Fish Oil Samples Research Articles

A novel perspective with characterized nanoliposomes: Limitation of lipid oxidation in fish oil

Fish oil-loaded brown and green macroalgae-based nanoliposomes (NS: Sargassum boveanum, NP: Padina distromatica, and NC: Caulerpa sertularioides) were successfully obtained. Release profile (<35%), zeta particles size (130.3–266.3 nm), encapsulation efficiency (99.9%) were revealed besides morphological characterizations (NS: 157.55, NP: 129.02, NC: 335.304 nm) of nanoliposomes (p < 0.05). Oxidation tests demonstrated that the use of NS more successfully limited the rapid increase in peroxide value of fish oil stored at 30 °C. FFA value of control group samples reached 6.8 from 3.4% (change: 100%) while NS (change: 12.5%) and NP (change: 12.5%) had better stability during 42 days' storage (p < 0.05). The anisidine values of control group samples were in the range of 8.8 and 22.8. But, NS, NP, and NC samples possessed 11.0, 9.9, and 10.0, respectively. Totox value in the control group fish oil samples reached 85.2. Yet, the highest values for NS, NP, and NC samples were found to be 33.1, 54.5, and 67.3 (p < 0.05). Also, color values in the samples treated with nanoliposomes had higher stability. The use of nanolioposme technique effectively retarded the oxidation in fish oil stored at 30 °C. Thus, this applied novel methodology provided with a nanoliposomal approach can guide further food treatments in the food industry.

Fast and Specific Screening of EPA/DHA-Enriched Phospholipids in Fish Oil Extracted from Different Species by HILIC-MS.

Eicosapentaenoic acid- and docosahexaenoic acid-enriched phospholipids (PLEPA/DHA) have versatile health-beneficial functions and can be well absorbed in the intestine. Herein, a precursor ion scan-driven hydrophilic interaction chromatography mass spectrometry (PreIS-HILIC-MS) method with the fatty acyl moieties of m/z 301.6 and 327.6 locked was established to specifically and selectively screen PLEPA/DHA in different fish oil samples, including saury, grass carp, hairtail, and yellow croaker. Taking saury oil as an example, a total of 24 PLEPA/DHA were successfully identified and quantified, including 20 PCEPA/DHA and 4 PEEPA/DHA. Finally, this method was validated in terms of sensitivity (limit of detection ≤ 4.15 μg·mL-1), linearity (≥0.9979), precision (RSDintraday ≤ 4.65%), and recovery (≥78.6%). The performance of the PreIS-HILIC-MS method was also compared with that of the traditional full-scan mode, and the former demonstrated its unique superiority in targeted screening of PLEPA/DHA in fish oils.

THE EFFECTS OF ADSORBENT MATERIALS ON THE LIPID QUALITY OF LEMURU FISH OIL AND THE ENRICHMENT OF OMEGA-3 USING LIPASE

Sardinella (lemuru) is a genus of fish that is widespread in the East Java Sea region, especially in Muncar, Banyuwangi in East Java Province, Indonesia. Marine fishes are rich in essential fatty acids, including omega-3. This study aims to improve the quality of lemuru fish oil through the use of adsorbent materials such as bentonite and activated carbon. The sample was analyzed to determine its free fatty acid (FFA) content and peroxide value (PV) using the titrimetric method. The clarity of the oil was determined using the spectrophotometric method (λ 440 nm). The omega 3 content from the fish oil sample was then enriched through enzymatic reactions using lipase. Enzymatic reactions were carried out for 5, 10, 15, 20, 35, and 47 hours with a lipase concentration of 500, 1000, 1500, and 2000 units respectively. The omega-3 content of the fish oil products was analyzed using the GC-FID method. The results show that the addition of 3% activated carbon and bentonite in the fish oil reduced the FFA and PV results. Moreover, the absorbance value at λ 440 nm was also reduced from 0.883 to 0.559. The highest content of omega-3 was obtained through hydrolysis with lipase at a concentration of 1000 units for 35 hours with alpha-methyl linolenate (ALA), methyl all-cis-5,8,11,14,17-eicosapentanoate (EPA) and cis-4,7,10,13,16,19-docosa-hexaenoic acid methyl ester (DHA) at 0.78, 1.06, and 0.29% respectively.

