Identification and genetic characterization of Anisakis larvae from marine fishes in the South China Sea using an electrophoretic‐guided approach

From 35 species of marine fishes (n = 327) from the South China Sea, 237 nematode larvae were collected and identified morphologically as Anisakis. Genomic DNA was isolated from each larva and subjected to PCR‐based RFLP and targeted sequencing of a nuclear ribosomal DNA region between the 3′‐end of the small subunit and 5′‐end of the large subunit of the rRNA genes (= internal transcribed spacers, ITS+). Four different RFLP profile combinations (sets) were detected for all restriction endonucleases (HinfI, HhaI, and TaqI), of which three were characteristic of Anisakis typica, A. pegreffii, and A. physeteris, respectively. One profile set (for sample CA‐2012) was linked to an ITS+ sequence that was identical to a previously published sequence of Anisakis sp. (sample HC‐2005; originating from the African shelf) and another sequence (PH‐2010; Madeira, Portugal). Phylogenetic analysis was carried out using the ITS+ sequence data from this study and reference sequences from the GenBank database. Neighbor joining and maximum parsimony trees displayed three clades. Clades I and II included nine described species of Anisakis, including all type I and type II larvae; clade III represented some undescribed species of Anisakis. Morphological comparison showed that Anisakis sp. CA‐2012 was distinct from type I and type II larvae based on its tail shape and ratio of tail length to body length. The phylogenetic analysis and morphological characters suggest that Anisakis sp. CA‐2012 represents a new record, now called Anisakis type III larvae.     

Elucidating the identity of Anisakis larvae from a broad range of marine fishes from the Yellow Sea,China, using a combined electrophoretic‐sequencing approach

Larval nematodes were collected from marine fishes from the Yellow Sea, China. Specimens (n=1731) of Anisakis type I from 311 fishes (representing 40 species) were each identified based on morphological characters. From the genomic DNA from individual specimens, a region of nuclear ribosomal DNA was amplified by PCR, followed by digestion with restriction endonuclease HinfI, TaqI or HhaI. Subsequently, the ITS‐1 and ITS‐2 regions of selected samples were sequenced. The results revealed three species of Anisakis, namely Anisakis pegreffii (n=1709), A. typica (n=3) and a genotype (n=19) proposed, also based on comparison with previous studies, to be a “hybrid” between A. pegreffii and A. simplex sensu stricto. Thus, A. pegreffii was the dominant species, accounting for 98.7% of the total number of specimens examined herein. This is the first report of A. typica and the “hybrid” genotype from fishes from the Yellow Sea. This study provides important basic information on Anisakis in this region and suggests that the genus Anisakis has substantial host and geographical distributions.     

Phenolic compounds: antioxidant and larvicidal potential of Smilax brasiliensis Sprengel leaves

Abstract

Smilax brasiliensis is a medicinal species of the Brazilian Cerrado. The extract and fractions of this plant were analysed by LC-DAD-MS. Identified constituents included glycosylated and non-glycosylated flavonoids, especially quercetin, and phenylpropanoids, such as chlorogenic acids. The antioxidant activity was significantly more pronounced for the methanol extract and fractions than that of the commercial antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT). Maximum larvicidal activity of 85.83% was recorded in the dichloromethane fraction (LC₅₀ = 469.78?µg mL^?1). The methanol extract and fractions presented low toxicity to larvae of the shrimp brine Artemia salina, indicating selectivity for C. quinquefasciatus. These results contribute to the phytochemical study of S. brasiliensis. These compounds were identified for the first time in this species and encourage additional work on the isolation of compounds present in the extract and fractions of S. brasiliensis to evaluate the possibility of using them as natural sources of antioxidants, since cytotoxic effects were not demonstrated.     

Mercury in fish products: what’s the best for consumers between bluefin tuna and yellowfin tuna?

A total of 205 bluefin and yellowfin tuna samples were examined for mercury detection in order to verify possible differences and have a detailed risk assessment of the two tuna species. The results showed significant higher mercury concentration in muscle tissue of bluefin tuna respect yellowfin tuna (p < 0.001) with mean concentration of 0.84 mg/kg and maximum value of 1.94 mg/kg. These differences can be due the different biological and ecological aspects of the two tuna species and to different oceanographic aspects between Atlantic Ocean and Mediterranean sea. The results obtained in this study suggest an advisable containment of the sources of pollution and further studies on the closed-loop farming of bluefin tuna, in order to ensure the product safety.     

