Application of a Weight of Evidence Approach for Monitoring Complex Environmental Scenarios: the Case-Study of Off-Shore Platforms

Multidisciplinary investigations based on integration of chemical and biological measurements, represent an added value to monitoring and management protocols, and their use is recommended by European Directives to evaluate the environmental status of aquatic ecosystems. However, assessing the overall significance of results obtained in different typologies of studies is often a difficult challenge. The aim of this work was to present a quantitative Weight Of Evidence (WOE) model (Sediqualsoft) to integrate huge amounts of heterogeneous data and to validate this approach in complex monitoring scenarios. Using the case-study of an off-shore platform field in the Adriatic Sea, procedures are presented to elaborate different typologies of data (lines of evidence, LOEs), including chemical characterization of sediments, bioavailability, biomarkers, ecotoxicological bioassays and benthic communities around three platforms. These data are initially evaluated by logical flowcharts and mathematical algorithms, which provide specific hazard indices for each considered LOE, before their different weighting and overall integration in an environmental risk index. The monitoring study selected for the WOE elaboration consisted on chemical analyses of trace metals, aliphatic hydrocarbons, polycyclic aromatic hydrocarbons carried out on 60 sediment samples; the same samples were also characterized for the status of benthic communities; bioavailability of metals from sediments was assessed in laboratory conditions on the polychaete Hediste diversicolor, while bioaccumulation of inorganic and organic chemicals and biomarker responses were measured in native and transplanted mussels; ecotoxicological properties of sediments were evaluated through a battery of bioassays determining algal growth of the diatom Phaeodactylum tricornutum, bioluminescence of the marine bacterium Vibrio fischeri, survival of the copepod Acartia tonsa and embryotoxicity of sea urchin Paracentrotus lividus. Overall, almost 7000 analytical results were elaborated and summarized in specific hazard indices. The WOE integration of multiple typologies of data allowed more robust and weighted conclusions compared to the use of individual LOEs, highlighting the feasibility of this procedure for multidisciplinary monitoring and risk assessment approaches. On a practical side, the WOE evidences also suggested a revision of actual monitoring procedures. Overall, the proposed WOE model appeared as a useful tool to summarize large datasets of complex data in integrative indices, and to simplify the interpretation for stakeholders and decision makers, thus supporting a more comprehensive process of "site-oriented" management decisions

Ecotoxicological potential of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) in marine organisms: bioavailability, biomarkers and natural occurrence in Mytilus galloprovincialis

