Stress on stress response of wild mussels,Mytilus edulis and Mytilus trossulus,as an indicator of ecosystem health

Mussels' health as indicated by the survival time of 50% of sampled animals (LT(50)) when maintained in air at 15 degrees C was examined at three sites in Halifax Harbour with expected differing levels of contamination. Condition and gonad indices, lipid content and the body burden of polycyclic aromatic compounds (PACs) were compared with this stress response in 60 groups of mussels covering two species. At each sampling time, the bioaccumulation of PACs, lipid content and condition indices were higher within Mytilus edulis and Mytilus trossulus displaying shorter survival than at the other sites. M. edulis was generally more tolerant than M. trossulus (for n=11, LT(50) of 9.3 and 7.9 days), with indications of shorter and later gonad development in M. trossulus. Minimum and maximum tolerance was apparent in June and October (LT(50) spanning 3-14 days), respectively. Our results indicate that the stress on stress response provides a simple and sensitive indicator of environmental health, which could be integrated with mussel watch studies.     

Biological effects of palytoxin-like compounds from Ostreopsis cf. ovata: A multibiomarkers approach with mussels Mytilus galloprovincialis

Massive blooms of the harmful benthic dinoflagellate Ostreopsis cf. ovata are of growing environmental concern in the Mediterranean, having recently caused adverse effects on benthic invertebrates and also some intoxication episodes to humans.The toxicological potential of produced palytoxin-like compounds was investigated in the present study on a typical marine sentinel species, the mussel Mytilus galloprovincialis. Organisms were sampled during various phases of a O. cf. ovata bloom, in two differently impacted sites. The presence of the algal toxins was indirectly assessed in mussels tissues (mouse test and hemolysis neutralization assay), while biological and toxicological effects were evaluated through the measurement of osmoregulatory and neurotoxic alterations (Na⁺/K⁺-ATPase and acetylcholinesterase activities), oxidative stress responses (antioxidant defences and total oxyradical scavenging capacity), lipid peroxidation processes (level of malondialdehyde), peroxisomal proliferation, organelle dysfunctions (lysosomal membrane stability, accumulation of lipofuscin and neutral lipids), immunological impairment (granulocytes percentage).Obtained results demonstrated a significant accumulation of algal toxins in mussels exposed to O. cf. ovata. These organisms exhibited a marked inhibition of the Na⁺/K⁺-ATPase activity and alterations of immunological, lysosomal and neurotoxic responses. Markers of oxidative stress showed more limited variations suggesting that toxicity of the O. cf. ovata toxins is not primarily mediated by an over production of reactive oxygen species. This study provided preliminary results on the usefulness of a multi-biomarker approach to assess biological alterations and toxicological events associated to blooms of O. cf. ovata in marine organisms.     

Lysosomal and lipid alterations in the digestive gland of mussels, Mytilus galloprovincialis (L.) as biomarkers of environmental stress

Lysosomal and lipid alterations in the digestive gland cells of the common mussel Mytilus galloprovincialis collected from six stations along Thermaikos Gulf (Northern Greece) were monitored through a 1-year period. The ultrastructural observations of the digestive cells of sampled mussels indicated a higher incidence of "granular" or "dark" residual bodies in the relatively more polluted stations compared to less polluted ones. Alterations in the lysosomal system of digestive cells were assessed: (1) using the lysosomal membrane stability test (2) quantifying lysosomal structural alterations of the residual bodies and the heterolysosomes of the digestive cells at the ultrastructural level, and (3) determining the elemental composition of lysosomes by means of X-ray microanalysis on epoxy resin sections. Evaluation of neutral lipids was realized using image analysis on histochemical resin sections. Mussels from the most heavily polluted stations exhibited reduced lysosomal membrane stability, higher volume density and diameter of heterolysosomes and residual bodies, and, in some cases, relatively higher lysosomal heavy metal content compared to mussels from less polluted stations. The results showed that the lysosomal membrane stability and the volume density of the residual bodies are more sensitive to general stress compared to the other biomarkers used, and could be used successfully as early warning tools in biomonitoring programs.     

