Molecular and biochemical effects of the antifouling DCOIT in the mussel Perna perna.

Biological fouling is an unwanted phenomenon that results in economic losses to the shipping industry. To prevent fouling, antifouling paints are used. DCOIT (4,5- dichloro-2-n-octyl-4-isothiazolin-3-one) is a biocide present in many antifouling paint formulations, and is toxic to a wide range of organisms. The aim of the present study was to evaluate the effects of DCOIT on oxidative stress indicators of the brown mussel, Perna perna. Molecular (SOD-like, GSTO-like and MGST-like mRNA levels) and biochemical (activities of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST), and levels of glutathione (GSH), reactive oxygen species (ROS) and protein carbonyls (PCO)) components were evaluated. Further, levels of biomarkers were assessed in the gills and digestive glands of mussels. Bivalves were exposed to DCOIT (control, 0.1 µg/L and 10 µg/L) for up to 96 h. DCOIT exposure decreased GSH content in gills. Moreover, exposure to DCOIT also decreased CAT activity in the gills and digestive glands of mussels. GST activity increased in digestive gland after exposure for 24 h to both concentrations of DCOIT tested. SOD activity, ROS levels and PCO content were not affected by exposure to the contaminant. Regarding the molecular biomarkers evaluated, DCOIT exposure altered mRNA levels of SOD-like in both tissues after 24 and 96 h of exposure, and decreased MGST-like mRNA levels in the digestive gland after 96 h of exposure to the chemical. These findings suggested that exposure to DCOIT may alter the biochemical and molecular functioning of P. perna, which may harm the species.

Marine contamination and cytogenotoxic effects of fluoxetine in the tropical brown mussel Perna perna.

Concerns are growing about the presence of fluoxetine (FLX) in environmental matrices, as well as its harmful effects on non-target organisms. FLX in aquatic ecosystems has been detected in a range varying from pg/L to ng/L, while adverse effects have been reported in several organisms inhabiting freshwater and marine environments. The present study quantifies FLX concentrations in seawater samples from Santos Bay, Brazil and assesses metabolic responses and sublethal effects on the tropical brown mussel Perna perna. Levels of ethoxyresorufin­O­deethylase, dibenzylfluorescein dealkylase, glutathione S-transferase, glutathione peroxidase, cholinesterase, lipoperoxidation, and DNA damage were assessed in the gills and digestive gland of these animals, and lysosomal membrane stability was also assessed in hemocytes. FLX altered phase I and II enzyme activities, caused cytogenotoxic effects, and negatively impacted the overall health of mussels exposed to environmentally relevant concentrations. These findings contribute to characterize the risks of introducing this drug into the marine environment.

Hypoxia effects on oxidative stress and immunocompetence biomarkers in the mussel Perna perna (Mytilidae, Bivalvia).

This study investigated the effects of hypoxia on oxidative stress response and immune function in mussels Perna perna exposed to air for 6, 12, 24 and 48 h. In air-exposed mussels, the antioxidant enzymes superoxide dismutase (SOD), catalase, and glutathione reductase (GR) activities were lower in gill tissues (24-48 h) and digestive gland (12 h), while the glutathione peroxidase and GR activities were increased in the digestive gland (48 h). In both tissues, aerial exposure promoted a rapid (6 h) and persistent (up to 48 h) increase of glutathione levels. Decreased hemocyte count and viability, as well as increased phagocytic activity and cellular adhesion capacity were detected after prolonged aerial exposure (>12 h). In summary, induction of thiol pools, altered antioxidant enzyme activities, and activation of immune responses were detected in hypoxia exposed brown mussels, indicating hypoxia induced tissue-specific responses in both antioxidant and immune systems.

The effects of ocean acidification and a carbon dioxide capture and storage leak on the early life stages of the marine mussel Perna perna (Linneaus, 1758) and metal bioavailability.

