Rozprawy doktorskie na temat „Estuarine invertebrates”

The importance of different sources of carbon (energy) supporting secondary productivity in estuaries and coastal waters is still rather uncertain, despite decades of research on this topic. One of the major uncertainties is the relative importance of carbon from macrophyte detritus and microalgae, particularly diatoms and cyanobacteria within sediments (microphytobenthos, MPB). Existing methods have been unable to determine their relative contributions to consumer nutrition. This thesis addresses some of the limitations of existing methods, and then uses an innovative combination of techniques to determine the relative contributions of detritus and MPB to the nutrition of consumers within a mangrove forest in southeast Queensland, Australia. To ensure wide applicability, techniques were developed for both mud and sand, encompassing the range of sediments encountered in estuaries and protected coastal waters. Stable isotope analysis is commonly used to resolve food web issues. Difficulty extracting MPB from sediment to obtain a pure carbon isotopic signature (δ13C), however, has hampered efforts to determine the importance of MPB as a carbon source for consumers. I showed that compound-specific isotope analysis of phytol, part of the chlorophyll molecule, can be used to estimate MPB δ13C with enough precision for most food web studies: within 1-1.8‰ of actual values (95% CI) at normal levels of replication (n = 5 or 10). Although the majority of phytol in sediments is produced in situ by MPB, part of the phytol pool is derived from detritus (e.g. 33% for mud, 17% for sand in the current study). In some situations, this could lead to errors in estimating MPBδ13C, realistically by about 1‰ (in sand) or 2‰ (in mud), but an adjustment can be made where necessary. Compound-specific isotope analysis of phytol (the phytol method) can therefore be confidently used to estimate MPB δ13C values in estuarine environments. Where δ13C values of detritus and MPB are similar, natural abundance stable isotopes are unable to resolve their contribution to consumer nutrition. Enrichment of producers with the heavy 13C isotope generates distinct δ13C values that can be traced into consumers. Studies aiming to enrich MPB using 13C-enriched sodium bicarbonate have, however, observed simultaneous enrichment of bacteria. This may simply be due to use of MPB-derived carbon by bacteria, but if bacteria acquire the label directly, enrichment of consumers would indicate use of bacteria, rather than MPB, as a primary carbon source. Application of 13C-enriched bicarbonate resulted in enrichment of MPB in both mud and sand and subsequent secondary enrichment of bacteria. Direct bacterial uptake was trivial in comparison to MPB uptake. Any labelling of animals in subsequent enrichment experiments could therefore be attributed to use of MPB as a primary carbon source. I used a combination of the phytol method, stable isotope enrichment, and compartment modelling within a mangrove forest to examine use of detritus and MPB as carbon sources for common consumers; crabs (Australoplax tridentata and Parasesarma erythrodactyla) and foraminifera (Ammonia beccarii and Trochammina inflata). Compartment modelling quantified the extent of use through comparison of producer and consumer enrichment over time. The main carbon source for T. inflata was unable to be established, but all other species acquired virtually all of their carbon locally (within a 1 m radius), from MPB and/or mangrove detritus. The majority of carbon for A. tridentata was derived from MPB (99%), whereas P. erythrodactyla and A. beccarii predominantly utilised mangrove detritus (88 and 84%, respectively). For the latter two species, the remainder of carbon was derived from MPB. The different strategies in carbon utilisation by the crab species indicate feeding selectivity that may be a strategy to avoid interspecific competition for food resources. The high abundance of benthic invertebrates and their rapid assimilation of local carbon sources highlight their potential importance in influencing carbon cycling and productivity in estuaries. Consumption of benthic invertebrates by more mobile, higher consumers may also allow for transport of locally-produced carbon to adjacent waters via trophic relay. The methods demonstrated in this thesis have potential application in resolving food web issues in a variety of habitats. Further application of these techniques at a variety of temporal and spatial scales would help to develop a broader understanding of the importance of macrophyte detritus and MPB, and establish general patterns in utilisation of carbon sources in estuarine systems.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment
Faculty of Environmental Sciences
Full Text

Between 1988 and 2014, otter trawls, seine nets, and plankton nets were deployed along the salinity gradients of 18 estuaries by the University of South Florida and the Florida Fish and Wildlife Research Institute (FWRI, a research branch of the Florida Fish and Wildlife Conservation Commission). The purpose of these surveys was to document the responses of aquatic estuarine biota to variation in the quantity and quality of freshwater inflows that were being managed by the Southwest Florida Water Management District (SWFWMD). In the present analyses, four community types collected by these gears were compared with a diversity of habitat factors to identify the factors with the greatest influence on beta diversity, and also to identify the factors that were most influential to important prey species and economically important species. The four community types were (1) plankton-net invertebrates, (2) plankton-net ichthyoplankton, (3) seine nekton, and (4) trawl nekton. The habitat factors were (1) vertical profiles of salinity, dissolved oxygen, pH, and water temperature taken at the time of the biological collections, (2) various characterizations of local habitat associated with seine and trawl deployments, (3) chlorophyll a, color, and turbidity data obtained from the STORET database (US Environmental Protection Agency), and (4) data that characterize the effects of freshwater inflow on different estuarine zones, including factors for freshwater inflow, freshwater turnover time, and temporal instability in freshwater inflow (flashiness). Only 13 of the 18 estuaries had data that were comprehensive enough to allow habitat-factor analysis. An existing study had performed distance-based redundancy analysis (dbRDA) and principle component analysis (PCA) for these data within 78 estuarine survey zones that were composited together (i.e., regardless of estuary of origin). Based on that study’s