M. Oliveira

Contamination assessment of a coastal lagoon (Ria de Aveiro, Portugal) using defence and damage biochemical indicators in gill of Liza aurata - an integrated biomarker approach.

Fish gill importance in toxicants uptake, bioconcentration and excretion allied to meagre knowledge on branchial damage/protection responses substantiate this study. Five critical sites in Ria de Aveiro (Portugal) were assessed in comparison with a reference site (Torreira), focusing on Liza aurata gill antioxidant defences versus damage (oxidative and genetic). Only in Barra fish displayed damage (lipid peroxidation) though no differences were found in antioxidants. In all other sites, except Rio, antioxidant alterations were found. Thus, fish from Gafanha, Laranjo and Vagos showed higher total glutathione, glutathione peroxidase and catalase. Higher glutathione reductase and glutathione S-transferase activity was also found in the first and the last sites, respectively. In Laranjo, metallothionein levels were higher though lower in Gafanha and Vagos. In general, damage was not accompanied by defences weakening confirming that predicting damage based on antioxidants depletion is not straightforward. The integrated biomarker response index ranked sites as: Gafanha>Barra>Laranjo>Vagos>Rio>Torreira.

Organ specific antioxidant responses in golden grey mullet (Liza aurata) following a short-term exposure to phenanthrene

Phenanthrene (Phe) is among the most abundant and ubiquitous polycyclic aromatic hydrocarbons (PAHs) in the aquatic environment as a result of human activities. Even so, the knowledge about its impact on fish health is still limited. In this study, the teleost Liza aurata was exposed to 0.1, 0.3, 0.9 and 2.7 microM Phe concentrations during 16 h. Enzymatic antioxidants such as selenium dependent glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (GR) and catalase (CAT), as well as a non-enzymatic antioxidant (glutathione - GSH) were quantified in three target organs - gill, kidney and liver. The lipid peroxidation (LPO) was also assessed as a marker of oxidative damage. GPx activity was decreased in gill (0.1 and 0.9 microM), whereas in the liver it was increased (0.3-2.7 microM). GST activity was decreased in kidney (0.3-2.7 microM) and CAT activity was increased in gill after 0.9 microM exposure. GSH content was significantly increased in gill by the lowest concentration and in liver by all Phe concentrations. Despite the antioxidant defense responses, LPO increased in gill (0.3-2.7 microM), kidney (0.1 microM) and liver (0.1 and 2.7 microM). These results revealed organ specific antioxidant defenses depending on the Phe concentration. Liver demonstrated a higher adaptive competence expressed as antioxidant defenses activation, namely GSH and GPX. The lower vulnerability of the kidney to oxidative damage (compared to gill and liver) seems to be related to its higher antioxidant basal levels. Globally, current data highlight the Phe potential to induce oxidative stress and, consequently, to affect the well-being of fish.

Does the presence of microplastics influence the acute toxicity of chromium(VI) to early juveniles of the common goby (Pomatoschistus microps)? A study with juveniles from two wild estuarine populations

Toxicological interactions between microplastics (MP) and other environmental contaminants are of grave concern. Here, the potential influence of MP in the short-term toxicity of chromium to early juveniles of Pomatoschistus microps was investigated. Three null hypotheses were tested: (1) exposure to Cr(VI) concentrations in the low ppm range does not induce toxic effects on juveniles; (2) the presence of microplastics in the water does not influence the acute toxicity of Cr(VI) to juveniles; (3) the environmental conditions of the natural habitat where fish developed do not influence their sensitivity to Cr(VI)-induced acute stress. Fish were collected in the estuaries of Minho (M-est) and Lima (L-est) Rivers (NW Iberian Peninsula) that have several abiotic differences, including in the water and sediment concentrations of various environmental contaminants. After acclimatization to laboratory conditions, two 96h acute bioassays were carried out with juveniles from both estuaries to: (i) investigate the effects of Cr(VI) alone; (ii) investigate the effects of Cr(VI) in the presence of MP (polyethylene spheres 1-5μm ∅). Cr(VI) alone induced mortality (96h-LC50s: 14.4-30.5mg/l) and significantly decreased fish predatory performance (≤74%). Thus, in the range of concentrations tested (5.6-28.4mg/l) Cr(VI) was found to be toxic to P. microps early juveniles, therefore, we rejected hypothesis 1. Under simultaneous exposure to Cr(VI) and MP, a significant decrease of the predatory performance (≤67%) and a significant inhibition of AChE activity (≤31%) were found. AChE inhibition was not observed in the test with Cr(VI) alone and MP alone caused an AChE inhibition ≤21%. Mixture treatments containing Cr(VI) concentration ≥3.9mg/l significantly increased LPO levels in L-est fish, an effect that was not observed under Cr(VI) or MP single exposures. Thus, toxicological interactions between Cr(VI) and MP occurred, therefore, we rejected hypothesis 2. In the presence of MP, the negative effect caused by high concentrations of Cr(VI) on the predatory performance was significantly reduced in L-est fish but not in M-est fish, and Cr(VI) concentrations higher than 3.9mg/l caused oxidative damage in L-est fish but not in M-est fish. The acclimatization and test conditions were similar for fish from the two estuaries and these ecosystems have environmental differences. Thus, long-term exposure to distinct environmental conditions in the natural habitat during previous developmental phases influenced the sensitivity and responses of juveniles to Cr(VI), therefore, we rejected hypothesis 3. Overall, the results of this study indicate toxicological interactions between MP and Cr(VI) highlighting the importance of further investigating the combined effects of MP and other common contaminants.

