Composition and seasonal variation of brachyura and anomura (Crustacea, Decapoda) associated with brown mussel farms at Praia da Cocanha, Brazil

The composition and seasonal variation of brachyuran and anomuran species associated with mussel farms were evaluated at Praia da Cocanha, São Paulo between May 2007 and February 2008. Nine mussel ropes were sampled at random in each quarter, and 1,208 organisms were identified, comprising five families and 28 species. The most numerous species was the porcellanid Pachycheles laevidactylus (18.5%), followed by the xanthids Acantholobulus schmitti (16.6%), Hexapanopeus paulensis (11.3%), Panopeus americanus (10.2%), and Menippe nodifrons (8.4%). The exotic crab Charybdis hellerii was recorded throughout the study period. The ecological descriptors, except Pielou evenness index, varied significantly over the time. The highest abundance and diversity of the species were recorded during November and February. This pattern was reversed for Berger-Parker dominance, with the lowest values recorded in February. The development of epifauna was correlated with the different stages of the mussel farms, since the mean size of mussels and consequently the abundance of epibiotic organisms and the structural complexity on the mussel ropes increased from May (seeding) until February (harvest). Despite this, the temporal population variations in recruitment patterns of the different epibionts should not be overlooked. The results indicated that the mussel farms provided favorable conditions for the development of these crustacean groups, which could be used in environmental monitoring programs and / or be exploited for the aquarium trade.

Role of food availability in the bathymetric distribution of the starfish "Marthasterias glacialis" (Lamk.) on reefs of northern Portugal

[EN] We examined whether the abundance and size of the starfish Marthasterias glacialis (Lamk.) exhibit a depth-dependent partitioning on subtidal reefs. We tested the hypothesis that differences in food availability can result in habitat partitioning along a depth gradient. The abundance and size of M. glacialis was registered at 4 depth strata: 0-4 m, 4-8 m, 8-12 m, and >12 m; we also recorded the number of food items that they were preying on. The abundance and size of M. glacialis decreased with depth. Mussels (Mytilus galloprivincialis) were the most preyed food item across all depth strata, followed by gastropods, sea urchins and barnacles; M. glacialis also consumed a significantly larger amount of mussels in feeding experiments compared with sea urchins and gastropods. The abundance of M. galloprivincialis beds decreased with depth. The clear link between the decrease in abundance and size of M. glacialis with depth and the decay of the most consumed prey (mussels) suggest that food availability may play an important role in the vertical distribution of this starfish, though wave-associated turbulence in the first few metres of the subtidal could also limit the abundance of M. glacialis.

Organismi acquatici e ambiente: meccanismi biochimici di interazione, risposta e adattamento

The research is focused on the relationship between some Mg2+-dependent ATPase activities of plasma- and mitochondrial membranes from tissues of cultured marine bivalve molluscs and potentially stressful environmental conditions, such as the exposure to contaminants both of natural origin (ammonia nitrogen, the main contaminant of aquaculture plants) and of anthropic source (alkyltins). The two filter-feeding bivalve species selected colonize different habitats: the common mussel Mytilus galloprovincialis binds to hard substrates and the Philippine clam Tapes philippinarum burrows into sea bottom sandy beds. The choice of typical species of coastal waters, extremely suitable for environmental studies due to their features of poor motility, resistance to transport and great filtering efficiency, may constitute a model to evaluate responses to contaminants of membrane-bound enzyme activities involved in key biochemical mechanisms, namely cell ionic regulation and mitochondrial energy production. In vitro and in vitro approaches have been pursued. In vitro assays were carried out by adding the contaminants (NH4Cl and alkyltins) directly to the ATPase reaction media. In vivo experiments were carried out by exposing mussels to various tributyl tin (TBT) concentrations under controlled conditions in aquaria. ATPase activities were determined spectrophotometrically according to the principles of the method of Fiske and Subbarow (1925). The main results obtained are detailed below. In Tapes philippinarum the interaction of NH4 +, the main form of ammonia nitrogen at physiological and seawater pHs, with the Na,K-ATPase and the ouabaininsensitive Na-ATPase was investigated in vitro on gill and mantle microsomal membranes. The proven replacement by NH4 +of K+ in the activation of the Na,KATPase and of Na+ in the activation of the ouabain-insensitive ATPase displayed similar enzyme affinity for the substituted cation. on the one hand this finding may represent one of the possible mechanisms of ammonia toxicity and, on the other, it supports the hypothesis that NH4 + can be transported across the plasma membrane through the two ATPases. In this case both microsomal ATPases may be involved and co-operate, at least under peculiar circumstances, to nitrogen excretion and ammonia detoxification mechanisms in bivalve molluscs. The two ATPase activities stimulated by NH4 + maintained their typical response to the glycoside ouabain, specific inhibitor of the Na,K-ATPase, being the Na++ NH4 +-activated ATPase even more susceptive to the inhibitor and the ouabain-insensitive ATPase activity activated indifferently by Na+ or NH4 + unaffected by up to 10-2 M ouabain. In vitro assays were carried out to evaluate the response of the two Na-dependent ATPases to organotins in clams and mussels and to investigate the interaction of TBT with mussel mitochondrial oligomycin-sensitive Mg-ATPase. Since no literature data were available, the optimal assay conditions and oligomycin sensitivity of mussel mitochondrial MgATPase were determined. In T. philippinarum the ouabain-insensitive Na-ATPase was found to be refractory to TBT both in the gills and in the mantle, whereas the Na,K-ATPase was progressively inhibited by increasing TBT doses; the enzyme inhibition was more pronounced in the gills than in the mantle. In both tissues of M. galloprovincialis the Na,K-ATPase inhibition by alkyltins decreased in the order TBT>DBT(dibutyltin)>>MBT(monobutyltin)=TeET(tetraethyltin) (no effect). Mussel Na-ATPase confirmed its refractorimess to TBT and derivatives both in the gills and in the mantle. These results indicate that the Na,K-ATPase inhibition decreases as the number of alkyl chains bound to tin decreases; however a certain polarity of the organotin molecule is required to yield Na,K-ATPase inhibition, since no enzyme inhibition occurred in the presence of tetraalkyl-substituted derivatives such as TeET . Assays carried out in the presence of the dithioerythritol (DTE) pointed out that the sulphhydrylic agent is capable to prevent the Na,K-ATPase inhibition by TBT, thus suggesting that the inhibitor may link to -SH groups of the enzyme complex.. Finally, the different effect of alkyltins on the two Na-dependent ATPases may constitute a further tool to differentiate between the two enzyme activities. These results add to the wealth of literature data describing different responses of the two enzyme activities to endogenous and exogenous modulators . Mussel mitochondrial Mg-ATPase was also found to be in vitro inhibited by TBT both in the gills and in the mantle: the enzyme inhibition followed non competitive kinetics. The failed effect of DTE pointed out that in this case the interaction of TBT with the enzyme complex is probably different from that with the Na,K-ATPase. The results are consistent with literature data showing that alkyltin may interact with enzyme structures with different mechanisms. Mussel exposure to different TBT sublethal doses in aquaria was carried out for 120 hours. Two samplings (after 24 and 120 hrs) were performed in order to evaluate a short-term response of gill and mantle Na,K-ATPase, ouabain-insensitive Na-ATPase and Mg-ATPase activities. The in vivo response to the contaminants of the enzyme activities under study was shown to be partially different from that pointed out in the in vitro assays. Mitochondrial Mg-ATPase activity appeared to be activated in TBTexposed mussels with respect to control ones, thus confirming the complexity of evaluating in vivo responses of the enzyme activities to contaminants, due to possible interactions of toxicants with molluscan metabolism. Concluding, the whole of data point out that microsomal and mitochondrial ATPase activities of bivalve molluscs are generally responsive to environmental contaminants and suggest that in some cases membrane-bound enzyme activities may represent the molecular target of their toxicity. Since the Na,K-ATPase, the Na-ATPase and the Mg-ATPase activities are poorly studied in marine bivalves, this research may contribute to enlarge knowledge in this quite unexplored field.

