General Stress Biomarker Research Articles

Biomarker responsiveness in Norwegian Sea mussels, Mytilus edulis, exposed at low temperatures to aqueous fractions of crude oil alone and combined with dispersant

Biological effects of aqueous fractions of a crude oil, alone or in combination with dispersant, were investigated in mussels, Mytilus edulis, exposed at three temperatures (5, 10 and 15 °C). Polycyclic aromatic hydrocarbons (PAHs) tissue concentrations were determined, together with genotoxicity, oxidative stress and general stress biomarkers and the Integrated Biological Response (IBR) index. The bioaccumulation of individual PAHs varied depending on the exposure temperature, with relevant bioaccumulation of phenantrene and fluoranthene at 5 °C and heavier (e.g. 5-rings) PAHs at 15 °C. The values and response profiles of each particular biomarker varied with exposure time, concentration of the oil aqueous fraction and dispersant addition, as well as with exposure temperature. Indeed, PAH bioaccumulation and biomarker responsiveness exhibited specific recognizable patterns in mussels exposed at low temperatures. Thus, genotoxicity was recorded early and transient at 5 °C and delayed but unremitting at 10–15 °C. Catalase activity presented a temperature-dependent response profile similar to the genotoxicity biomarker; however, glutathione-S-transferase responsiveness was more intricate. Lysosomal membrane stability in digestive cells decreased more markedly at 5 °C than at higher temperatures and the histological appearance of the digestive gland tissue was temperature-specific, which was interpreted as the combined effects of PAH toxicity and cold stress. It can be concluded that the profile and level of the biological effects are definitely different at low temperatures naturally occurring in the Arctic/Subarctic region (e.g. 5 °C) than at higher temperatures closer to the thermal optimum of this species (10–15 °C).

Energy Status of Stygophilous Amphipod Synurella ambulans as a Promising Biomarker of Environmental Stress in the Hyporheic Zone

The hyporheic zone (HZ) is a sensitive ecotone in river ecosystems because of its biodiversity and susceptibility to human activities. Hyporheic fauna are exposed to multiple stressors that affect the physiology and metabolism of organisms and ultimately ecosystem functioning and biodiversity. This study aimed to quantify adenine nucleotides in the stygophilous freshwater amphipod Synurella ambulans and to evaluate the potential of adenylate energy charge (AEC) as a physiological biomarker of general stress in the HZ of the Sava River at sites with different intensities of environmental and anthropogenic stress. Field studies were conducted seasonally (in December 2018 and April, July, and October 2019) at two sampling sites, one upstream (1-UP) and the other downstream (2-DOWN) of the discharge from the wastewater treatment plant using the standard piston pump. The amphipod population from site 1-UP had significantly higher AEC values than the population from site 2-DOWN in all seasons except summer. Coordinated changes in AEC values with the ATP/ADP ratio indicated differences in energy status between the two populations. However, no changes in the apparent equilibrium constant of adenylate kinase were observed, except in spring at site 1-UP. Multiple linear regression models showed the strongest associations of AEC with Fe and Zn accumulated in amphipods, followed by environmental factors (conductivity, dissolved O2, and concentrations of nitrites and phosphates in the interstitial water). AEC was shown to be a useful index of environmental stress in S. ambulans because it can directly measure the change in available energy and thus the metabolic stress to which the organism is exposed. Finally, seasonal and spatial variations in AEC values reflected ecological status in the HZ.

Pollution impact on metal and biomarker responses in intestinal cytosol of freshwater fish.