findings, the communities of primarily spring-fed and primarily surface-fed estuaries were analyzed separately in the present study. Analysis was also performed with the habitat factors grouped into three categories (water management, restoration, and water quality) based on their ability to be directly modified by different management sectors. For an analysis of beta diversity interactions with habitat factors, dbRDA (called distance-based linear modeling (DistLM) in the PRIMER software) was performed using PRIMER 7 software (Quest Research Limited, Auckland, NZ). The dbRDA indicated pH, salinity, and distance to the Gulf of Mexico (distance-to-GOM) usually explained the most variation in the biotic data. These results were compared with partial dbRDA using the Akaike Information Criterion (AIC) as the model selection criterion with distance-to-GOM held as a covariate to reduce the effect of differences in the connectivity of marine-derived organisms to the different estuaries; distance-to-GOM explained between 8.46% and 32.4% of the variation in beta diversity. Even with the variation from distance-to-GOM removed, salinity was still selected as most influential factor, explaining up to an additional 23.7% of the variation in beta diversity. Factors associated with the water-management sector were most influential (primarily salinity), followed by factors associated with the restoration sector (primarily factors that describe shoreline type and bottom type). For the analysis of individual species, canonical analysis of principal coordinates (CAP) was performed to test for significant difference in community structure between groups of sites that represented high and low levels of each factor. For those communities that were significantly different, an indicator value (IndVal) was calculated for each species for high and low levels of each factor. Among species with significant IndVal for high or low levels of at least one factor, emphasis was given to important prey species (polychaetes, copepods, mysids, shrimps, bay anchovy juveniles, and gammaridean amphipods) and to species of economic importance, including adults, larvae and juveniles of commercial and recreational fishes, pink shrimp, and blue crab. Shrimps, copepods and mysids were all associated with estuarine zones that had low percentages of wooded or lawn-type shoreline, a factor that may serve as a proxy for flood conditions, as lawns or trees were usually only sampled with seines at high water elevations and in the freshwater reaches of the estuaries. Many copepod and shrimp species were strongly associated with high flushing times, which suggests that if flushing times were too short in an estuarine zone, then these species or their prey would be flushed out. Multiple regression analysis was performed on each of the selected indicator species, using AIC as a selection criterion and distance-to-GOM as a covariate. As might be expected, the apparent influences of different habitat factors varied from species to species, but there were some general patterns. For prey species in both spring-fed and surface-fed estuaries, pH and flushing time explained a significant amount of variation. In surface-fed estuaries, the presence of oysters on the bottom also had a positive effect for many prey species. For economically important species, depth was important in both spring-fed and surface-fed estuaries. This suggested the importance of maintaining large, shallow areas, particularly in surface-fed estuaries. Another important factor in spring-fed estuaries was the percent coverage of the bottom with sand; however, a mixture of positive and negative coefficients on this factor suggested the importance of substrate variety. In surface-fed estuaries, flashiness also often explained substantial variation for many economically important species, usually with positive coefficients, possibly due to the importance of alternation between nutrient-loading and high-primary-productivity periods. When comparing the three management sectors, the restoration sector was the most explanatory. Several factors were averaged over entire estuaries due to data scarcity or due to the nature of the factors themselves. Specifically, the STORET data for chlorophyll, color, and turbidity was inconsistently distributed with in the survey areas and was not collected at the same time as the biological samples. Moreover, certain water-management factors such as freshwater-inflow rate and flashiness are inherently less dimensional than other factors, and could only be represented by a single observation (i.e., no spatial variation) at any point in time. Due to concern that reduced spatiotemporal concurrence/dimensionality was masking the influence of habitat factors, the community analysis was repeated after representing each estuary with a single value for each habitat factor. We found that far fewer factors were selected in this analysis; salinity was only factor selected from the water-management factors. Overall, the factor that explained the most variation most often was the presence of emergent vegetation on the shoreline. This factor is a good proxy for urban development (more developed areas have lower levels of emergent vegetation on the shoreline). Unlike the previous analysis, the restoration sector overwhelmingly had the highest R2 values compared with other management sectors. In general, these results indicate the seeming importance of salinity in the previous analysis was likely because it had a higher resolution compared with many other factors, and that the lack of resolution homogeneity did influence the results. Of the habitat factors determined to be most influential with the analysis of communities and individual species (salinity, pH, emergent vegetation and lawn-and-trees shoreline types, oyster and sand bottom types, depth, flashiness, and flushing time) most were part of an estuarine gradient with high values at one end of the estuary with a gradual shift to low values at the other end. Since many of the analyzed species also showed a gradient distribution across the estuary, the abundance and community patterns could be explained by any of the habitat factors with that same gradient pattern. Therefore, there is a certain limitation to determining which factors are most influential in estuaries using this type of regression-based analysis. Three selected factors that do not have a strong estuarine gradient pattern are the sand bottom type, depth, and flashiness. In particular, flashiness has a single value for each estuary so it is incapable of following the estuarine gradient. This suggests that flashiness has an important process-based role that merits further investigation of its effect on estuarine species.

