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The effects of polystyrene microbeads (micro-PS; mix of 2 and 6 μm; final concentration: 32 μg L(-1)) alone or in combination with fluoranthene (30 μg L(-1)) on marine mussels Mytilus spp. were investigated after 7 days of exposure and 7 days of depuration under controlled laboratory conditions. Overall, fluoranthene was mostly associated to algae Chaetoceros muelleri (partition coefficient Log Kp = 4.8) used as a food source for mussels during the experiment. When micro-PS were added in the system, a fraction of FLU transferred from the algae to the microbeads as suggested by the higher partition coefficient of micro-PS (Log Kp = 6.6), which confirmed a high affinity of fluoranthene for polystyrene microparticles. However, this did not lead to a modification of fluoranthene bioaccumulation in exposed individuals, suggesting that micro-PS had a minor role in transferring fluoranthene to mussels tissues in comparison with waterborne and foodborne exposures. After depuration, a higher fluoranthene concentration was detected in mussels exposed to micro-PS and fluoranthene, as compared to mussels exposed to fluoranthene alone. This may be related to direct effect of micro-PS on detoxification mechanisms, as suggested by a down regulation of a P-glycoprotein involved in pollutant excretion, but other factors such as an impairment of the filtration activity or presence of remaining beads in the gut cannot be excluded. Micro-PS alone led to an increase in hemocyte mortality and triggered substantial modulation of cellular oxidative balance: increase in reactive oxygen species production in hemocytes and enhancement of anti-oxidant and glutathione-related enzymes in mussel tissues. Highest histopathological damages and levels of anti-oxidant markers were observed in mussels exposed to micro-PS together with fluoranthene. Overall these results suggest that under the experimental conditions of our study micro-PS led to direct toxic effects at tissue, cellular and molecular levels, and modulated fluoranthene kinetics and toxicity in marine mussels., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Biomarkers are useful tools to assess biological effects of pollutants and have been extensively used in monitoring programs to determine ecosystem health. In these programs, a wide range of environmental conditions are covered and sometimes, obtained data are difficult to interpret because of natural variables are affecting biomarker responses. Among these variables, musseĺs reproductive status has been considered one of the most changing variables between sites in a monitoring survey. Thus, the main aim of this work was to identify the effect that mussel reproductive status has on biomarker responses. For that purpose, mussels sampled at two periods in the reproductive cycle (reproductive and resting stages) were conditioned to the same laboratory conditions and exposed to fluoranthene (FLU) for three weeks. Studied biomarkers covering a wide range of organism responses were included: bioaccumulation, physiological rates (clearance rate -CR-, absorption efficiency -AE-, respiration rate -RR- and their integration in the scope for growth -SFG-), antioxidant enzyme activities (superoxide-dismutase -SOD-, catalase -CAT-, glutathione reductase -GR-, glutathione peroxidase -GPx-, glutathione-S-tranferase -GST-) and biochemical damage responses (lipid membrane peroxidation -LPO-). The results obtained evidenced that the levels of the biomarkers studied (RR, SOD, CAT and GPx) were higher at reproductive than at resting stage. On the other hand, the effect of toxicant was observed in SFG, CAT and GPx but this effect was only detected during the resting period. Moreover, there was a deterioration of mussel gonadal tissue with FLU exposure during reproductive stage. FLU accumulation in mussel tissues was also dependent of the reproductive status with higher internal concentrations during resting than reproductive period. In conclusion, there was a strong effect of reproductive status on studied biomarkers which seems to mask the effect of FLU at reproductive stage. The present study evidences the need to include the measurement of mussel biological status in marine pollution monitoring programs for a correct interpretation of biomarker data., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The effect of the quality of two microalgal species on select biological and biochemical responses used as indicators of pollution were assessed. Mussels were conditioned for 6 weeks with the diatom Chaetoceros neogracile and the dinoflagellate Heterocapsa triquetra, chosen for being two clearly different types of primary production quality that differ in both biometric and biochemical characteristics. After dietary conditioning, the mussels were exposed to a polycyclic aromatic hydrocarbon, fluoranthene (FLU), for 1 week followed by 1 week of depuration. Results showed higher