A novel vortex assisted dispersive solid phase extraction of some trace elements in essential oils and fish oil

In this paper, a novel vortex assisted solid phase extraction procedure combined with inductively coupled plasma optic emission spectrometry was presented for determination of Mn, Cu, Ni, Cr, Cd and Pb directly in oily matrix. The suggested separation and preconcentration method was based on simultaneous sorption of the target analytes from real essential oil matrix onto γ-Al2O3 functionalized with fluorescein (Al-Flr) and transfer to aqueous eluent prior to quantification. The effect of different parameters including sorption and elution time, sample volume, eluent type and concentration and interfering ions were investigated. The working conditions were given as follows, vortex agitation for sorption and elution 40 s, sample volume 20 mL, eluent type HCl, eluent concentration 0.3 mol L−1. The detection limits were found to be 0.7 μg L−1 for Mn, 4.1 μg L−1 for Cu, 1.0 μg L−1 for Ni, 1.6 μg L−1 for Cr, 0.7 μg L−1 Cd and 1.0 μg L−1 Pb. The evaluation of the accuracy and precision of the method was tested via metallo-organic certified reference material. The recovery values have warranted the accuracy and found between 98.4 and 103.3% with 2.6–6.4% relative standard deviation (RSD, %). The sampling frequency of the procedure was calculated as 11.25 h−1.Finally, the proposed analytical method was successfully applied on analyte spiked and unspiked apricot, black cumin, pine turpentine, thyme, mint essential oils and fish oil samples without any need of performing mineralization pretreatments.

Determination of Inorganic Arsenic in Fish Oil and Fish Oil Capsules by LC-ICP-MS.

As inorganic arsenic is a highly toxic compound, its concentration in foods should be determined. Develop an analytical method for determining inorganic arsenic in fish oil and fish oil capsules. Inorganic arsenic was extracted from fish oil by heating at 80°C in 1.6% tetramethylammonium hydroxide-ethanol. The concentration of inorganic arsenic in fish oil was determined by liquid chromatography (LC) inductively coupled plasma (ICP) MS using an octadecylsilane (ODS) column with a mobile phase containing an ion-pair reagent. The LOD (0.005, 0.004 mg/kg), LOQ (0.016, 0.011 mg/kg), repeatability (4.2, 3.5%), intermediate precision (5.4, 3.5%), and trueness (recoveries 94-109% based on spiked samples) of the proposed method were satisfactory for inorganic arsenic in fish oil and fish oil capsules. A low level of inorganic arsenic was detected only in anchovy oil among all fish oil samples that were used in this study. Inorganic arsenic levels were below the quantitation limit in all fish oil capsules. Inorganic arsenic was determined after extraction from fish oil by heating at 80°C in 1.6% tetramethylammonium hydroxide-ethanol. The level of inorganic arsenic in all fish oil samples examined in this study was lower than 0.1 mg/kg of the maximum level defined in the Codex. Arsenic speciation in fish oil and fish oil capsules was analyzed by LC-ICP-MS using an ODS column with a mobile phase containing an ion-pair reagent. A low level of inorganic arsenic was detected only in anchovy oil. No inorganic arsenic was detected in fish oil capsules.

A Miniaturized Gas-Liquid Separator for Use in Liquid-Phase Microextraction Procedures: Determination of Mercury in Food

A method for the preconcentration of mercury using vortex-assisted temperature-controlled dispersive liquid-phase microextraction (VA-TC-DLPME) is proposed. A miniaturized gas-liquid separator (m-GLS) was developed and applied to the determination of mercury after VA-TC-DLPME. The detection was performed using cold vapor atomic absorption spectrometry (CV AAS). Ammonium pyrrolidine dithiocarbamate (APDC) reagent was used as a complexing agent for Hg(II). The VA-TC-DLPME method consists in dispersing the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF6]) in the aqueous phase by vigorous vortexing, followed by heating and cooling in an ice bath. The mixture was submitted to centrifugation, and the sedimented rich phase was then dissolved in an acid solution to reduce viscosity. Mercury was quantified in the final solution using m-GLS and CV AAS. Under optimized conditions, the method presents a limit of detection of 4.5 × 10−2 μg L−1, and an enrichment factor of 54. The accuracy was evaluated by the determination of mercury in reference material-certified ERM-CE 278, mussel tissue. The method was applied to the determination of mercury in fish oil samples. The developed m-GLS can be tested for use after other LPME procedures.

