Mussel Tissue Samples Research Articles

Determination of vanadium species in sediment, mussel and fish muscle tissue samples by liquid chromatography–inductively coupled plasma-mass spectrometry

Vanadium is introduced into the environment during the extraction of petrochemical products and in the production of steels and insecticides. In this study, a liquid chromatographic method for the separation of V(IV) and V(V) as ethylenediaminetetra acetic acid (EDTA) complexes was developed using reversed-phase ion-pair liquid chromatography with inductively coupled plasma-mass spectrometry detection. A C-8 reversed-phase column, 15 cm long, was used to separate the species. A solution containing ammonium acetate 0.06 M, tetrabutylammonium hydroxide 10 mM, ammonium di-phosphate 10 mM and EDTA 2.5 mM at pH 6 was used as the mobile phase in order to avoid the use of organic solvents that reduce the sensitivity of the determination. To prevent changes in distribution of the vanadium species, samples should be prepared freshly. The method developed was applied to the study the vanadium speciation in sediment, mussel and fish muscle samples collected from Lake Maracaibo, Venezuela. The concentration ranges of V(IV) and V(V) in sediment samples were 0.7–61 and 1.4–2.3 μg g −1, respectively. The method is simple and has adequate sensitivity for these practical applications.

Mercury Contamination of Marine Sediments and Blue Mussels (Mytilus edulis) in the Vicinity of a Mercury Cell Chlor-Alkali Plant in Dalhousie, New Brunswick, Canada

Abstract This report examines mercury concentrations in the marine environment of the Restigouche River, near a mercury cell chloralkali plant operated by Pioneer Chemicals Canada Inc. (PCI) in Dalhousie, New Brunswick. The PCI plant is a known point source of mercury in the area, but the extent of marine contamination in the Baie des Chaleurs as a result of those discharges has not been recently evaluated. Marine sediments (five transects), mussel tissue (six locations) and effluent samples (four locations) were obtained in the vicinity of the PCI plant and analyzed for total mercury. Mussel tissues were also analyzed for condition index, metallothioneins and zinc in the digestive gland and gonad as well as gonad vitellin-like proteins and digestive gland lipid peroxidation. Results indicated that the area of the Restigouche River around the PCI plant had elevated concentrations of mercury in sediment and mussel tissue compared with local background concentrations. Mercury concentrations in sediments decreased with distance from the chlor-alkali plant. Concentrations in sediments ranged from 0.03 mg/kg at a distance of 4.6 km to 1.96 mg/kg at a distance of 20 m from the shoreline at the plant. Nine sites had mercury concentrations exceeding the CCME Interim Sediment Quality Guideline (0.13 mg/kg), the furthest of which was 1090 m from PCI. Mercury concentrations in water discharges from four of the five observed outfalls ranged from 0.84 to 4.32 µg/L. Mercury concentrations in mussel tissue ranged from 0.02 mg/kg at a distance 4.6 km from the plant to 1.40 mg/kg directly in front of the plant. Condition index and zinc concentrations in mussels close to the plant were correlated with mercury levels; however, some abnormal biomarker results were found at locations far from the plant as well, indicating other sources of contaminants. Further studies are recommended to define the area of contamination near the PCI plant, as well as to determine the potential impact of other sources of mercury in the area. The environmental risk of the mercury contamination should also be more clearly established.

Assessment of different commercial RNA-extraction and RT-PCR kits for detection of hepatitis A virus in mussel tissues

In the present study, the efficiency of several nucleic acid extraction and RT-PCR commercial kits for the detection of hepatitis A virus (HAV) from seeded mussel tissue samples was evaluated in comparison with the “in-house” method used currently in our laboratory. The best results were achieved with Total Quick RNA Cells & Tissues version mini (Talent) for RNA extraction and the Superscript™ One-Step RT-PCR System (Life Technologies) for the RT-PCR reaction, obtaining a detection limit of 0.1–1 pfu/mg of mussel tissue. A slightly lower sensitivity (in 1 log unit) was achieved using the Rneasy ® plant mini kit (Qiagen) and the Total Quick RNA Cells & Tissues version maxi in combination with the Superscript RT-PCR system. The conventional method usually employed in our laboratory resulted in a sensitivity of 300 pfu/mg of tissue. Taken together, these findings indicate that the combination of Total Quick RNA Cells & Tissues version mini and Superscript™ One-Step RT-PCR System cannot only improve significantly the sensitivity for the HAV detection from mussel, but are also labor and time saving and easy to standardize.