The study assesses the effects of carbon dioxide capture and storage (CCS) leaks and ocean acidification (OA) on the metal bioavailability and reproduction of the mytilid Perna perna. In laboratory-scale experiments, CCS leakage scenarios (pH 7.0, 6.5, 6.0) and one OA (pH 7.6) scenario were tested using metal-contaminated sediment elutriates and seawater from Santos Bay. The OA treatment did not have an effect on fertilisation, while significant effects were observed in larval-development bioassays where only 16 to 27 % of larva developed normally. In treatments that simulated CO2 leaks, when compared with control, fertilisation success gradually decreased and no larva developed to the D-shaped stage. A fall in pH increased the bioavailability of metals to marine mussels. Larva shell size was significantly affected by both elutriates when compared with seawater; moreover, a significant difference occurred at pH 6.5 between elutriates in the fertilisation bioassay.

Investigation of thermostable metalloproteins in Perna perna mussels from differentially contaminated areas in Southeastern Brazil by bioanalytical techniques.

Metallomic studies regarding environmental contamination by metals are of value in elucidating metal uptake, trafficking, accumulation and metabolism in biological systems. Many proven bioindicator species, such as bivalves, have not yet, however, been well-characterized regarding their metalloprotein expression in response to environmental contaminants. In this context, the aim of the present study was to investigate metalloprotein expressions in the thermostable protein fraction of muscle tissue and digestive glands from mussels (Perna perna) from three differentially metal-contaminated sites in Southeastern Brazil in comparison with a reference site. The thermostable protein fractions were analyzed by SDS-PAGE and SEC-HPLC-ICP-MS. Metal content was also determined in both the crude and the purified extracts. Several inter-organ differences were observed, which is to be expected, while inter-site differences regarding thermostable protein content were also verified, indicating accumulation of these elements in muscle tissue and digestive glands and disruption of homeostasis of essential elements, with detoxification attempts by metal-bound proteins, since all metalloproteins present in both matrices eluted bound to at least one non-essential metal. These results are also noteworthy with regard to the adopted reference site, that also seems to be contaminated by toxic metals.

The brown mussel Perna perna (L., 1758) as a sentinel species for chlorinated pesticide and dioxin-like compounds.

To contribute to the use of the tropical brown mussel Perna perna as a sentinel species for organochlorine pesticides (OCP) and polychlorinated biphenyls (PCB), the present study reports data on the toxicokinetics of these compounds in P. perna. Specifically, the authors present data on OCP and PCB bioaccumulation for eight sampling months from three bays (SE Brazil) and two transplant experiments (each 1 month long). Although seasonality is observed in the total lipid content of the whole soft tissue, with summer samples showing higher values, no such seasonality is observed in the OCP and PCB concentrations bioaccumulated by the mussel P. perna. Because no seasonal effect is observed in the annual OCP and PCB concentrations bioaccumulated by P. perna, the use of this species as a sentinel organism to monitor organochlorinated compounds is encouraged. One month of transplantation is not enough to allow the transplanted specimens to reach the concentrations observed in animals reared at the destination site. Nevertheless, P. perna showed a clear tendency to depurate the DDT metabolites p,p'-DDD and p,p'-DDE after 1 month of transplantation.

Temporal variations of heavy metals levels in Perna viridis, on the Chacopata-Bocaripo lagoon axis, Sucre State, Venezuela.

Perna viridis was used as biomonitor to assess heavy metal levels in the Chacopata-Bocaripo lagoon axis, Venezuela, during rain and drought seasons. The mussels were weighed and measured. The metal concentrations were determined by atomic absorption spectrophotometry. For rain period, the order of bioavailability was: Cu>Ni>Mn>Co>Cd>Pb, and for drought: Cu>Mn>Ni>Co>Pb>Cd. The concentrations of Ni, Co, Cd and Pb showed significant differences (P<0.05) in both periods. There was higher metal accumulation during drought season, possibly related to upwelling, since it produces an increase in primary productivity, which translates more food into organisms, making metals bioavailable for mussels. Only Cu and Mn showed significant relationships between the size and metal concentration, during drought period, it may be because of the organisms need for these essential metals in different physiological processes.