Shelf-life and quality of chicken nuggets fortified with encapsulated fish oil and garlic essential oil during refrigerated storage.

Fish oil (FO) is a rich source of long-chain omega-3 polyunsaturated fatty acids (ω-3 LCPUFA) which are important for human health. This research investigated the fortification of chicken nuggets with encapsulated FO-Garlic essential oil (GEO) as a possible way for delivery of ω-3 LCPUFA. Five different chicken nugget samples were prepared according to different treatments: Control sample (without fish oil and encapsulated FO-GEO), bulk fish oil samples (0.4% and 0.8%, w/w), and encapsulated FO-GEO samples (4% and 8%, w/w). The quality of the chicken nugget samples were monitored during a 20-day refrigerated storage. Results showed that the addition of encapsulated FO-GEO could significantly delay lipid oxidation and microbiological spoilage of the samples during refrigerated storage. This is reflected by the pH, PV, TBARS and TVBN data (P < 0.05). Samples fortified with encapsulated FO-GEO also showed significantly higher sensory quality and overall acceptability (P < 0.05). The use of 8% encapsulated FO-GEO gave the best antioxidative and antimicrobial properties during storage. However, the best sensory scores were observed in the 4% encapsulated FO-GEO up to 20days of storage. This study demonstrated that the encapsulated FO-GEO could be used for fortifying and extending shelf-life of food products.

Reverse phase dispersive liquid–liquid microextraction coupled to slotted quartz tube flame atomic absorption spectrometry as a new analytical strategy for trace determination of cadmium in fish and olive oil samples

In the present study, trace level of cadmium was determined by a new, sensitive and simple analytical method called as reverse phase dispersive liquid–liquid microextraction (RP-DLLME) combined to slotted quartz tube flame atomic absorption spectrometry (SQT-FAAS) system. Microextraction parameters such as type and volume of disperser solvent, volume of extraction solvent, heating effect, initial sample volume, final volume, centrifugation period and type/period of mixing were optimized. FAAS and SQT-FAAS systems were also examined to get high signal to noise ratio. After optimizing all significant parameters, the limit of detection (LOD) and limit of quantification (LOQ) of the developed analytical strategy were calculated as 0.74 and 2.48 μg/kg, respectively. In order to determine the applicability and accuracy of the RP-DLLME-SQT-FAAS method, recovery studies were performed in fish oil syrup. Matrix matching calibration strategy was used to eliminate heavy matrix effects on analytical signals, and percent recovery results were found between 95.6 and 105.7 %.

Miniature 3D-printed integrated electrochemical cell for trace voltammetric Hg(II) determination

This work describes an integrated miniature all-3D-printed device for the determination of trace Hg(II) by anodic stripping voltammetry (ASV). The device is fabricated through a single-step printing process using a dual-extruder 3D printer and is composed of a mini vessel (printed from a polylactic acid (PLA) filament) and of 3 thermoplastic electrodes printed from a carbon-loaded PLA conductive filament and integrated at the bottom of the vessel. The working electrode surface is modified with a thin gold film electroplated in situ. The formation of the gold deposit was studied by linear sweep voltammetry and optical microscopy and the electrode materials as well as the chemical and instrumental conditions for Hg(II) determination were optimized. The limit of detection for Hg(II) is 0.52 μg L−1, the within-sensor repeatability (expressed as the coefficient of variation of 8 measurements of 20 μg L−1 Hg(II) at the same device) is 3.9 % and the between-cell reproducibility (expressed as the coefficient of variation 5 measurements of 20 μg L−1 Hg(II) conducted at different cells) is 8.9 %. The 3D-printed device was successfully applied to the determination of Hg(II) in bottled water and fish oil samples.

Separation of high-purity eicosapentaenoic acid and docosahexaenoic acid from fish oil by pH-zone-refining countercurrent chromatography.