In the present study, essential and nonessential metal content and biomarker responses were investigated in the intestine of fish collected from the areas polluted by mining. Our objective was to determine metal and biomarker levels in tissue responsible for dietary intake, which is rarely studied in water pollution research. The study was conducted in the Bregalnica River, reference location, and in the Zletovska and Kriva Rivers (the Republic of North Macedonia), which are directly influenced by the active mines Zletovo and Toranica, respectively. Biological responses were analyzed in Vardar chub (Squalius vardarensis; Karaman, 1928), using for the first time intestinal cytosol as a potentially toxic cell fraction, since metal sensitivity is mostly associated with cytosol. Cytosolic metal levels were higher in fish under the influence of mining (Tl, Li, Cs, Mo, Sr, Cd, Rb, and Cu in the Zletovska River and Cr, Pb, and Se in the Kriva River compared to the Bregalnica River in both seasons). The same trend was evident for total proteins, biomarkers of general stress, and metallothioneins, biomarkers of metal exposure, indicating cellular disturbances in the intestine, the primary site of dietary metal uptake. The association of cytosolic Cu and Cd at all locations pointed to similar pathways and homeostasis of these metallothionein-binding metals. Comparison with other indicator tissues showed that metal concentrations were higher in the intestine of fish from mining-affected areas than in the liver and gills. In general, these results indicated the importance of dietary metal pathways, and cytosolic metal fraction in assessing pollution impacts in freshwater ecosystems.

Integration assay of bacteriological risks in marine environment using salmonella spp and multimarker response in the bivalve Donax trunculus: Novel biomonitoring approach

Microbiological contamination is one of the riskiest forms of human contamination in seawater, which threaten the stability of ecosystems and human health. In this study, we study the accumulation of a pathogenic bacteria Salmonella spp; isolated from the marine environment, in the soft tissue of Donax trunculus (Mollusca, Bivalvia), a commonly used as a bioindicators species for aquatic ecosystems monitoring, under laboratory conditions during both exposure and recovery periods. These bacteria were added in seawater at three concentrations previously determined against sentinel specie at three exposure periods (24, 48 and 96 h). In a second series of experiments, exposed specimens were afterward transplanted to clean water to assess the recovery pattern. The mortality rate of bivalves was determined as biomarker of general stress. Our findings suggest that microbiological contamination by Salmonella spp was gradually incorporated into the body of D. trunculus causing a significant induction of enzymatic activity of acetylcholinesterase (AChE), Catalase (CAT), glutathione-S-transferase (GST) and malondialdehyde (MDA) levels, as a function of time and concentration. Exposure to a bacterial concentration of 5.104 bacteria/liter resulted in the mortality of more than 80% of the specimens. This study is to test the pathogenicity of Salmonella strains at concentrations close to those of the marine environment, and their effects on biomarkers, thus deducing the existence of an exponential relationship between bacterial concentrations and enzymatic response. The principal component analysis shows that the four biomarkers had similar variation with bacterial concentrations, while two groups were obtained to change following the exposure time (CAT-GST and AChE-MDA). This study provides new findings on the potential accumulation of pathogenic bacteria associated with neurotoxicity and oxidative stress in the wedge clam Donax trunculus.

Clozapine modulation of zebrafish swimming behavior and gene expression as a case study to investigate effects of atypical drugs on aquatic organisms

Mental illnesses affect more than 150 million people in Europe and lead to an increasing consumption of neuroactive drugs during the last twenty years. The antipsychotic compound, clozapine, is one of the most used psychotropic drugs worldwide, with potentially negative consequences for the aquatic environment. Hence, the objectives of the study presented here were the quantification of clozapine induced changes in swimming behavior of exposed Danio rerio embryos and the elucidation of the molecular effects on the serotonergic and dopaminergic systems. Yolk-sac larvae were exposed to different concentrations (0.2 mg/L, 0.4 mg/L, 0.8 mg/L, 1.6 mg/L, 3.2 mg/L and 6.4 mg/L) of clozapine for 116 h post-fertilization, and changes in the swimming behavior of the larvae were assessed. Further, quantitative real-time PCR was performed to analyze the expression of selected genes. The qualitative evaluation of changes in the swimming behavior of D. rerio larvae revealed a significant decrease of the average swimming distance and velocity in the light-dark transition test, with more than a 36% reduction at the highest exposure concentration of 6.4 mg/L. Furthermore, the total larval body length was reduced at the highest concentration. An in-depth analysis based on expression of selected target genes of the serotonin (slc6a4a) and dopamine (drd2a) system showed an upregulation at a concentration of 1.6 mg/L and above. In addition, a lower increase in expression was detected for biomarkers of general stress (adra1a and cyp1a2). Our data show that exposure to clozapine during development inhibits swimming activity of zebrafish larvae, which could, in part, be due to disruption of the serotonin- and dopamine system.

