Fate of microbial contamination in a South European Coastal Lagoon (Ria Formosa) under the influence of treated effluents dispersal.

AIM: Assessment of the fate of microbial contamination driven from treated wastewater disposal at a highly productive zone on a South European coastal lagoon (Ria Formosa). METHODS AND RESULTS: Microbial indicators of contamination (Total coliforms, Escherichia coli, and Enterococci) were evaluated monthly during September 2018-September 2020 at three study areas (Faro, Olhão, and Tavira) under different wastewater discharge flows and hydrodynamic conditions. Additional data on E. coli monitoring in bivalves, available from the national institution responsible for their surveillance was also considered. The maximum microbial contamination was found at Faro, the highest-load and less-flushed study area, contrasting the lowest contamination at Olhão, a lower-load and strongly flushed area. The wastewater impact decreased along the spatial dispersal gradients and during high water, particularly at Faro and Tavira study areas, due to a considerable dilution effect. Microbial contamination at Olhão increased during the summer, while at the other study areas seasonal evidence was not clear. Data also indicate that E. coli in bivalves from bivalve production zones next to the three study areas reflected the differentiated impact of the wastewater treatment plants effluents on the water quality of those areas. CONCLUSIONS: Effluent loads together with local hydrodynamics, water temperature, solar radiation, precipitation, and land runoff as well as seabirds populations and environmentally adapted faecal or renaturelized bacterial communities, contributed to microbial contamination of the study areas.

Understanding the bioaccumulation of pharmaceutical active compounds by clams Ruditapes decussatus exposed to a UWWTP discharge.

Twenty-four pharmaceutical active compounds (PhACs) were evaluated in the soft tissues of clams Ruditappes decussatus exposed along a 1.5-km dispersal gradient of the treated effluent from an urban wastewater treatment plant discharging in Ria Formosa, and compared with those in the marine waters and discharged effluents. The clams were exposed for 1 month, in June-July 2016, 2017 and 2018. PhACs were quantified by high performance liquid chromatography coupled to tandem mass spectrometry after the quick, easy, cheap, effective, rugged and safe (QuEChERS) method (clams) or solid-phase extraction (water samples). The most representative PhACs in the effluents and receiving waters (regardless of the tidal dilution effect) were diclofenac, carbamazepine and caffeine (on average ≤ 2 µg/L) and only caffeine exhibited significant inter-annual differences, with higher values in 2017. In turn, the most bioaccumulated PhACs in clams were caffeine (0.54-27 ng/g wet weight, significantly higher in 2016) and acetaminophen (0.37-3.7 ng/g wet weight, significant lower in 2016). A multivariate principal component analysis showed (i) PhAC bioaccumulation primarily depended on biotic factors (clams length and weight), (ii) PhAC physicochemical properties Log Kow, pKa and water solubility interplaying with water abiotic variables were more relevant for explaining data variability in water than the physical dilution/tidal mixing, (iii) this process, reflected by the salinity gradient, had a tertiary role in data variation, responsible for spatial discrimination of marine waters. This study provides a better understanding of PhACs bioaccumulation by clams Ruditapes decussatus in real environmental conditions, under the influence of urban treated effluent dispersal in Ria Formosa coastal lagoon, a major producer of bivalves, ultimately disentangling key factors of PhAC bioaccumulation.

Spatial and seasonal biomarker responses in the clam Ruditapes decussatus.

The clam Ruditapes decussatus is an important resource to preserve in coastal lagoon systems around the Mediterranean including the South Portugal. To assess spatial and temporal biomarker responses to contamination in the species, a multibiomarker approach was conducted using antioxidant enzymes, MFO system phase I and II; acetylcholinesterase, metallothionein (MT), δ-aminolevulinic acid dehydratase and lipid peroxidation (LPO). The condition index (CI), metals and polycyclic aromatic hydrocarbons (PAHs) were also determined. The levels of contaminants were not particularly high and the antioxidant enzymes, acetylcholinesterase (AChE), MT in the digestive gland, and δ-aminolevulinic acid dehydratase (ALAD) do not provide a suitable seasonal and spatial discrimination reversely to that regarding CYP450, glutathione-S-transferase (GST), MT in the gills, and LPO in both tissues. However, even those could vary with natural variables that may act as confounding factors. Thus, seasonal variability and natural range of biomarker responses must be carefully and accurately taken into account in ecotoxicological approaches of environmental quality assessment programmes.

A multi-biomarker approach in cross-transplanted mussels Mytilus galloprovincialis.

