Towards the standardisation of the neuroblastoma (neuro-2a) cell-based assay for ciguatoxin-like toxicity detection in fish: application to fish caught in the Canary Islands

The ouabain/veratridine-dependent neuroblastoma (neuro-2a) cell-based assay (CBA) was applied for the determination of the presence of ciguatoxin (CTX)-like compounds in ciguatera-suspected fish samples caught in the Canary Islands. In order to avoid matrix interferences the maximal concentration of wet weight fish tissue exposed to the neuro-2a cells was set at 20 mg tissue equivalent?(TE) ml(-1) according to the sample preparation procedure applied. In the present study, the limit of quantification (LOQ) of CTX1B equivalents in fish extract was set at the limit of detection (LOD), being defined as the concentration of CTX1B equivalents inhibiting 20% cell viability (IC(20)). The LOQ was estimated as 0.0096 ng CTX1B eq.g TE(-1) with 23-31% variability between experiments. These values were deemed sufficient even though quantification given at the IC(50) (the concentration of CTX1B equivalents inhibiting 50% cell viability) is more accurate with a variability of 17-19% between experiments. Among the 13 fish samples tested, four fish samples were toxic to the neuro-2a cells with estimations of the content in CTX1B?g(-1) of TE ranging from 0.058 (± 0.012) to 6.23 (± 0.713) ng CTX1B eq.g TE(-1). The high sensitivity and specificity of the assay for CTX1B confirmed its suitability as a screening tool of CTX-like compounds in fish extracts at levels that may cause ciguatera fish poisoning. Species identification of fish samples by DNA sequence analysis was conducted in order to confirm tentatively the identity of ciguatera risk species and it revealed some evidence of inadvertent misidentification. Results presented in this study are a contribution to the standardisation of the neuro-2a CBA and to the risk analysis for ciguatera in the Canary Islands.     

Polycyclic aromatic hydrocarbons and halogenated persistent organic pollutants in canned fish and seafood products: smoked versus non-smoked products

In this study, levels of several groups of environmental contaminants represented by PAHs, PCBs, organochlorine pesticides and polybrominated diphenyl ethers were determined in various types of canned smoked and non-smoked fish and seafood products (54 samples) obtained from the Czech market. PAHs were detected in all of the studied samples, and at least one of the target halogenated persistent organic pollutants was present above the LOQ in 85% of the samples. The levels of PAHs, PCBs, organochlorine pesticides (mainly DDTs) and polybrominated diphenyl ethers found in the canned products varied in the range of 1.4–116?µg?kg^?1, 0.6–59.6?µg?kg^?1, 0.6–82.7?µg?kg^?1 and 0.1–2.1?µg?kg^?1 can content, respectively. Smoked sprats were the most contaminated fish product (n?=?12) in which the highest levels of both PAHs and persistent organic pollutants were found. In 67% of the samples of smoked sprats in oil, the level of benzo[a]pyrene exceeded the maximum level of 5?µg?kg^?1 established for smoked fish by European Union legislation. The distribution of target analytes between oil and fish fractions was also assessed. Significantly higher levels of PAHs were measured in the oil fraction.     

Caribbean ciguatoxin profile in raw and cooked fish implicated in ciguatera

A cooked meal remnant and uncooked portion of a Caribbean barracuda suspected in ciguatera fish poisoning were examined for the presence of ciguatoxins (CTX). Samples were analysed using a tiered method of CTX analysis consisting of in vitro cell (N2a) assay to assess composite toxicity and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for structural confirmation. Meal remnant and uncooked fish extracts were cytotoxic by N2a cell assay and Caribbean ciguatoxin congener C-CTX-1 was structurally confirmed. Sample extracts were fractionated by LC and fractions analysed by the cell assay. The cytotoxicity profiles of cooked meal remnant and uncooked fish were similar. Cytotoxicity-guided LC-MS/MS analyses identified several CTX congeners contributing to the composite toxicity of the samples. C-CTX-1 was a major contributor, supporting its utility as a biomarker of Caribbean ciguatoxic fish.     