Bioremediation of PAHs - Limitations and soultions

Introduction 1.1 Occurrence of polycyclic aromatic hydrocarbons (PAH) in the environment Worldwide industrial and agricultural developments have released a large number of natural and synthetic hazardous compounds into the environment due to careless waste disposal, illegal waste dumping and accidental spills. As a result, there are numerous sites in the world that require cleanup of soils and groundwater. Polycyclic aromatic hydrocarbons (PAHs) are one of the major groups of these contaminants (Da Silva et al., 2003). PAHs constitute a diverse class of organic compounds consisting of two or more aromatic rings with various structural configurations (Prabhu and Phale, 2003). Being a derivative of benzene, PAHs are thermodynamically stable. In addition, these chemicals tend to adhere to particle surfaces, such as soils, because of their low water solubility and strong hydrophobicity, and this results in greater persistence under natural conditions. This persistence coupled with their potential carcinogenicity makes PAHs problematic environmental contaminants (Cerniglia, 1992; Sutherland, 1992). PAHs are widely found in high concentrations at many industrial sites, particularly those associated with petroleum, gas production and wood preserving industries (Wilson and Jones, 1993). 1.2 Remediation technologies Conventional techniques used for the remediation of soil polluted with organic contaminants include excavation of the contaminated soil and disposal to a landfill or capping - containment - of the contaminated areas of a site. These methods have some drawbacks. The first method simply moves the contamination elsewhere and may create significant risks in the excavation, handling and transport of hazardous material. Additionally, it is very difficult and increasingly expensive to find new landfill sites for the final disposal of the material. The cap and containment method is only an interim solution since the contamination remains on site, requiring monitoring and maintenance of the isolation barriers long into the future, with all the associated costs and potential liability. A better approach than these traditional methods is to completely destroy the pollutants, if possible, or transform them into harmless substances. Some technologies that have been used are high-temperature incineration and various types of chemical decomposition (for example, base-catalyzed dechlorination, UV oxidation). However, these methods have significant disadvantages, principally their technological complexity, high cost , and the lack of public acceptance. Bioremediation, on the contrast, is a promising option for the complete removal and destruction of contaminants. 1.3 Bioremediation of PAH contaminated soil & groundwater Bioremediation is the use of living organisms, primarily microorganisms, to degrade or detoxify hazardous wastes into harmless substances such as carbon dioxide, water and cell biomass Most PAHs are biodegradable unter natural conditions (Da Silva et al., 2003; Meysami and Baheri, 2003) and bioremediation for cleanup of PAH wastes has been extensively studied at both laboratory and commercial levels- It has been implemented at a number of contaminated sites, including the cleanup of the Exxon Valdez oil spill in Prince William Sound, Alaska in 1989, the Mega Borg spill off the Texas coast in 1990 and the Burgan Oil Field, Kuwait in 1994 (Purwaningsih, 2002). Different strategies for PAH bioremediation, such as in situ , ex situ or on site bioremediation were developed in recent years. In situ bioremediation is a technique that is applied to soil and groundwater at the site without removing the contaminated soil or groundwater, based on the provision of optimum conditions for microbiological contaminant breakdown.. Ex situ bioremediation of PAHs, on the other hand, is a technique applied to soil and groundwater which has been removed from the site via excavation (soil) or pumping (water). Hazardous contaminants are converted in controlled bioreactors into harmless compounds in an efficient manner. 1.4 Bioavailability of PAH in the subsurface Frequently, PAH contamination in the environment is occurs as contaminants that are sorbed onto soilparticles rather than in phase (NAPL, non aqueous phase liquids). It is known that the biodegradation rate of most PAHs sorbed onto soil is far lower than rates measured in solution cultures of microorganisms with pure solid pollutants (Alexander and Scow, 1989; Hamaker, 1972). It is generally believed that only that fraction of PAHs dissolved in the solution can be metabolized by microorganisms in soil. The amount of contaminant that can be readily taken up and degraded by microorganisms is defined as bioavailability (Bosma et al., 1997; Maier, 2000). Two phenomena have been suggested to cause the low bioavailability of PAHs in soil (Danielsson, 2000). The first one is strong adsorption of the contaminants to the soil constituents which then leads to very slow release rates of contaminants to the aqueous phase. Sorption is often well correlated with soil organic matter content (Means, 1980) and significantly reduces biodegradation (Manilal and Alexander, 1991). The second phenomenon is slow mass transfer of pollutants, such as pore diffusion in the soil aggregates or diffusion in the organic matter in the soil. The complex set of these physical, chemical and biological processes is schematically illustrated in Figure 1. As shown in Figure 1, biodegradation processes are taking place in the soil solution while diffusion processes occur in the narrow pores in and between soil aggregates (Danielsson, 2000). Seemingly contradictory studies can be found in the literature that indicate the rate and final extent of metabolism may be either lower or higher for sorbed PAHs by soil than those for pure PAHs (Van Loosdrecht et al., 1990). These contrasting results demonstrate that the bioavailability of organic contaminants sorbed onto soil is far from being well understood. Besides bioavailability, there are several other factors influencing the rate and extent of biodegradation of PAHs in soil including microbial population characteristics, physical and chemical properties of PAHs and environmental factors (temperature, moisture, pH, degree of contamination). Figure 1: Schematic diagram showing possible rate-limiting processes during bioremediation of hydrophobic organic contaminants in a contaminated soil-water system (not to scale) (Danielsson, 2000). 1.5 Increasing the bioavailability of PAH in soil Attempts to improve the biodegradation of PAHs in soil by increasing their bioavailability include the use of surfactants , solvents or solubility enhancers.. However, introduction of synthetic surfactant may result in the addition of one more pollutant. (Wang and Brusseau, 1993).A study conducted by Mulder et al. showed that the introduction of hydropropyl-ß-cyclodextrin (HPCD), a well-known PAH solubility enhancer, significantly increased the solubilization of PAHs although it did not improve the biodegradation rate of PAHs (Mulder et al., 1998), indicating that further research is required in order to develop a feasible and efficient remediation method. Enhancing the extent of PAHs mass transfer from the soil phase to the liquid might prove an efficient and environmentally low-risk alternative way of addressing the problem of slow PAH biodegradation in soil.