Development of quantitative vitellogenin-ELISAs for fish test species used in endocrine disruptor screening

The yolk protein precursor vitellogenin (Vtg) in plasma has proved to be a simple and sensitive biomarker for assessing exposure of fish to environmental estrogens. Within international bodies such as the Organization for Economic Cooperation and Development (OECD) work is ongoing to develop screening and testing programmes for endocrine disrupting effects of new chemicals, and in the focus of this development are the fish test species common carp (Cyprinus carpio), fathead minnow (Pimephales promelas), zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes). In this study we have developed quantitative enzyme linked immunosorbent assays (ELISAs) for Vtg in common carp/fathead minnow, zebrafish and Japanese medaka. The assays were developed using a combination of monoclonal and polyclonal fish Vtg antibodies in a sandwich format, using stabilized Vtg from the test species as a standard. The carp Vtg ELISA has a working range of 1–63 ng/mL, a minimal detection limit of 0.6 ng/mL, and may also be used for quantification of Vtg in fathead minnow. In fathead minnow whole-body homogenate samples, the practical detection limit is 400 ng/mL due to the matrix effect. The zebrafish Vtg ELISA has a working range of 0.5–63 ng/mL, a minimal detection limit of 0.4 ng/mL, and a practical detection limit of 200 ng/mL in whole-body homogenate samples. The medaka Vtg ELISA has a working range of 0.25–16 ng/mL, a minimal detection limit of 0.1 ng/mL, and a practical detection limit of 125 ng/mL in whole-body homogenate samples. The intra- and inter-assay variations were below 20% for all assays. The assays were evaluated with sets of representative samples spanning the wide dynamic range of Vtg-levels found in fish exposed to environmental estrogens, and all three assays are currently undergoing international inter-laboratory validation.     

Heavy Metal Concentrations in Water,Sediment and Fish from Izmit Bay,Turkey

Abstract

Mercury, cadmium and lead levels in water, sediment and fish samples from Izmit Bay, Turkey have been determined. Sampling and analysis methods are described. Variations of heavy metal concentrations from different sampling stations are discussed. Results indicate that the levels of mercury and cadmium were highest in the vicinity of a chlor-alkali plant while the highest concentration of lead was near a metallic pipe factory. The amounts of heavy metals found in the shoreline sediment samples were similar to those found in fish species from the bay.     

Vertical Distributions of Nonmethane Hydrocarbons Over Western Europe: Seasonal Cycles of Mixing Ratios and Source Strengths

Abstract

Airborne measurements of the vertical and horizontal distributions of nonmethane hydrocarbons and methyl chloride over western Europe are reported. Annual emission cycles have been calculated from the seasonal variations of mixing ratios using a simple one-dimensional two-box model. They indicate natural sources of ethene, propene, i-butane, i-pentane, and methyl chloride. Grab samples were analysed by subambient temperature programmed gas chromatography.     

Determination of Lead,Cadmium and Mercury in Surface Marine Sediments and Mussels

Abstract

Lead, cadmium and mercury were determined in sediments and mussels, and the ability of these indicators to record metal variations in coastal marine environment is described in this work. The results of an extended investigation of the status of three gulfs at Northern Greece are given, regarding the content of these metals in surface sediments and Mytilus galloprovincialis. The samples were collected during a four-year period. The total concentration of the above heavy metals was determined after digestion of the samples by suitable mixtures of acids, including nitric, perchloric and hydrofluoric acid. The digestion was carried out in a steel pressurised bomb with closed teflon vessels. Lead and cadmium were determined by means of electrothermal atomic absorption spectrometry (ETAAS), and mercury by cold vapour atomic absorption spectrometry (CVAAS). The results were statistically evaluated by analysis of variance, and emphasis was given to annual, seasonal and spatial sources of variation. The annual changes during the last four years and the spatial distribution of heavy metals load is also discussed.     