Assessment of the mechanisms of detoxification of chemical compounds and antioxidant enzymes in the digestive gland of mussels, Mytilus galloprovincialis, from Mediterranean coastal sites

In this study the effects of the main marine pollutants (metals, PAHs, PCBs and DDTs) were assessed in native mussels from the Mediterranean coast of Spain. For this purpose several biomarkers such as benzo[a]pyrene hydroxylase (BPH), DT-diaphorase (DTD), glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPs), glutathione reductase (GR), metallothionein (MT) and lipid peroxidation (LPO) were measured in the digestive gland. Results showed increased LPO levels in mussels which accumulated high loads of organic compounds and arsenic in their tissues. BPH levels correlated to the concentrations of organic compounds in mussel tissues, though the range of BPH response was low in relation to the high gradient of accumulation of organic pollutants. Increased BPH levels, concomitant to low DTD and GST activities, were detected in mussels which presented high levels of organic pollutants in their tissues. This suggests that signs of LPO present in these organisms are related to the imbalance between phase I and phase II biotransformation processes. Furthermore, the increased levels of MT and CAT detected in mussels which showed high levels of Cd in their tissues appear to reflect a coordinated response which protects against the toxicity of this metal. The application of these biomarkers in environmental assessment is discussed.     

Effect of physiological conditions and biochemical factors of mussels Mytilus galloprovincialis in radioactivity monitoring programs along the Algerian coast

Environmental Science and Pollution Research - A study was carried out to determine the activities of radionuclides (137Cs, 40K, 226Ra, 214Bi, 214Pb, and 210Pb) and the levels of biochemical...     

Distribution of organotin compounds in tissues of mussels Mytilus edulis and clams Mya arenaria

Concentrations and composition of butyltin (BT) and phenyltin (PT) compounds were compared in tissues of mussels Mytilus edulis and clams Mya arenaria collected from the Mersey Estuary. Tributyltin (TBT) concentrations were consistently highest in digestive gland and low in muscular tissues such as siphons (Mya) and adductor muscle (Mytilus). Lowest concentrations were found in the periostracum surrounding the siphons of Mya. Ratios of monobutyltin (MBT), dibutyltin (DBT) and TBT to total BT content of Mytilus tissues were in the ranges 25-34%, 21-28% and 44-51%, respectively. Much higher proportions of TBT:total BT were characteristic of Mya (>80% in the digestive gland, remaining tissue and gonad) implying that slow degradation rates account for the relatively high levels of TBT in this species. TPT was the only phenyltin compound detected (albeit at relatively low levels), and, like TBT, was present in highest concentrations in the digestive gland of clams--suggesting an important dietary component in the accumulation of OTs in these bivalves.     

Yessotoxin determination in Mytilus galloprovincialis revealed by an in vitro functional assay

Yessotoxins (YTXs) are polycyclic ether compounds produced by phytoplanktonic dinoflagellates and accumulated in filter-feeding shellfish. Mouse bioassay is still the official method to detect these toxins, even if it is lacking of specificity and sensitivity. Moreover, there is growing resistance against the use of animal experiments. Many efforts have been made to determine YTXs with other methods. The detection of YTX using a functional assay allows its quantification with an automated and repetitive technique at concentrations in the range of the 1 mg of YTX equivalent/kg European regulatory limit. In this study, an in vitro functional assay based on YTX treatment of MCF-7 cells and resulting in the accumulation of a 100-kDa fragment of E-cadherin was developed on samples of Mytilus galloprovincialis collected from the Adriatic Sea, Italy, along the coasts of Abruzzo, Molise, and Emilia Romagna regions. The YTX concentrations ranged from 0.2 to 1.8 mg of YTX equivalent/kg. The occurrence of levels exceeding the above mentioned limit was observed only in samples of Emilia Romagna region. This last result could represent a risk for human health, but these shellfish were not intended to consumers, because they belonged to a preventive monitoring program.     