Natural patterns and anthropogenic disturbance in north Adriatic marine benthic assemblages: descriptive and methodological studies

Marine soft bottom systems show a high variability across multiple spatial and temporal scales. Both natural and anthropogenic sources of disturbance act together in affecting benthic sedimentary characteristics and species distribution. The description of such spatial variability is required to understand the ecological processes behind them. However, in order to have a better estimate of spatial patterns, methods that take into account the complexity of the sedimentary system are required. This PhD thesis aims to give a significant contribution both in improving the methodological approaches to the study of biological variability in soft bottom habitats and in increasing the knowledge of the effect that different process (both natural and anthropogenic) could have on the benthic communities of a large area in the North Adriatic Sea. Beta diversity is a measure of the variability in species composition, and Whittaker’s index has become the most widely used measure of beta-diversity. However, application of the Whittaker index to soft bottom assemblages of the Adriatic Sea highlighted its sensitivity to rare species (species recorded in a single sample). This over-weighting of rare species induces biased estimates of the heterogeneity, thus it becomes difficult to compare assemblages containing a high proportion of rare species. In benthic communities, the unusual large number of rare species is frequently attributed to a combination of sampling errors and insufficient sampling effort. In order to reduce the influence of rare species on the measure of beta diversity, I have developed an alternative index based on simple probabilistic considerations. It turns out that this probability index is an ordinary Michaelis-Menten transformation of Whittaker's index but behaves more favourably when species heterogeneity increases. The suggested index therefore seems appropriate when comparing patterns of complexity in marine benthic assemblages. Although the new index makes an important contribution to the study of biodiversity in sedimentary environment, it remains to be seen which processes, and at what scales, influence benthic patterns. The ability to predict the effects of ecological phenomena on benthic fauna highly depends on both spatial and temporal scales of variation. Once defined, implicitly or explicitly, these scales influence the questions asked, the methodological approaches and the interpretation of results. Problem often arise when representative samples are not taken and results are over-generalized, as can happen when results from small-scale experiments are used for resource planning and management. Such issues, although globally recognized, are far from been resolved in the North Adriatic Sea. This area is potentially affected by both natural (e.g. river inflow, eutrophication) and anthropogenic (e.g. gas extraction, fish-trawling) sources of disturbance. Although few studies in this area aimed at understanding which of these processes mainly affect macrobenthos, these have been conducted at a small spatial scale, as they were designated to examine local changes in benthic communities or particular species. However, in order to better describe all the putative processes occurring in the entire area, a high sampling effort performed at a large spatial scale is required. The sedimentary environment of the western part of the Adriatic Sea was extensively studied in this thesis. I have described, in detail, spatial patterns both in terms of sedimentary characteristics and macrobenthic organisms and have suggested putative processes (natural or of human origin) that might affect the benthic environment of the entire area. In particular I have examined the effect of off shore gas platforms on benthic diversity and tested their effect over a background of natural spatial variability. The results obtained suggest that natural processes in the North Adriatic such as river outflow and euthrophication show an inter-annual variability that might have important consequences on benthic assemblages, affecting for example their spatial pattern moving away from the coast and along a North to South gradient. Depth-related factors, such as food supply, light, temperature and salinity play an important role in explaining large scale benthic spatial variability (i.e., affecting both the abundance patterns and beta diversity). Nonetheless, more locally, effects probably related to an organic enrichment or pollution from Po river input has been observed. All these processes, together with few human-induced sources of variability (e.g. fishing disturbance), have a higher effect on macrofauna distribution than any effect related to the presence of gas platforms. The main effect of gas platforms is restricted mainly to small spatial scales and related to a change in habitat complexity due to a natural dislodgement or structure cleaning of mussels that colonize their legs. The accumulation of mussels on the sediment reasonably affects benthic infauna composition. All the components of the study presented in this thesis highlight the need to carefully consider methodological aspects related to the study of sedimentary habitats. With particular regards to the North Adriatic Sea, a multi-scale analysis along natural and anthopogenic gradients was useful for detecting the influence of all the processes affecting the sedimentary environment. In the future, applying a similar approach may lead to an unambiguous assessment of the state of the benthic community in the North Adriatic Sea. Such assessment may be useful in understanding if any anthropogenic source of disturbance has a negative effect on the marine environment, and if so, planning sustainable strategies for a proper management of the affected area.