Estuarine ecosystems are among the most biologically productive areas and they provide important ecosystem services such as erosion control, habitat and refugia for several species. These environments are characterized by the presence of a variety of organic matter sources due to their transitional position between rivers and the sea. The biotic compositions can undergo spatial and seasonal changes along the estuary due to the spatial and temporal fluctuations of environmental factors such as salinity, temperature and seston loads. Therefore, the different combinations of biotic and abiotic factors make each estuary a unique ecosystem. Because of this spatial and temporal complexity, the understanding of estuarine food web structure and which factors affect the trophic relationships within the ecosystem through space and time represent challenging tasks. Furthermore, estuaries are under an increasing number of anthropogenic perturbations because of the growing concentration of human populations in coastal areas. Knowledge of ecosystem structure and functioning is essential for effective conservation and management planning of coastal areas.In this dissertation, I combine the utilization of biological tracers to examine spatial and temporal variability in the food web structure within a small temperate and microtidal estuary located in South Africa. To this end, fatty acid profiles and stable isotope signatures were measured in several primary organic matter sources and consumers (including zooplankton, fishes and benthic invertebrates) during four consecutive seasons and in three different estuarine regions: upper, middle, and lower reaches. The three reaches had distinct habitat features of vegetation type and morphology, and in particular the lower reaches were colonized by the marsh grass Spartina maritima. Isotopic mixing models were used to estimate the relative contribution of each food source to the diets of invertebrates and fishes within the estuarine food web. The isotopic and fatty acid data showed similar results. In general, the lower reaches of the estuary were characterized by a higher deposition and assimilation by brachyuran crabs of carbon derived from marsh grass detritus, whiletowards the upper reaches a mixture of microphytobenthos and particulate organic matter (phytoplankton and detritus) was deposited and sustained the pelagic and benthic fauna. The highest deposition and assimilation of marsh grass detritus in the lower reaches of the estuary occurred during periods of low freshwater discharge (autumn and winter). In the upper reaches, microphytobenthos and suspended particulate organic matter were dominant basal food resources for the food web during all seasons. These results indicated that benthic consumers incorporated mainly local carbon sources from their local habitat.To clarify isotopic and fatty acid patterns I examined the trophic behaviour of the sesarmid crab Sesarma catenata through laboratory feeding experiments. Results from these experiments validated that decomposed leaves of riparian trees and the salt marsh plant S. maritima were the preferred food of the sesarmid crabs, potentially due to high bacterial loads. The remaining leaf material not assimilated by crabs, together with faecal material, are likely important subsidies for adjacent environments, hence representing an important energy pathway involving the microbial food chain. Furthermore, this dissertation showed the importance of mobile top predators as vectors energetically connecting distinct food chains within the estuary (i.e. littoral, benthic and pelagic). I concluded that a combination of physical (i.e. patterns of freshwater discharge and estuary morphology) and biological factors (i.e. organism feeding behaviour, mobility, primary productivity, the local vegetation type) influence the pattern of dominant primary organic matter sources, and therefore the food web structure along the estuarine environment. In particular, marsh grass detritus contributed substantially to the diets of estuarine fauna during periods of low freshwater discharge. Given the importance of the salt marsh habitat in providing trophic resources, it is important to preserve this environment to sustain the natural biota and ecosystem functioning.

There is an increasing concern over the safety of engineered nanoparticles (ENPs) to humans and the environment. It is important that the environmental risks of these particles to be tested under research and regulatory schemes, e.g. Nano Risks to the environment & Human Health (NanoReTox) under Seventh Framework Programme (FP7) in EU. Due to their unique properties and the fact that their detection and characterization in complex matrices is challenging, classic analytical methods and test approaches for assessing environmental risk may not be appropriate for ENPs. In this paper I present the challenges associated with ENPs exposure to the estuarine environment and the testing of a chosen ENPs to generate data on ecotoxicity in the test estuarine organisms for further consideration of risk assessment of marine environment. Careful consideration was given to the selection of the test materials (benthic organisms Nereis diversicolor and Scrobicularia plana), the test system and the test exposure conditions (CdS ENPs and aqueous CdS added to filtered natural seawater with a concentration of 10 µg L-1). Evaluation of the exposure effects was carried out by behavioral tests (burrowing kinetics and feeding rate) and biochemical responses (quantification of biomarkers). Multispecies Freshwater Biomonitor®  (MFB) tests and GST analysis results show significant differences in between control group and CdS NPs exposure one, indicating that CdS NPs are potential to cause sublethal effects in test organisms. Our knowledge in environmental risk assessment of ENPs is still limited. Coordinated research is required to gain a better understanding of the factors and processes affecting ENP fate and effects in the environment as well as to develop more usable, robust and sensitive methods for characterization and detection of ENPs in environmental systems.
NanoReTox, FP7 NMP Work Programme

Waterfowl are known to be capable of influencing wetland ecology in a number of ways, sometimes to the detriment of other species that also inhabit this type of environment. Western Golden Bay including Farewell Spit is one of the largest areas of intertidal sand flat habitat in New Zealand and supports a wide array of species including internationally important populations of bar-tailed godwits (Limosa lapponica) and red knot (Calidris canutus). These species, particularly red knot, have declined in number over the last the 25 years at this site. Another numerous species at this site, the black swan (Cygnus atratus), has been suggested as a possible contributor to the observed decline in wader numbers through their impact on the habitat. This thesis presents the findings of a research project on the role of black swans in the tidal seagrass (Zostera muelleri) ecosystem in western Golden Bay carried out between October 2007 and October 2008. In an effort create a clear picture of what role the black swans play in this environment the project focused on four major aspects of swan-ecosystem interactions. The first of these looked at the activity patterns of black swan. This showed the swans’ activity is largely dictated by the tidal cycle with foraging occurring during the intertidal period when the seagrass is accessible while roosting is mostly confined to around high and low tides. The second part of the project explored the influence black swans have on the tidal seagrass landscape through their foraging habits. This showed that while swan foraging occurs across the tide flats it is concentrated on denser patches, on both small (meters) and large (hectares) scales. Experimental grubbings showed that the grubbing activity of swans is capable of forming and expanding bare sand patches within seagrass beds and that these bare patches can persist for at least two months. The third part of the project focused on the direct impacts of swan foraging on the seagrass and associated benthic invertebrates. Exclusion plots showed that at some sites swan foraging can significantly reduce Zostera biomass and invertebrate biodiversity. The final aspect examined was the role of swan in biomass and nutrient cycling. A faecal deposition survey showed swans consume 23.40 g DW ha-1 day-1 of Zostera. The average intake rate was 27.25 g DW ha-1 day-1. Nutrient analysis of seagrass 4 showed that shoot material has significantly higher N, P, Ca and fibre than rhizome and that rhizome has significantly more soluble carbohydrates than shoots. On the basis of the swans’ direct and/or indirect influences on Zostera muelleri beds and the associated invertebrate fauna, swans could arguably be considered to be a major ecosystem engineer in the intertidal sandflats of Golden Bay.