The Role of Petroleum Geochemistry in Defining Oil Spill Recovery: Examples from the Exxon Valdez Spill in Prince William Sound, Alaska

ABSTRACT The application of petroleum geochemistry to determining the fate and effects of oil spills in the marine environment requires high quality data to allow source identification through fingerprinting methods and unbiased sampling designs that allow valid comparisons to be made to detect spill related effects in the presence of other sources of environmental stress. This is particularly important in long-term studies, where sources of hydrocarbons and other pollutants unrelated to the spill event and non-spill related environmental effects become far more important in defining the environmental status of the spill zone. In studies of the Exxon Valdez oil spill, petroleum geochemistry is used to identify the many sources contributing polycyclic aromatic hydrocarbons (PAH) to the marine environment of Prince William Sound (PWS), including the Exxon Valdez oil spill, oil seep residues, particulates from eroding organic shales and coal formations, forest fire fallout, recent biogenic inputs and past human and industrial activities. A sediment quality triad-based shoreline ecology program (SEP), begun in 1990, includes a random sampling component and worst-case non-random site component that provides chemistry data through 2001 to determine the fate and bioavailability of spill oil residues on the shorelines. Environmental half-life values for the PAH at oiled sites range from 2.0 – 7.4 months for upper intertidal areas. Analysis of mussel tissue samples collected at worst-case spill locations find PAH concentrations in the same range as reference site samples. The results of concurrent sediment toxicity measurements using standard bioassays defines a dose-response relationship for total PAH concentration (TPAH) having a threshold of effect TPAH of 2,600 ng/g (dry wt.) that agrees well with the onset of sublethal ecological stress as defined by reductions in infaunal community structure parameters.

Evaluation of two flow injection systems for mercury speciation analysis in fish tissue samples by slurry sampling cold vapor atomic absorption spectrometry

Two simple flow injection (FI) systems were used for mercury speciation analysis in slurried fish tissue samples by cold vapor-atomic absorption spectrometry (CV-AAS). The solid samples were first suspended in hydrochloric acid containing Triton X-100 as dispersing agent, and then the slurries were injected in an acid carrier stream, which was either sequentially mixed with sulfuric acid, potassium persulfate oxidizing agent and stannous chloride reducing agent streams or merged with sodium borohydride reducing agent stream. Mercury speciation analysis was carried out as function of the oxidation coil temperature or sodium borohydride concentration, respectively. The detection limit of the method was 57 and 67 ng g−1, referred to dry weight, using the FI system with on-line oxidation for inorganic mercury and total mercury determination, respectively. Nevertheless, the FI system without on-line oxidation resulted in being simpler, faster and more sensitive for inorganic mercury and total mercury determination. Furthermore, the last system permitted the separate determination of methylmercury and inorganic mercury by selecting an adequate hydrochloric acid concentration in the suspension medium and the suitable sodium borohydride reducing agent. The detection limit of the method was 4.04, 0.977 and 11.0 ng g−1, referred to dry weight, for total mercury, inorganic mercury and methylmercury determination, respectively. Both FI methodologies were validated using two fish tissue certified reference materials, NRC DOLT-2 and NRC DORM-2, obtaining results in good agreement with the certified values, and were applied to the analysis of two real mussel tissue samples.