FLU accumulation in mussels fed on C. neogracile compared to those fed on H. triquetra. Concomitantly, a greater impact of this toxicant was observed in the biomarker responses of mussels fed on C. neogracile. These mussels showed an increase in the percentage of dead hemocytes, an activation of phagocytosis and ROS production of hemocytes after exposure. Some enzymatic activities also increased upon FLU exposure (superoxide dismutase -SOD-, catalase -CAT-, and glutathione reductases -GR-) and after depuration (glutathione-s-transferase -GST-). Results suggest that FLU exposure as well as food quality influence biomarker responses, with higher values of SOD, CAT and GR in non-exposed mussels fed on C. neogracile. In addition, upon exposure to the same FLU concentration, GR response varied according to dietary conditioning, suggesting that diet could act as a confounding factor in biomarker responses to pollution. Consequently, trophic conditions should be considered in marine pollution monitoring programs for a better interpretation of biomarker responses., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Biomarkers have been extensively used in monitoring programs with the aim of assessing the biological effects of pollutants on marine organisms and determining environmental status. Data obtained from these programs are sometimes difficult to interpret due to the large amount of natural variables affecting biological processes, which could act as confounding factors on biomarker responses. The main aim of this work was to identify the effect of one of these variables, the food availability, and consequently, the mussel nutritive status, on biomarker responses. For that purpose, mussels (Mytilus galloprovincialis) were conditioned to three different food rations for 2 months in order to create three mussel nutritive statuses and afterwards, each status was exposed to three nominal concentrations of fluoranthene (FLU) for 3 weeks. A battery of biomarkers was considered in this study to cover a wide range of organism responses, both physiological (scope for growth - SFG) and biochemical (superoxide dismutase - SOD, catalase - CAT, glutathione reductase - GR, glutathione peroxidase - GPx, glutathione-S-transferase - GST and phenoloxidase - PO activities, and lipid membrane peroxidation - LPO). The results obtained, evidenced that most of the studied biomarkers (SFG, SOD, CAT, GPx, and PO) were strongly affected by mussel nutritive status, showing higher values at lower status, whereas the effect of toxicant was not always evident, masked by the nutritive status effect. This paper demonstrates that toxicants are not the only source of variability modulating pollution biomarkers, and confirms nutritive status as a major factor altering biochemical and physiological biomarkers., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The Mar Menor is a coastal lagoon affected by the growth of intensive agriculture and urban development in the surrounding area. Large amounts of chemical pollutants from these areas are discharged into El Albujón, a permanent water-course flowing into the lagoon. Biomarkers such as the activity of acetylcholinesterase or antioxidant enzymes have been previously tested in this lagoon demonstrating the presence of neurotoxicity and oxidative stress in clams transplanted in sites affected by the dispersion of the effluent from El Albujón. To complete this traditional toxicology work, a metabolomic profiling of these transplanted organisms has been carried out for the detection of metabolic biomarkers induced by agricultural/urban pollutants. More than 70 metabolites have been quantified using a targeting metabolomics platform based on HPLC-MS. The intracellular metabolic pattern was analyzed by PCA from the digestive gland of clams after 7 and 22 days of transplantation. Results showed a different profile of metabolite between organisms collected from control and exposed sites. At the shorter exposure time, there was an increase in several metabolites in the latter when compared with those from control sites, whereas metabolic profiling at 22 days showed that those metabolites were drastically diminished, with even lower levels than at control sites. These metabolites included: (i) 12 amino acids from the 21 proteogenic and HomoSer, (ii) osmotic protectants such as γ-butyrobetaine and taurine and (iii) nucleotides such as ITP. Regarding sulfur-containing molecules, taurine could be highlighted as a potential biomarker since its concentration was reduced by more than 30 times after 22 days of exposure, whereas the antioxidant glutathione remained constant in the organisms from both control and exposed sites. Although targeted metabolomics has been shown as an early technique of pollutant effect detection, the two-phase pattern could highlight a more complicated metabolite response to pollutants than classical biomarkers., (Copyright © 2015 Elsevier B.V. All rights reserved.)