Assessment of heavy metal and organic contaminants levels along the Libyan coast using transplanted mussels (Mytilus galloprovincialis)

Within the framework of the MYTIOR project in 2009, heavy metals and organic compounds contaminations were assessed in transplanted mussels in 16 different stations along the coasts of Libya. These stations were located at miles offshore industrial/urban sources but in open sea providing original results related to the background contamination rather than linked to a specific coastal source of pollutants. Results indicated mercury (Hg, 0.045–0.066 mg/kg dry weight (dw)), lead (Pb, 0.44–0, 71 mg/kg dw) and copper (Cu, 3.56–4.21 mg/kg dw) were in the same range or at lower value than control for all stations. Chromium (Cr) in Meleta (3.08 mg/kg dw) and Bomba (3.80 mg/kg dw) and Cadmium values in all stations (1.21–2.41 mg/kg dw) were above control. Meleta, stations from the gulf of Syrt and the three eastern stations were the most affected stations by nickel (max at 5.83 mg/kg dw in Syrt) when zinc was in the same range (141–197 mg/kg dw) and above the control (92 mg/kg dw) at all stations. Polycyclic aromatic hydrocarbon (PAH) levels were found in the range of 16.8–42.8 mg/kg (dry weight) indicating low levels along the Libyan coast with acenaphthene and benzo (a, b, k) pyrenes detected mainly in western Libya. The study of PAH ratios indicated a mixed petrogenic/pyrolytic origin. The only polychlorinated biphenyls (PCBs) found in Libya were PCB 101 in one location and PCB 153 in Tripoli, Garrapoli, Syrt, Ras Lanuf and Benghazi (1.2–1.9 μg/kg dw). Insecticides were lower than control in all stations except DDT, only detected in Misratah (3.5 μg/kg dw). Overall, the results indicated a low background contamination and a low pollution extent according to the environmental pressure occurring offshore the Libyan coast.     

Effects of increasing temperatures on biomarker responses and accumulation of hazardous substances in rope mussels (Mytilus galloprovincialis) from Bizerte lagoon

This study examined the influence of increasing temperatures in spring and summer on biochemical biomarkers in Mytilus galloprovincialis mussels sampled from Bizerte lagoon (northern Tunisia). Spatial and seasonal variations in a battery of seven biomarkers were analyzed in relation to environmental parameters (temperature, salinity, and pH), physiological status (condition and gonad indexes), stress on stress (SoS), and chemical contaminant levels (heavy metals, polycyclic aromatic hydrocarbons (PAHs), and PCBs) in digestive glands. Integrated biological response (IBR) was calculated using seven biomarkers (acetylcholinesterase (AChE), benzo[a]pyrene hydroxylase (BPH), multixenobiotic resistance (MXR), glutathione S-transferase (GST), catalase (CAT), malondialdehyde (MDA), and metallothioneins (MT). Seasonal variations in biological response were determined during a critical period between spring and summer at two sites, where chemical contamination varies by a factor of 2 for heavy metals and a factor 2.5 for PAHs. The analysis of a battery of biomarkers was combined with the measurement of physiological parameters at both sites, in order to quantify a maximum range of metabolic regulation with a temperature increase of 11 °C between May and August. According to our results, the MT, MDA, CAT, and AChE biomarkers showed the highest amplitude during the 11 °C rise, while the BPH, GST, and MXR biomarkers showed the lowest amplitude. Metabolic amplitude measured with the IBR at Menzel Abdelrahmen—the most severely contaminated station—revealed the highest metabolic stress in Bizerte lagoon in August, when temperatures were highest 29.1 °C. This high metabolic rate was quantified for each biomarker in the North African lagoon area and confirmed in August, when the highest IBR index values were obtained at the least contaminated site 2 (IBR = 9.6) and the most contaminated site 1 (IBR = 19.6). The combined effects of chemical contamination and increased salinity and temperatures in summer appear to induce a highest metabolic adaptation response and can therefore be used to determine thresholds of effectiveness and facilitate the interpretation of monitoring biomarkers. This approach, applied during substantial temperature increases at two sites with differing chemical contamination, is a first step toward determining an environmental assessment criteria (EAC) threshold in a North African lagoon.     