The evidence for unique effects of eicosapentaenoic acid and docosahexaenoic acid is growing. Further understanding and exploration of their independent effects in the nutraceutical and pharmaceutical industry is calling for the more efficient separation techniques to overcome the equivalent chain length rule of fatty acids. In this study, free eicosapentaenoic and docosahexaenoic acid were successfully separated by pH-zone-refining countercurrent chromatography for the first time. The different solvent systems and the influence of retainer and eluter concentration on the separation efficiency were investigated. A two-phase solvent system composed of n-heptane/methanol/water (100:55:45, v/v) was selected with 50mM of trifluoroacetic acid as retainer in the organic phase and 40mM of ammonium hydroxide as an eluter in the aqueous phase for the separation of 500mg of free fatty acids from a refined fish oil sample. 79.6mg of eicosapentaenoic acid and 328.3mg docosahexaenoic acid were obtained with the purities of 95.5 and 96.9% respectively determined by gas chromatography with mass spectrometry after methyl esterification. The scale-up separation of 1g of samples from both refined and crude fish oil after urea complexation were also achieved successfully with a markedly increased concentration 150mM of retainer, producing satisfactory yields and purities of targets.

Lignin-Based Biopolymeric Active Packaging System for Oil Products.

Antioxidant activity of enzymatically modified soybean protein film with two different forms of added lignin (alkali lignin and lignosulfonate) was investigated using two stimulated food systems involving direct and indirect contact with soybean oil and fish fatty acid ethyl ester (FAEE). For the direct system, control and lignin-doped films were added to oil vials which were stored at dark under 40 °C whereas for indirect, films were used to cover oil-containing glass vials stored under standard commercial lighting conditions. Autoxidation of oil samples in the direct contact system was determined by peroxide value (PV), color, headspace oxygen, and volatile compounds, while for the indirect contact system photoxidation was determined by using PV and color. For the direct contact system with soybean oil, the PV was significantly lower during storage for both lignins used compared to the control (packaging system without lignin film). There was not a significant effect of lignin on the color of the oils (P > 0.05). Modified films tested in this study did not have a significant effect on headspace oxygen contents of oil samples; however, it resulted in reduced volatile compounds for both soybean oil and fish oil samples. Based on our observation, soybean protein films with lignin showed a greater impact on soybean oil than fish oil, possibly because of high initial oxidation levels in the fish oil. Enzymatic modified soy protein films with lignin are alternative active packaging materials for highly sensitive to oxidation by radical and UV light. PRACTICAL APPLICATION: Plastic packaging materials require the use of petroleum oil and are not biodegradable. Packaging materials made from renewable, biodegradable biopolymers are of great interest but often suffer from performance problems, such as weak mechanical properties compared to petroleum-based plastics. Applying modified biopolymeric film with lignin in the inner layer of food packaging system improved some aspects of their performance during storage, not only by preventing the migration of chemical compounds from the package to the food but also by radical scavenging activity and UV-blocking ability of the packaging system.

Integrated on-chip sensor with sputtered Ag-Au-Au electrodes for the voltammetric determination of trace Hg(II)

This work describes a planar sensor chip with three integrated electrodes for the determination of Hg(II) by anodic stripping voltammetry (ASV). The sensor features Au-film working and auxiliary electrodes and a Ag-film reference electrode; all three metal-film electrodes are deposited on a silicon substrate by sputtering. Definition of the geometrical pattern of the sensor chip involves appropriate application of simple plastic masks during deposition of the metal electrodes making this approach simpler, faster and less laborious than photolithography. The Au-film working electrode is characterized by optical techniques and cyclic voltammetry and the chemical and instrumental conditions for Hg(II) determination by ASV are studied in detail. The limit of detection of Hg(II) is 0.6 μg L−1, the within-sensor repeatability (expressed as the coefficient of variation of 20 measurements at the 20 μg L−1 Hg(II) level) is 3.7% and the between-sensor reproducibility (expressed as the coefficient of variation of 6 measurements at different sensors at the 20 μg L−1 Hg(II) level) is 11.8%. The sensor chip is successfully applied to the determination of Hg(II) in a fish oil sample. The proposed fabrication approach offers scope for the mass-production of robust integrated sensors at low cost which are applicable to on-site analysis.