Rapid sample preparation procedure for As speciation in food samples by LC-ICP-MS

This paper describes a rapid method for arsenic (As) speciation by LC-ICP-MS in several types of food samples. Prior to analysis, samples were milled and the As species extracted from biological tissues by sonication in only 2?min with a solution containing MeOH (10%, v/v) plus HNO₃ (2%, v/v). As species were separated by LC using an anion exchange column. Method detection limits for AsB, As³⁺, DMA, MMA and As⁵⁺ were 1.3, 0.9, 0.6, 0.7 and 0.8?ng?g^?1, respectively. Method accuracy and precision were traceable to Certified Reference Materials SRM1577 bovine liver from the National Institute of Standards and Technology, CE278 mussel tissue from the Institute of Reference Materials and Measurements and DOLT-3 dogfish liver tissue and DORM-3 fish protein from the National Research Council of Canada. Finally, the method was applied to speciate As in food samples (egg, fish muscle, beef and chicken) purchased in Brazilian markets.     

Trimethylamine oxide,dimethylamine, trimethylamine and formaldehyde levels in main traded fish species in Hong Kong

Levels of trimethylamine oxide (TMAO), dimethylamine (DMA), trimethylamine (TMA) and formaldehyde (FA) were studied in 266 different fishes, including fresh/frozen raw whole fishes of 89 different species that traded in Hong Kong, China. Determination of TMAO can confirm the source of DMA and FA if present in the sample. These samples were purchased from different commercial outlets between April and August 2007. All samples of raw whole fish were identified for their species by the Agriculture, Fisheries and Conservation Department. The content of TMAO was determined by high-performance liquid chromatography (HPLC) coupled with a chemiluminescent nitrogen detector. The possible decomposition products of TMAO, DMA and TMA were analysed by headspace solid-phase micro-extraction gas chromatography-mass spectrometry (HS-SPME-GC-MS), while FA was conducted by steam distillation then quantified by a HPLC. The range for TMAO of all samples was <5–3800 mg kg^?1 with median of 970 mg kg^?1, while the endogenous enzymatic cleavage products DMA, TMA and FA were in the range of <2–320, <1–190 and <1–160 mg kg^?1, respectively. These cleavage products were mainly found in three fish species, Harpadon nehereus, Saurida elongata and Saurida tumbil, that belong to the family Synodontidae (Lizardfishes) and subfamily Harpadontinae. Besides, freshwater fish species, namely, Micropterus salmoides, Oreochromis niloticus niloticus and Siniperca chuatsi, were found to contain TMAO in the range of 510–760, 85–720 and 400–640 mg kg^?1, respectively.     

Determination of lasalocid residues in the tissues of broiler chickens by liquid chromatography-tandem mass spectrometry

Lasalocid is a polyether ionophoric coccidiostat used for the prevention of coccidiosis in poultry at a prescribed concentration and during a certain time interval. Due to a public health concern about the presence of coccidiostat residues in poultry, the aim of the present study was to determine the levels of lasalocid residues in the edible tissues of broiler chickens (breast muscle, thigh muscle, heart, liver, gizzard, kidneys and skin/fat) fed commercially produced feed containing 100?mg?kg^?1 of lasalocid in complete feed throughout the 5-day withdrawal period (WP). The residues were investigated by liquid chromatography coupled with electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) with triple quadrupole. The limit of detection (LOD) and the limit of quantification (LOQ) of the method were 0.47 and 1.44?µg?kg^?1, respectively. The average recovery based on the matrix-fortified calibrations for chicken tissues ranged between 79% and 98%. Lasalocid was found to accumulate in the liver, followed by the heart, skin/fat, kidneys, thigh muscle and gizzard. The lowest concentrations of lasalocid residues were found in the breast muscle. On day 5 of the WP, residue concentrations of lasalocid did not decline below the LOQ of the method, but were far below the maximum residue level (MRL) established for lasalocid in poultry from 20 to 100?µg kg^?1 by European Commission Regulation (EU) No. 37/2010. The results confirmed that the WP established for lasalocid is sufficient to ensure the decline of its residues in the tissues of broiler chickens to the safe residue level.     