The present work integrates the active biomonitoring (ABM) concept in mussels Mytilus galloprovincialis from the South coast of Portugal transplanted during 28 days between two sites with different sources of contamination, and vice versa, in order to assess biological effects in these mussels. For that purpose a multibiomarker approach was used. The suit of biomarkers indicative of metal contamination were metallothioneins (MT) and the enzyme δ-aminolevulinic acid dehydratase (ALAD), for organic contamination mixed function oxidase system (MFO), glutathione-S-transferase (GST) and acetylcholinesterase (AChE), as oxidative stress biomarkers superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and lipid peroxidation (LPO). These biomarkers were used to determine an index to evaluate the stress levels in these two sites. Site A is strongly influenced by metallic contamination, with higher Cu, Cr and Pb in M. galloprovincialis, as well as higher MT levels, antioxidant enzymes activities and LPO concentrations, and lower ALAD activity. In site B organic compounds (PAHs) are prevalent and native mussels show higher activities of the MFO system components and GST. Transplanted mussels had significant alterations in some biomarkers that reflect the type of contaminants present in each site, which demonstrates the primary role of the environment in determining the physiological characteristics of resident mussels. Therefore the application of ABM using a battery of biomarkers turns out to be a useful approach in sites where usually complex mixtures of contaminants occurs. In this study the biomarkers that better differentiate the impact of different contaminants at each site were MT, CYP450, SOD and CAT.

DNA damage as a biomarker of genotoxic contamination in Mytilus galloprovincialis from the south coast of Portugal.

DNA damage was evaluated in the haemolymph of Mytilus galloprovincialis from nine sites along the south coast of Portugal using the comet assay. DNA damage was low, in the same range of sites considered to suffer low impact from genotoxic contaminants. Even so, differences between sites, seasons and genders were found. Highest values were in mussels from the main estuaries and the fishery harbour, reflecting higher genotoxin levels, whereas the lowest values can be used as a baseline for future work. Non-contaminant related factors (e.g. temperature and oxygen) were also shown to influence DNA damage. Between seasons, highest values were in summer related not only to the increase of tourism in this region (∼10-fold), but also to temperature. Between genders, males were found to be more sensitive. The condition index was also generally higher in summer. Lipid peroxidation, another damage biomarker, was measured in gills to assess if there is any association between the responses of both biomarkers and if they are similarly affected by the same environmental conditions. LPO like DNA damage was higher in summer. This work confirms that DNA damage is a sensitive biomarker to discriminate genotoxic contamination, even in areas considered to suffer low impact from genotoxins.

Effects of climate change and anthropogenic pressures in the water quality of a coastal lagoon (Ria Formosa, Portugal).

Understanding how climatic and anthropogenic drivers will influence coastal lagoons is fundamental to guarantee their preservation and sustainability. The Ria Formosa (coastal lagoon, South coast of Portugal) is a very important ecosystem that supports diverse economic activities in the region. The 3D coupled hydrodynamic-biogeochemical model SCHISM was validated and used to assess the influence of climate change and anthropogenic pressures on the water quality of the Ria Formosa. Five scenarios were simulated: reference scenario (S0), mean sea level rise (SLR) of 0.5 m (S1), increase of the air temperature of 1.68 °C (S2), increase of the outflow from the wastewater treatment plants (WWTP) by 50% (S3) and a combined scenario (S4). Results suggest that SLR of 0.5 m promotes an increase of 0.5-3 in the salinity near the area of influence of the WWTP. SLR decreases the inorganic nutrient concentrations in these areas by about 40-60%, due to an increase of the dilution. In contrast, the increase of the outflow from the WWTP by 50% increases the nutrients concentrations by about 20-40%. The increase of the air temperature alone by 1.68 °C increases the water temperature by 0-1 °C. The combined scenario suggests antagonist effects in the nutrient concentrations. Overall, the trophic index (TRIX) of the lagoon calculated for the scenarios exhibits only minor differences relative to the reference scenario, except in some areas near the WWTP discharges. In these areas, TRIX tends to increase with the increase of the outflow from the WWTP in scenario S3. These results provide further insight into the response of coastal lagoons, and the Ria Formosa in particular, to future changes and contribute to support their management.

Biomarker Responses of the Clam Ruditapes decussatus Exposed to a Complex Mixture of Environmental Stressors under the Influence of an Urban Wastewater-Treatment Plant.

To evaluate the potential impact of an urban wastewater-treatment plant on Ria Formosa coastal lagoon, a sentinel species, the clam Ruditapes decussatus, was exposed along a gradient of the effluent's dispersal for 1 mo. Three exposure sites were selected to study the responses of 3 biomarkers: electron transport system, acetylcholinesterase, and lipid peroxidation. As complementary data, morphometric measurements, condition index, and lipid and protein content were considered together with in situ physicochemical characterization of the sites (temperature, salinity, pH, and dissolved oxygen). Electron transport system activity levels were between 35.7 and 50.5 nmol O2 /min g protein, acetylcholinesterase activity levels ranged from 2.6 to 3.8 nmol/min g protein, and lipid peroxidation ranged from 174.7 to 246.4 nmol malondialdehyde/g protein. The exposure sites shaped the response not only of biomarkers but also of "health" parameters (protein, lipids, and condition index). Lipid peroxidation was the most responsive biomarker also associated with electron transport system, especially at the closest site to the urban wastewater-treatment plant. Because of the presence of complex mixtures of contaminants in urban effluents, biomarker responses can provide valuable information in environmental assessment. However, it is vital to identify all biological and ecological factors induced by the natural life cycle of clams. Abiotic factors can mask or overlap the response of biomarkers and should be considered in a multibiomarker approach. Environ Toxicol Chem 2021;40:272-283. © 2020 SETAC.