I residui dei composti farmaceutici presenti in ambiente rappresentano una problematica emergente dato che le informazioni riguardo la loro presenza, distribuzione e potenziale ecotossicologico sono molto limitate, specialmente per le aree costiere. In questa tesi è stata valutata la sensibilità del mitilo Mediterraneo Mytilus galloprovincialis nei confronti di diversi farmaci anti infiammatori non steroidei (FANS), applicando un approccio integrato che ha previsto sia attività di laboratorio che indagini di campo. In condizioni di laboratorio i mitili sono stati esposti a diverse concentrazioni ambientalmente realistiche (25, 2.5 e 0.5 μg/L) di acetaminofene AMP, diclofenac DIC, ibuprofene IBU, ketoprofene KET e nimesulide NIM, per diversi tempi di esposizione (da 14 a 60 giorni). Il potenziale ecotossicologico dei FANS è stato valutato combinando le analisi chimiche del bioaccumulo dei farmaci con un approccio multi-biomarker, basato sullo studio di un ampio numero di risposte subcellulari che rappresentano dei segnali di allerta precoce di alterazione cellulare e di tossicità. Per alcune condizioni sperimentali, le alterazioni funzionali misurate a livello cellulare sono state integrate con modificazioni trascrittomiche a livello molecolare attraverso la tecnica di microarray a DNA. I risultati ottenuti hanno dimostrato che i mitili sono in grado di bioaccumulare DIC, IBU e NIM non seguendo, tuttavia, una cinetica dose dipendente, mentre AMP e KET non sono mai stati misurati indipendentemente dalla dose e dal tempo di esposizione. Ciononostante, tutte le molecole testate e tutte le condizioni sperimentali hanno determinato l’insorgenza di alterazioni a carico dei parametri immunitari, modulazione del metabolismo lipidico e danno genotossico. Le analisi trascrittomiche hanno rivelato modificazioni a carico degli organismi esposti alle dosi più basse, sia nel breve (KET e NIM) che nel lungo termine (KET). I risultati ottenuti a livello molecolare supportano le alterazioni misurate a livello cellulare e suggeriscono che il meccanismo di azione dei FANS negli invertebrati risulta essere molto simile a quello ampiamente documentato nei mammiferi. Le indagini a lungo termine hanno permesso di comprendere che l’effetto dei FANS si mantiene costantemente per 60 giorni. Le indagini in campo hanno rivelato, per la prima volta, la presenza di DIC, IBU e NIM nei mitili naturali campionati in primavera e in estate da tipiche aree turistiche del Mare Adriatico centrale. Complessivamente tutti i risultati hanno dimostrato che il M. galloprovincialis è una buona specie sentinella per i FANS, e il reale pericolo ecotossicologico dei farmaci nel Mediterraneo.Pharmaceuticals represent a major environmental concern since the knowledge on their occurrence, distribution and ecotoxicological potential is still limited particularly in coastal areas. In this thesis the sensitivity of the Mediterranean mussels Mytilus galloprovincialis toward different Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) was assessed, applying an integrated approach which combined laboratory studies with field investigation. In laboratory conditions mussels were exposed to different environmental realistic concentrations (25, 2.5 and 0.5 μg/L) of acetaminophen AMP, diclofenac DIC, ibuprofen IBU, ketoprofen KET and nimesulide NIM, for different periods (from 14 to 60 days). The ecotoxicological potential of NSAIDs was evaluated combining chemical analyses on pharmaceuticals bioaccumulation with a multi-biomarker approach, based on a wide array of molecular and subcellular responses reflecting early warning signals of biological disturbance, modulation of specific cellular pathways, onset of various typologies of cellular damages and toxicity. For some experimental condition, functional alteration at cellular level were further integrated with transcriptomic changes at molecular level using DNA microarray. Obtained results demonstrated that mussels are able to bioconcentrate DIC, IBU and NIM without dose dependent response, while AMP and KET are never detected independently from the doses and the exposure period. Nonetheless, for all tested NSAIDs and in all experimental conditions, measurement of a large panel of ecotoxicological biomarkers highlighted impairment of immunological parameters, modulation of lipid metabolism and genotoxic effects. The analyses on transcriptomic profile highlighted changes at molecular level for organisms exposed to lower doses, both in short (for KET and NIM) and long-term condition (for KET). Molecular results supported changes obtained at cellular level and suggest similar mechanisms of action of NSAIDs in mammals and vertebrates. Long-term responses allowed to determine that the effects of anti-inflammatory pharmaceuticals were constantly maintained over 60 days. Field studies provided the first evidence on the occurrence of DIC, IBU and NIM in tissues of wild mussels sampled during summer and spring periods from typical, touristic areas of Central Adriatic Sea. Overall results demonstrated M. galloprovincialis as a good sentinel species toward anti inflammatory pharmaceuticals and the actual ecotoxicological hazard of pharmaceuticals in the Mediterranean

The Biological Effects of Pharmaceuticals in the Marine Environment

Environmental pharmaceuticals represent a threat of emerging concern for marine ecosystems. Widely distributed and bioaccumulated, these contaminants could provoke adverse effects on aquatic organisms through modes of action like those reported for target species. In contrast to pharmacological uses, organisms in field conditions are exposed to complex mixtures of compounds with similar, different, or even opposing therapeutic effects. This review summarizes current knowledge of the main cellular pathways modulated by the most common classes of environmental pharmaceuticals occurring in marine ecosystems and accumulated by nontarget species—including nonsteroidal anti-inflammatory drugs, psychiatric drugs, cardiovascular and lipid regulator agents, steroidal hormones, and antibiotics—and describes an intricate network of possible interactions with both synergistic and antagonistic effects on the same cellular targets and metabolic pathways. This complexity reveals the intrinsic limits of the single-chemical approach to predict the long-term consequences and future impact of pharmaceuticals at organismal, population, and community levels

Pharmaceuticals in the aquatic environments: Evidence of emerged threat and future challenges for marine organisms