Estuarine environments are threatened by the synergistic effects of anthropogenic disturbance and bioinvasion. The construction of artificial structures (such as pilings and pontoons) provides a habitat resource in close proximity to vessel hulls that may be carrying a wide range of non-indigenous fouling species. In addition, the release and accumulation of toxicants from antifouling (AF) paints on vessel hulls creates a chemical disturbance that may reduce the invasion resistance of native communities. This thesis examines how shipping-related disturbances affect sessile communities, and in particular what role AF paints and artificial structures play in the invasion of estuaries. Using a series of field-based experiments, I found that copper and tributyltin have the potential to influence both the transport of species to a new region, via the application of AF paints on vessel hulls, and their subsequent establishment, via the accumulation of AF biocides in estuaries. Temperature, pH, salinity and turbidity were also related to species distributions. During subtidal surveys of artificial and natural structures I found more non-indigenous species (NIS) on pilings and pontoons than on rocky reef, and shallow floating structures were identified as hotspots for invaders. These findings suggest that artificial structures play an important role in the initial establishment of sessile non-indigenous species in new regions. A subset of NIS were also present on the reefs sampled during the survey and I conducted manipulative experiments to determine factors affecting the invasibility of turf and canopy-forming algal assemblages. The resident assemblage provided a barrier to most invaders, particularly when light and sedimentation levels were also high (i.e. on horizontal substrate). My results suggest that the areas of reef most susceptible to invasion are vertical rock walls and those subjected to disturbances that release space. In summary, this is the first study to relate copper and tributyltin contamination in the field to NIS distributions. My research has also highlighted the potential role of artificial structures in facilitating the establishment of NIS in estuaries and identified invasion threats to rock wall communities and disturbed reefs in estuarine systems.

Este trabalho consistiu em avaliar a influência dos moluscos bivalves na especiação de metais pesados (MPs) (Co, Ni, Cu, Zn, Cd, Pb, Cr) em sedimento estuarino. Neste estudo foi utilizado o molusco bivalve Scrobicularia plana (da Costa, 1778), uma espécie bentónica predominantemente filtradora, que se distribui por toda costa atlântica e Sul da Europa, e constitui um recurso haliêutico com alguma importância em Portugal. Estudos têm demonstrado que S. plana é um bom bioindicador, razão pela qual tem sido muito utilizada nos programas de biomonitorização, principalmente quando se pretende investigar a presença de metais pesados em substrato estuarino. Os sapais escolhidos para a recolha dos animais e sedimentos situam-se no estuário do Tejo: o sapal das Hortas (Alcochete), considerado de baixo nível de contaminação por metais pesados e onde foram recolhidos os animais; e o sapal do Rosário (Moita), seleccionado pelas suas características e considerável pressão antropogénica, contendo níveis elevados de metais pesados, e onde foram recolhidas as amostras de sedimento. Esta experiência decorreu num mesocosmos em ambiente laboratorial, tendo sido monitorizados os parâmetros abióticos considerados relevantes para o sucesso do ensaio. Ao todo foram utilizados 420 espécimes adultos da S. plana e 40 litros de sedimento, ambos previamente preparados. Os animais foram mantidos em tanques com água salobra artificial e sedimento estuarino, aí permanecendo por 48h, tempo necessário para se alimentarem, sendo depois colocados em tanques apenas com água salobra artificial, para permitir a libertação e recolha dos pellets fecais. Após a recolha, amostras dos pellets, do sedimento inicial (SI) e sedimento controlo (SC) de cada tanque foram centrifugadas, liofilizadas e armazenadas a 4°C. No processo de extracção sequencial, foi utilizado o procedimento descrito por Tessier et al. (1979) e modificado posteriormente por Hullebush et al. (2005). A determinação dos metais pesados foi feita através de espectrometria de massa com indução de plasma (ICP­ MS). Foi determinado o teor de matéria orgânica (MO) por análise termogravimétrica (TGA) e a distribuição de partículas (PSD) pela técnica de foto-sedimentação (HORIBA). Neste trabalho foi avaliada a alteração na especiação química dos metais pesados induzida pela acção da S. plana através da comparação da concentração dos metais pesados nas diferentes fracções definidas operacionalmente, no SI, nos pellets e no se. Nos resultados, o teor de MO variou entre 7.5% e 9.3°/o, tendo os valores maiores ocorridos nos pellets, mas as diferenças não foram estatisticamente significativas (p>0.05.), tal como não foi significativa a correlação entre a MO e a concentração de nas vármetais pesados nas francções. Os pellets apresentaram maior concentração de ed, Cu, Pb e Zn, no entanto, esta diferença não foi estatisticamente significativa. A maioria dos metais pesados está ligada principalmente à fracção residual do sedimento. O teor de metais pesados nas amostras ocorreu na seguinte ordem: fracção residual > fracção ligada ao complexo MO e sulfuretos > fracção ligada aos carbonatos > fracção de troca. Na fracção de troca as diferenças não foram significativas para a maioria dos metais, excepto para o Cu e Zn entre o SC e os outros dois grupos de amostras; nesta fracção houve aumento de três (Cu) a dezassete vezes (Zn) nos pellets em relação ao SI e SC. Na fraccão ligada aos carbonatos houve diferenças entre os pellets e se e entre SI eSC para o Cd, o eo, o Cu e o Pb; o Cd foi cerca de dez vezes superior nos pellets; o Ni apresentou diferenças significativas entre os pellets e o SI e o SC. Para o Zn houve diferenças significativas entre os pellets e o SI. Na fracção ligada à MO e sulfuretos houve diferenças entre o SI e os pellets para o Co, Ni e Zn, e para Cd e Cu entre os pellets e SC. Na fracção residual, o ed, Cu, Pb e Zn apresentaram diferenças entre os pellets e SC. Os resultados obtidos evidenciaram a importância da S. plana na biodisponibilização de determinados elementos, principalmente o Ni, não parecendo ter a mesma relevância no caso do Co, Cr