Determination of inorganic arsenic in white fish using microwave-assisted alkaline alcoholic sample dissolution and HPLC-ICP-MS

An analytical method for the determination of inorganic arsenic in fish samples using HPLC-ICP-MS has been developed. The fresh homogenised sample was subjected to microwave-assisted dissolution by sodium hydroxide in ethanol, which dissolved the sample and quantitatively oxidised arsenite (As(III)) to arsenate (As(V)). This allowed for the determination of inorganic arsenic as a single species, i.e. As(V), by anion-exchange HPLC-ICP-MS. The completeness of the oxidation was verified by recovery of As(V) which was added to the samples as As(III) prior to the dissolution procedure. The full recovery of As(V) at 104±7% (n=5) indicated good analytical accuracy. The uncertified inorganic arsenic content in the certified reference material TORT-2 was 0.186±0.014 ng g^–1 (n=6). The method was employed for the determination of total arsenic and inorganic arsenic in 60 fish samples including salmon from fresh and saline waters and in plaice. The majority of the results for inorganic arsenic were lower than the LOD of 3 ng g^–1, which corresponded to less than one per thousand of the total arsenic content in the fish samples. For mackerel, however, the recovery of As(III) was incomplete and the method was not suited for this fat-rich fish.     

Determination of heavy metals by inductively coupled plasma-optical emission spectrometry in fish from the Piracicaba River in Southern Brazil

A total of 202 fish, representing 16 species, were collected during 2008 (March–October) in the Tanquan region of the Piracicaba River using nets. Flesh samples were collected and analyzed, using inductively coupled plasma-optical emission spectroscopy for Al, As, Cd, Co Cr, Cu, Mn, Mo, Ni, Pb, Se, Sn, Sr, and Zn. The results showed that the flesh of these fish all contained extremely high levels of Al and Sr, and moderately high levels of Cr, As, Zn, Ni, Mn and Pb. The metals were higher in these fish during rainy season, with fish collected during the months of March and October being the highest. In addition, the accumulation of metals was species-dependent. Cascudos (Hypostomus punctatus) and piranhas (Serrasalmus spilopleura) exhibited high levels of almost all of the metals, while curimbata (Prochilodus lineatus) had moderate levels. A few species, including pacu (Piaractus mesopotamicus) and dourado (Salminus maxillosus), had very low levels of most metals. The results show that the Piracicaba River Basin is widely contaminated with high levels of many toxic heavy metals, and that human consumption of some fish species is a human health concern.     

Specific immunoassays for endocrine disruptor monitoring using recombinant antigens cloned by degenerated primer PCR

Vitellogenin (VTG) and choriogenin (CHO) are valuable biomarkers of endocrine-disrupting compound (EDC) exposure in fish. Existing immunoassays are limited to a few species, which restricts their use for the analysis of local wildlife sentinels. Using C. facetum as a relevant South American model fish, this work presents a new strategy for the preparation of antibodies to VTG and CHO, with zero cross-reactivity with fish serum components. Recombinant fragments of Cichlasoma facetum VTG (280-mer) and CHO (223-mer) were prepared by degenerate primer RT-PCR and expression in E. coli. Polyclonal and monoclonal antibodies prepared with these antigens were used to develop rapid dotblot assays for VTG and CHO. Both the polyclonal and monoclonal antibodies prepared with the recombinant antigens reacted against the native proteins adsorbed on to nitrocellulose allowing the set up of sensitive dotblot assays. The VTG assay was further validated with spiked samples and purified native VTG. Exposure experiments with several estrogenic compounds revealed the potential of C. facetum as a sensitive biomonitor that produced measurable responses at concentrations of 100 ng L⁻¹ of 17-beta-estradiol, 100 ng L⁻¹ of ethynylestradiol, and 6.6 μg L⁻¹ of nonylphenol. The approach described here may be applied to other native species to produce highly specific and sensitive rapid tests. It may be particularly advantageous for species that cannot be kept in captivity or when homogeneous purification of the immunizing proteins is particularly challenging. In conclusion, we present a novel approach to develop a strategy for the generation of immunoassay reagents for vitellogenin (VTG) and choriogenin (CHO), which will facilitate regional studies on the impact of endocrine-disrupting chemicals on local wildlife.     