Composti organostannici nell'ambiente marino e membrane biologiche: risposte molecolari e biochimiche nei molluschi bivalvi

Organotin compounds are worldwide diffused environmental contaminants, mainly as consequence of their extensive past use as biocides in antifouling paints. In spite of law restrictions, due to unwanted effects, organotin still persist in waters, being poorly degraded, easily resuspended from sediments and bioaccumulated in exposed organisms. The widespread toxicity and the possible threat to humans, likely to be organotin-exposed through contaminated seafood, make organotin interactions with biomolecules an intriguing biochemical topic, apart from a matter of ecotoxicological concern. Among organotins, tributyltin (TBT) is long known as the most dangerous and abundant chemical species in the Mediterranean Sea. Due to its amphiphilic nature, provided by three lipophilic arms and an electrophilic tin core, TBT can be easily incorporated in biomembranes and affect their functionality. Accordingly, it is known as a membrane-active toxicant and a mitochondrial poison. Up to now the molecular action modes of TBT are still partially unclear and poorly explored in bivalve mollusks, even if the latter play a not neglectable role in the marine trophic chain and efficiently accumulate organotins. The bivalve mollusk Mytilus galloprovincialis, selected for all experiments, is widely cultivated in the Mediterranean and currently used in ecotoxicological studies. Most work of this thesis was devoted to TBT effects on mussel mitochondria, but other possible targets of TBT were also considered. A great deal of literature points out TBT as endocrine disrupter and the masculinization of female marine gastropods, the so-called imposex, currently signals environmental organotin contamination. The hormonal status of TBT-exposed mussels and the possible interaction between hormones and contaminants in modulating microsomal hydroxilases, involved in steroid hormone and organotin detoxification, were the research topics in the period spent in Barcelona (Marco Polo fellowship). The variegated experimental approach, which consisted of two exposure experiments and in vitro tests, and the choice of selected tissues of M. galloprovincialis, the midgut gland for mitochondrial and microsomal preparations for subsequent laboratory assays and the gonads for the endocrine evaluations, aimed at drawing a clarifying pattern on the molecular mechanisms involved in organotin toxicity. TBT was promptly incorporated in midgut gland mitochondria of adult mussels exposed to 0.5 and 1.0 μg/L TBT, and partially degraded to DBT. TBT incorporation was accompanied by a decrease in the mitochondrial oligomycin-sensitive Mg-ATPase activity, while the coexistent oligomycin-insensitive fraction was unaffected. Mitochondrial fatty acids showed a clear rise in n-3 polyunsaturated fatty acids after 120 hr of TBT exposure, mainly referable to an increase in 22:6 level. TBT was also shown to inhibit the ATP hydrolytic activity of the mitochondrial F1FO complex in vitro and to promote an apparent loss of oligomycin sensitivity at higher than 1.0 μM concentration. The complex dose-dependent profile of the inhibition curve lead to the hypothesis of multiple TBT binding sites. At lower than 1.0 μM TBT concentrations the non competitive enzyme inhibition by TBT was ascribed to the non covalent binding of TBT to FO subunit. On the other hand the observed drop in oligomycin sensitivity at higher than 1.0 μM TBT could be related to the onset of covalent bonds involving thiolic groups on the enzyme structure, apparently reached only at high TBT levels. The mitochondrial respiratory complexes were in vitro affected by TBT, apart from the cytocrome c oxidase which was apparently refractory to the contaminant. The most striking inhibitory effect was shown on complex I, and ascribed to possible covalent bonds of TBT with –SH groups on the enzyme complexes. This mechanism, shouldered by the progressive decrease of free cystein residues in the presence of increasing TBT concentrations, suggests that the onset of covalent tin-sulphur bonds in distinct protein structures may constitute the molecular basis of widespread TBT effects on mitochondrial complexes. Energy production disturbances, in turn affecting energy consuming mechanisms, could be involved in other cellular changes. Mussels exposed to a wide range of TBT concentrations (20 - 200 and 2000 ng/L respectively) did not show any change in testosterone and estrogen levels in mature gonads. Most hormones were in the non-biologically active esterified form both in control and in TBT-treated mussels. Probably the endocrine status of sexually mature mussels could be refractory even to high TBT doses. In mussel digestive gland the high biological variability of microsomal 7-benzyloxy-4-trifluoromethylcoumarin-O-Debenzyloxylase (BFCOD) activity, taken as a measure of CYP3A-like efficiency, probably concealed any enzyme response to TBT exposure. On the other hand the TBT-driven enhancement of BFCOD activity in vitro was once again ascribed to covalent binding to thiol groups which, in this case, would stimulate the enzyme activity. In mussels from Barcelona harbour, a highly contaminated site, the enzyme showed a decreased affinity for the 7-benzyloxy-4-trifluoromethylcoumarin (BCF) substrate with respect to mussel sampled from Ebro Delta, a non-polluted marine site. Contaminant exposure may thus alter the kinetic features of enzymes involved in detoxification mechanisms. Contaminants and steroid hormones were clearly shown to mutually interact in the modulation of detoxification mechanisms. The xenoestrogen 17α-ethylenyl estradiol (EE2) displayed a non-competitive mixed inhibition of CYP3A-like activity by a preferential bond to the free enzyme both in Barcelona harbour and Ebro Delta mussels. The possible interaction with co-present contaminants in Barcelona harbour mussels apparently lessened the formation of the ternary complex enzyme-EE2-BCF. The whole of data confirms TBT as membrane toxicant in mussels as in other species and stresses TBT covalent binding to protein thiols as a widespread mechanism of membrane-bound-enzyme activity modulation by the contaminant.