Analysing organochlorine pesticides in strawberry jams using GC-ECD,GC-MS/MS and QuEChERS sample preparation

This paper describes a comparison of adaptations of the QuEChERS (quick, easy, cheap, effective, rugged and safe) approach for the determination of 14 organochlorine pesticide (OCP) residues in strawberry jam by concurrent use of gas chromatography (GC) coupled to electron capture detector (ECD) and GC tandem mass spectrometry (GC-MS/MS). Three versions were tested based on the original QuEChERS method. The results were good (overall average of 89% recoveries with 15% RSD) using the ultrasonic bath at five spiked levels. Performance characteristics, such as accuracy, precision, linear range, limits of detection (LOD) and quantification (LOQ), were determined for each pesticide. LOD ranged from 0.8 to 8.9?µg?kg^?1; LOQ was in the range of 2.5–29.8?µg?kg^?1; and calibration curves were linear (r ^2?>?0.9970) in the whole range of the explored concentrations (5–100?µg?kg^?1). The LODs of these pesticides were much lower than the maximum residue levels (MRLs) allowed in Europe for strawberries. The method was successfully applied to the quantification of OCP in commercially available jams. The OCPs were detected lower than the LOD.     

Mercury and methylmercury levels in the main traded fish species in Hong Kong

This survey was undertaken to determine the levels of aflatoxins in melon seeds. Among 65 samples analyzed by liquid chromatography (LC), the results showed that aflatoxin B₁ (AFB₁) was the major toxins in melon seeds, detected in 58 samples (89.2% of the total) at an average concentration of 8.5?ng?g^?1. The level of AFB₁ in 12 samples exceeded the maximum tolerated level for AFB₁ in Iranian (5?ng?g^?1) regulations; in other words, 18.5% of samples were unfit for human consumption.     

Survey of aflatoxins in maize tortillas from Mexico City

In Mexico, maize tortillas are consumed on a daily basis, leading to possible aflatoxin exposure. In a survey of 396 2-kg samples, taken over four sampling days in 2006 and 2007 from tortilla shops and supermarkets in Mexico City, aflatoxin levels were quantified by HPLC. In Mexico, the regulatory limit is 12?µg?kg^?1 total aflatoxins for maize tortillas. In this survey, 17% of tortillas contained aflatoxins at levels of 3–385?µg?kg^?1 or values below the limit of quantification (<LOQ) and, of these, 13% were >12?µg?kg^?1 and 87% were below the regulatory limit. Average aflatoxin concentrations in 56 contaminated samples were: AFB₁ (12.1?µg?kg^?1); AFB₂ (2.7?µg?kg^?1); AFG₁ (64.1?µg?kg^?1) and AFG₂ (3.7?µg?kg^?1), and total AF (20.3?µg?kg^?1).     

Polycyclic aromatic hydrocarbons in Brazilian commercial soybean oils and dietary exposure

In this study the 13 polycyclic aromatic hydrocarbons (PAHs) identified as being genotoxic and carcinogenic by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) were determined in different brands of soybean oils available on the Brazilian market, totalling 42 samples. A solid-phase extraction (SPE) method for sample clean-up with a C₁₈ cartridge, followed by reversed-phase HPLC with fluorescence detection, was used for determination. The method showed good recoveries for most PAHs studied with values between 74% and 111%. Good intra- and inter-day precisions (0.5?<?RSD?<?11.9) and high correlation coefficients (r ^2?>?0.999) were obtained. The presence of PAHs was detected in all 42 samples with mean summed PAH levels ranging from 10.4 to 112.0?µg?kg^?1. The mean and maximum dietary exposures for total PAHs were estimated as 12.4 and 19.1?ng?kg?bw^?1?day^?1, respectively.     

Survey of fumonisins B1, B2 and B3 in conventional and organic retail corn products in Spain and Italy and estimated dietary exposure