LC-MS analyses of microcystins in fish tissues overestimate toxin levels—critical comparison with LC-MS/MS

Microcystins are cyclic peptide toxins with hepatotoxic and tumour-promoting properties which are produced in high quantities in freshwater cyanobacterial water blooms, and several studies have reported microcystin accumulation in fish with possible food transfer to humans. In this study, we provide the first comparison of liquid chromatography with single mass-spectrometric and with tandem mass-spectrometric detection for analyses of microcystins in complex fish tissue samples. Use of traditional single mass spectrometry (i.e. monitoring of ions with m/z 519.5 for microcystin-RR and m/z 995.5 for microcystin-LR) was found to provide false-positive responses, thus overestimating the concentrations of microcystins in the tissue samples. More selective tandem mass spectrometry seems to provide more reliable results. The concentrations of microcystins detected by tandem mass spectrometry in fish from controlled-exposure experiments were more than 50% lower in comparison with concentrations obtained by single mass spectrometry. Extensive analyses of edible fish parts—muscles (148 fish specimens from eight different species from five natural reservoirs with dense cyanobacterial water blooms)—showed negligible microcystin concentrations (all analyses below the limit of detection; limit of detection of 1.2–5.4 ng/g fresh weight for microcystin-RR, microcystin-YR and microcystin-LR in multiple reaction monitoring mode). Our findings have practical consequences for critical re-evaluation of the health risks of microcystins accumulated in fish.     

Biomethylation of Arsenic in an Arsenic-rich Freshwater Environment

Arsenic circulation in an arsenic-rich freshwater ecosystem was elucidated to detect arsenic species in the river water and in biological samples living in the freshwater environment. Water-soluble arsenic compounds in biological samples were extracted with 70% methanol. Samples containing arsenic compounds in the extracts were treated with 2 mol dm³ of sodium hydroxide and reduced with sodium borohydride. The detection of arsenic species was accomplished using a hydride generation/cold trap/cryofocus/gas chromatography-mass spectrometry (HG/CT/CF/GC-MS) system. The major arsenic species in the river water, freshwater algae and fish are inorganic arsenic, dimethylarsenic and trimethylarsenic compounds, respectively. Trimethylarsenic compounds are also detected in aquatic macro-invertebrates. The freshwater unicellular alga Chlorella vulgaris, in a growth medium containing arsenate, accumulated arsenic and converted it to a dimethylarsenic compound. The water flea Daphnia magna, which was fed on arsenic-containing algae, converted it to a trimethylarsenic species. © 1997 by John Wiley & Sons, Ltd.     

Identification of anti-nociceptive constituents from the pollen of Typha angustifolia L. using effect-directed fractionation

Abstract

The pollen of T. angustifolia, also known as Pu huang in Chinese, has been used for treatment of stranguria, hematuria, dysmenorrhea, metrorrhagia and injuries in China for a long time. Extensive efforts have been directed toward its phytochemical and biological aspects. However, little is known about its anti-nociceptive implication and material basis. This work presented the investigation of the anti-nociceptive effect of Typhae Pollen using an effect-directed fractionation strategy, thereby leading to identification of isorhamnetin-3-O-neohesperidin (1) and typhaneoside (2), together with other minor flavonoid glycoside congeners, as the main anti-nociceptive constituents. This work not only unveils the anti-nociceptive potential of Typhae Pollen, but also establishes a method to enrich and identify the anti-nociceptive constitutes of Typhae Pollen. Moreover, this work is a successful example of effect-directed fractionation strategy, which represents a powerful tool in TCM-based drug discovery and development.     

Specificity of Lichen Species in Respect to 137Cs Binding

Abstract

The aim of this work was to investigate the significance of species specificity on the efficiency of ¹³⁷Cs isolation from lichens. It was shown that a 5% solution of both ammonium oxalate and phosphoric acid was able to solubilize 77.5% of ¹³⁷Cs from Cetraria islandica, 47.6% from Cladonia fimbriata and 46.4% from Usnea barbata. Since the tested lichen species had similar specific radioactivities (i.e. amount of ¹³⁷Cs) the difference could be explained by the existence of different types of bonds between radiocesium and the corresponding binding sites. Crystals precipitated from these extracts incorporated most of the soluble ¹³⁷Cs. The amount and specific radioactivity of the crystals varied between lichen species which could also be interpreted as the presence of specific ions in each lichen that either participated in crystal formation or inhibited the process. The potential of a tested solution to extract and “concentrate” ¹³⁷Cs in crystalline form may be a tool to correlate mass and radioactivity of ¹³⁷Cs.     