The Response of the Planorbid Snail Isidorella newcombi to Chronic Copper Exposure Over a 28-Day Period: Linking Mortality, Cellular Biomarkers, and Reproductive Responses.

The native freshwater gastropod Isidorella newcombi attacks the roots of developing rice plants in southern Australia and is controlled using copper sulphate. The apparent tolerance of this species to moderate levels of copper (Cu) exposure led us to investigate its potential usefulness as a biomonitor species. To assess its response to chronic Cu exposure, adult I. newcombi were exposed to 0-120µgL-1 of Cu for 28days. Lethal and sublethal responses were investigated. The relationships between subcellular biomarkers and life history traits also were explored. At exposure concentrations of 60µgL-1 Cu and above, 100% mortality was observed during the 28-day exposure period. In these treatments, there was an exposure concentration dependent decrease in the time that the snails survived. In the surviving snails, there was an exposure concentration-dependent increase in tissue Cu concentration. In the snails exposed to Cu concentrations above 15µgL-1, no eggs were produced during the final week of copper exposure, indicating that populations would not persist at Cu concentrations above 15µgL-1. The general stress biomarker lysosomal membrane destabilisation (LD) indicated organisms exposed to 10µgL-1 Cu and above were experiencing Cu induced stress. This suggests that LD could act as an early warning system for responses at higher levels of biological organisation in I. newcombi exposed to copper.

Fish biomarkers from a different perspective: evidence of adaptive strategy of Abramis brama (L.) to chemical stress

BackgroundA prerequisite for long-term survival of populations under multi-stress conditions is their capacity to set up efficient adaptive strategies. However, changes in the activity of molecular biomarkers have been for decades considered as early signals of the deterioration of the fish health and evidence of stress-related adverse biological effects. The aim of this study was to show that such changes actually represent adaptive response of fish to chemical stress. Gene expression and enzyme activity level in liver and brain of specimens from two populations of Abramis brama from contrasted habitats (nature reserve and urban) were examined. Selected parameters included biomarkers of general stress, antioxidative defence, xenobiotic metabolism, endocrine disruption, glucose homeostasis, iron homeostasis, and neurotoxicity.ResultsExposure of A. brama population from urban area to chemical stress was confirmed by assessment of chronic toxic pressure at fish habitats using Toxic Unit approach. The most pronounced response to chemical stress is observed through the activation of antioxidative defence mechanisms in brain and liver at gene and enzyme activity level, high biotransformation capacity of liver, and activation of mechanisms that will meet energy demands and compensate for the metabolic costs of the response to toxicants (higher expression of genes related to glucose homeostasis in the exposed population). Higher hepatosomatic index in the exposed population implies liver hypertrophy due to increased functional load caused by pollution. Body condition factors indicate good overall condition of both fish populations and confirm high efficacy of mobilized adaptation mechanisms in the exposed population.ConclusionsThe study provided the first data on basal expression of a number of genes in A. brama, potentially valuable for biomonitoring studies in absence of clear pollution gradient and/or reference sites (conditions). The study highlights importance of newly identified roles of various genes and proteins, typically considered as biomarkers of effects, and shows that changes in these parameters do not necessarily indicate the deterioration of the fish health. Such changes should be considered as adaptive response to chemical stress, rather than direct proof of ecological impact of pollution in situ.

Oxidative stress, biochemical, lipid peroxidation, and antioxidant responses in Clarias gariepinus exposed to acute concentrations of ivermectin.