This study deals with the identification and characterization of biological variables that may affect some of the biological responses used as pollution biomarkers. With this aim, during the 2012 mussel survey of the Spanish Marine Pollution monitoring program (SMP), at the North-Atlantic coast, several quantitative and qualitative biological variables were measured (corporal and shell indices, gonadal development and reserves composition). Studied biomarkers were antioxidant enzymatic activities (CAT, GST, GR), lipid peroxidation (LPO) and the physiological rates integrated in the SFG biomarker (CR, AE, RR). Site pollution was considered as the chemical concentration in the whole tissues of mussels. A great geographical variability was observed for the biological variables, which was mainly linked to the differences in food availability along the studied region. An inverse relationship between antioxidant enzymes and the nutritional status of the organism was evidenced, whereas LPO was positively related to nutritional status and, therefore, with higher metabolic costs, with their associated ROS generation. Mussel condition was also inversely related to CR, and therefore to SFG, suggesting that mussels keep an "ecological memory" from the habitat where they have been collected. No overall relationship was observed between pollution and biomarkers, but a significant overall effect of biological variables on both biochemical and physiological biomarkers was evidenced. It was concluded that when a wide range of certain environmental factors, as food availability, coexist in the same monitoring program, it determines a great variability in mussel populations which mask the effect of contaminants on biomarkers., (Copyright © 2014 Elsevier Inc. All rights reserved.)

This study undertakes an overall assessment of pollution in a large region (over 2500 km of coastline) of the N-NW Spanish coast, by combining the use of biochemical (AChE, GST, GPx) and physiological (SFG) responses to pollution, with chemical analyses in wild mussel populations (Mytilus galloprovincialis). The application of chemical analysis and biological techniques identified polluted sites and quantified the level of toxicity. High levels of pollutants were found in mussel populations located close to major cities and industrialized areas and, in general, average concentrations were higher in the Cantabrian than in the Iberian Atlantic coast. AChE activities ranged between 5.8 and 27.1 nmol/min/mg prot, showing inhibition in 12 sampling sites, according to available ecotoxicological criteria. GST activities ranged between 29.5 and 112.7 nmol/min/mg prot, and extreme variability was observed in GPx, showing activities between 2.6 and 64.5 nmol/min/mg prot. Regarding SFG, only 5 sites showed 'moderate stress' (SFG value below 20 J/g/h), and most sites presented a 'high potential growth' (>35 J/g/h) corresponding to a 'healthy state'. Multivariate statistical techniques applied to the chemical and biological data identified PCBs, organochlorine pesticides and BDEs as the main responsible of the observed toxicity. However, the alteration of biological responses caused by pollutants seems to be, in general, masked by biological variables, namely age and mussel condition, which have an effect on the mussels' response to pollutant exposure., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The Mar Menor is a coastal lagoon threatened by the development of intensive agriculture in the surrounding areas. Large amounts of pesticides from these areas are discharged into El Albujón, a permanent watercourse flowing into the lagoon. We have used a multi-biomarker approach to assess the biological effects of agricultural pollution on a bivalve species. Biomarkers indicative of neurotoxicity (acetylcholinesterase, AChE), oxidative stress (catalase, CAT; glutathione reductase, GR and lipid peroxidation, LPO), phase II biotransformation of xenobiotics (glutathione S-transferase, GST) and physiological stress (scope for growth, SFG) were measured in clams transplanted to four sites of the lagoon (two reference sites and two sites affected by the dispersion of the effluent of the El Albujón), for exposure periods of 7 and 22 days. The hazards of this effluent were also examined by simultaneously measuring up to 83 contaminants (pesticides, PCBs, PAHs and others) in samples of fresh water from the watercourse mouth and seawater from the deployed sites, as well as the bioaccumulation of organochlorinated compounds and PAHs in the transplanted animals. Biomarker responses showed marked differences between reference and affected sites after 7 and 22 days. However it was only after 22 days that principal component analysis (PCA) of the biomarker responses distinguished between clams deployed in sites affected by the dispersion of the effluent of the watercourse and those from the reference sites. The chemical analysis of water showed high concentrations of pesticides close to El Albujón watercourse mouth, with the greatest input flux corresponding to the organophosphate chlorpyrifos, followed by pendimethalin and naphthalene, and at lower levels acenaphthene, terbuthylazine-desethyl and chlorpyrifos-methyl. In this regard, PCA analysis showed that the biological effects of the mixture of pesticides in caged clams after 22 days were reduced levels of AchE and SFG and increased levels of GR and phase II GST