LIPID AUTOXIDATION OF FISH, LARD, CORN AND LINSEED OILS BY ISOTHERMAL CALORIMETRY

This work proposes an approach to characterize lipid oxidation of oils based on the measurement of isothermal calorimetry data, including information on the duration of the monomolecular, bimolecular and termination periods. The approach has been exploited with fish and lard oil samples at temperatures from 40 to 80°C and with corn and linseed oils at 80°C. The length of the monomolecular period was the most sensitive to the variation of temperature. Accordingly, the monomolecular period was used as index of oxidative stability of oils. Thus, the highest oxidative stability was observed for corn oil samples at 80°C (tmono = 2060 min), followed by linseed (tmono = 390 min), fish (tmono = 40 min) and lard oils (tmono = 30 min). The different stability of the samples was discussed with their fatty acids profile and antioxidant activity. The results confirmed that the content in natural antioxidants is the key responsible for the final oxidative stability of the samples.

Green determination of brominated flame retardants and organochloride pollutants in fish oils by vortex assisted liquid-liquid microextraction and gas chromatography-tandem mass spectrometry

A "green", simple, and low-cost sample extraction procedure involving the use of a deep eutectic solvent (DES) in a vortex assisted liquid-liquid microextraction (VALLME) technique followed by gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis was developed for the simultaneous determination of different PBDEs congeners and OCPs residues in fish oils. After evaluation of different eutectic mixtures, the extraction parameters (volume of DES, amount of oil sample and extraction time) were optimized by means of experimental design in order to maximise extraction efficiency. The developed method was validated in terms of linearity, accuracy and precision, presenting limits of detection in the low ng g−1 level. Its application in the analysis of five fish oil samples, allowed the detection of all the target analytes at levels up 21.5 ng g−1. Fish oils used in animal feed showed to be more contaminated than fish oils for human consumption.

Quantitative Analysis of Polycyclic Aromatic Hydrocarbons at Part Per Billion Levels in Fish Oil by Headspace Solid-Phase Microextraction and Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS).

Headspace solid-phase microextraction (HS-SPME) was used in combination with gas chromatography-mass spectrometry (GC-MS) for the analysis of polycyclic aromatic hydrocarbons (PAH) at part per billion levels in fish oil samples collected from menhaden fish. The method was initially developed using fish oil from capsules spiked with a standard PAH mixture. The final HS-SPME-GC-MS method presented a linear range from 3 to 1,500 ng/g, with precision for most analytes <10% relative standard deviation. The limits of detection varied from 1 to 7 ng/g depending on the analyte. Real sample analysis was done on menhaden fish oil extracted from fish collected off the coasts of New Jersey and Louisiana. Naphthalene, fluorene, fluoranthene, pyrene, anthracene were detected at low levels of 70-180 ng/g in the real samples. The concentrations of PAHs detected in the real samples were well below established levels of concern for PAHs in finfish.

Endocrine disrupting potency of organic pollutant mixtures isolated from commercial fish oil evaluated in yeast-based bioassays.

The aim of this work was to evaluate the activity of xenobiotic mixtures containing persistent organic pollutants isolated from commercial fish oil samples against sex hormone receptors, including estrogen and androgen. The applied bioassay was based on transgenic yeast strains. The mixtures were extracted from the samples using the semi-permeable membrane dialysis technique and analyzed with gas chromatography/ion trap mass spectrometry. It turned out that mixtures of chemicals isolated from fish oil may interact with human steroid sex hormone receptors in various ways: the tested samples showed both estrogenic and anti-androgenic activity. Calculated 17β-estradiol equivalents for the tested samples ranged between 0.003 and 0.073 pg g–1 (fat). Anti-androgenic activity expressed as the flutamide equivalent concentration was in the 18.58–216.21 ng g–1 (fat) range. Polychlorinated biphenyls and various DDT metabolites were the main fish oil pollutants influencing the receptors. Additivity and/or synergy between chemicals was observed in the ER/AR mediated response.

Trans Lipid Library: Synthesis of Docosahexaenoic Acid (DHA) Monotrans Isomers and Regioisomer Identification in DHA-Containing Supplements.