The short-term effects of ivermectin (IVMT) on the oxidative stress and biochemical parameters of Clarias gariepinus juvenile was assessed under semi-static conditions at concentrations of 9 to 25μgL-1 for up to 4days. Juveniles were highly sensitive to ivermectin, with an LC50 of 15μgL-1.The antioxidant enzyme profile assessed included glutathione reductase (GR), superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT). General stress biomarkers such as serum glucose, protein, alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were also determined at 24-h, 48-h, 72-h, and 96-h exposure durations. Lipid peroxidation showed significant (p < 0.05) decreases in higher concentrations (21μgL-1and 25μgL-1) and durations of exposure (72h and 96h). Significant concentration-dependent increases (p < 0.05) were recorded in the liver function enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) when compared to the control. GPx decreased significantly (p < 0.05) in higher concentrations (21μgL-1and 25μgL-1) and durations of exposure (48-96h). Protein showed significant concentration-dependent decreases, while glucose recorded a mixed trend. The changes in the hepatic antioxidant enzyme activities and serum metabolites were indicative of oxidative stress induced by IVMT. This showed that IVMT is toxic to fish and should be used with utmost caution.

The effects of ZnO nanostructures of different morphology on bioenergetics and stress response biomarkers of the blue mussels Mytilus edulis

Biofouling causes massive economical losses in the maritime sector creating an urgent need for effective and ecologically non-harmful antifouling materials. Zinc oxide (ZnO) nanorod coatings show promise as an antifouling material; however, the toxicity of ZnO nanorods to marine organisms is not known. We compared the toxicity of suspended ZnO nanorods (NR) with that of ZnO nanoparticles (NP) and ionic Zn2+ in a marine bivalve Mytilus edulis exposed for two weeks to 10 or 100 μg Zn L−1 of ZnO NPs, NRs or Zn2+, or to immobilized NRs. The multi-biomarker assessment included bioenergetics markers (tissue energy reserves, activity of mitochondrial electron transport system and autophagic enzymes), expression of apoptotic and inflammatory genes, and general stress biomarkers (oxidative lesions, lysosomal membrane stability and metallothionein expression). Exposure to ZnO NPs, NRs and Zn2+ caused accumulation of oxidative lesions in proteins and lipids, stimulated autophagy, and led to lysosomal membrane destabilization indicating toxicity. However, these responses were not specific for the form of Zn (NPs, NR or Zn2+) and showed no monotonous increase with increasing Zn concentrations in the experimental exposures. No major disturbance of the energy status was found in the mussels exposed to ZnO NPs, NRs, or Zn2+. Exposure to ZnO NPs and NRs led to a strong induction of apoptosis- and inflammation-related genes, which was not seen in Zn2+ exposures. Based on the integrated biomarker response, the overall toxicity as well as the pro-apoptotic and pro-inflammatory action was stronger in ZnO NPs compared with the NRs. Given the stability of ZnO NR coatings and the relatively low toxicity of suspended ZnO NR, ZnO NR coating might be considered a promising low-toxicity material for antifouling paints.

Multiple-biomarker approach in the assessment of the health status of a novel sentinel mussel Brachidontes rodriguezii in a harbor area

The objective of this study is to analyze whether a combination of biomarkers at different levels of biological complexity could be used to assess the health status of a population of Brachidontes rodriguezii associated to a harbor area in Mar del Plata, Argentina. A battery of biomarkers of general stress was measured in mussels collected from impacted and non-impacted areas. This included: condition index; shell analysis; histopathological alterations; atrophy and integrity of the digestive gland tissue; changes in cell type composition of the digestive gland, and glycogen accumulation. The studied biomarkers were integrated into the Integrative Biological Response (IBR/n) index. Overall, the IBR/n indicated a higher level of stress in mussels located in the harbor area. Thus, this biomarker index is a sensitive analytical tool that could be used to classify the ecotoxicological risk in coastal sites.

Biomarkers of general stress in mussels as common indicators for marine biomonitoring programmes in Europe: The ICON experience

This study investigated whether general stress biomarkers in mussels can be applied as common first-tier biomarkers in regional biomonitoring programmes in the North Sea (including Iceland) and western Mediterranean Sea. Stress on Stress (SoS) and lysosomal membrane stability (LMS) biomarkers were analysed in resident mussels (Mytilus sp.) from 8 coastal sites and in transplanted mussels (Mytilus galloprovincialis) from two Spanish Mediterranean coastal sites. The assessment of results, as input to pollution monitoring strategies, was performed jointly for LMS and SoS data from the two regions. Contaminant body burden of the mussels was compared with biomarker results. The results demonstrated that these two general and non-expensive stress biomarkers in mussel can be applied throughout European waters, providing a cost-effective and harmonised approach to screen contaminant-related biological effects within the framework of wide-scale pollution biomonitoring programmes, such as that proposed by the European Union, i.e. the Marine Strategy Framework Directive.