Pharmaceutical residues in aquatic systems: mode of action and effects on mussel physiology

Pharmaceutical residues contaminate aquatic ecosystems as a result of their widespread human and veterinary usage. Since continuously released and not efficiently removed, certain pharmaceuticals exhibit pseudo-persistence thus generating concerns for the health of aquatic wildlife. This work aimed at assessing on mussels Mytilus galloprovincialis, under laboratory conditions, the effects of three pharmaceuticals, carbamazepine (antiepileptic), propranolol (β-blocker) and oxytetracycline (antibiotic), to evaluate if the human-based mode of action of these molecules is conserved in invertebrates. Furthermore, in the framework of the European MEECE Programme, mussels were exposed to oxytetracycline and copper at increasing temperatures, simulating variations due to climate changes. The effects of these compounds were assessed evaluating a battery of biomarkers, the expression of HSP70 proteins and changes in cAMP-related parameters. A decrease in lysosomal membrane stability, induction of oxidative stress, alterations of cAMP-dependent pathway and the induction of defense mechanisms were observed indicating the development of a stress syndrome, and a worsening in mussels health status. Data obtained in MEECE Programme confirmed that the toxicity of substances can be enhanced following changes in temperature. The alterations observed were obtained after exposure to pharmaceuticals at concentrations sometimes lower than those detected in the aquatic environment. Hence, further research is advisable regarding subtle effects of pharmaceuticals on non-target organisms. Furthermore, results obtained during a research stay in the laboratories of Cádiz University (Spain) are presented. The project aimed at measuring possible effects of polluted sediments in Algeciras Bay (Spain) and in Cádiz Bay, by assessing different physiological parameters in caged crabs Carcinus maenas and clams Ruditapes decussatus exposed in situ for 28 days. The neutral red retention assay was adapted to these species and proved to be a sensitive screening tool for the assessment of sediment quality.

Effects of artificial defences and flooding on coastal habitats and assemblages

Since large stretches of European coasts are already retreating and projected scenarios are worsening, many artificial structures, such as breakwaters and seawalls, are built as tool against coastal erosion. However artificial structures produce widespread changes that alter the coastal zones and affect the biological communities. My doctoral thesis analyses the consequences of different options for coastal protection, namely hard engineering ‘artificial defences’ (i.e. impact of human-made structures) and ‘no-defence’ (i.e. impact of seawater inundation). I investigated two new aspects of the potential impact of coastal defences. The first was the effect of artificial hard substrates on the fish communities structure. In particular I was interested to test if the differences among breakwaters and natural rocky reef would change depending on the nature of the surrounding habitat of the artificial structure (prevalent sandy rather than rocky). The second was the effect on the native natural sandy habitats of the organic detritus derived from hard-bottom species (green algae and mussels) detached from breakwaters. Furthermore, I investigated the ecological implication of the “no-defend” option, which allow the inundation of coastal habitats. The focus of this study was the potential effect of seawater intrusion on the degradation process of marine, salt-marsh and terrestrial detritus, including changes on the breakdown rates and the associated macrofauna. The PhD research was conducted in three areas along European coasts: North Adriatic sea, Sicilian coast and South-West England where different habitats (coastal, estuarine), biological communities (soft-bottom macro-benthos; rocky-coastal fishes; estuarine macro-invertebrates) and processes (organic enrichment; assemblage structure; leaf-litter breakdown) were analyzed. The research was carried out through manipulative and descriptive field-experiments in which specific hypothesis were tested by univariate and multivariate analyses.