e Pb. == ABSTRACT: The objective of this work was to evaluate the influence of bivalve molluscs in the speciation of heavy metals, namely cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), cadmium (Cd), lead (Pb) and chrome (Cr) in estuarine sediment. For this study we used the bivalve Scrobicularia plana (da Costa, 1778), a predominantly benthic filter­ feeding species. lt is geographically distributed throughout the Atlantic coast and southern part of Europe and constitutes a fishery resource of some importance in Portugal. Research has shown that Scrobicularia plana is a good bio-indicator, reason for which it has been widely used in biomonitoring programs, especially to investigate the presence of heavy metals in estuarine substrate. The salt marshes chosen to collect the specimens and sediments used in the experiment are located in the Tagus estuary: Hortas saltmarsh (Alcochete), considered a place with low-level of heavy metals contamination and where the animals were collected, and Rosário saltmarsh (Moita), presenting high levels of heavy metals contamination and where the sediment samples were collected. This experience took place in macrocosm environment in the laboratory and environmental conditions monitored. Altogether 420 adult specimens of Scrobicularia plana and 40 liters of sediment were used, both previously treated. The animals were placed into tanks with artificial brackish water and estuarine sediment and remained there for 48 hours, which was considered to be enough time for feeding to occur; after that period the animals were placed in tanks with artificial brackish water for elimination and collection of fecal pellets. After collection, the pellets and sediment samples from each tank were centrifuged, freeze dried and stored at 4°C. ln the sequential extraction procedure, we used the method described by Tessier et a/. (1979) and later modified by Hullebusch et al. (2005). Mass spectrometry with induced coupled plasma (ICP-MS) was used to determine the heavy metals. The content of organic matter (OM) was determined by Thermo gravimetric Analysis (TGA), and particle size distribution (PSD) was also determined using the of photo-sedimentation technique by HORIBA. The change in chemical speciation of heavy metals induced by the action of Scrobicularia plana was assessed by comparing the concentration of heavy metals in different fractions of the initial sediment, of pellets and control sediment. The relationship between the concentration of heavy metals and organic matter (OM) was also studied. The OM content ranged between 7.5°/o and 9.30°/o, the higher values occurred in the pellets, but the differences were not statistically significant (p>O.OS) nor was a significant correlation between OM and heavy metal fractions in all samples. The pellets had higher concentrations of Cd, Cu, Pb and Zn, however, this difference between groups was not statistically significant, the concentration of heavy metals in the pellets differ from other samples for Cd, Co, Cu, Ni, Pb and Zn. Most heavy metals are primarily bonded to residual fraction. The content of heavy metais in the fractions occurred in the following order: residual fraction> organic matter and sulfide complex > carbonates fraction > exchange fraction. ln the exchangeable fraction the differences were generally not significant, except for Cu and Zn, where differences were found between initial sediment and the other two groups of samples; metals in this fraction increased by three times (Cu) to 17 times (Zn) in the pellets when compared to the initial and control sediment. ln the fraction bound to carbonates differences were found between the pellets and control, and the control and the initial sediment for Cd, Co, Cu and Pb; Cd was about ten times higher in pellets; Ni presented differences between the pellets and the initial and control sediments. For Zn there were significant differences between the pellets and initial sediment. ln the fraction bound to organic matter and sulfides there were differences between the initial sediment and the pellets for Co, Ni, Zn and Cd and Cu between the pellets and control. ln the residual fraction, Cd, Cu, Pb and Zn showed differences between the pellets and control. The results obtained in this work have evidenced the importance of S. plana in the bioavailability of certain elements, particularly Ni; in the case of Co, Cr and Pb this species does not seem to affect the availability to the biota.

Tese de doutoramento, Biologia (Ecologia), Universidade de Lisboa, Faculdade de Ciências, 2015
Estuaries face different anthropogenic pressures as a consequence of their privileged location and high productivity, and thus a diverse array of pollutants enter the ecosystem. Metals are of particular concern, due to their persistent and non-degradable character and pernicious effects exerted on the biota. Metals are found in several compartments of the estuarine ecosystem. They may be in dissolved or particulate forms in the water column, sorbeb on the sediments or accumulated in the biota. This thesis aimed to determine the effect of the sediment-organism interactions in metals’ cycling in salt marsh and intertidal sediments. Special attention was given to metal speciation, to assess the mobility and bioavailability of such elements. Total metal concentration was determined in tissues of two fish species occupying different levels in the estuarine trophic web, as well as in bottom sediments, to assess metal exposure and accumulation in fish tissues. Sequential extractions were made in rhizosediments of three halophytes and adjacent bare mud flat sediments from two different salt marshes. Operationally defined fractions were obtained using solution of increasing strength and acidity, to evaluate the effect of halophytes on metal availability. Two laboratory trials were conducted in which metal fractionation was assessed in on sediments before and after passing through the gut of the two species. The results of these works indicate that metal accumulation in estuarine sediments can affect the accumulation in fish tissues. Sediment-organism interactions alter metal mobility in the sediments. Salt marsh plants tend to immobilize metals in their rhizosediments, while the ingestion of sediment by and detritivorous species, and subsequent excretion of fecal pellets, makes some metals more bioavailable to the estuarine trophic web.