Biomonitoring of water and soil quality: a case study of ecotoxicological methodology application to the assessment of reclaimed agroindustrial wastewaters used for irrigation

The aim of the work was to evaluate the ecotoxicity of reclaimed agroindustrial wastewaters used for irrigation through ecotoxicological bioassays and biomarkers. The ecotoxicological monitoring was addressed on both treated wastewaters and irrigated soils. Wastewater biomonitoring was performed by the acute test with Daphnia magna, the Microtox® test, and a new in vitro patented method. Soil quality monitoring was performed by the acute and chronic tests with the earthworm Eisenia fetida and biomarker analysis, such as lysosomal membrane stability, general stress biomarker of chemical pollution, and metallothionein, specific biomarker of exposure to heavy metals. Overall the integrated ecotoxicological analysis excluded the presence of ecotoxicity both in the reclaimed waters resulting from tertiary treatment and in the irrigated soils. In particular, the analysis of metallothionein allowed to exclude the accumulation of bioavailable heavy metals in the soil. This study suggests the suitability of ecotoxicological methods for the biomonitoring of water and soil during the reclaimed wastewaters reuse for irrigation, contributing to improving the process of agricultural re-use of wastewater in terms of assessment of the toxicological safety of the waters for the environment, for traders and consumers.

Establishment of toxicity thresholds in subpopulations of coelomocytes (amoebocytes vs. eleocytes) of Eisenia fetida exposed in vitro to a variety of metals: implications for biomarker measurements.

Coelomocytes comprise the immune system of earthworms and due to their sensitivity responding to a wide range of pollutants have been widely used as target cells in soil ecotoxicology. Recently, in vitro assays with primary cultures of coelomocytes based in the neutral red uptake (NRU) assay have been developed as promising tools for toxicity assessment chemical in a reproducible and cost-effective manner. However, NRU showed a bimodal dose-response curve previously described after in vivo and in vitro exposure of earthworm coelomocytes to pollutants. This response could be related with alterations in the relative proportion of coelomocyte subpopulations, amoebocytes and eleocytes. Thus, the aims of the present work were, first, to establish the toxicity thresholds that could be governed by different cell-specific sensitivities of coelomocytes subpopulations against a series of metals (Cu, Cd, Pb, Ni), and second to understand the implication that coelomocyte population dynamics (eleocytes vs. amoebocytes) after exposure to pollutants can have on the viability of coelomocytes (measured by NRU assay) as biomarker of general stress in soil health assessment. Complementarily flow cytometric analyses were applied to obtain correlative information about single cells (amoebocytes and eleocytes) in terms of size and complexity, changes in their relative proportion and mortality rates. The results indicated a clear difference in sensitivity of eleocytes and amoebocytes against metal exposure, being eleocytes more sensitive. The bimodal dose-response curve of NRU after in vitro exposure of primary cultures of coelomocytes to metals revealed an initial mortality of eleocytes (decreased NRU), followed by an increased complexity of amoebocytes (enhanced phagocytosis) and massive mortality of eleocytes (increased NRU), to give raise to a massive mortality of amoebocytes (decrease NRU). A synergistic effect on NRU was exerted by the exposure to high Cu concentrations and acidic pH (elicited by the metal itself), whereas the effects on NRU produced after exposure to Cd, Ni and Pb were due solely to the presence of metals, being the acidification of culture medium meaningless.