European eel (Anguilla anguilla L.) metallothionein, endocrine, metabolic and genotoxic responses to copper exposure.

This study investigated Anguilla anguilla (European eel) physiological and genotoxic responses to copper (Cu) and their relation with metallothionein (MT) protection. Eels were exposed during 7 days to Cu 0.2 micromol/L. MT induction was assessed in gill and liver, revealing significant response only in liver. Endocrine responses displayed a plasma free triiodothyronine (T3) and cortisol significant decrease, though the thyroid-stimulating hormone (TSH) and free thyroxine (T4) concentrations were unaltered. A significant plasma glucose increase was observed whereas lactate was significantly decreased. Despite the absence of DNA integrity decrease in blood, gill, liver and kidney, erythrocytic nuclear abnormalities (ENA) frequency significantly increased in Cu exposed group. MT induction was insufficient to prevent endocrine and metabolic alterations as well as genotoxicity/clastogenicity in blood. However, MT protection was evident in liver by preventing DNA integrity loss. Globally, it was demonstrated that Cu environmentally realistic levels may pose a serious ecological risk to fish.

Wild juvenile Dicentrarchus labrax L. liver antioxidant and damage responses at Aveiro Lagoon, Portugal

The Aveiro Lagoon, at the north-western coast of Portugal, has been under considerable anthropogenic pressure for the last 5 decades. In order to perform an adequate survey of the effects induced by the contaminants in presence, wild juveniles Dicentrarchus labrax (sea bass) were selected. Thus, sea bass was captured at five sites: Torreira (TOR, as reference site), Gafanha (GAF), Rio Novo Príncipe (RIO), Laranjo (LAR) and Vagos (VAG) in autumn 2005. Liver defence responses such as catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), total glutathione (GSHt), total non-protein thiols (NP-SH) and metallothioneins (MT) were measured. Liver damage was determined as lipid peroxidation (LPO) and DNA integrity loss. RIO, LAR and VAG presented lower CAT, GR, GST activities and NP-SH and GSHt depletion in comparison to TOR. VAG and LAR showed higher GPx activity when compared to TOR. The highest MT level was found at GAF and VAG. The NP-SH and DNA integrity decreased at GAF compared to TOR. This field study demonstrated that not only antioxidant induction but also inhibitory responses must be considered as a signal of contamination.

DNA damage and lipid peroxidation vs. protection responses in the gill of Dicentrarchus labrax L. from a contaminated coastal lagoon (Ria de Aveiro, Portugal)

The present research work aimed to investigate the damage vs. protection responses in gill of European sea bass (Dicentrarchus labrax) captured at a polluted coastal lagoon, Ria de Aveiro (Portugal), as a tool to evaluate the human impacts on environmental health. Damage was assessed as DNA strand breakage and lipid peroxidation (LPO) whereas protection was evaluated by measuring catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), glutathione S-transferase (GST), total glutathione (GSHt), thiols and metallothioneins (MT). Fish were caught at five locations: Gafanha (GAF), Rio Novo do Príncipe (RIO), Laranjo (LAR) and Vagos (VAG) presenting each a different recognized source of contamination, and Torreira (TOR), assumed as reference site. Among the surveyed sites, gill damage was observed only at GAF, as measured by DNA integrity loss and LPO increase. An overall induction in enzymatic antioxidant protection was perceptible in fish from GAF and VAG, expressed as higher GPX, GR and GST activities. In addition, LAR fish showed elevated GST and CAT activities. Thiols content was higher in all study sites irrespective to the pollution spectrum, whereas GSHt increase was only observed at GAF and VAG. The highest MT level was detected in fish from VAG and the lowest level from RIO suggesting a low contamination degree on this particular site. Globally, the results expressed site-specific response patterns, signalling two critical areas - GAF and VAG. Additionally, a joint analysis (damage vs. protection) allowed the following ordering of surveyed sites according to the contamination degree and risk to fish health; GAF>VAG>LAR>RIO>TOR. D. labrax gill responses demonstrated their efficacy as early warning signals of the contaminants presence. Moreover, the adopted approach, considering simultaneously protection responses and damaging effects, also revealed its usefulness on the pollution extent assessment.

Hepatic metallothionein concentrations in the golden grey mullet (Liza aurata) - relationship with environmental metal concentrations in a metal-contaminated coastal system in Portugal.

