spring and summer, and were found quite altered in terms of floristic composition of the riparian communities. The riparian forest and the forbs fringe were dominated by several alien woody species and nitrophilous communities, respectively. Based on a multidisciplinary approach, this study provides an in-depth description of the ecological status of two small Mediterranean rivers located in sites of European interest, as well as a sound basis for the management of the aquatic environments. Mitigation of diffuse pollution and restoration of the riparian zones are a priority to improve their ecological status.

Assessing the ecological status of fluvial ecosystems employing a macroinvertebrate multi-taxon and multi-biomarker approach.

Biomarkers are recognised sensitive early-warning tools of biological effects in aquatic organisms. In this scope, the main aim of this study was to investigate the potential usefulness of a battery of biomarkers, evaluated in different benthic macroinvertebrate taxa, to discriminate aquatic ecosystems with different levels of ecological status and to provide further clues supporting environmental management. The study took place during the autumn of 2013 and the spring and summer of 2014, and the study cases were two Mediterranean rivers (Âncora and Ferreira rivers), differing in their ecological status. The biomarkers determined are widely employed and comprise a large set of biochemical responses: the activity of enzymes (cholinesterases, glutathione S-transferases, catalase and lactate dehydrogenase) and the levels of lipid peroxidation. They were assessed seasonally and in different macroinvertebrate taxa. Thirteen water physico-chemical parameters were also seasonally determined, and the concentration of seven organophosphorus pesticides and the percentage of 32 trace metals in sediments were determined in the spring. This is particularly useful for water management. Based on this, authorities can take actions to prevent further damage in the ecological status. Multivariate analyses showed distinct patterns of biological response for the Calopteryx spp., Chironomidae and Baetis spp. taxa. Calopteryx spp. and Chironomidae, in particular, showed distinct response patterns for the two rivers, which were fairly stable across seasons. This study sets the foundations for future cost-effective biomonitoring campaigns in Mediterranean rivers, allowing to establish historical data important to understand ecosystem evolution, as well as baseline levels of diagnostic biomarkers in informative macroinvertebrate taxa.

Lymnaea stagnalis as a freshwater model invertebrate for ecotoxicological studies.

Lymnaea stagnalis, also referred to as great or common pond snail, is an abundant and widespread invertebrate species colonizing temperate limnic systems. Given the species importance, studies involving L. stagnalis have the potential to produce scientifically relevant information, leading to a better understanding of the damage caused by aquatic contamination, as well as the modes of action of toxicants. Lymnaea stagnalis individuals are easily maintained in laboratory conditions, with a lifespan of about two years. The snails are hermaphrodites and sexual maturity occurs about three months after egg laying. Importantly, they can produce a high number of offspring all year round and are considered well suited for use in investigations targeting the identification of developmental and reproductive impairments. The primary aims of this review were two-fold: i) to provide an updated and insightful compilation of established toxicological measures determined in both chronic and acute toxicity assays, as useful tool to the design and development of future research; and ii) to provide a state of the art related to direct toxicant exposure and its potentially negative effects on this species. Relevant and informative studies were analysed and discussed. Knowledge gaps in need to be addressed in the near future were further identified.

Data for the analysis of interactive multibiomarker responses of a marine crustacean to long-term exposure to aquatic contaminants.

The data presented herein relates to the article entitled "Multibiomarker interactions to diagnose and follow-up chronic exposure of a marine crustacean to hazardous and noxious substances (HNS)" (Abreu et al., 2018). Multibiomarker approaches, including molecular, biochemical, physiological and behaviour parameters, are recognised as valuable and cost-effective to employ in integrated chemical and biological effects monitoring of aquatic contamination. Many biomarkers assessed in such programmes share common physiological pathways, showing concomitant or interdependent responses, which can reflect in increased energy costs related to physiological acclimation. Though, routine single biomarker data analysis, and exploratory principal component analysis, limit information obtained from the data collected and their functional interpretation. Ultimately, this influences the type of management actions taken to protect an affected ecosystem. This article presents data employed to develop an analytical approach accounting for multibiomarker interactions. The method was useful to diagnose and follow-up long-term exposure of the marine green crab (Carcinus maenas) to Hazardous and Noxious Substances (HNS).

Multibiomarker interactions to diagnose and follow-up chronic exposure of a marine crustacean to Hazardous and Noxious Substances (HNS).

Integrated compensatory responses of physiological systems towards homeostasis are generally overlooked when it comes to analysing alterations in biochemical parameters indicative of such processes. Here an hypothesis-driven multivariate analysis accounting for interactive multibiomarker responses was used to investigate effects of long-term exposure of Carcinus maenas to Hazardous and Noxious Substances (HNS). Adult male crabs were exposed to low and high post-spill levels of acrylonitrile (ACN) or aniline (ANL) for 21d. Bioaccumulation, feeding behaviour, and biomarkers related to mode-of-action (MoA) (detoxification, neurotransmission and energy production) were evaluated over time. Distinct temporal patterns of response to low and high exposure concentrations were depicted, with a main set of interactive multibiomarker predictors identified for each HNS (five for ACN and three for ANL), useful to follow coupled evolvement of biomarker responses. ACN caused peripheral neurotoxic effects coupled with enhanced biotransformation and significant oxidative damage particularly relevant in gills. ANL elicited alterations in central neurotransmission affecting ventilation coupled with very low levels of oxidative damage in gills. Results indicate chronic toxicity data are determinant to improve HNS hazard assessment if the aim is to obtain reliable risk calculations, and develop effective predictive models avoiding overestimation but sufficiently protective. Accounting for multibiomarker interactions brought otherwise overlooked information about C. maenas responses and MoA of ACN and ANL.