Partition of organochlorine concentrations among suspended solids, sediments and brown mussel Perna perna, in tropical bays.

For evaluating the brown mussel Perna perna as a sentinel organism regarding environmental concentrations of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs), the present study reports original data on the relationship between the concentrations of these chemicals in bottom surface sediments, suspended solids (SS) and concentrations bioaccumulated by this bivalve. Three P. perna cultivation areas, located at three bays in southeastern Brazil were used in this study. The three estuaries are under different degrees of environmental impact. Variations in the OCP and PCB concentrations bioaccumulated by the bivalves tended to be similar to those observed in the sediment, but differed from those found in SS. This latter difference might suggest that the SS trapping apparatuses should have been left in place for approximately 60 days (not only 15 days). This longer period would allow the integration of the environmental variability of the OCP and PCB burden adsorbed to this compartment. Authors encourage future studies to evaluate P. perna exposure to OCPs and PCBs through the evaluation of sediment concentrations.

Zinc causes acute impairment of glutathione metabolism followed by coordinated antioxidant defenses amplification in gills of brown mussels Perna perna.

Zinc demonstrates protective and antioxidant properties at physiological levels, although these characteristics are not attributed at moderate or high concentrations. Zinc toxicity has been related to a number of factors, including interference with antioxidant defenses. In particular, the inhibition of glutathione reductase (GR) has been suggested as a possible mechanism for acute zinc toxicity in bivalves. The present work investigates the biochemical effects of a non-lethal zinc concentration on antioxidant-related parameters in gills of brown mussels Perna perna exposed for 21 days to 2.6 µM zinc chloride. After 2 days of exposure, zinc caused impairment of the antioxidant system, decreasing GR activity and glutathione levels. An increase in antioxidant defenses became evident at 7 and 21 days of exposure, as an increase in superoxide dismutase and glutathione peroxidase activity along with restoration of glutathione levels and GR activity. After 7 and 21 days, an increase in cellular peroxides and lipid peroxidation end products were also detected, which are indicative of oxidative damage. Changes in GR activity contrasts with protein immunoblotting data, suggesting that zinc produces a long lasting inhibition of GR. Contrary to the general trend in antioxidants, levels of peroxiredoxin 6 decreased after 21 days of exposure. The data presented here support the hypothesis that zinc can impair thiol homeostasis, causes an increase in lipid peroxidation and inhibits GR, imposing a pro-oxidant status, which seems to trigger homeostatic mechanisms leading to a subsequent increase on antioxidant-related defenses.

Polybrominated diphenyl ethers (PBDES) and polychlorinated biphenyls (PCBS) in mussels and two fish species from the estuary of the Guanabara Bay, Southeastern Brazil.

The present investigation aimed to analyze PBDE and PCB contamination in mussels (Perna perna) and two commercially important fish species, croaker (Micropogonias furnieri) and mullet (Mugil liza), in the Guanabara Bay, the most important Brazilian estuary, by gas chromatography coupled to mass spectrometry, in order to further knowledge regarding these compounds in the southern hemisphere. This is also the first report of PBDE in this mussel species in the Guanabara Bay. Fish were captured in September (dry season, winter) and March (wet season, summer) 2007 and September 2008. Mussels were collected in August (dry season, winter) 2006, in February (wet season, summer) 2007, and in August 2007 (winter). The results show that all samples showed higher PCB contamination when compared to other ecosystems around the world. On the other hand, PBDEs presented lower concentrations in 41 % of the samples. Croakers presented the highest PCB and PBDE levels, with mullet showing intermediary values and mussels, the lowest.

Effects of glyphosate on cholinesterase activity of the mussel Perna perna and the fish Danio rerio and Jenynsia multidentata: in vitro studies.