This field survey was designed to assess the environmental metal contamination status of Ria de Aveiro (Portugal). To achieve that goal, the concentrations of Cd, Hg, Cu and Zn in the sediments and water were assessed and Liza aurata hepatic metallothionein (MT) determined. The relationships between MT and environmental metal concentrations and hydrological factors were examined. Results revealed a wide distribution of metals both in water and sediments throughout the lagoon, mainly at Rio Novo do Principe (RIO) and Laranjo (LAR), at concentrations that may affect biota. MT concentrations were higher at the sites with high metal content (RIO and LAR). A significant positive correlation was found between MT and Cd in the sediments as well as with MT and Hg and Cu in the water. Moreover, a negative correlation between MT and salinity was found. Thus, the current data support MT use as a biomarker of metal exposure emphasizing the importance of hydrological parameters in its concentrations. Results suggest the continued monitoring of this lagoon system.

Monitoring pollution of coastal lagoon using Liza aurata kidney oxidative stress and genetic endpoints: an integrated biomarker approach

Despite the importance of fish kidney in several functions (immune, metabolism and excretion of xenobiotics) its use in coastal water biomonitoring focusing on protection and damage is scarce. Five critical sites in Ria de Aveiro (Portugal; Barra—BAR, Gafanha—GAF, Rio Novo do Principe—RIO; Laranjo—LAR and Vagos—VAG) were assessed in comparison to a reference site (Torreira—TOR), focusing on Liza aurata kidney antioxidant defences versus damage responses. Non protein thiols were higher at RIO (near a former bleached kraft pulp mill effluent) and total glutathione at RIO, LAR (mercury contaminated) and VAG (polycyclic aromatic hydrocarbons contaminated). Catalase and glutathione S-transferase activities were higher at RIO and LAR whereas no differences were found in glutathione peroxidase activity. However, glutathione reductase was higher at BAR (subject to naval traffic), GAF (harbour water area), RIO and LAR. No peroxidative damage was observed despite the decreased DNA integrity at RIO and VAG. The integrated biomarker response index ranked impacted sites as: LAR>RIO>BAR>GAF>VAG>TOR.

Fish thyroidal and stress responses in contamination monitoring: An integrated biomarker approach

Xenobiotics may affect fish endocrine system, making endocrine responses relevant in pollution monitoring. Five critical sites in Ria de Aveiro (Portugal), having different contamination profiles, were assessed in comparison to a reference site (Torreira), focusing on Liza aurata stress (plasma cortisol, glucose and lactate) and thyroidal (plasma thyroid stimulating hormone-TSH, thyroxine-T4 and triiodothyronine-T3) responses. L. aurata at Vagos (PAHs contaminated) and Laranjo (mercury contaminated), respectively, displayed, low cortisol and high glucose as well as lactate levels. Considering thyroidal responses, high TSH levels were found at Laranjo; low T4 levels were found at Barra (subject to naval traffic) and low T3 levels were found at Rio Novo do Principe (near a former bleached kraft pulp mill effluent), Laranjo and Vagos. The integrated biomarker index ranked impacted sites as: Laranjo>Vagos>Gafanha>Rio Novo do Principe>Barra>Torreira. Endocrine responses reflected xenobiotics exposure suggesting environmental risk to fish at Ria de Aveiro. However no clear relation was found between stress and thyroidal responses.

Effects of the lipid regulator drug gemfibrozil: A toxicological and behavioral perspective.

Pharmaceuticals are emerging contaminants as their worldwide consumption increases. Fibrates such as gemfibrozil (GEM) are used in human medicine to reduce blood concentrations of cholesterol and triacylglycerol and also are some of the most frequently reported pharmaceuticals in waste waters and surface waters. Despite some studies have already demonstrated the negative impact in physiological and/or reproductive endpoints in adult fish, data on survival and behavioral effects in fish larvae are lacking. This study aimed to assess the effects of GEM on zebrafish eleutheroembryo development and locomotor behavior. A fish embryo toxicity (FET) test was undertaken to evaluate GEM acute toxicity by exposing embryos to 0, 6.58, 9.87, 14.81, 22.22, 33.33 and 50mg/L. Developmental endpoints such as hatching success, edemas and malformations were recorded. A second test was undertaken by exposing embryos to 0, 1.5, 3 and 6mg/L in order to evaluate the effects of GEM on 120 and 144h post fertilization (hpf) larvae locomotor activity by video tracking, using a Zebrabox(®) (Viewpoint, France) device. From the data recorded, several parameters such as total swimming distance (TSD) and total swimming time (TST) in each 120s integration time were calculated. Data showed that this compound has a moderate toxic effect on fish embryo development, affecting both survival and hatching success with a calculated 96h LC50 of 11.01mg/L and no effects at the developmental level at 6mg/L. GEM seems to impair locomotor activity, even at concentrations where developmental abnormalities were unperceived, at concentrations as low as 1.5mg/L. Both TSD and TST were sensitive to GEM exposure. These effects do not seem to be independent of the developmental stage as 120hpf larvae seem to present a development bias with repercussions in locomotor behavior. This study highlights the need to include behavioral endpoints in ecotoxicological assays as this seems to be a more sensitive endpoint often disregarded.