Docosahexaenoic acid (DHA) is a semiessential polyunsaturated fatty acid (PUFA) for eukaryotic cells that is found in natural sources such as fish and algal oils and widely used as an ingredient for omega-3 containing foods or supplements. DHA effects are connected to its natural structure with six cis double bonds, but geometrical monotrans isomers can be formed during distillation or deodorization processes, as an unwanted event that alters molecular characteristics and annihilates health benefits. The characterization of the six monotrans DHA regioisomers is an open issue to address for analytical, biological, and nutraceutical applications. Here we report the preparation, separation, and first identification of each isomer by a dual approach consisting of the following: (i) the direct thiyl radical-catalyzed isomerization of cis-DHA methyl ester and (ii) the two-step synthesis from cis-DHA methyl ester via monoepoxides as intermediates, which are separated and identified by nuclear magnetic resonance spectroscopy, followed by elimination for the unequivocal assignment of the double bond position. This monotrans DHA isomer library with NMR and GC analytical characterization was also used to examine the products of thiyl-radical-catalyzed isomerization of a fish oil sample and to evaluate the trans isomer content in omega-3 containing supplements commercially available in Italy and Spain.

Optimized Gas Chromatography-Mass Spectrometric Method to Profile Esterified Fatty Acids in Fish Roe and Fish Oil

A gas chromatography-mass spectrometric method was optimized and applied to profile the major esterified fatty acids in eight lyophilized fish roe species and three marketed fish oil capsules. The method was able to separate efficiently 36 standard fatty acids as methyl esters using Teknokroma fused silica capillary column, 60 m×0.25 mm×0.2 μm. However, only 20 major esterified fatty acids were determined in fish roe and fish oil samples. The total amount of esterified fatty acids in salmon wild oil, salmon cold water oil and cod liver oil capsules were 235.5, 145.8, and 195.5 mg/g, respectively. However, the total amount of esterified fatty acids in fish roe species ranged from 40 to 61 mg/g. The quantitation data were treated with statistical principal component analysis, and showed a characteristic score value for each type of fish roe. Each type of fish roe showed a characteristic content profile of 20 major esterified fatty acids. The highest concentration of esterified omega-3, was found in salmon fish roe (15.34 mg/g) and Mosa fish roe (13.15 mg/g). The maximum amount of esterified α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid were found in salmon wild oil (7.73 mg/g), salmon fish roe (6.09 mg/g), and Mosa fish roe (11.00 mg/g), respectively. The developed method was found to be applicable for qualification and characterization of fish roe and fish oil.

Stripping Voltammetric Determination of Mercury in Fish Oil Capsules Using a Screen‐printed Gold Electrode

AbstractThis work presents a simple electrochemical method for Hg determination in fish oil capsules using anodic stripping voltammetry (ASV) on a screen‐printed gold electrode (SPGE). Samples were treated in an ultrasonic bath for 15 min in the presence of a 1 : 1 (v/v) concentrated HCl/H2O2 mixture at room temperature. The limit of detection (LOD) was estimated as 0.25 μg L−1 (corresponding to 7.6 μg kg−1 of oil) with a linear range between 5 and 400 μg L−1 (90 s of deposition time). The analyzed fish oil samples presented Hg concentration below the LOD value. Recovery values for samples spiked with oil standard containing Hg ranged from 95 to 105 %. The method is precise (inter‐day and intra‐day RSD&lt;4 % for n=3) as well as the SPGE sensors (RSD=4.3 %, n=3), which were used continuously (around 100 analyses without replacement) under such oxidation media. The proposed method is feasible to be applied for on‐site determinations due to the portability of sensing and sample treatment approaches.

Speciation of trace mercury impurities in fish oil supplements

Fish oil supplement is becoming increasingly popular worldwide because of beneficial long-chain omega-3 polyunsaturated fatty acids. However, mercury (Hg) impurity causes considerable concern because of its toxicity and bioaccumulation in the food chain. In this work, Hg impurities were extracted from fish oil by liquid-liquid partitioning. The sample solution was then mixed with a reductant (0.4% anthranilic acid-20% formic acid) and sequentially exposed to 311 and 254 nm UV radiation. The resulting Hg0 vapor was detected by atomic fluorescence spectrometry. Speciation was fulfilled by solving a set of two linear equations. Recovery of MeHg+ was 73%; total Hg was validated by ICP-MS. This method achieved 0.50 and 0.63 ng mL−1 limits of detection for Hg++ and MeHg+, respectively. Average Hg++ and MeHg+ contents in fish oil samples (n = 38), 0.67 ± 0.45 and 1.1 ± 1.3 ng mL−1, respectively, were 2-3 orders of magnitude lower than those in fish.