Although the herbicide glyphosate [N-(phosphonomethyl)glycine] is not classified as an acethylcholinesterase inhibitor, some studies have reported reduction in the acethylcolinesterase activity after in vivo exposure to both its pure form and its commercial formulations. Considering this controversy, the objective of the present study was to investigate, in vitro, the effects of glyphosate exposure on cholinesterase activity of the brown mussel Perna perna and of two fish species: zebrafish Danio rerio and onesided livebearer Jenynsia multidentata. For this purpose, samples of different tissues (brain and muscle for fish; gills and muscle for mussel) were homogenized and pre-incubated with different glyphosate concentrations before cholinesterase activity determination. Results demonstrated that cholinesterase from different fractions of all species tested was inhibited by glyphosate. The concentrations of glyphosate that inhibits 50% of cholinesterase activity (IC50) ranged from 0.62 mM for P. perna muscle to 8.43 mM for J. multidentata brain. According to this, cholinesterase from mussel seems to be more sensitive to glyphosate exposure than those from the fish D. rerio and J. multidentata.

Organochlorine pesticides in green mussel, Perna viridis, from the Cienfuegos Bay, Cuba.

The green mussel, Perna viridis, was used to measure bioaccumulated levels of organochlorine pesticides in the marine environment of Cuba. Samples were collected in the Cienfuegos Bay between January and December 2010. The organochlorine pesticides (i.e. DDT, Dieldrin, Chlordane, Endosulfan, HCB, Aldrin, Heptachlor and Lindane) were quantified by gas chromatography. The sum of all organochlorine pesticides in P. viridis was 6.31 ng g(-1). The concentration ranged from 3.53 to 4.42 ng g(-1) dry weight (dw) for DDTs (i.e. sum of pp' DDT, pp' DDD, op' DDE and pp' DDE); 1.7-1.9 ng g(-1) dw for Dieldrin; 0.17-0.20 ng g(-1) dw for Chlordanes; 0.14-0.16 ng g(-1) dw for Endosulfan; 0.11-0.17 ng g(-1) dw for HCB; 0.07-0.11 ng g(-1) dw for Aldrin; 0.046-0.054 ng g(-1) dw for Heptachlor and 0.035-0.039 ng g(-1) dw for Lindane. These levels can be considered as low when compared to reported values from similar studies conducted elsewhere in the world. The concentrations of all organochlorines residues detected in this study fell below the EU Maximum Residue Limits.

Regional assessment of PAHs contamination in SE Brazil using brown mussels (Perna perna Linnaeus 1758).

Cultured and native brown mussels (Perna perna Linnaeus 1758) were used to evaluate the contamination by PAHs in the SE Brazilian coastal zone. Mean concentrations of total PAH (38 parental and alkylated compounds) and cluster analysis revealed that: (i) mussel farms (32.7-70.1 ng g(-1)) are located in uncontaminated areas; (ii) coastal rockyshore areas (89.7 ± 25.8 ng g(-1)) have low level of contamination; and (iii) the Guanabara Bay (760.9 ± 456.3 ng g(-1)) is chronically contaminated by hydrocarbons, which is consistent with previous results. PAH profiles and diagnostic ratios suggested mixed petrogenic and pyrolitic sources for group (ii) and petrogenic sources for group (iii). The levels of PAH in the cultured and native mussels are below threshold values for safe food, even in Guanabara Bay, and thus in such cases other contaminants and pathogens should be considered in order to evaluate the quality of shellfish for human consumption.

Chronic contamination assessment integrating biomarkers' responses in transplanted mussels--a seasonal monitoring.