A multibiomarker approach in Mytilus galloprovincialis to assess environmental quality.

A multibiomarker approach was carried out for the first time in the South Portuguese Coast using Mytilus galloprovincialis, to assess environmental quality, establish if there are adverse biological responses associated to different sources of anthropogenic contamination and to determine spatial and seasonal trends. For this purpose the battery of biomarkers selected was: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx total and Se dependent), Cytochrome P450 component system, Glutathione-S-transferase (GST) and acetylcholinesterase (AChE), metallothionein (MT) and lead-delta-aminolevulinic acid dehydratase (ALAD), lipid peroxidation (LPO) and Condition Index (CI) along with the determination of PAHs and metals (Cd, Cu, Ni, Pb and Zn). Results show that despite the levels of both organic and metallic contaminants in these eight spots in the South Coast of Portugal not being particularly high compared with other contaminated/polluted sites worldwide, the selected battery of biomarkers responded efficiently to the environmental changes and allowed an environmental assessment between seasons and sites. Different spatial and seasonal responses were evident along the South Coast of Portugal, meaning that the contamination is not homogeneous. This does not only reflect different competition, origin and intensity of contamination, but also different environmental conditions (e.g. temperature, salinity). Along the South Portuguese Coast site 8 was the most contaminated, while site 2 was considered the least contaminated. Despite environmental factors possibly causing difficulties in the general interpretation of biomarker data, those that better responded to environmental contamination were CYP450, SOD-mit and T-GPx for the summation SigmaPAHs, MT (digestive gland) for metals (especially Cu), ALAD for Pb and LPO for both organic and metallic contamination. These biomarkers were also positively correlated with temperature in summer, revealing this as a more stressful/critical season. In future environmental contamination assessments there is no need to analyse the components b5, P418, NADH and NADPH of phase I MFO system, and MT in the gills, since their responses are not evident.

Determining the footprint of sewage discharges in a coastal lagoon in South-Western Europe.

Ria Formosa is a highly productive lagoon in South-Western Europe, supporting 90% of Portuguese clam production. Decreases in shellfish production have been ascribed to deterioration of water quality due to sewage discharges. Nevertheless, a thorough study considering their impact on the whole lagoon system has been missing. This work determined the sewage footprint from the major sewage treatment plants (STP) regarding eutrophication and microbial contamination within a two-year monitoring program. This focused on salinity, oxygen, nutrients, chlorophyll-a and faecal coliforms. Areas closer to sewage discharges showed an evident impact with maximum effects detected at the major STP. However, globally, the Ria Formosa did not show clear eutrophication problems due to high tidal flushing. Ammonium, oxygen, chlorophyll-a and faecal coliforms, unlike the other parameters, showed no seasonality. Microbiological contamination was of great concern and public health issues could be avoided by settling shellfish beds at least 500 m away from discharge points.

Genotoxicity of copper oxide and silver nanoparticles in the mussel Mytilus galloprovincialis.

Though there is some information on cytotoxicity of copper nanoparticles and silver nanoparticles on human cell lines, there is no information on their genotoxic and cytotoxic behaviour in bivalve molluscs. The aim of this study was to investigate the genotoxic impact of copper oxide and silver nanoparticles using mussels Mytilus galloprovincialis. Mussels were exposed to 10 µg L⁻¹ of CuO nanoparticles and Cu²âº and Ag nanoparticles and Ag⁺ for 15 days to assess genotoxic effects in hemocytes using the comet assay. The results obtained indicated that copper and silver forms (nanoparticles and ionic) induced DNA damage in hemolymph cells and a time-response effect was evident when compared to unexposed mussels. Ionic forms presented higher genotoxicity than nanoparticles, suggesting different mechanisms of action that may be mediated through oxidative stress. DNA strand breaks proved to be a useful biomarker of exposure to genotoxic effects of CuO and Ag nanoparticles in marine molluscs.

Genotoxicity in two bivalve species from a coastal lagoon in the south of Portugal.

DNA damage was evaluated by comet assay in the haemolymph of two bivalve species Ruditapes decussatus and Mytilus galloprovincialis from the Ria Formosa lagoon (south Coast of Portugal). Clams and mussels were sampled from sites close to each other to determine interspecific responses to similar environmental conditions, considering also seasonal and gender differences. Coupled with genotoxic effect, another damage biomarker (lipid peroxidation) was analysed to verify if the conditions that instigate DNA damage can be related with injury to cell membranes. For both species, DNA damage was low, reflecting the low levels of genotoxic contaminants in the lagoon, and no interspecific differences were found. However, seasonal differences were established for both bivalve species, reflecting higher environmental stress in summer. Regarding gender susceptibility, only clams showed differences in percentage of Tail DNA, with females more sensitive to DNA damage than males. Additionally, results for clams point out that factors responsible for LPO may not be the same as those causing genotoxicity. This study demonstrated that DNA damage is a sensitive biomarker to discriminate spatial, temporal and gender differences, being an appropriate biomarker for genotoxicity evaluation even in places of low contamination, such as the Ria Formosa lagoon.