Stability of Polycyclic Aromatic Hydrocarbons in Frozen Mussel Tissue

The stability of polycyclic aromatic hydrocarbons (PAHs) in frozen mussel tissue (stored at -80 °C and -120 °C was assessed by analyzing samples of SRM 1974 and SRM 1974a, Organics in Mussel Tissue (Mytilus edulis), since the initial certification analyses. Results of analyses of SRM 1974 and SRM 1974a after 10 years and 6 years of storage, respectively, indicate that the PAH concentrations have not changed. Comparison of results from the analyses of frozen versus freeze-dried mussel tissue samples indicate significant losses of the more volatile PAH such as naphthalene and methylnaphthalenes during the freeze-drying process.

Polycyclic Aromatic Hydrocarbon Pollution in Native and Caged Mussels

During 1999, a biological monitoring study was conducted at four sites along the Ligurian coast (Cornigliano, Voltri, Vado Ligure and Sanremo). At each site the concentration and composition of polycyclic aromatic hydrocarbons (PAH) were investigated in native and caged mussels. The mussels ( Mytilus galloprovincialis), sampled in the Spring and the Autumn, showed different accumulation patterns according to the source of pollution they were exposed to. The PAH concentrations were higher in the native than in the caged mussels. The coastal sites were classified according to PAH concentrations found in mussel tissue samples: Native mussels: Vado Ligure<Voltri<Sanremo<=Cornigliano, Caged mussels: Vado Ligure = Voltri = Sanremo ≪ Cornigliano. The different classification is explained by the different location of the organisms: native mussels were located near the air–water interface, while caged mussels were situated at – 3 m from the water surface. The PAH concentrations in the native and caged mussels showed a similar seasonal variability, and can provide the same information about the sources of PAHs.

Use of the Zebra Mussel (Dreissena polymorpha) as a Bioindicator for Aromatic Hydrocarbons in Hamilton Harbour

Abstract Three methods for the extraction of polycyclic aromatic hydrocarbons (PAHs) from zebra mussels (Dreissena polymorpha) sampled from Hamilton Harbour were compared. Replicate freeze-dried mussel tissue samples were extracted using acid digestion, tissue homogenization (mechanical extraction) and ultra-sonication. Each extract was submitted to a cleanup procedure (alumina chro-matography and Sephadex LH20 gel chromatography), followed by analysis using gas chromatography-mass spectrometry (GC-MS). The three extraction methods were equally efficient, based on a statistical comparison of mean concentrations of individual PAHs. Mussel extracts, when subjected to bioassays with Salmonella typhimurium strain YG1029 (TA100-like) in the presence of an exogenous metabolic activation system (S9), exhibited significant mutagenic responses; these responses varied with the PAH content of the mussel extracts. Sources of PAHs in mussel extracts were determined by examining the profiles of sulfur-containing polycyclic aromatic compounds (thia-arenes). Comparison of the ratios of certain thia-arenes with ratios in source samples enabled identification of vehicular emissions and coal tar-contaminated sediment as two sources of PAH contamination in Hamilton Harbour.

Selective Reduction Method for Separate Determination of Inorganic and Total Mercury in Mussel Tissue by Flow-Injection Cold Vapor Technique

A flow-injection cold vapor atomic absorption spectrometry (FI-CV-AAS) method was developed to determine inorganic mercury and total mercury in mussel samples obtained from the Galicia coasts. The mussel samples were digested in a microwave oven using an HNO3/H2O2mixture and then total mercury was determined using sodium borohydride as reducing agent. In a separate subsample, following ultrasonic extraction in hydrochloric acid medium, inorganic mercury was determined by selective reduction using stannous chloride in acid medium as reducing agent. The accuracy of the digestion method was checked by analyzing BCR Reference Material No. 278 Mussel Tissue (Mytilus edulis). There were no significant differences between the certified and found concentration values. As a certified reference material of mussel tissue containing both methylmercury and inorganic mercury was not available, recovery studies on mussel tissue samples spiked with inorganic mercury and methylmercury were done to check the reliability of the method. The results revealed that the mercury contained in mussel samples was methylmercury.