activity. An integrated biomarker response index was calculated from the combination of these biomarkers, proving useful for the assessment of the impact of agricultural pollution in caged clams., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The present study was aimed to establish the relationship between the functionality of the digestive gland and physiological rates including SFG (scope for growth) in wild mussels, Mytilus galloprovincilis. The experimental set-up consisted in the evaluation of changes in the morphology of the gland, as well as in the activity of some key digestive enzymes (amylase, laminarinase, cellulase and protease) within a broad range of SFG obtained through manipulation of food ration. The higher SFG values were correlated to an increase in both the size of the digestive gland and the activities of enzymes when expressed in relation to individual. In contrast, no clear relations were observed when the activity of enzymes was expressed in relation to soluble protein, with the exception to amylase. The higher protease activities measured in mussels showing lower SFG may reflect an initial stage of catabolic processes intended to compensate the energy deficit produced by food restriction. The potential use of parameters measured in digestive glands in studies of marine pollution was discussed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

SFG and physiological rates were measured in wild mussels from the Spanish Marine Pollution monitoring program (SMP) in order to determine seawater quality. It consists of 41 stations, covering almost 2500 km of coast, making the SMP the widest-ranging monitoring network in the Iberian Peninsula's Atlantic region. Results of the 2007 and 2008 surveys when 39 sites were sampled: (20 in 2007 and 19 in 2008, being 8 sites sampled both years) were presented. Chemical analyses were carried out to determine the relationships between physiological rates and the accumulation of toxic compounds. Data presented are the first to become available on the use of SFG as a biomarker of the marine environment on a large spatial scale (>1000 km) along Spain's Atlantic seaboard. SFG values enable significant differences to be established between the areas sampled and between the two years surveyed. The integration of biological and chemical data suggests that certain organochlorine compounds, namely chlordanes and DDTs, may have a negative effect on SFG, although such an effect is of a lesser magnitude than that associated with certain biological parameters such as condition index and mussel age. These variables act as confounding factors when attempting to determine the effect of chemical compounds present in the marine environment on mussel SFG. Further research is therefore needed on the relation between these confounding factors and SFG in order to apply the relevant corrective strategies to enable this index to be used in monitoring programs. The effect of these confounding factors is more clearly revealed in studies that cover a wide-ranging spatial and time scale, such as those carried out within the SMP. These results do not invalidate the use of biological data in monitoring programs, but rather point to the need to analyze all the factors affecting each biological process., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The aim of this study was to assess environmental quality at some of the sites most severely affected by the Prestige oil spill off 2 years after the spillage (April and November 2004). For this purpose analyses of polycyclic aromatic hydrocarbons (PAH) and several biochemical (antioxidant enzymes catalase, glutathione reductase, glutathione peroxidase, superoxide dismutase and DT-diaphorase and lipid peroxidation) and physiological [scope for growth (SFG)] biomarkers were determined on wild mussel populations (Mytilus galloprovincialis) collected at four points along the Costa da Morte and compared with those of a reference site not affected by the oil spill. Results showed that PAH contents had markedly decreased 17 months after the accident, although they were higher in April than in November, when they showed values similar to background levels reported for this area. Nevertheless, the predominance of chrysene on PAH profiles, similarly to findings obtained immediately after the spill, indicated the Prestige as their main source. In spite of the low PAH levels recorded, antioxidant activity levels (explained through the integrated antioxidant response-IAR) were higher in the Costa da Morte than at the reference site either in April and November. In April IAR seems to be related to PAH levels found 3 months after the accident (February 2003), suggesting the persistence in the environment of oxidative stress-producing components from the spill. However, evidence of oxidative stress was not reflected at physiological level by scope for growth, with only very slight differences being observed between values from the reference site and those from Costa da Morte sites. In conclusion, although 2 years after the spill PAHs bioaccumulated by mussels from the Costa da Morte had decreased to background levels, biochemical parameters showed signals of oxidative stress in mussels from this area. However, SFG reflected a good health status for the mussel populations studied and did not reveal evidence of physiological disturbance either 17 or 24 months after the Prestige spill.