This study aimed to provide the first biomonitoring integrating biomarkers and bioaccumulation data in São Paulo coast, Brazil and, for this purpose, a battery of biomarkers of defense mechanisms was analyzed and linked to contaminants' body burden in a weigh-of-evidence approach. The brown mussel Perna perna was selected to be transplanted from a farming area (Caraguatatuba) to four possibly polluted sites: Engenho D'Água, DTCS (Dutos e Terminais do Centro-Oeste de São Paulo) oil terminal (Sao Sebastiao zone), Palmas Island, and Itaipu (It; Santos Bay zone). After 3 months of exposure in each season, mussels were recollected and the cytochrome P4501A (CYP1A)- and CYP3A-like activities, glutathione-S-transferase and antioxidants enzymes (catalase, glutathione peroxidase, and glutathione reductase) were analyzed in gills. The concentrations of polycyclic aromatic hydrocarbons, linear alkylbenzenes, and nonessential metals (Cr, Cd, Pb, and Hg) in whole tissue were also analyzed and data were linked to biomarkers' responses by multivariate analysis (principal component analysis-factor analysis). A representation of estimated factor scores was performed to confirm the factor descriptions and to characterize the studied stations. Biomarkers exhibited most significant alterations all year long in mussels transplanted to It, located at Santos Bay zone, where bioaccumulation of organic and inorganic compounds was detected. This integrated approach using transplanted mussels showed satisfactory results, pointing out differences between sites, seasons, and critical areas, which could be related to land-based contaminants' sources. The influence of natural factors and other contaminants (e.g., pharmaceuticals) on biomarkers' responses are also discussed.

Assessing the effects of Cu, Cd, and exposure period on metallothionein production in gills of the Brazilian brown mussel Perna perna by using factorial design.

Factorial design plan was experimented in Perna perna in order to find out the contribution of day exposure, Cd and Cu concentrations in water, and their interactions on metallothioneins (MT), Cd, and Cu (the dependent measured variables) in the gills. The picture obtained is more adequate than by studying the factor effect separately. Compared with the control group, the MT concentration after 22 days exposure period in the mixture of 100 µg/mL Cu and Cd is increased almost two times, showing that P. perna might be used as a biomonitor. Cd showed stronger effect than Cu on MT inducing.

Induction of attachment of the Mussel Perna perna by natural products from the brown seaweed Stypopodium zonale.

Marine invertebrates settle, attach, and/or metamorphose in response to signals from several sources, including seaweeds. In response to the aquaculture challenge of producing constant numbers of juveniles from cultured species, natural inducers have been screened for their ability to improve those processes. However, few chemical inducers of attachment of invertebrates have been identified, and even less of these were secondary metabolites. The goal of this work was to isolate the natural products responsible for induction activity using bioassay-guided fractionation of the organic extract of the brown seaweed Stypopodium zonale and the attachment of juveniles of the common brown mussel, Perna perna, as a model. The meroditerpene epitaondiol, identified by comparison of spectral data with the literature, promoted as much as 4.7 times more mussel attachment compared to controls at the natural concentration found in this alga (0.041% of the crude extract or 0.012% of algal dry weight). This is the first report showing that a seaweed produces terpenoid compounds as cues for invertebrate attachment, and future studies evaluating this action on settlement of mussels in the field are expected to improve aquaculture technology by increasing mussel spat production.

Food availability and reproduction affects lipid and fatty acid composition of the brown mussel, Perna perna, raised in suspension culture.

We examined the influence of the reproductive cycle and environmental factors on variations of the condition index (CI), tissue dry mass, shell size, total lipid content, and relative percent of fatty acids in the mussel, Perna perna. Spat or juveniles were reared to commercial size (70 mm) in suspension culture in the Golfo de Cariaco, Venezuela between May and October 2004. The dry mass of soft tissues and shell, a visual assessment of gonadal status and the organism lipid profile were established every fortnight. In parallel, we measured the environmental conditions, following chlorophyll a, salinity, temperature and seston levels. After an initial decrease, the CI rose and remained high until August after which it decreased continuously until October. Total lipid values also decreased initially, after which they showed two periods of rapid recuperation and depletion, the first between May and August and the second between August and October. Similar tendencies were noted in the fatty acids, C18:3n-3, C18:4n-3 and C22:6n-3. Correlation analysis found no significant relationships between environmental parameters and the variations in total lipids. However, significant correlations were noted between fatty acids and specific environmental parameters. In particular, temperature was inversely correlated with C14:0, C16:1n-7, C18:0, C18:1n-9 and 20:5n-3. Chlorophyll a was positively correlated with C14:0, C16:1n-7, C18:1n-7, C18:4n-3 and 20:4n-6. On the other hand, gametogenesis had an effect on C14:0, C16:1n-7, C18:1n-9 and C18:1n-7, while spawned and gonadal regression states had an effect on fatty acid 20:4n-6. Temperature and chlorophyll a levels strongly influenced the proportion of mussels spawning, suggesting that their influence upon lipid composition may be secondary to their impact upon reproduction. Despite the thermal stability of this tropical system, the lipid composition of mussels changed markedly during the study, reflecting the central role of diet and reproductive investment upon lipid composition.