Dose-dependent hepatic transcriptional responses in Atlantic salmon (Salmo salar) exposed to sublethal doses of gamma radiation

Due to the production of free radicals, gamma radiation may pose a hazard to living organisms. The high-dose radiation effects have been extensively studied, whereas the ecotoxicity data on low-dose gamma radiation is still limited. The present study was therefore performed using Atlantic salmon (Salmo salar) to characterize effects of low-dose (15, 70 and 280mGy) gamma radiation after short-term (48h) exposure. Global transcriptional changes were studied using a combination of high-density oligonucleotide microarrays and quantitative real-time reverse transcription polymerase chain reaction (qPCR). Differentially expressed genes (DEGs; in this article the phrase gene expression is taken as a synonym of gene transcription, although it is acknowledged that gene expression can also be regulated, e.g., at protein stability and translational level) were determined and linked to their biological meanings predicted using both Gene Ontology (GO) and mammalian ortholog-based functional analyses. The plasma glucose level was also measured as a general stress biomarker at the organism level. Results from the microarray analysis revealed a dose-dependent pattern of global transcriptional responses, with 222, 495 and 909 DEGs regulated by 15, 70 and 280mGy gamma radiation, respectively. Among these DEGs, only 34 were commonly regulated by all radiation doses, whereas the majority of differences were dose-specific. No GO functions were identified at low or medium doses, but repression of DEGs associated with GO functions such as DNA replication, cell cycle regulation and response to reactive oxygen species (ROS) were observed after 280mGy gamma exposure. Ortholog-based toxicity pathway analysis further showed that 15mGy radiation affected DEGs associated with cellular signaling and immune response; 70mGy radiation affected cell cycle regulation and DNA damage repair, cellular energy production; and 280mGy radiation affected pathways related to cell cycle regulation and DNA repair, mitochondrial dysfunction and immune functions. Twelve genes representative of key pathways found in this study were verified by qPCR. Potential common MoAs of low-dose gamma radiation may include induction of oxidative stress, DNA damage and disturbance of oxidative phosphorylation (OXPHOS). Although common MoAs were proposed, a number of DEGs and pathways were still found to be dose-specific, potentially indicating multiple mechanisms of action (MOAs) of low-dose gamma radiation in fish. In addition, plasma glucose displayed an apparent increase with increasing radiation doses, although the results were not significantly different from the control. These findings suggested that sublethal doses of gamma radiation may cause dose-dependent transcriptional changes in the liver of Atlantic salmon after short-term exposure. The current study predicted multiple MoA for gamma radiation and may aid future impact assessment of environmental radioactivity in fish.

Assessment of the effects of a marine urban outfall discharge on caged mussels using chemical and biomarker analysis

To assess the presence of endocrine disruptors in treated marine outfall discharges and their possible effects, mussels (Mytilus galloprovincialis) were caged in the environmental mixing zone of the outfall of the Santander sanitation system and in one control area. After 30, 60 and 90days, samples were collected to perform chemical analyses (metals, anionic surfactants, alkylphenols, bisphenol A, phthalates and estrogenic hormones), biomarkers of general stress (lysosomal membrane stability-LMS, histopathology) and biomarkers of endocrine disruption (vitellogenin-like proteins and gonad index). There were no significant differences between outfall and control sites on contaminant levels, except for 4-tert-octylphenol which was higher in the outfall site. Bacteriological counts were higher in the outfall area. No relevant differences in biomarkers were detected between treated and control mussels. A significant reduction in LMS occurred in both groups after 90days caging, indicating a stress situation possibly related to caging or to post-spawning reproductive state.

Effect of air exposure on lysosomal tissues of Mytilus edulis L. from natural intertidal wild beds and submerged culture ropes