Slurry sampling combined with ultrasonic pretreatment for total mercury determination in samples containing inorganic and methylmercury by flow injection-cold vapor-atomic absorption spectrometry

A method for the determination of total mercury in solid biological and environmental samples by slurry sampling combined with flow injection-cold vapor-atomic absorption spectrometry (FI-CV-AAS) was developed. The slurries were prepared in 15% m/m nitric acid (mussel tissue, aquatic plant) or a (9 + 1) 15% m/m nitric acid–15% m/m hydrochloric acid mixture (river sediment, sewage sludge) containing 0.02% v/v Triton X-100 as dispersing agent, and subjected to ultrasonic pretreatment before being injected into the FI manifold. Ultrasonic pretreatment of slurries was proved to be necessary in order to improve the low mercury recoveries obtained. The effect of several FI parameters on the effectiveness of cold vapor generation for total mercury determination in slurries of mussel tissue was investigated. Mercury concentrations obtained by the proposed method were compared with those obtained after microwave-assisted digestion of mussel tissue samples. The method was validated against CRM BCR 278 mussel tissue, CRM BCR 60 aquatic plant, CRM BCR 320 river sediment and CRM BCR 145R sewage sludge. LODs were 3, 26, 28 and 8 ng g–1 for mussel tissue, river sediment, sewage sludge and aquatic plant, respectively. The precision, expressed as relative standard deviation [RSD (%)], ranged from 4 to 7% for three replicates of each sample analysed by the standard additions method.

Seminested RT-PCR systems for small round structured viruses and detection of enteric viruses in seafood

Highly sensitive seminested RT-PCR systems for the specific detection of genotype I and II small round structured viruses (SRSVs) were developed based on the nucleic acid information deposited in the databanks. SRSVs could be detected in 10 7-fold dilutions of three different stool samples. In addition, a rapid and simple purification protocol for enteric viruses from seafood tissues was elaborated using poliovirus (PV) as model. The virus isolation and viral RNA purification include the following steps: elution of the viruses from the seafood tissue with glycine buffer, their concentration by PEG-precipitation, lysis of viral particles with guanidine hydrochloride and viral RNA isolation using a silica based membrane. The detection limit was 3 to 30 TCID 50 of poliovirus in 1.25 g of seeded seafood tissues without marked food matrix differences, whereas SRSV viruses were 10- and 100-fold better detected in mussels than in shrimps and oysters, respectively. The newly developed purification method, which was shown to remove potential RT-PCR inhibitors present in mussel tissue samples, was applied in a small market survey. 15 mussels, 15 oysters and 12 shrimps were examined for the presence of Hepatitis A virus (HAV), Enterovirus (EV), Rotavirus (RV) and SRSV using specific RT-PCR detection systems. The finding of three oyster samples positive for Rotavirus demonstrated the successful application of our method for the detection of enteric viruses in naturally contaminated seafood samples. The rapid isolation method might be suitable for application in routine testing laboratories and will help to improve public health controls for seafood.

Sensitive HPLC-fluorometric and HPLC-MS determination of diarrhetic shellfish poisoning (SP)-toxins as 4-bromomethyl-7-methoxycoumarin esters

Extracts containing the diarrhetic shellfish poisoning (DSP) toxins okadaic acid (OA), dinophysistoxin-2 (DTX2), and dinophysistoxin-1 (DTX1) were purified on a silica gel cartridge and derivatized with 4-bromomethyl-7 methoxycoumarin (BrMmc). After pre-column derivatization the BrMmc derivatives of the DSP toxins were directly injected into an HPLC system, isocratically eluted, and quantified by fluorescence detection. The signals of the esters showed good linearity in the fluorescence detector within the examined contamination range of 0.03 mg DSP/kg to 2.5 mg DSP/kg. The detection limits for the DSP toxins as 7-Mmc esters were 0.04 ng (corresponding to 0.05 mg DSP/kg). The chromatographic conditions allow to couple the HPLC device with mass spectrometry. The method was tested with various mussel tissue samples.