Os estuários enfrentam diferentes pressões antropogénicas inerentes à sua localização privilegiada e elevada produtividade, e têm como consequência a presença mais ou menos acentuada de diversos tipos de poluentes. A persistência e o carácter não degradável dos metais no ambiente é particularmente preocupante, tendo em conta os efeitos nocivos que podem exercer no biota. Os metais podem ocupar vários compartimentos num estuário, e.g., na coluna de água (dissolvidos ou particulados), adsorvidos ao sedimento ou acumulados nos organismos. A presente tese teve como objetivo avaliar o efeito das interações organismo-sedimento na dinâmica de metais em sedimentos de sapal e áreas intertidais adjacentes, incidindo em particular na especiação dos metais e na sua disponibilização para a teia trófica estuarina. Para avaliar a exposição de duas espécies de peixes de diferentes níveis da teia trófica estuarina à contaminação por metais no sedimento, determinaram-se as concentrações totais em tecidos e em sedimentos superficiais. Analisou-se também a especiação dos metais nos sedimentos entre raízes de três halófitas e nos sedimentos sem coberto vegetal, em dois sapais. Extraíram-se sequencialmente frações operacionais com soluções de força e/ou acidez crescente, para avaliar o impacto dos organismos na mobilidade dos metais. Realizaram-se ainda duas experiências em que se determinou a especiação dos metais no sedimento antes e depois da ingestão por duas espécies de diferentes grupos taxonómicos. Concluiu-se que a acumulação de metais no sedimento estuarino pode afetar a acumulação nos tecidos das espécies selecionadas. A interação sedimento-organismo conduz a alterações da dinâmica dos metais; a ação das plantas de sapal potencia a imobilização de alguns elementos, sendo o efeito da ingestão de sedimento por organismos detritívoros aparentemente contrário, disponibilizando os metais sob formas mais acessíveis à teia trófica estuarina
Fundação para a Ciência e a Tecnologia (FCT), SFRH/BD/37926/2007, projetos PEst-OE/MAR/UI0199/2014, POCI/MAR/58548/2004

I examine the influences of three key factors (habitat type, climatic regimes, floods and associated salinity regime changes) on the structure of benthic invertebrate assemblages of tropical estuaries. I compared the habitat-specific invertebrate assemblages of the full spectrum of habitat types available in one region of Deluge Inlet, a near-pristine estuary. There were distinct faunal assemblages associated with particular habitat types that related to sediment size and presence/absence of seagrass. The results of this study laid the foundation for the following studies in establishing the need to sample a single habitat type to facilitate spatial comparisons. Consequently, I selected bare sand for subsequent sampling because this substrate type was available in all estuaries and estuary regions studied. I then examined the influenced of climatic regimes (wet tropics, dry tropics and dry sub-tropics) on estuarine benthic invertebrate assemblages. The benthic assemblage in wet tropic estuaries differed from assemblages in dry sub-tropic estuaries, with many taxa only recorded in wet tropics estuaries and vice versa. There was little overlap of species between climatic regimes, particularly in upstream transitional zones (i.e. areas at the estuary/freshwater interface subject to substantial seasonal variation in physical conditions). However, a common pattern seen in all ten estuaries studied was distinct differences in faunal assemblages between the mouth and upstream transitional assemblages. In the third part of this thesis I examined the effect of wet-season floods and how changes in salinity regimes along the downstream gradient of estuaries affected invertebrate assemblages. Following the seasonal flood, upstream assemblages underwent radical restructuring ranging from the total loss of taxa from some sites to significant decreases in diversity and abundances at other sites. The recovery of faunal assemblages after the flood varied between sites and appeared to be mainly a function of the location of the site in relation to freshwater input (i.e., sites closest to freshwater input were more adversely affected than sites further away from the source of the freshwater input). Clearly, although flooding is a natural occurrence in tropical estuaries, it can have a profound effect on macrobenthic invertebrate assemblages by greatly reducing and/or eliminating taxa, particularly in the upstream reaches at times of low salinity. These studies provide detailed understanding of benthic assemblages in a range of North Queensland estuaries, of the differences in mouth and transitional zones and of how assemblages respond and recover after a flood. Understanding these patterns and processes is essential in order to understand the ecological functioning of the systems and as a precursor to effective management.