Pharmaceuticals are nowadays recognized as a threat for aquatic ecosystems. The growing consumption of these compounds and the enhancement of human health in the past two decades have been paralleled by the continuous input of such biologically active molecules in natural environments. Waste water treatment plants (WWTPs) have been identified as a major route for release of pharmaceuticals in aquatic bodies where concentrations ranging from ng/L to µg/L are ubiquitously detected. Since medicines principles are designed to be effective at very low concentrations, they have the potential to interfere with biochemical and physiological processes of aquatic species over their entire life cycle. Investigations on occurrence, bioaccumulation and effects in non target organisms are fragmentary, particularly for marine ecosystems, and related to only a limited number over the 4000 substances classified as pharmaceuticals: hence, there is a urgent need to prioritize the environmental sustainability of the most relevant compounds. The aim of this review is to summarize the main adverse effects documented for marine species exposed in both field and laboratory conditions to different classes of pharmaceuticals including non-steroidal anti-inflammatory drugs, psychiatric, cardiovascular, hypocholesterolaemic drugs, steroid hormones and antibiotics. Despite a great scientific advancement has been achieved, our knowledge is still limited on pharmaceuticals behavior in chemical mixtures, as well as their interactions with other environmental stressors. Complex ecotoxicological effects are increasingly documented and multidisciplinary, integrated approaches will be helpful to clarify the environmental hazard of these “emerged” pollutants in marine environment

Mixtures of environmental pharmaceuticals in marine organisms: Mechanistic evidence of carbamazepine and valsartan effects on Mytilus galloprovincialis

Unravelling the adverse outcomes of pharmaceuticals mixture represents a research priority to characterize the risk for marine ecosystems. The present study investigated, for the first time, the interactions between two of the most largely detected pharmaceuticals in marine species: carbamazepine (CBZ) and valsartan (VAL), elucidating mechanisms that can modulate bioaccumulation, excretion and the onset of toxicity. Mytilus galloprovincialis were exposed to environmental levels of CBZ and VAL dosed alone or in combination: measurement of drug bioaccumulation was integrated with changes in the whole transcriptome and responsiveness of various biochemical and cellular biomarkers. Interactive and competing mechanisms between tested drugs were revealed by the much higher CBZ accumulation in mussels exposed to this compound alone, while an opposite trend was observed for VAL. A complex network of responses was observed as variations of gene expression, functional effects on neurotransmission, cell cycle, immune responses and redox homeostasis. The elaboration of results through a quantitative Weight of Evidence model summarized a greater biological reactivity of CBZ compared to VAL and antagonistic interactions between these compounds, resulting in a reduced effect of the antiepileptic when combined with valsartan. Overall, new perspectives are highlighted for a more comprehensive risk assessment of environmental mixtures of pharmaceuticals

Seawater carbonate chemistry and carbamazepine bioaccumulation of Mytilus galloprovincialis

Contaminants of emerging concern and ocean changes are key environmental stressors for marine species with possibly synergistic, but still unexplored, deleterious effects. In the present study the influence of a simulated ocean acidification scenario (pH = 7.6) was investigated on metabolism and sub-lethal effects of carbamazepine, CBZ (1 µg/L), chosen as one of the most widely diffused pharmaceuticals in marine organisms. A multidisciplinary approach was applied on mussels, M. galloprovincialis, integrating measurement of drug bioaccumulation with changes in the whole transcriptome, responsiveness of various biochemical and cellular biomarkers including immunological parameters, lipid and oxidative metabolism, onset of genotoxic effects. Chemical analyses revealed a limited influence of hypercapnia on accumulation and excretion of CBZ, while a complex network of biological responses was observed in gene expression profile and functional changes at cellular level. The modulation of gamma-aminobutyric acid (GABA) pathway suggested similarities with the Mechanism of Action known for vertebrates: immune responses, cellular homeostasis and oxidative system represented the processes targeted by combined stressors. The overall elaboration of results through a quantitative Weight of Evidence model, revealed clearly increased cellular hazard due to interactions of CBZ with acidification compared to single stressors

The Comet Assay in Marine Animals

Comet assay is a quick and versatile technique for assessing DNA damage in individual cells. It allows for the detection of DNA single- and double-strand breaks, as well as the presence of alkali labile sites and cross-links. Here we describe protocols for the single-cell gel electrophoresis (Comet assay) in its alkaline (pH > 13), mild alkaline (pH = 12.1) and neutral (pH = 8) versions when applied in marine animals