Entwicklung eines immunologischen Testsystems zur Detektion des marinen Biotoxins Domoinsäure

Domoinsäure ist ein von mehreren Arten mariner Kieselalgen der Gattung Pseudonitzschia produziertes Toxin, welches während einer Algenblüte in Molluscen wie z.B. der Miesmuschel Mytilus sp. akkumuliert werden kann. Beim Verzehr solch kontaminierter Muscheln können sowohl beim Menschen als auch bei Tieren erhebliche Vergiftungserscheinungen auftreten, die von Übelkeit, Kopfschmerzen und Orientierungsstörungen bis hin zum Verlust des Kurzzeitgedächtnisses (daher auch als amnesic shellfish poisoning bekannt) reichen und in einigen Fällen tödlich enden. rnDie heute gängigen Methoden zur Detektion von Domoinsäure in Muschelgewebe wie Flüssigkeitschromatographie und Maus-Bioassay sind zeit- und kostenintensiv bzw. in Anbetracht einer Verbesserung des Tierschutzes aus ethischer Sicht nicht zu vertreten. Immunologische Testsysteme stellen eine erstrebenswerte Alternative dar, da sie sich durch eine vergleichsweise einfache Handhabung, hohe Selektivität und Reproduzierbarkeit auszeichnen.rnDas Ziel der vorliegenden Arbeit war es, ein solches immunologisches Testsystem zur Detektion von Domoinsäure zu entwickeln. Hierfür wurden zunächst Antikörper gegen Domoinsäure gewonnen, wofür das Toxin wiederum als erstes über die Carbodiimid-Methode an das Trägerprotein keyhole limpet hemocyanin (KLH) gekoppelt wurde, um eine Immunantwort auslösen zu können. Kaninchen und Mäuse wurden mit KLH-DO-Konjugaten nach vorgegebenen Immunisierungsschemata immunisiert. Nach vier Blutabnahmen zeigte das polyklonale Kaninchenantiserum eine ausreichend hohe Sensitivität zum Antigen; das nachfolgende Detektionssystem wurde mit Hilfe dieses polyklonalen Antikörpers aufgebaut. Zwar ist es gegen Ende der Arbeit auch gelungen, einen spezifischen monoklonalen Antikörper aus der Maus zu gewinnen, jedoch konnte dieser aus zeitlichen Gründen nicht mehr im Detektionssystem etabliert werden, was durchaus wünschenswert gewesen wäre. rnWeiterhin wurde Domoinsäure im Zuge der Entwicklung eines neuartigen Testsystems an die Trägerproteine Ovalbumin, Trypsininhibitor und Casein sowie an Biotin konjugiert. Die Kopplungserfolge wurden im ELISA, Western Blot bzw. Dot Blot nachgewiesen. Die Ovalbumin-gekoppelte sowie die biotinylierte Domoinsäure dienten im Folgenden als die zu messenden Größen in den Detektionsassays- die in einer zu untersuchenden Probe vorhandende, kompetitierende Domoinsäure wurde somit indirekt nachgewiesen. rnDer zulässige Höchstwert für Domoinsäure liegt bei 20 µg/g Muschelgewebe. Sowohl mit Biotin-DO als auch mit OVA-DO als den zu messenden Größen waren Domoinsäurekonzentrationen unterhalb dieses Grenzwertes nachweisbar; allerdings erwies sich der Aufbau mit Biotin-DO um das ca. 20-fache empfindlicher als jener mit OVA-DO. rnDie in dieser Arbeit präsentierten Ergebnisse könnten als Grundlage zur Etablierung eines kommerzialisierbaren immunologischen Testsystems zur Detektion von Domoinsäure und anderen Biotoxinen dienen. Nach erfolgreicher Validierung wäre ein solches Testsystem in seiner Handhabung einfacher als die gängige Flüssigkeitschromatographie und besser reproduzierbar als der Maus-Bioassay.rn

Ricerca e applicazione di metodologie ecotossicologiche nel monitoraggio di ambienti marino-costieri: Sviluppo di nuovi bioassay e biomarker

Obiettivo del lavoro è stato lo sviluppo e la validazione di nuovi bioassay e biomarker quali strumenti da utilizzare in un approccio ecotossicologico integrato per il biomonitoraggio di ambienti marino-costieri interessati da impatto antropico negli organismi che vivono in tali ambienti. L’ambiente reale impiegato per l’applicazione in campo è la Rada di Augusta (Siracusa, Italia). Una batteria di bioassay in vivo e in vitro è stata indagata quale strumento di screening per la misura della tossicità dei sedimenti. La batteria selezionata ha dimostrato di possedere i requisiti necessari ad un applicazione di routine nel monitoraggio di ambienti marino costieri. L’approccio multimarker basato sull’impiego dell’organismo bioindicatore Mytilus galloprovincialis in esperimenti di traslocazione ha consentito di valutare il potenziale applicativo di nuovi biomarker citologici e molecolari di stress chimico parallelamente a biomarker standardizzati di danno genotossico ed esposizione a metalli pesanti. I mitili sono stati traslocati per 45 giorni nei siti di Brucoli (SR) e Rada di Augusta, rispettivamente sito di controllo e sito impattato. I risultati ottenuti supportano l’applicabilità delle alterazioni morfometriche dei granulociti quale biomarker di effetto, direttamente correlato allo stato di salute degli organismi che vivono in un dato ambiente. Il significativo incremento dell’area dei lisosomi osservato contestualmente potrebbe riflettere un incremento dei processi degradativi e dei processi autofagici. I dati sulla sensibilità in campo suggeriscono una valida applicazione della misura dell’attività di anidrasi carbonica in ghiandola digestiva come biomarker di stress in ambiente marino costiero. L’utilizzo delle due metodologie d’indagine (bioassay e biomarker) in un approccio ecotossicologico integrato al biomonitoraggio di ambienti marino-costieri offre uno strumento sensibile e specifico per la valutazione dell’esposizione ad inquinanti e del danno potenziale esercitato dagli inquinanti sugli organismi che vivono in un dato ambiente, permettendo interventi a breve termine e la messa a punto di adeguati programmi di gestione sostenibile dell’ambiente.