Tese de mestrado, Ecologia Marinha, Universidade de Lisboa, Faculdade de Ciências, 2016
Estuaries are highly productive areas of high ecological and economic importance, providing various benefits and services for mankind. The estuarine watershed is a preferential location for human settlement and associated anthropogenic activities, such as industrial and agricultural development, resource exploitation and shipping activities, which result in continuous pressures inevitably leading to environmental degradation. Biomarkers are considered early-warning signs able to provide a predictive perspective of the long-term effects of exposure to pollutants in organisms. Therefore, biomarkers are considered useful tools for environmental quality assessment that integrate biological responses and the degree of stressors, usually applied in a single taxa context. Exposure to xenobiotics compounds and their metabolites lead to enhanced oxidative stress and potentially to major molecular damages such as oxidation of proteins, DNA and peroxidation of unsaturated lipids in cell membranes. Hence, defence mechanisms play a major role in preventing damages and include several enzymes such as antioxidant enzymes superoxide dismutase enzymes (SOD) and catalase (CAT), as well as phase I ethoxyresorufin-O-deethylase (EROD) and phase II glutathione S-transferase (GST) biotransformation enzymes. Accordingly, these enzymes activities as well as both biomarkers of effects lipid peroxidation (LPO) and DNA damage (DNAd) were determined in the present study aiming at assessing overall contamination impacts on various organisms. In this context, the aim of this work is to quantify multiple biomarker responses in a multi-taxa approach, in order to assess the diversity in response patterns among species from two differently impacted estuarine systems. Two Portuguese estuaries, Tejo and Ria de Aveiro, were sampled in two months, June and September 2015, specifically in two sites in each estuary: Alcochete (ALC) and Vila Franca de Xira (VFX) in Tejo and Mira channel (AVSUL) and Murtosa (MUR) in Ria de Aveiro. Several species were selected based on their abundance and estuarine occurrence as well as on their prior use as bioindicator species. Two fish species were considered, the European sea bass Dicentrarchus labrax, a marine migrant species whose juveniles use estuarine areas as nursery areas; and the common goby Pomatoschistus microps a resident estuarine species. Four invertebrate species were also sampled, two common infaunal species, the bivalve Scrobicularia plana and the ragworm Hediste diversicolor and two epibenthic crustaceans, the brown shrimp Crangon crangon and the green shore crab Carcinus maenas. Overall, biomarker responses signaled environmental chemical exposure and some degree of deleterious effects for all species. Low variability among sites and months was observed in antioxidant enzymes responses for most species and no clear pattern was discernible amongst species. In fish species, induction of both biotransformation enzymes was observed with significant spatial variability, with lower variability of GST comparing to EROD activity, though with overall concordant higher levels in Tejo estuarine sites, especially in ALC. Less marked responses in biotransformation enzymes were observed in invertebrate species. A similar GST response pattern was observed for H. diversicolor, S. plana and C. maenas, identifying higher contamination levels in AVSUL, considered the least contaminated site in this study. Very low or even incipient spatial and temporal variability in EROD activity was observed in all invertebrates. Nevertheless, these species signaled mostly Tejo sites according to phase I enzyme responses, especially VFX, which is in agreement with previously reported environmental contamination levels. Concerning monthly variation in general all species showed higher enzyme activities and effects in September, which may reflect the effects of continuous exposure. Notwithstanding the significant activity of detoxification enzymes, deleterious effects were reported for all species, suggesting an overall contamination level above the capacity of the molecular defence mechanisms to limit exposure effects in these species. Biomarkers of effects response patterns differed amongst species, yet akin LPO variability patterns were observed among species, namely between C. crangon, C. maenas and P. microps, signalling VFX and for D. labrax, H. diversicolor and S. plana, signalling MUR. Most species also showed higher mean DNAd values in VFX, except for C. crangon and C. maenas that signalled MUR. A positive correlation between biomarkers of effects was observed for all invertebrate species (except for H. diversicolor), highlighting site-specific contamination. IBR indices also varied throughout sampled sites for all species studied, yet the major pattern observed was the opposed response pattern between a highly mobile fish species D. labrax and both infaunal invertebrate species (S. plana and H. diversicolor). Species-specific responses were evident from lack of concordance among species biomarker responses and IBR scores, most likely due to biological differences (in terms of physiology, vulnerability and overall capacity of defence mechanisms), but also to ecological differences such as differential habitat use, feeding habits, life-strategies and consequently differential contaminants exposure. Overall, Tejo sites were considered more impacted than Ria de Aveiro, yet significant responses were also found in the latter for all species studied. In conclusion, this multi-biomarker and multi-taxa approach provided important insights into the variability of species responses to contaminants exposure in estuaries. The complexity of biomarker response patterns for all species in this study evidenced species differential response and differential exposure to environmental contamination, on top of the complex environmental stimuli, such as pollutants’ mixtures and natural variability characteristic of the estuarine environment. This emphasizes the difficulties associated with effective multi-species ecological risk assessment, and application must carefully consider the potential added ecological value of a multispecific approach (similar to a multi-biomarker approach) versus more complex results interpretation and assessment of the environmental quality.
Os estuários são sistemas de elevada importância ecológica onde, por exemplo, muitas espécies encontram as condições favoráveis à sobrevivência e rápido desenvolvimento, bem como de elevado potencial económico, constituindo uma fonte importante de recursos e serviços para o Homem. Consequentemente, estes sistemas tornam-se locais preferenciais para o desenvolvimento de centros urbanos, de que resultam inúmeras pressões associadas às atividades antropogénicas, desde a pesca, aquacultura, à indústria, agricultura e navegação, o que inevitavelmente conduz à degradação da qualidade ambiental. Os biomarcadores são frequentemente utilizados com o intuito de avaliar de que forma as pressões nestes sistemas afetam os organismos que os habitam. Constituem desta forma uma ferramenta útil em estudos de avaliação da qualidade ambiental, ao fornecerem uma perspetiva integrada da pressão existente e das respostas ao nível biológico. A exposição dos organismos a compostos xenobióticos resulta no aumento do stress oxidativo que propicia a ocorrência de danos ao nível da célula, nomeadamente a oxidação de proteínas e do DNA (DNAd) e peroxidação lipídica (LPO). Não obstante, os mecanismos de defesa das células desempenham um papel fundamental na prevenção desses