1. Limited information is available on how changes in horizontal and vertical space within enriched or furnished layer cages (as defined by Directive 1999/74/EC) influence hen behaviour. This study evaluated the effects of varying minimum cage heights and space allowances on the behaviour of laying hens housed in furnished cages. It was conducted on two flocks of medium brown hybrid hens housed in furnished cages with access to perches and nest boxes on a semi-commercial scale at ADAS Gleadthorpe. 2. Flock 1 consisted of two layer strains (ISA Brown and Babcock 380), housed at two minimum cage heights (38 and 45 cm) and 5 stocking densities between 609 and 870 cm2/bird, with 12 replicates of each of the 20 strain/cage height/stocking density treatment combinations. Stocking density was varied by varying the number of birds per cage from 10 to 7 in standard full-width cages or housing 7 hens in a narrower cage. As a consequence stocking density, group size and trough width per bird co-varied for 4 out of 5 stocking density treatments. 3. Behaviour of flock 1 was sampled at 33 to 36, 46 and 68 weeks of age. At each age one top-tier, one middle-tier and one bottom-tier cage was sampled for each treatment. 4. Few behavioural differences due to cage treatments were detected. Hens at 870 cm2 had shorter feeding bouts than hens at 609 and 762 cm2. Yawning was more common in the cages with greater cage height. 5. Video recordings of flock 1 examined cage height effects on hens' use of vertical space and provided additional data on stretching and self-maintenance activities. No differences in behaviour between 38 and 45 cm cages were found except that scratching head was more common in cages with greater cage height. 6. Flock 2 consisted of two layer strains (Shaver Brown and Hy-Line Brown), housed at 38 and 45 cm and 609, 762 and 1016 cm2/bird, with 18 replicates of each of the 12 strain/cage height/stocking density treatment combinations. Stocking density was varied by housing 10, 8 or 6 hens in standard full-width cages. Behaviour of flock 2 was sampled at 30, 48, 60 and 67 weeks from video recordings. Three cages per treatment from middle-tiers only were sampled at each age. 7. Hens housed at 609 cm2/hen had the longest mean feeding bout, greater than for hens at 762 cm2/hen but not hens at 1016 cm2/hen. More unsuccessful attempts to reach the feeder and sideways and backwards displacements from the feeder occurred at 762 and 609 cm2/hen than at 1016 cm2/hen. A maximum of 8 hens were observed feeding synchronously. 8. These results suggest that changes in horizontal and vertical space over the ranges we studied had little effect on behaviour other than feeding behaviour. Specifying a minimum useable trough space per hen, rather than calculating feeder space from total length of feeder per cage, irrespective of accessibility, might help avoid crowding at the feeder and associated disturbance of feeding bouts.

Immunocytochemical confocal laser scanning microscopy and ultrastructural analysis, including immunoelectron microscopy, reveals the distribution of structures in human term amniochorion similar in some respects to basement membranes but with unusually restricted dimensions. On the basis of their immunoreactivity, these trabecular structures, found on the fibroblast layer side of the spongy layer of human term amniochorion and adjacent reticular layer, have been shown to contain type IV collagen, laminin, and nidogen. The origin of these components may be from primitive epithelial structures which pumped fluid into the lakes that eventually coalesced to form the extraembryonic coelom separating the extraembryonic somatic mesoderm from the extraembryonic splanchnic mesoderm. Such a theory of their origin might link them with the mysterious 'cellular layer', a single-cell-thick layer of cells which is usually no longer present in fetal membranes at term. The similarity in composition but not in size of these structures to anchoring plaques for type VII collagen is possible support for the view that these structures are integrators of extracellular matrix polymeric proteins. The 'pseudobasement' membrane associated with the trophoblast layer, on investigation, appears to be typical by six criteria. 'Coiled' fibrous structures in the extracellular matrix of the spongy layer may aid adjustments under tension at this shear surface by a detachable 'Velcro' or 'two spring' fastening system. The coils are rich in fibronectin. The suggestion is made that the compact layer is a giant lamina reticularis associated with the amniotic epithelial basement membrane.