Stress biomarkers and alkali-labile phosphate level in mussels (Mytilus galloprovincialis) collected in the urban area of Venice (Venice Lagoon, Italy)

In this study, a spatial and temporal survey at three sites located in the "canals" of the Venice historic centre (Italy) and at a reference site was undertaken to evaluate stress effects on mussels sampled in the Venice urban area, where raw sewage is discharged without treatment directly into the water. A battery of biomarkers (metallothionein, micronuclei, condition index and survival in air) was used to evaluate the stress condition of the animals. At the same time the alkali-labile phosphate assay (ALP) was performed in mussel' hemolymph with the aim to find an estrogenic effect biomarker in this mussel species. Biomarker results showed an impairment of the general health condition in the mussels coming from the urban area, in agreement with the chemical analysis. Significantly higher level of the ALP was found in male mussels sampled in April in the urban area, in comparison with the ones from the reference site (P<0.001). Finally, the PCA proved an easy and useful tool to summarize the obtained results, also able to classify the data to indicate a pollution gradient in the Venice urban area.     

Seasonal and inter-annual variability of DNA integrity in mussels Mytilus galloprovincialis: A possible role for natural fluctuations of trace metal concentrations and oxidative biomarkers

The DNA strand fragmentation as analyzed by the Comet assay is a common biomarker widely used to assess the genotoxic potential of chemical pollutants in marine organisms. However, basal levels of DNA strand breaks can be influenced also by factors unrelated to chemical exposure, and a better knowledge on the natural variability of such response would thus be important to discriminate the effects of anthropogenic activities. In this respect, the aim of the present work was to characterize the fluctuations of DNA strand breaks in mussels, Mytilus galloprovincialis, seasonally sampled over a 3 year period from a reference site along the Adriatic coast. Parameters reflecting DNA integrity exhibited significant seasonal changes in mussels haemocytes, with considerable differences between various sampling years. Comparing such results with those previously obtained on the natural fluctuations of trace metal concentrations and oxidative stress biomarkers in mussels tissues, significant correlations were obtained between DNA strand breaks with levels of Ba, Cd, Cu, Hg, Ni, Se, activity of catalase and content of malondialdehyde. Although mechanisms remain to be demonstrated, the overall results of this work suggest the potential role of natural prooxidant factors in affecting the baseline levels of DNA integrity in mussels, and confirm that natural variability of such responses should be carefully considered when monitoring the impact of genotoxins in marine ecosystems.     

PIXE trace element analysis of an aquatic plant as indicator of heavy metal pollution

A new way of detecting trace elements in water is developed by using charged $\text{P}$article $\text{I}$nduced $\text{X}$-ray $\text{E}$mission (PIXE) analysis of cell walls of $\text{Chara corallina}$, a fresh water alga.     

PCBs in wild mussels (Mytilus galloprovincialis) from the N-NW Spanish coast: current levels and long-term trends during the period 1991-2009

Concentrations of polychlorinated biphenyls (PCBs) were determined in wild mussels (Mytilus galloprovincialis) from seven areas of the N-NW Spanish coast, during the period 1991-2009. The studied area is comprised of highly productive ecosystems which support important commercial fishing and shellfishing activities. The seven PCBs indicator congeners recommended by ICES (IUPAC nos. 28, 52, 101, 118, 138, 153 and 180), were analysed by gas chromatography with an electron capture detector (GC-ECD). Lowest average concentrations of PCBs were recorded in Arousa (1.04 μg kg(-1) wet weight), a low populated area with no industry; while the highest levels were found in areas near cities and/or showing a high industrial activity such as: Bilbao Ziérbena (139 μg kg(-1) ww) or A Coru?a (55 μg kg(-1) ww). The hexachlorinated congeners CB153 and CB138 dominated the profiles in all wild mussel populations, although a geographical pattern was found in which the relative contributions of the lower chlorinated congeners were higher in the west coast (Rías of Vigo, Pontevedra and Arousa), which may reflect the occurrence of recent inputs of those compounds in the area. Finally, the application of the Mann-Kendall's test demonstrate the existence of a significant decrease in PCBs levels since 1991 up to 1996 in the studied area, whereas either a slow decrease or stable concentrations were registered over the past decade.     