efeitos, de que são exemplos: enzimas antioxidantes, como a catalase (CAT) e a superóxido dismutase (SOD), e de biotransformação, como a 7-etoxiresorufina-O-deetilase (EROD) e a glutationa-S-transferase (GST). O objetivo deste trabalho consiste no estudo comparativo das respostas biológicas de várias espécies às pressões antropogénicas a que estão sujeitas no seu ambiente natural, através da quantificação de diferentes biomarcadores, em estuários com níveis de pressões distintos, o estuário do Tejo e a Ria de Aveiro. A amostragem foi realizada em dois meses, em junho e em setembro de 2015, e em dois locais em cada estuário: Alcochete (ALC) e Vila Franca de Xira (VFX) no estuário do Tejo e o canal de Mira (AVSUL) e Murtosa (MUR) na Ria de Aveiro, cujo nível de contaminação, fontes de pressão, função de viveiro e abundância de espécies foi previamente descrito. Para tal, foram selecionadas várias espécies que utilizam ambos os estuários e consideradas, em trabalhos anteriores, bioindicadoras da qualidade ambiental, nomeadamente: duas espécies de peixes, o robalo-legítimo Dicentrarchus labrax e o caboz-comum Pomatoschistus microps, e quatro espécies de invertebrados, nomeadamente duas espécies endobentónicas, o poliqueta Hediste diversicolor e o bivalve Scrobicularia plana, e duas espécies de crustáceos epibentónicos, o camarão-mouro Crangon crangon e o caranguejo-verde Carcinus maenas. As atividades das enzimas SOD, CAT, EROD e GST bem como os níveis de LPO e DNAd foram analisados nos tecidos apropriados para cada uma das espécies consideradas. De um modo geral, as respostas dos biomarcadores evidenciaram a exposição a contaminantes no ambiente, passível de induzir alguns mecanismos de defesa e destoxificação e de produzir efeitos ao nível molecular em todas as espécies estudadas. A atividade das enzimas antioxidantes apresentou reduzida variabilidade entre locais e meses e não se observou nenhum padrão específico entre todas as espécies. As enzimas de biotransformação apresentaram maior variabilidade espacial e temporal que as antioxidantes, e maior nos peixes do que nos invertebrados. Os efeitos a nível molecular também foram evidentes, em todas as espécies, evidenciando um nível de contaminação global considerável que ultrapassa a capacidade de defesa e metabolização de xenobióticos destas espécies. Na generalidade, as respostas dos biomarcadores sinalizaram maiores níveis de contaminação nos locais do estuário do Tejo, assim como uma tendência de respostas mais elevadas em setembro, entre todas as espécies, quer para as enzimas antioxidantes e de biotransformação como para os biomarcadores de efeitos, possivelmente evidenciando um efeito de exposição continuada a stresses ambientais. De um modo geral, as respostas biológicas bem como os valores obtidos através do índice de IBR variaram bastante entre espécies e de acordo com os locais amostrados, tendo no entanto sido possível observar alguns padrões de resposta semelhantes entre espécies, particularmente no que diz respeito ao uso do habitat. Além disso, apesar dos locais do estuário do Tejo serem considerados mais impactados que os locais da Ria de Aveiro, nestes foram igualmente observados respostas significativas em várias espécies. A complexidade dos padrões de resposta dos vários biomarcadores analisados evidencia diferenças entre espécies em termos da sua fisiologia, vulnerabilidade aos contaminantes e capacidades dos mecanismos de defesa, bem como ao nível da ecologia, como o uso do habitat, e hábitos alimentares de que naturalmente decorrem diferentes níveis de exposição. A aplicação de uma abordagem multiespecífica e de vários biomarcadores pode constituir uma ferramenta útil para a compreensão de diferentes vias de exposição e para uma análise mais completa dos efeitos em diferentes componentes biológicos do ecossistema. No entanto, apesar de pertinente a nível ecológico, é evidente a complexidade de interpretação que lhe está associada, e para a qual a aplicação em programas de monitorização e de avaliação da qualidade ambiental requer maior planeamento. Além disso, a seleção das espécies deverá ser ponderada de acordo com o objetivo do estudo, sendo que diferentes espécies irão gerar avaliações de impacto diferentes para os mesmos locais.

Knowledge of the interactions among species and communities is vital for their management and protection. Increasingly, the role of marine protected areas in this regard is being recognised, primarily because of the potential for previously-harvested species to increase in density and biomass, and the linkages among species to be restored. Monitoring and research was conducted within and surrounding two marine reserves on the east coast of the North Island of New Zealand – Te Tapuwae o Rongokako, near Gisborne, and Te Angiangi, south of Napier. The aim was to describe the biological characteristics of spiny lobsters (Jasus edwardsii) in the absence of fishing, and to describe the effects of fishing and protection on lobster populations and the communities of which they are a component. Diver and pot surveys showed that lobsters were significantly more abundant within the reserves than in the surrounding fisheries and that the populations were comprised of a larger proportion of legal-sized individuals. Higher female fecundity within Te Tapuwae o Rongokako Marine Reserve compared with the surrounding fishery was proposed to be primarily a result of increased availability of large males within the reserve. The impact of the fishery on lobsters was also evidenced in the lower tail width to carapace length ratio of the fished population compared to the population within Te Tapuwae o Rongokako Marine Reserve, due to the minimum legal size for Jasus edwardsii in New Zealand being based on tail width. The largest tagging study ever to be conducted in a New Zealand marine reserve showed that sublegal male lobsters within Te Tapuwae o Rongokako Marine Reserve were growing on average faster than the same sized individuals outside the reserve, potentially as a result of the effects of handling and / or size-selective fishing. A decrease in growth rate over time was recorded in male lobsters within the reserve, which coincided with an increase in catch per unit effort and may indicate that density-dependent effects are operating. Distinct seasonal movements of tagged lobsters were recorded, with the vast majority of movements taking place within reefs. Where the boundaries of the reserve crossed reef habitat, significant movement of lobsters across the boundaries occurred. Lobsters within Te Tapuwae o Rongokako Marine Reserve not only exhibited cannibalistic behaviour but foraged during the day, including on intertidal reef platforms at high tide, potentially as a behavioural response to increased competition for food – behaviour not previously reported for this species. Outside the reserve, lobster bait apparently provided an alternative protein source but despite this supplementation of diet, these lobsters were in poorer nutritional condition, as evidenced by their lower body weights relative to carapace length and tail width for both sexes. Lobsters outside the reserve were also significantly more affected by a bacterial infection associated with handling, than lobsters within the reserve. These findings have significant implications for fisheries management and for the design and management of marine protected areas (MPAs). This study demonstrates that many of the biological parameters used in evaluating harvest strategies in the New Zealand lobster fishery may be biased unless collected from populations with a natural size structure, such as may occur within marine protected areas.