Matrix effects in the determination of β‐receptor agonists in animal‐derived foodstuffs by ultra‐performance liquid chromatography tandem mass spectrometry with immunoaffinity solid‐phase extraction

Matrix effects in determination of three β‐receptor agonists including salbutamol (SAL), clenbuterol, and terbutaline in animal‐derived foodstuffs were studied by ultra‐performance LC‐MS/MS with cleanup of immunoaffinity SPE column (IAC). Some animal tissue samples including pig liver, swine muscle, and fish muscle were hydrolyzed by the mixed enzyme solution or HCl solution, and the cleanup efficiencies with SAL IAC, MCX SPE column, and C₁₈‐SCX tandem columns were examined and compared by using spiked experiments. The results showed that the matrix effects in the determination of SAL and terbutaline can be eliminated with SAL IAC cleanup, and the average recoveries of SAL were 77.4～81.5%, 79.0～80.3%, and 85.0～87.2% in pig liver, swine muscle, and fish muscle, respectively. The decision limit (ccα) and detection capability (ccβ) for SAL in pig liver were 0.02 and 0.05 μg/kg, respectively.     

Quality Control of Krill Oil by Nuclear Magnetic Resonance (NMR) Spectroscopy: Composition and Detection of Foreign Species

Krill oil is currently among the most highly promoted products in the dietary supplement market, which, due to its high price, can be potentially adulterated with fish species and artificial oil. For a holistic control of krill oil quality, ¹H, ¹³C, and ³¹P nuclear magnetic resonance (NMR) spectroscopies were used. The fatty acid and phospholipid composition as well as secondary ingredients, such as homarine, amino acids, and chitin, were examined. The following phospholipid species were detected: phosphatidylcholine (75–85?mol %), phosphatidylethanolamine (4–7?mol%) and their lyso derivatives 1-lysophosphatidylcholine (1–2?mol%)–2-lysophosphatidylcholine (10–16?mol%) and lysophosphatidylethanolamine (1?mol%). In the -2 position of phospholipids, the content of eicosapentaenoic acid (mean 68.23%; relative standard deviation 2.23%) was twice as high as the content of docosahexaenoic acid (mean 31.77%; relative standard deviation 4.79%). ¹³C NMR spectroscopy was used to distinguish between krill and fish oil-based dietary supplements. The adulteration of krill oil can be detected by fatty acid distribution in the sn-2 triacylglycerol position. The sensitivity of the method is about 10% (w/w) of fish content in blends, which is enough to detect deliberate adulteration. The same methodology can be used to recognize synthetically modified krill oil. The method was successfully applied to 30 commercially available krill and fish oil supplements.     

Organochlorine Pesticides in Water,Sediment and Fish from the Nile River and Manzala Lake in Egypt

Abstract

To assess the risks of organochlorine pesticides discharged into the hydrospheric environment of Egypt, river and lake water, drinking water, suspended solids, sediments and fish were collected during 1993—1994 from the Nile River and Manzala Lake in Egypt and were transported to Japan for chemical analysis. Among different organochlorine pesticides analyzed, p,p′-DDE was the most predominant in fish (7.6 to 67 ng/g wet wt.), sediments (3.2 to 432 ng/g dry wt.) and suspended solids (5.3 tc 138 pg/L). However, in the dissolved phase of water samples HCH compounds predominated (α-HCH, 71 to 2,815 pg/L). Concentrations of organochlorine pesticides, except chlordane, were higher in Manzala Lake than in the River Nile. Concentrations of organochlorine pesticides in fish corresponded with those in sediments from each location. Comparison of organochlorine concentrations in Nile River water with those reported in earlier studies suggested a decrease in concentrations during the last decade. However, concentrations of p,p′-DDE has increased in fish. It appears that the release of this metabolite from contaminated sediment is the major source of p,p′-DDE in fish during recent years.