A survey was conducted to determine the occurrence of fumonisin B₁, B₂ and B₃ during 2007 in 186 samples of organic and conventional locally available corn products. Samples included baby food (n = 62), corn flour (11), cornflakes (23), pasta (14), cookies (17) and other corn products (59) were obtained from popular markets of Valencia (Spain) and Perugia (Italy). The analytical method used pressurized liquid extraction and liquid chromatography/electrospray ionization tandem mass spectrometry with a triple quadrupole (QqQ) analyser. Of the 104 Spanish samples, 22% contained levels in the range of 2–449 µg kg^?1, 2–229 µg kg^?1 and 6–105 µg kg^?1 for FB₁, FB₂ and FB₃, respectively, while 19 (23%) of the 82 Italian samples were positive with quantifiable levels between 2–235 µg kg^?1, 3–187 µg kg^?1, and 4–40 µg kg^?1 for fumonisins B₁, B₂ and B₃, respectively. Overall, none of the Italian samples and only one organic baby food sample from a Spanish market was above the maximum permitted levels established by European legislation. Fumonisins were found mostly in corn flour followed by cookies and cornflakes. Eleven samples from Spain and nine samples from Italy were organic products, being contaminated the 72% and 77% of the samples, respectively. Analysis of the results showed that levels of fumonisins in corn products were similar in Italy and Spain. The safety of fumonisin intake through corn products was demonstrated by the calculation of the estimated daily intake of both populations considering organic and conventional products separately, which ranged from 1.7 × 10^?3 to 0.72 µg kg^?1 bw day^?1 and comparing them with the provisional maximum total daily intake (PMTDI) of 2 µg kg^?1 bw day^?1 established by the European Union.     

Antioxidant properties of garden strawberry leaf extract and its effect on fish oil oxidation

The present study characterises garden strawberry leaf extract and reports its effect on shelf life and quality characteristics of fish oil. Radical scavenging capacity of the extract in DPPH^? and ABTS^?+ assays was equivalent to 1207 and 1579 μmol g^?1 of trolox equivalents, respectively, total phenolic content was 257 mg g^?1 of gallic acid equivalents. Phenolic and volatile constituents were analysed by liquid chromatography/mass spectrometry (LC/MS) and gas chromatography/mass spectrometry (GC/MS). The extract was added to fish oil and its oxidation was followed during 42 days: peroxide, p‐anisidine values and hexanal concentration were lower in the samples with extract, particularly at the end of storage. The effect of extract on fatty acid composition was not significant, however the changes in the percentages of some individual acids were observed. Fish oil containing 5% of extract had lower levels of lipid oxidation in comparison to other samples. The results indicate that strawberry leaves could be a potential source of natural antioxidants.     

Fast and isocratic HPLC-method for steviol glycosides analysis from Stevia rebaudiana leaves

A fast isocratic HPLC method for analysis of steviol glycosides from Stevia rebaudiana leaves was developed with a high selectivity for nine known steviol glycosides and low eluent consumption. The analytical method was performed on a Purospher^? STAR RP-18 endcapped 3???m Hibar^? RT 250-4.6 column at 50?°C with an eluent composition of water (65 vol.?%) and acetonitrile (35 vol.?%). With a flow rate of 1?mL?min^?1, nine known steviol glycosides were detected selectively after 15?min. Method validation for rebaudioside A showed a LOD of 0.0004?mg?mL^?1 and a LOQ of 0.0038?mg?mL^?1. Particularly with regard to moderate solubility, the method is linear up to a concentration of rebaudioside?A of 4.8?mg?mL^?1. The linear calibration curve was obtained with a coefficient of determination of 0.9997?±?0.0002 and a total error of 2.01?% RSD (n?=?12). The accuracy of the method was determined by the percentage mean recovery rate to 100.99?±?2.01?%. The intra-day precision was in a range of 0.12 to 1.96?%?RSD and the inter-day precision varies from 0.02 to 1.89?% RSD. Small changes in operating conditions like eluent composition (65?±?2 vol.?%), temperature (50?±?10?°C) or flow rate (1?±?0.2?mL?min^?1) do not affect the performance of the analytical method. The reliable and robust proposed HPLC method can be applied for quantification of steviol glycosides in Stevia rebaudiana Bertoni leaves in laboratories and quality control in industry.     