Impact of anthropogenic stressors on the population genetics of the common bioindicator Dreissena polymorpha (Pallas, 1771)

Toxicant inputs from agriculture, industry and human settlements have been shown to severely affect freshwater ecosystems. Pollution can lead to changes in population genetic patterns through various genetic and stochastic processes. In my thesis, I investigated the impact of anthropogenic stressors on the population genetics of the zebra mussel Dreissena polymorpha. In order to analyze the genetics of zebra mussel populations, I isolated five new highly polymorphic microsatellite loci. Out of those and other already existing microsatellite markers for this species, I established a robust marker set of six microsatellite loci for D. polymorpha. rnMonitoring the biogeographical background is an important requirement when integrating population genetic measures into ecotoxicological studies. I analyzed the biogeographical background of eleven populations in a section of the River Danube (in Hungary and Croatia) and some of its tributaries, and another population in the River Rhine as genetic outgroup. Moreover, I measured abiotic water parameters at the sampling sites and analyzed if they were correlated with the genetic parameters of the populations. The genetic differentiation was basically consistent with the overall biogeographical history of the populations in the study region. However, the genetic diversity of the populations was not influenced by the geographical distance between the populations, but by the environmental factors oxygen and temperature and also by other unidentified factors. I found strong evidence that genetic adaptation of zebra mussel populations to local habitat conditions had influenced the genetic constitution of the populations. Moreover, by establishing the biogeographical baseline of molecular variance in the study area, I laid the foundation for interpreting population genetic results in ecotoxicological experiments in this region.rnIn a cooperation project with the Department of Zoology of the University of Zagreb, I elaborated an integrated approach in biomonitoring with D. polymorpha by combining the analysis techniques of microsatellite analysis, Comet assay and micronucleus test (MNT). This approach was applied in a case study on freshwater contamination by an effluent of a wastewater treatment plant (WWTP) in the River Drava (Croatia) and a complementary laboratory experiment. I assessed and compared the genetic status of two zebra mussel populations from a contaminated and a reference site. Microsatellite analysis suggested that the contaminated population had undergone a genetic bottleneck, caused by random genetic drift and selection, whereas a bottleneck was not detected in the reference population. The Comet assay did not indicate any difference in DNA damage between the two populations, but MNT revealed that the contaminated population had an increased percentage of micronuclei in hemocytes in comparison to the reference population. The laboratory experiment with mussels exposed to municipal wastewater revealed that mussels from the contaminated site had a lower percentage of tail DNA and a higher percentage of micronuclei than the reference population. These differences between populations were probably caused by an overall decreased fitness of mussels from the contaminated site due to genetic drift and by an enhanced DNA repair mechanism due to adaptation to pollution in the source habitat. Overall, the combination of the three biomarkers provided sufficient information on the impact of both treated and non-treated municipal wastewater on the genetics of zebra mussels at different levels of biological organization.rnIn my thesis, I could show that the newly established marker set of six microsatellite loci provided reliable and informative data for population genetic analyses of D. polymorpha. The adaptation of the analyzed zebra mussel populations to the local conditions of their habitat had a strong influence on their genetic constitution. We found evidence that the different genetic constitutions of two populations had influenced the outcome of our ecotoxicological experiment. Overall, the integrated approach in biomonitoring gave comprehensive information about the impact of both treated and non-treated municipal wastewater on the genetics of zebra mussels at different levels of biological organization and was well practicable in a first case study.

Assessment of marine litter in the Western Adriatic Sea and its impact on marine biota

Due to its environmental, safety, health and socio-economic impacts, marine litter has been recognized as a 21st century global challenge, so that it has been included in Descriptor 10 of the EU MSFD. For its morphological features and anthropogenic pressures, the Adriatic Sea is very sensitive to the accumulation of debris, but data are inconsistent and fragmented. This thesis, in the framework of DeFishGear project, intents to assess marine litter on beaches and on seafloor in the Western Adriatic sea, and test if debris ingestion by fish occurs. Three beaches were sampled during two surveys in 2015. Benthic litter monitoring was carried out in the FAO GSA17 during fall 2014, using a rapido trawl. Litter ingestion was investigated through gut contents analysis of 260 fish belonging to 8 commercial species collected in Western Gulf of Venice. Average litter density on beaches was 1.5 items/m2 during spring, and decreased to 0.8 items/m2 in summer. Litter composition was heterogeneous and varied among sites, even if no significant differences were found. Most of debris consisted of plastic sheets, fragments, polystyrene pieces, mussels nets and cottons bud sticks, showing that sources are many and include aquaculture, land-based activities and local users of beaches. Average density of benthic litter was 913 items/Km2 (82 Kg/Km2). Plastic dominated in terms of numbers and weight, and consisted mainly of bags, sheets and mussel nets. The highest density was found close to the coast, and sources driving the major differences in litter distribution were mussel farms and shipping lanes. Litter ingestion occurred in 47% of examined fish, mainly consisting of fibers. Among species, S. pilchardus swallowed almost all debris categories. Findinds may provide a baseline to set the necessary measures to manage and minimize marine litter in the Western Adriatic region and to protect aquatic life from plastic pollution, even accounting the possible implications on human health.