Analysis of Diarrhetic Shellfish Poisoning Toxins in Shellfish Tissue by Liquid Chromatography with Fluorometric and Mass Spectrometric Detection

Diarrhetic shellfish poisoning (DSP) is a severe gastrointestinal illness caused by consumption of shellfish contaminated with toxigenic dinoflagellates. The main toxins responsible for DSP are okadaic acid (OA), DTX-1, DTX-2, and DTX-3, the latter being a complex mixture of 7-O-acyl derivatives of the first 3. In this study, existing methods based on liquid chromatography (LC) combined with mass spectrometry (LC-MS) and LC with fluorometric detection (LC-FLD) of anthryldiazomethane (ADAM) derivatives were improved upon to achieve a high degree of accuracy and precision for the determination of DSP toxins in a new mussel tissue reference material (MUS-2). All experimental parameters were examined comprehensively, and a new internal standard and a new solid-phase extraction cleanup method were introduced. Quantitative extraction of DSP toxins from shellfish tissue was achieved by exhaustive extraction with aqueous 80% methanol. Cleanup was accomplished by partitioning the crude aqueous methanol extract with hexane to remove lipids and then with chloroform to isolate the toxins. A further cleanup based on an aminopropylsilica column was useful for LC-MS and looks promising for the ADAM/LC-FLD method. The internal standard, 7-O-acetylokadaic acid, synthesized by partial acetylation of OA, improved accuracy and precision by correcting for incomplete recoveries in extraction, cleanup, and derivatization steps and for volumetric errors and instrumental drift. An improved silica cleanup after ADAM derivatization also was developed by controlling the activities of both sorbent and solvents. The methods were tested with various mussel tissue samples. The resulting improved methods will be useful to analysts involved in routine monitoring of DSP toxins.

Bioaccumulation of PCB congeners by blue mussels (Mytilus edulis) deployed in New Bedford Harbor, Massachusetts

AbstractBlue mussels (Mytilus edulis) were deployed to monitor the levels of bioavailable contaminants during a pilot dredging project in New Bedford Harbor (NBH), Massachusetts. Dissolved and particulate seawater samples also were collected periodically during these deployments. Poly‐chlorinated biphenyl (PCB) congener concentrations in both mussel tissue and seawater samples were among the quantified contaminants. Large differences in the dissolved and particulate PCB concentrations in seawater and tissue residue concentrations in mussels were observed along a gradient from the upper harbor to Buzzards Bay. Bioconcentration factors (BCFs) were calculated for six PCB congeners at two stations in NBH and a reference site in Buzzards Bay. Equations were generated relating the log BCF to the log octanol/water partition coefficient (Kow) for these congeners. A consistent relationship was found between dissolved PCB congener concentrations and tissue concentrations in the mussels. This study demonstrates the utility of the blue mussel as a monitoring organism for quantifying bioavailable contaminants in seawater and for relating PCB tissue residues with seawater concentrations.

Comparison of dry mineralization and microwave-oven digestion for the determination of arsenic in mussel products by platform in furnace Zeeman-effect atomic absorption spectrometry

Two digestion procedures were compared in order to obtain an accurate method for the determination of As in mussel products using platform in furnace Zeeman-effect atomic absorption spectrometry. One procedure was based on dry mineralization of the samples and the other on microwave-oven sample digestion in closed polytetrafluoroethylene reactors. Microwave-oven digestion with HNO3 and H2O2 allows the accurate determination of As in certified oyster and mussel tissue samples and provides results comparable to those found by dry mineralization of real mussel product samples, with a sensitivity of 3.9 absorbance units per µg ml–1 and a relative standard deviation of 5% in the analysis of samples containing 8 µg g–1 dry mass of As. Experimental conditions for the determination of As in real samples were optimized and a series of real samples analysed in order to determine the analytical characteristics of the proposed procedure. Sample treatment time was reduced from 2–3 d using dry mineralization, to 20 min per sample using microwave-oven digestion.