Blue mussels collected from suspended culture ropes and from three natural intertidal wild beds from different areas of the German Bight were tested for their ability to cope with hypoxic conditions. During the experiment mussels were exposed to air from 0 to 72 h. Mussels from all sampling sites displayed high tolerance to aerial exposure with moderate levels of mortality after 12 to 48 h of exposure. Lysosomal membrane stability (LMS), a biomarker of general stress, changed notably between minimum values after 12 h and maximum values after 24 h of aerial exposure in intertidal mussels. In contrast, labilization times of mussels from the hanging culture increased continuously up to 48 h of exposure. Intertidal mussels from the island of Heligoland exhibited significantly decreased membrane stability after 72 h of air exposure, correlating to higher mortality rates. Intertidal mussels, although adapted to daily aerial exposure in their natural environment, showed a similar pattern of mortality and lower LMS values during the experiment than mussels from the suspended culture site. The increase of LMS values of mussels under hypoxic conditions at the beginning of the experiment at all sites was tested for the influence of macro-autophagic processes using immune labelling techniques. With this approach it could be demonstrated that high LMS values significantly correlate with low autophagic activity. However, hypoxic conditions do not enhance autophagic processes during the early periods of aerial exposure. Only at the end of the experiment, high values for autophagy were measured in mussels from an intertidal site accompanied with high mortalities. The results indicate that autophagic processes are not involved in the early adaptive processes that enable the mussel to cope with periods of aerial exposure.

Application of a battery of biomarkers in mussel digestive gland to assess long-term effects of the Prestige oil spill in Galicia and the Bay of Biscay: Lysosomal responses

In order to assess the long-term lysosomal responses to the Prestige oil spill (POS), mussels, Mytilus galloprovincialis, were collected in 22 localities from Galicia and the Bay of Biscay (North Iberian peninsula) in July, and September 2003, April, July, and October 2004-2005 and April 2006. Lysosomal membrane stability (labilisation period, LP) and lysosomal structural changes (lysosomal volume density, Vv(L) and lysosomal surface-to-volume ratio, S/V(L)) were measured as general stress biomarkers. The most remarkable long-term effects after the POS were drastic changes in lysosomal size (lysosomal enlargement) and membrane stability (extremely low LP values) up to April-04. Later on, a recovery trend was envisaged all along the studied area after July-04, albeit membrane stability continued to be below 20 min throughout the studied period up to April-06, which indicates a "distress-to-moderate-stress" condition. Lysosomal Response Index (LRI) revealed that environmental stress was more marked in Galicia than in the Bay of Biscay, mainly in the first sampling year, although a "moderate-to-high-stress" condition persisted until July-05. Overall, although lysosomal size returned to reference values, membrane stability was not fully recovered indicating a stress situation throughout the studied period.

Biomonitoring of environmental pollution on the Algerian west coast using caged mussels Mytilus galloprovincialis:

Abstract An active biomonitoring study was carried out on the Algerian west coast usingwild reference mussels ( Mytilus galloprovincialis ) sampled from the Kristel (K)site and transplanted in net cages during one month (between May and June2007) to Oran Harbour (OH) and Mostaganem Harbour (MH), areas characterisedby high levels of urban and industrial pollution. The biological response ofthe mussels was evaluated by their condition index and the use of a generalstress biomarker (evaluation of lysosomal membrane stability: the neutral redretention time (NRRT) method), a genotoxic effects biomarker (determination ofmicronuclei (MN) frequency) and a neurotoxic effects biomarker (determination ofthe acetylcholinesterase (AChE) concentration).Compared to the K reference specimens, OH and MH caged mussels presenteda significant decrease of NRRT in lysosomal haemocytes (56.45±26.48 minand 67.25±22.77 min, respectively) (78±16.97 min for K mussels), an MNfrequency respectively 7.3 and 9 times higher in the haemocytes and the gillcells of the OH caged mussels, and 7.2 and 6.4 times higher in the twotissues of the MH caged mussels. Significant inhibition of AChE activity wasnoted in the gills (16.93±3.1 nmol min

Translational responses of Mytilus galloprovincialis to environmental pollution: Integrating the responses to oxidative stress and other biomarker responses into a general stress index