Bioconcentration of two pharmaceuticals (benzodiazepines) and two personal care products (UV filters) in marine mussels (Mytilus galloprovincialis) under controlled laboratory conditions

Bioaccumulation is essential for gaining insight into the impact of exposure to organic micropollutants in aquatic fauna. Data are currently available on the bioaccumulation of persistent organic pollutants, but there is very little documentation on the bioaccumulation of pharmaceuticals and personal care products (PPCPs). The bioconcentration of selected PPCPs was studied in marine mussels (Mytilus galloprovincialis). The selected PPCPs were two organic UV filters, i.e., 2-ethylhexyl-4-trimethoxycinnamate (EHMC) and octocrylene (OC), and two benzodiazepines (BZP), i.e., diazepam (DZP) and tetrazepam (TZP). Laboratory experiments were performed in which M. galloprovincialis was exposed to these compounds either directly from water, for the less lipophilic substances (BZP) or via spiked food for lipophilic UV filters. M. galloprovincialis uptook and eliminated BZP following first-order kinetics. The biological half-life (t (1/2)) of TZP was 1.4?days, resulting in a bioconcentration factor of 64 and 99?mL?g(-1) dry weight (dw), respectively, for 2.3 and 14.5?μg?L(-1) of exposure, while the biological half-life (t (1/2)) of DZP was 0.4?days, resulting in a bioconcentration factor of 51?mL?g(-1) dw for 13.2?μg?L(-1) of exposure. The uptake of UV filter was rapid in mussels, followed by elimination within 24?h. EHMC increased from 15 to 138?ng?g(-1) dw in 1?h and decreased to 25?ng?g(-1) after 24?h for 11.9?μg?L(-1) exposure. OC reached 839?ng?g(-1) dw after 1?h and decreased to 33?ng?g(-1) after 24?h for 11.6?μg?L(-1) exposure. However, EHMC and OC were slightly accumulated in 48?h, i.e., 38 and 60?ng?g(-1) dw, respectively.     

Pesticide residues in Indian raw honeys,an indicator of environmental pollution

Honey has multifaceted beneficial properties, but polluted environment and unapproved apicultural practices have led to its contamination. In this study, QuEChERS method followed by chromatographic analysis by GC-μECD/FTD and GC-MS was validated and used for determination of 24 pesticides in 100 raw honey samples from various floral origins of Northern India. Matrix-matched calibrations showed that the method was selective and linear (r²?>?0.99) with detection limit <?9.1 ng g^?1 for all the studied pesticides except for monocrotophos (21.3 ng g^?1). The average recoveries at different fortification levels ranged from 86.0 to 107.7% with relative standard deviation <?20%. Pesticide residues were detected in 19.0% samples, and most prevalent compounds detected were dichlorvos in 6.0% samples followed by monocrotophos (5.0%), profenofos (5.0%), permethrin (4.0%), ethion (3.0%), and lindane (3.0%) with concentrations ranging from 58.8 to 225.5, from 96.0 to 430.1, from 14.6 to 43.2, from 27.8 to 39.6, from 25.6 to 28.0, and from 19.6 to 99.2 ng g^?1, respectively. Honey samples originating from cotton, sunflower, and mustard crops (33.3%) that tested positive for pesticide residues were found to be significantly higher (p?<?0.05) than the honey originating from natural and fruity vegetation (13.5%). Therefore, considering the contamination of environmental compartments due to extensive application of pesticides in the study area and their potential for subsequent transfer to honey by the expeditious bees, the results of present study proclaim that honey may be used as an indicator of environmental pollution. Further, estimated daily intakes of all contaminants were found to be at levels well below their acceptable daily intakes suggesting that consumption of honeys at current levels does not pose deleterious effects on human health. However, precautionary measures should always be taken considering the customary honey feeding in infants and cumulative effect of these chemicals in the foreseeable future.     