Effect of zinc and benzene on respiration and excretion of mussel larvae (Perna perna) (Linnaeus, 1758) (Mollusca; Bivalvia).

The presence of pollutants in the ocean may affect different physiological parameters of animals. Oxygen consumption and ammonia excretion were evaluated in D-shaped larvae of mussels (Perna perna) exposed to zinc sulphate (ZnSO(4)) and benzene (C(6)H(6)). When compared to the control group, both pollutants presented a significant reduction in oxygen consumption. A reduction in the ammonia excretion was also observed, both for ZnSO(4) and C(6)H(6) and also in the oxygen consumption. The results indicate that anaerobic metabolism may occur at the beginning of P. perna mussels development, as observed in veliger larvae. The O:N ratio under experimental conditions showed low values indicating that catabolism in veliger larvae was predominantly proteic.

Effect of zinc and benzene on respiration and excretion of mussel larvae (Perna perna) (Linnaeus, 1758) (Mollusca; Bivalvia)

The presence of pollutants in the ocean may affect different physiological parameters of animals. Oxygen consumption and ammonia excretion were evaluated in D-shaped larvae of mussels (Perna perna) exposed to zinc sulphate (ZnSO4) and benzene (C6H6). When compared to the control group, both pollutants presented a significant reduction in oxygen consumption. A reduction in the ammonia excretion was also observed, both for ZnSO4 and C6H6 and also in the oxygen consumption. The results indicate that anaerobic metabolism may occur at the beginning of P. perna mussels development, as observed in veliger larvae. The O:N ratio under experimental conditions showed low values indicating that catabolism in veliger larvae was predominantly proteic.

A presença de poluentes no oceano pode afetar diferentes parâmetros fisiológicos. O consumo de oxigênio e a excreção de amônia foram avaliados nas larvas D de mexilhão (Perna perna) expostas ao sulfato de zinco (ZnSO4) e ao benzeno (C6H6). Quando comparados ao grupo controle, ambos os poluentes apresentaram uma significativa redução no consumo de oxigênio. A redução na excreção de amônia também foi observada para o ZnSO4 e para o C6H6, assim como para o consumo de oxigênio. Os resultados indicam que o metabolismo anaeróbico pode ocorrer no início do desenvolvimento do mexilhão P. perna, como foi observado na larva véliger. A razão O:N apresentou baixos valores, indicando que o catabolismo nas larvas véliger foi predominantemente protéico.

Physiological rates in different classes of sizes of Perna perna (Linnaeus, 1758) submitted to experimental laboratory conditions.

Physiological studies of the mussel Perna perna in Brazil are almost 30 years behind those of other, more exhaustively investigated species, such as Mytilus edulis. Little is known about the variations in physiological rates due to size and the consequences of maintaining P. perna in laboratory conditions. This work investigated the variations in respiration, clearance, excretion and absorption efficiency rates of P. perna, classified by size and acclimatized in a laboratory, monitoring the mussels respiration rates and biometry over a period of 30 days, in laboratory conditions. The respiration, clearance and excretion rates presented an allometric relation with the dry weight of the organisms, with b values of 0.66, 0.48 and 0.91 respectively. On the other hand, these same rates, when considered by weight (specific rates) showed a relationship that was inverse to the size of the organisms. Only the absorption efficiency was independent of the weight of the mussel. In terms of acclimatization, it was observed that it takes 10 days for the respiration rate of the mussel P. perna to stabilize in laboratory conditions, after which it follows a routine metabolism.