Heavy metals are commonly associated with the generation of reactive oxygen species (ROS), which may cause oxidative damage to several cellular macromolecules and organelles. In an attempt to correlate biomarker responses to oxidative stress, caged mussels ( Mytilus galloprovincialis) were exposed for 30 days in a relatively clean site and two areas (Stations 1 and 2) unevenly polluted by heavy metals in Gulf of Patras (Greece). Three periods of caging were: one in winter, the second in spring, and the third in autumn. Heavy metal content was determined in digestive glands of the exposed mussels as a measure of metal pollution, metallothionein content as an adaptive and detoxifying index, lysosomal membrane stability as a biomarker of general stress, superoxide radical production and lipid peroxidation as indicators of oxidative stress, and micronucleus frequency in gill cells as an index of chromosomal damage. Considering that protein-synthesizing machinery is one of the candidate targets for ROS, the in vivo activity of ribosomes in digestive glands was also tested. Compared with the reference samples, mussels transplanted to Station 1 showed increased levels of heavy metals and metallothionein in digestive glands, lower lysosomal membrane stability, higher values in oxidative stress indices, reduced activity of ribosomes, and increased chromosomal damage in gill cells. In addition, run-off ribosomes isolated from mussels transplanted to Station 1 were less efficient at initiating protein synthesis in a cell-free system than those from mussels in the reference site. Mussels transplanted to Station 2 exhibited similar but less pronounced responses. Statistical analysis revealed a strong positive correlation of ribosomal activity with lysosomal membrane stability, as well as a significant negative correlation with the oxidative stress indices, metallothionein content, micronucleus frequency, and the digestive gland content in Cr, Cu and Mn. Integration of all the measured biomarker responses into one general “stress index” demonstrated a clear distinction between the sampling sites, allowing classification along a pollution gradient (reference site < Station 2 < Station 1), independently from the season. Moreover, this analysis allowed us to compare responses between sampling campaigns and showed that the biomarker responses show best integration in winter. It was also evident that climatic or metabolic changes could modulate bioavailability of pollutants and priming of cellular defence processes.

DNA adducts, benzo( a)pyrene monooxygenase activity, and lysosomal membrane stability in Mytilus galloprovincialis from different areas in Taranto coastal waters (Italy)

The aim of this study was to investigate the impact of environmental pollution at different stations along the Taranto coastline (Ionian Sea, Puglia, Italy) using several biomarkers of exposure and the effect on mussels, Mytilus galloprovincialis, collected in October 2001 and October 2002. Five sampling sites were compared with a “cleaner” reference site in the Aeronautics Area. In this study we also investigated the differences between adduct levels in gills and digestive gland. This Taranto area is the most significant industrial settlement on the Ionian Sea known to be contaminated by polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls, heavy metals, etc. Exposure to PAHs was evaluated by measuring DNA adduct levels and benzo( a)pyrene monooxygenase activity (B( a)PMO); DNA adducts were analyzed by 32P-postlabeling with nuclease P1 enhancement in both gills and digestive glands to evaluate differences between DNA adduct levels in the two tissues. B( a)PMO was assayed in the microsomal fraction of the digestive glands as a result of the high expression of P450-metabolizing enzymes in this tissue. Lysosomal membrane stability, a potential biomarker of anthropogenic stress, was also evaluated in the digestive glands of mussels, by measuring the latent activity of β- N-acetylhexosaminidase. Induction of DNA adducts was evident in both tissues, although the results revealed large tissue differences in DNA adduct formation. In fact, gills showed higher DNA adduct levels than did digestive gland. No significant differences were found in DNA adduct levels over time, with both tissues providing similar results in both years. DNA adduct levels were correlated with B( a)PMO activity in digestive gland in both years ( r=0.60 in 2001; r=0.73 in 2002). Increases were observed in B( a)PMO activity and DNA adduct levels at different stations; no statistical difference was observed in B( a)PMO activity over the two monitoring campaigns. The membrane labilization period in mussels from some stations was decreased in both years. No statistical differences were established in the membrane labilization times from 2001 to 2002. Our results suggest the existence of different sources and amounts of environmental contaminants at the stations investigated. The formation of DNA adducts confirms the existence of activation pathways in mussels and shows the importance of DNA adduct analysis in the gill tissue in addition to the more commonly used digestive gland; these results confirm the utility of lysosomal membrane stability as a biomarker of general stress. Overall, the integrated use of biomarkers of exposure and the effects of environmental contaminants on living marine organisms may help to better interpret the impact of pollutants in a marine coastal environment.