Dietary risk assessment of organophosphorus and dithiocarbamate pesticides in a total diet study at a Brazilian university restaurant

In this study ready-to-eat food samples were collected in the production line of the university restaurant of the University of Brasilia, Brazil, which serves non-vegetarian and vegetarian meals daily. Samples were analysed for the presence of ten organophosphorus insecticides (OPs) by GC/FPD, after extraction with ethyl acetate and anhydrous sodium sulfate (LOQ?=?0.002?mg?kg^–1), and for dithiocarbamate fungicides, as CS₂, using the spectrophotometric method (LOQ?=?0.05?mg?kg^?1). About 43% of the 175 samples analysed contained at least one OP compound at levels up to 1.83?mg?kg^?1. Methamidophos was the compound most detected (37.7%), present in most of the soup, soybean and salad samples. No OP residues were found in fruit juice, beans and bran rice samples. The cumulative acute intake of OPs was estimated using methamidophos and acephate as index compounds (IC). The total cumulative intake represented 9.1% and 47.7% of the methamidophos ARfD for the non-vegetarian and vegetarian diets, respectively. When acephate was used as IC, the total intakes represented 20.7% and 116% of the ARfD for the non-vegetarian and vegetarian diets, respectively. Dithiocarbamates were detected in 70% of the 177 samples analysed, at levels up to 0.51?mg?kg^?1 CS₂; all salad samples were positive and no residues were found in fruit juice. The chronic intake of dithiocarbamates represented 8.6 and 8.9% of the ADI (mancozeb) for the vegetarian and non vegetarian diets, respectively.     

Parvalbumin in fish skin-derived gelatin: is there a risk for fish allergic consumers?

The major allergen parvalbumin was purified from cod muscle tissues, and polyclonal antibodies were raised towards it. The antibodies were tested for specificity and an enzyme-linked immunosorbent assay (ELISA) was developed using these antibodies. The ELISA was applied to measure parvalbumin in cod skin, the starting material for fish gelatin made from deep sea, wild fish. The ELISA was sufficiently sensitive (LLOQ?=?0.8?ng?ml^?1 in extracts, corresponding to 0.02?µg of parvalbumin per g of tissue), and did not cross-react with common food constituents. Fish gelatin, wine and beer, matrices for the potential use of this ELISA, did not cause disturbance of the assay performance. The data show that the parvalbumin content in cod muscle tissue is 6.25?mg?g^?1, while the skins contained considerably less, 0.4?mg?g^?1. Washing of the skins, a common industrial procedure during the manufacturing of fish gelatin, reduced the level of parvalbumin about 1000-fold to 0.5?µg?g^?1, or 0.5?ppm. From 95 commercial lots of fish gelatin it is shown that 73 are below 0.02?µg?g^?1 parvalbumin. From the other 22 lots, the one with the highest concentration contained 0.15?µg?g^?1 of parvalbumin. These levels are generally assumed to be safe for fish-allergic individuals.     

Exposure of consumers to morphine from poppy seeds in Hungary

Poppy seed-containing foods are popular dishes in Hungary and some other Central European countries. The alkaloids of poppy are used in the production of medicines. Poppy seeds used as food may also contain considerable amounts of alkaloids, which raises the question of food safety. Morphine, codeine, thebaine and noscapine concentrations of poppy seed samples from the period 2001–2010 and consumption data from two Hungarian surveys, carried out in 2003 and 2009, were evaluated. Exposure calculations were made for morphine intake by both point estimate and probabilistic methods, and the uncertainty of the calculated values was estimated. The point estimate for the acute consumer exposure, calculated using the 97.5th percentiles of morphine concentration and of poppy seed consumption and taking into account the reduction of morphine content by processing, was 78.64?µg (kg?bw)^?1 day^?1 for adults, and 116.90?µg (kg?bw)^?1?day^?1 for children. Based on probabilistic estimations, the 97.5th and 99th percentile exposures ranged between 18.3–25.4 and 25.6–47.4?µg (kg?bw)^?1?day^?1 for adults, and between 32.9 and 66.4?µg (kg?bw)^?1 day^?1 for children, respectively. As a no observed effect level (NOEL) had not been established, the significance of exposure could not be assessed.     