Lipid-Filled semipermeable membrane devices and mussels as samplers of organochlorine compounds in lake water

Semipermeable membrane sampling devices (SPMDs) and caged lake mussels (Anodonta piscinalis) were simultaneously deployed at four lake watercourse sites in Central Finland four weeks in August 1992. This study was part of the regular annual monitoring of the organochlorine compounds (OCC) in pulp-mill recipient watercourses of Finland with bivalves. Chlorohydrocarbons (CHCs), chlorophenol compounds (PCPs), chloroanisoles (PCAs) and chloroveratroles (PCVs) were analyzed from lipid extract of mussels and from the synthetic triolein lipid of the SPMDs. Hexane-diethyl ether (9:1, v/v) dialysis using polyethylene membrane was applied in dean up of the SPMD lipids and, for comparison, to six sets of the mussel fat. Dialysis recovered CHCs but not PCPs from the mussel fat. CHCs, PCPs, PCAs and PCVs were all recovered in dialysis of the SPMD lipid. Handling of SPMDs in the transport and deployment operations caused significant OCC contamination for the blank SPMDs. Similar trends were revealed in the OCC profiles for mussels ans SPMDs. An exception was the lack of PCPs appearing in SPMDs that did appear in mussels and in a complementary manner the appearance of the PCAs and PCVs in SPMDs.     

Genotoxicity biomarkers and acetylcholinesterase activity in natural populations of Mytilus galloprovincialis along a pollution gradient in the Gulf of Oristano (Sardinia, western Mediterranean)

A year-round biomonitoring study on blue mussels (Mytilus galloprovincialis) was carried out in 4 selected sites along the Gulf of Oristano (Sardinia, Italy): a commercial port (Port), the outlet of the S'Ena Arrubia and Marceddì lagoons (in the catchment area of intensive agricultural and diary activities, and abandoned mining), and a reference site (North). Heavy metal concentrations in sediments from Marceddì were 2-3 to 10-20 times higher in Pb, Cd and Zn, respectively, than those found at North and S'Ena Arrubia. Higher values (P<0.05) of micronuclei frequency were detected in mussels from Marceddì and Port compared to those detected in mussels from North and S'Ena Arrubia. DNA damage in animals from North was significantly lower than that at the other sites. Results of acetylcholinesterase inhibition consistently showed the strongest effects in mussels from Port and Marceddì. Our results suggest that these biomarkers can be used in coastal marine biomonitoring as early signals of exposure and adverse effects along a pollution gradient.     

A global assessment of chromium pollution using sperm whales (Physeter macrocephalus) as an indicator species

Chromium (Cr) is a well-known human carcinogen and a potential reproductive toxicant, but its contribution to ocean pollution is poorly understood. The aim of this study was to provide a global baseline for Cr as a marine pollutant using the sperm whale (Physeter macrocephalus) as an indicator species. Biopsies were collected from free-ranging whales around the globe during the voyage of the research vessel The Odyssey. Total Cr levels were measured in 361 sperm whales collected from 16 regions around the globe detectable levels ranged from 0.9 to 122.6 μg Cr g tissue⁻¹ with a global mean of 8.8 ± 0.9 μg g⁻¹. Two whales had undetectable levels. The highest levels were found in sperm whales sampled in the waters near the Islands of Kiribati in the Pacific (mean = 44.3 ± 14.4) and the Seychelles in the Indian Ocean (mean = 19.5 ± 5.4 μg g⁻¹). The lowest mean levels were found in whales near the Canary Islands (mean = 3.7 ± 0.8 μg g⁻¹) and off of the coast of Sri Lanka (mean = 3.3 ± 0.4 μg g⁻¹). The global mean Cr level in whale skin was 28-times higher than mean Cr skin levels in humans without occupational exposure. The whale levels were more similar to levels only observed previously in human lung tissue from workers who died of Cr-induced lung cancer. We conclude that Cr pollution in the marine environment is significant and that further study is urgently needed.