Cladophora, mass transport, and the nearshore phosphorus shunt

Excessive Cladophora growth in the Great Lakes has led to beach fouling and the temporary closure of nuclear power plants and has been associated with avian botulism and the persistence of human pathogens. As the growth-limiting nutrient for Cladophora, phosphorus is the appropriate target for management efforts. Dreissenids (zebra and quagga mussels) have the ability to capture particulate phase phosphorus (otherwise unavailable to Cladophora) and release it in a soluble, available form. The significance of this potential nutrient source is, in part, influenced by the interplay between phosphorus flux from the mussel bed and turbulent mixing in establishing the phosphorus levels to which Cladophora is exposed. It is hypothesized that under quiescent conditions phosphorus will accumulate near the sediment-water interface, setting up vertical phosphorus gradients and favorable conditions for resource delivery to Cladophora. These gradients would be eliminated under conditions of wind mixing, reducing the significance of the dreissenid-mediated nutrient contribution. Soluble reactive phosphorus (SRP) levels were monitored over dreissenid beds (densities on the order of 350•m-2 and 3000∙m-2) at a site 8 m deep in Lake Michigan. Monitoring was based on the deployment of Modified Hesslein Samplers which collected samples for SRP analysis over a distance of 34 cm above the bottom in 2.5 cm intervals. Deployment intervals were established to capture a wind regime (calm, windy) that persisted for an interval consistent with the sampler equilibration time of 7 hours. Results indicate that increased mussel density leads to an increased concentration boundary layer; increased wind speed leads to entrainment of the concentration boundary layer; and increased duration of quiescent periods leads to an increased concentration boundary layer. This concentration boundary layer is of ecological significance and forms in the region inhabited by Cladophora

Energetics of byssus attachment and feeding in the green-lipped mussel Perna canaliculus.

In most animals, significant increases in metabolic rate are due to activity and to feeding (known as apparent specific dynamic action). We determined the energetic costs of activity and feeding in adult green-lipped mussels (Perna canaliculus). Maximal metabolic rate was determined, using closed-chamber respirometry, during byssus re-attachment, during specific dynamic action after 16 h of feeding with Isochrysis galbana, and for the two activities combined, in 23 mussels. Metabolic rate was significantly elevated above rest by about 1.9-fold during byssus attachment (17.1 ± 1.53 μg O(2) h(-1) g(-1) whole mussel wet weight at rest, increased to 27.9 ± 0.91 μg O(2) h(-1) g(-1)), and by 2.2-fold after feeding (31.4 ± 1.20 μg O(2) h(-1) g(-1)). Combined feeding and byssus attachment led to a still higher metabolic rate (34.0 ± 1.23 μg O(2) h(-1) g(-1)). Behavior was also significantly altered, with mussels being almost continuously open during attachment and after feeding (90%-99% of the time); however, the time spent open during the day decreased, reaching a minimum of 52% ± 9% 3 days after feeding, and remained low (67%-82%) for the following 45-day starvation period. Significant diurnal differences were observed, with mussels continuously (92%-100%) open at night. The key findings from this study are that green-lipped mussels (1) have an aerobic scope of approximately 2-fold; (2) reach a higher metabolic rate during feeding than during activity, and the two combined can raise the metabolic rate higher still; (3) display a marked diurnal behavior.

An Aquaculture-Based Method for Calibrated Bivalve Isotope Paleothermometry

To quantify species- specific relationships between bivalve carbonate isotope geochemistry ( delta O-18(c)) and water conditions ( temperature and salinity, related to water isotopic composition [delta O-18(w)]), an aquaculture-based methodology was developed and applied to Mytilus edulis ( blue mussel). The four- by- three factorial design consisted of four circulating temperature baths ( 7, 11, 15, and 19 degrees C) and three salinity ranges ( 23, 28, and 32 parts per thousand ( ppt); monitored for delta O-18(w) weekly). In mid- July of 2003, 4800 juvenile mussels were collected in Salt Bay, Damariscotta, Maine, and were placed in each configuration. The size distribution of harvested mussels, based on 105 specimens, ranged from 10.9 mm to 29.5 mm with a mean size of 19.8 mm. The mussels were grown in controlled conditions for up to 8.5 months, and a paleotemperature relationship based on juvenile M. edulis from Maine was developed from animals harvested at months 4, 5, and 8.5. This relationship [ T degrees C = 16.19 (+/- 0.14) - 4.69 (+/- 0.21) {delta O-18(c) VPBD - delta O-18(w) VSMOW} + 0.17 (+/- 0.13) {delta O-18(c) VPBD - delta O-18(w) VSMOW}(2); r(2) = 0.99; N = 105; P < 0.0001] is nearly identical to the Kim and O'Neil ( 1997) abiogenic calcite equation over the entire temperature range ( 7 - 19 degrees C), and it closely resembles the commonly used paleotemperature equations of Epstein et al. ( 1953) and Horibe and Oba ( 1972). Further, the comparison of the M. edulis paleotemperature equation with the Kim and O'Neil ( 1997) equilibrium- based equation indicates that M. edulis specimens used in this study precipitated their shell in isotopic equilibrium with ambient water within the experimental uncertainties of both studies. The aquaculture- based methodology described here allows similar species- specific isotope paleothermometer calibrations to be performed with other bivalve species and thus provides improved quantitative paleoenvironmental reconstructions.