Signal amplification using colloidal gold in a biolayer interferometry-based immunosensor for the mycotoxin deoxynivalenol

Deoxynivalenol (DON) is a toxin produced by certain species of Fusarium fungi that can infest wheat, barley and corn. The fungi cause diseases in crops worldwide and some of the secondary metabolites, such as DON, can adversely affect animal health and food safety. To monitor DON in wheat rapidly, a biosensor using the principle of biolayer interferometry (BLI) was developed. The signal from the sensor was substantially amplified through the use of a primary antibody–colloidal gold conjugate. The amplification was much greater in the presence of wheat matrix than in buffered solution, suggesting matrix components may have contributed to the enhancement. The improved signal provided by the amplification allowed for the development of rapid qualitative and quantitative assays. The limit of detection of the method was 0.09?mg?kg^?1; the limit of quantitation was 0.35?mg?kg^?1. Recovery from wheat spiked over the range from 0.2 to 5?mg?kg^?1 averaged 103% (RSD?=?12%). The quantitative assay compared favourably (r ^2?=?0.9698) with a reference chromatographic method for 40 naturally contaminated wheats. The qualitative assay was able to classify accurately the same group of 40 samples as either above or below a 0.5?mg?kg^?1 threshold. These results suggest that the BLI technique can be used to measure DON in wheat rapidly.     

Fumonisin B1 and B2 occurrence in dried fig fruits (Ficus carica L.) under Meander Valley's climatic conditions and relationship with fruit quality

Fusarium is the agent causing endosepsis (internal rot) in fig fruits and it is widespread in fig orchards in the Aegean region. This research was conducted to determine the natural occurrence of fumonisin B₁ (FB₁) and B₂ (FB₂) on dried fig fruits of Sarilop (syn. Calimyrna) variety which are mainly grown in the Big and Small Meander Basins in the Aegean region, representing 60% of world dried fig production. A total of 262 samples belonging to two quality classes, Class A and Class cull, were collected from 12 different locations during the two crop years in 2004 and 2005. The fumonisin detection method is based on extraction with methanol–acetonitrile–water, derivatization with o-phthaldehyde and quantification by reversed-phase high-performance liquid chromatography with fluorescence detection. The mean concentrations of FB₁ and FB₂ in fumonisin-positive samples were 0.080?±?0.047?µg?g^?1 and 0.055?±?0.031?µg?g^?1 and ranged from LOD to 0.332?µg?g^?1 and from LOD to 0.198?µg?g^?1, respectively. The incidence of fumonisins significantly differed between the two crop years. This difference can be attributed to the alteration in the rainfall regime from mid-May to mid-August (7.2?mm in 2004, 90.9?mm in 2005) and number of humid wind currents from a westerly direction (183 in 2004, 492 in 2005) from the end of July and mid-August that may have triggered a higher incidence of Fusarium spp. and thus fumonisin production.     

Survey of deoxynivalenol and its conjugates deoxynivalenol-3-glucoside and 3-acetyl-deoxynivalenol in 374 beer samples

Beer is one of the most popular beverages worldwide. Malted cereal grains are among the basic ingredients and hence mycotoxin contamination might occur. Previous studies reported the presence of the Fusarium mycotoxins deoxynivalenol (DON) and 3-acetyl-deoxynivalenol (3ADON), as well as of the masked mycotoxin deoxynivalenol-3-glucoside (D3G) in beer. In the present survey, 374?beer samples from 38?countries with a focus on Austrian (156) and German (64) beers were analysed for the presence of D3G, DON and 3ADON. Beers were assigned to the following six categories: pale (217), wheat (46), dark (47), bock (20), nonalcoholic beers (19) and shandies (25). In total, 348 and 289 beers (93 and 77%, respectively) contained D3G and DON at the levels above the limit of detection, whereas 3ADON was not detected in any of the samples. Average concentrations of all beers were 6.9?µg?L^?1 for D3G and 8.4?µg?L^?1 in the case of DON. Nonalcoholic beers and shandies showed the lowest contaminations, 1.5 and 3.2?µg?L^?1 for D3G and 2.7 and 4.4?µg?L^?1 for DON, respectively. In bock beers characterised by a higher gravity, a significant trichothecene load of 14.8?µg?L^?1 D3G and 12.4?µg?L^?1 DON was found. The highest contamination (81?µg?L^?1 D3G, 89?µg?L^?1 DON) was detected in a pale beer from Austria, underlining the importance of this study for food safety. The molar D3G to DON ratio ranged between 0.11 and 1.25 and was 0.56 on average. Concluding, the average contamination of beer is not of toxicological concern for moderate beer drinkers. However, in the case of heavy beer drinkers, beer consumption may considerably contribute to the overall intake of DON, which might even lead to exceeding the maximum tolerable limits established for this Fusarium toxin.